CA2215039A1 - Immunoisolation - Google Patents

Abstract

Living cells that produce a therapeutic substance are immunoisolated by preparing a composite microreactor (10) containing internal particles (20) containing cells (30). The cells are embedded in matrix (40) which optionally contains coating (50). The internal particles are embedded in matrix (60) to form the composite microreactor which optionally has outer coating (70). The matrices and coatings are made immunoisolative by controlling porosity to exclude large molecules. The composite microreactor may be embedded in a matrix to form a double composite microreactor which optionally has an outer coating. The composite microreactor may be prepared by gelling alginate droplets containing cells with calcium ions to form the internal particles and then mixing the internal particles with alginate to form spheres that are the composite microreactor. The double composite microreactor is formed by mixing the composite microreactor with alginate and forming larger spheres.

Description

CA 0221~039 1997-09-09 IMMUNOISOLATION
B~ck~ronn-l of the Tnvention The invention relates to gel particles such as beads or spheres and methods of their m~mlf~l~*lre and use.
Transplantation of donor tissue into a recipient can be used to treat a wide variety of disorders, including heart ~ e~ce7 neoplastic ~ e~ce7 and endocrine ~ e~ee The clinical application of transplantation-based therapies are, however, limited by several factors. These factors include immune rejection of tr~n~pl~ntecl allogeneic or xenogeneic tissue by the tr~n~pl~nt recipient, a shortage of allogeneic donor-tissue, and donor-prop~g~tç~l immlm~
attack of recipient tissue (graft-versus-host-disease).
Tmmnne rejection of transplanted donor-tissue may be the most serious barrier tomore widespread availability of the benefits of transplantation-based therapies. Implantation of allogeneic or xenogeneic donor-tissue into an immunocompetent recipient generally results in a vigorous and destructive immune response directed against the donor-graft. Efforts to prevent immune-based destruction of donor tissue have generally fallen into two categories.
In one approach, efforts have been directed to moderating the recipient's immune response, e.g., by the induction of specific immlmological tolerance to transplanted tissue, or much more frequently, by the ~lmini~tration of broad-spectrum immlme suppress~nt~, e.g., cyclosporin. In the other major approach, efforts to prolong the acceptance of a donor-graft have been directed to rendering the donor-graft less susceptible to attack, e.g., by immunoisolating the donor-tissue by çn~rslll~ting it in a way which minimi7es contact of elements of the recipient's immune system with the encapsulated donor tissue.
Tmmlln~isolation is particularly attractive for the trç~tment of endocrine disorders or in hormone or en_yme replacement therapies. For example. the implantation of immunoisolated pancreatic islet cells can be used to restore glucose-responsive insulin function in a diabetic recipient. Islets can be placed in a mechanical enclosure, or can be coated with a material, which allows relatively free diffusion of glucose, insulin, nutrients, and cellular waste products but which is impervious to components of the recipient's immune system.
A microcapsule typically includes an inner core in which living cells are embedded and optionally an outer semipermeable coating. The outer coating often has a porosity which prevents components of the implant recipient's immune system from entering and destroying ~ the cells within the microcapsule. Gel microcapsules co.. l;l;.. in~ a small number of living cells have been used to transplant both allogeneic and xenogeneic donor cells into recipient 35 ~nim~l~ Several methods for micro~on~pslll~tin~ cells, e.g., pancreatic islet cells, in an ~lgin~te gel have been investip;~terl These include the ~lgin~te-polylysine technique - described in Lim et al., U.S. Patent No. 4,391,909 and Soon-Shiong et al.~ Transplantation, 54:769-774 (1992). the ~lgin~te-chitosan system described in Rha et al., U.S. Patent No.

WO 96/28029 ~ 9610313~;

4,744,933, and the polyacrylate en~ps~ ti--n method described in Sefton, U.S. Patent No.
4,353,888.
Sl~mm~ry of the Invention The inventors have discovered that composite microreactors can be used to 5 immllnoisolate donor cells. Composite microreactors of the invention allow donor cells, e.g., porcine, bovine, canine, or human islet cells to be sllcces~.~fully kansplanted into a recipient animal, e.g., mouse, rat, dog, or human with little or no need for immlmn 7u~pl~,.7S~lL or anti-fibrotic drugs.
Accordingly, in one aspect, the invention features, a composite microreactor which 10 includes:
(a) one, or a plurality, of an internal particle which includes:
(i) a source of a therapeutic substance, e.g., an islet;
(ii) an internal particle matrix, e.g., a gel core or a solid particle, which contacts the source;
(iii) (optionally) an internal particle coating enclosing the internal particle makix; and (b) a super matrix, e.g., a gel super matrix, in which the internal particle (or particles) is embedded; and (c) (optionally) an outer coating enclosing the super matrix, 20 the composite microreactor preferably providing a molecular weight cutoff that prevents molecules larger than about 400.000 daltons from coming into contact with the source and wherein a component of the composite microreactor, e.g., the internal particle, the super matrix, or both, are geometrically stabilized.
In ~lc:r~;llcd embo-liment~ the internal particle matrix is or includes: a gel, e.g., a 25 hydrogel, e.g., an ~l~in~te or agarose gel; a solid particle, e.g., a glass bead; a particle having pores or interstices. In preferred embocliment~ the internal particle matrix is other than a liquid. The intern~l particle matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA). or polyornithine (PLO).
In ~l~r~ d embo~liment~ the internal particle matrix hinders the passage, and preferably e~centi~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa. preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement.
In ~l~r~l~d embodiments the internal particle coating is or includes: a poly~mino~cid, e.g., polylysine (PLL) or PLO: a naturally occl-~ring substance, e.g.. chitosan; a synthetic ~ polymer e.g., PAN-PVC. A particularly ~l~;Ç~lc;d coating is polyamino acid. e.g., polylysine CA 0221~039 1997-09-09 WO g6/28029 PCT/US96103135 or polyo. ~ 1il le7 having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly ~,er~..ed are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa, about 1 kDa-less than 4 kDa, e.g., 3.7 kDa, or about 5 kDa to less than about 15 kDa, or about 5 kDa to less than about 10 5 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~ler~ d are poly~mino~cid, e.g., PLL or PLO, co~tinE.~ in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
Preferred coatings are volume-re(ll~ ing co~tin~s In ~cfe~lcd embotliment~ the int~rn~l particle coating hinders the passage, and preferably essenti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement, or recipient-derived cells.
In preferred embodiments the super matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~te or agarose gel. In ,olef~,.-ed embor1imentc the super matrix is other than a liquid.
The super matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g.~ it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO). A high molecular weight, molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more. can be added to the super matrix to confer immunoisolating properties.
In preferred embodiments the super matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~Icfcllcd embo~iment~ the super matrix has little or no ability to exclude low molecular weight species. e.g., IgG or complement, with this property being relegated to other components of the microcapsule.
In ~lcf~llcd embodiments the outer coating (which is optional) is or includes: apoly~minozlcid, e.g., polylysine (PLL) or PLO, a naturally occurring substance, e.g., chitosan.
A particularly preferred coating is polyamino acid, e.g., polyk~sine or polyo",ill~ e, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly ~lcre.lcd are polyamino acids. e.g.. polylysines, with a molecular weight of about I kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa. or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 35 kDa, e.g., 9.7 kDa. Also ~lcrclled are polyaminoacid, e.g., PLL or PLO, co~tin3~s in the range of 1 or 2-10 kD. preferably in the range of 1-2, 1-3, or 1-4 kD.

CA 0221~039 1997-09-09 WO 96/28029 PCI'IUS96/03135 In pl~r~lled'embo~liment~ the outer coating hinders the passage, and preferably çc~Pnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immlme system components such 5 as Ig molecules or complement, or recipient-derived cells.
In pl~r.,-led emborliment~ the ~ meter of the internal particle, before application of a volume-recl~lcin~ coating, is bc;Lwt:en 50 and 700 microns, more preferably between 100 an 500 microns, more preferably between 200 and 400 microns, and most preferably about 300 microns in ~ mptpr. The diameter of the internal particles, after application of a volume-reducing coating, is preferably belw~ 35 and 500 microns, more preferably between 75 and 400 microns, more preferably between 100 and 300 microns, and most preferably about 200 microns in ~ metPr In ~lcrt:lled embo~limentc the diameter ofthe composite microreactor is between 100microns and 4 millimeters, between 300 and 1200 microns, between 300 and 1500 microns, between 400 and 1000 microns, or between 400 and 800 microns. More preferably the diameter is about 600 microns.
In ~l~ft:lled embo~lim~nt~ the composite microreactor includes a plurality of internal particles, e.g., between 2 and 100, e.g., 2 and 10, internal particles.
In preferred embodiments the composite microreactor is a component of a higher 20 order composite, e.ga double composite, or a third order composite.
In preferred embodiment one or more components of the composite is geometricallystabilized. For example: the internal particle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days, prior to coating it: the internal particle is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days prior to embedding it in the super matrix In ~l~r~ d embodiments the internal particle coating has a lower molecular weight exclusion number than does the super matrix, the outer coating (if present), or the combination of the super matrix and the outer coating, e.g.. the internal particle coating 30 excludes recipient immune molecules, e.g., IgG or complement, and the super matrix, the outer coating (if present), or the combination of the super matrix and the outer coating, allows immlme molecules. e.g., IgG or complement, to pass but excludes the passage of recipient cells.
In preferred embol1imentc: the outer surface of the composite is biologically 35 compatible, e.g.. it is sufficiently smooth that it inhibits fibrotic encapsulation of the composite; the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic ~ .s~ tion of the composite but the surface of the internal CA 0221~039 1997-09-09 particle is not biologically con.~dlible, e.g., it is not sufficiently smooth to inhibit fibrotic P.n~ rsnl~tion.
In ~lcr~ d embo-lim~onte: at least one of the super matrix and the outer coatingplc~t;llL~ contact of fibrotic cells with the internal particle coating.
S In plc;r~ d embo-limente the composite microreactor further includes:
(b) one, or a plurality, of a second int~rn~l particle which includes:
(i) a second source of a th~;;-d~uLic sllhst~nce, e.g., an islet or a cell other than and islet;
(ii) a second internal particle matrix which includes the second source, (iii) (optionally) a second internal particle coating enclosing the second int~rn~l particle;
In ~l~r.,lled emborlim~nt~: super matrix prevents contact of fibrotic cells with the intt?rn~l particle coating; the super matrix and the outer coating (if present) is free of defects which arise from the inclusion of non-geometrically stabilized components, e.g., non-15 geometrically stabilized internal particles; at least two, or three, or four, components chosen from the group of the internal particle matrix, the internal particle coating, the super matrix, and the outer coating (if present), provides a molecular weight cutoffthat prevents molecules larger than about 150,000 daltons from coming into contact with the sources; the intf rn~l particle molecular weight cutoff is provided by a pore structure of the intern~l particle matrix, 20 and that pore structure results, e.g., from cross-linking of the internal particle gel; the molecular weight cutoffthe super matrix is provided by a pore structure of the super matrix.
Preferred embodiments lack an outer coating.
In preferred embo-liment~ the outer surface of the composite microreactor is a gel, e.g., an zllgin~te gel. In more p.er~ d embo~1iment~ the outer surface of the composite 25 microreactor is a gel, e.g., an ~1f in~te gel, the outer surface of which has been modified, e.g., by cross-linking, to produce a covalently modified gel surface, e.g.~ to form a coating.
In pler~ ed embo~liment~ the outer component of the composite microreactor, i.e., the component in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
In ~.~rell~d embo-iim~-nt~ one or more components of the composite microreactor is 30 of sufficient diameter, or of sufficient thickness, such that it imposes a substantial lli~t~nre (or separation) between recipient cells, e.g., Iymphocytes, macrophages, or NK cells, and the source of a thel~ulic substance. In more plefelled embodiments the thickness (e.g.. the t~n~ e between its inner surface and its outer surface) of a component, e.g., a matrix. e.g., a particle matrix or super matrix, is: at least 5, 10. 20. 50. 75. 100, or 200 microns. In more 35 ~ ,r~ d embo~1iment~ the ~ t~nce between recipient cells and the source of a therapeutic substance is: at least 5, 10, 20, 50, 75, 100. or 200 microns, sufficient such that exposure of the source of a th~_ldl~u~ic substance to small molecules (e.g., molecules which are not CA 0221~039 1997-09-09 W O 96/28029 ~ ~ llU~5f.'03135 excluded by a component which excludes IgG, e.g., cytokinPs, nitric oxide (NO), and other toxic moieties) released by recipient cells is ~.~hsl~.-liRlly reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, or 90 %; sufficient such that the concentration of small molecules (e.g., molecules which are not ~xclll-letl by the ~ eable barrier-components of the composite microreactor, e.g., cytokines, NO, and other toxic moieties) released by recipient cells is ~ b~l~..l;Rlly reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, or 90 % at the source of a theld~; ~uLic substance. In more ~lc;r~,lled embot1imt?nt~: the distance is supplied by one or both of the particle matrix and the super matrix.In pl~r~ ,d embotlim~nt~ one or more components of the composite microreactor is10 of sufficient diameter, or of sufficient thickness, such that it imposes a substantial distance (or separation) between recipient cells, e.g., lymphocytes, macrophages, or NK cells, and one or both of the source of a therapeutic s~hstRnrc or donor antigen (other than the th~ ;u-ic substance) released by the source (e.g., donor proteins, which could stimlllRte a recipient response against donor tissue). In more ~lert~ ,d embo-limentc the thickness (e.g., the 15 distance between its inner surface and its outer surface) of a component~ e.g., a matrix, e.g., a particle matrix or super matrix, is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more ~l~fe~ d embotlimentc the ~ tRnce between recipient cells and the source of a tht;la~ulic substance is: at least 5, 10, 20, 50, 75, 1OO, or 200 microns; sufficient such that the amount, number, or concentration of a donor antigen, released into the recipient, or contR~tin~
recipient cells, is sllhstRntiRlly reduced (e.g., by diffusion, or by trapping in or exclusion by the component or components which supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient such that contact of cells of the recipient with donor Rntigen~, e.g., proteins, which protrude from or extend through the internal particle matrix or internal particle coating, or both, is substRntiRlly reduced (e.g., by diffusion, or by trapping in or 25 exclusion by the component or components which supply the separation), e.greduced by at least 10, 20, 50, 75, or 90 %; sufficient to inhibit acute release of donor Rntigen~ In more d embof1imentc: the ~ tRnre or separation is supplied by one or both of the particle matrix and the super matrix.
In another aspect, the invention features, a composite microreactor which includes:
(a) one, or a plurality, of an internal particle which includes:
(i) a source of a therapeutic sllhstRnee, e.g., an islet;
(ii) an internal particle matrix which contacts the first source: and (iii) an internal particle coating of a polyamino acid enclosing the internRl particle matrix.
(b) a super matrix in which the internal particle (or particles) is embedded;
(c) (optionally) an outer coating enclosing the super matrix, CA 0221~039 1997-09-09 the composite microreactor preferably providing a molecular weight cutoff that plc~c~
molecules larger than about 400,000 daltons from coming into contact with the source.
In plcfcllcd embo~limentc the int~rn~l particle matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~l~in~tt- or agarose gel; a solid particle, e.g., a glass bead, a particle having S pores or interstices. In plcr~lcd embo~limentc the intern~l particle matrix is other than a liquid. The intern~l particle matrix can include snbst~n~ec which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), poly~lylcne sulfonic acid (PSA), or polyc,. . ,;I~ e (PLO).
In plcrcllcd embo-limt-ntc the internal particle matrix hinders the passage, andpreferably eccenti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~lcÇ~ ,d embo(1imentc the internal particle coating is or includes: a poly~min~ iqcid, e.g., polylysine (PLL) or PLO. A particularly l~lcrcllcd coating is a polyamino acid, e.g., polylysine or polyornithine. having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly plcr~,llcd are polyamino acids, e.g., polylysines. with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~lcrcllcd are poly:~nnino~cid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Plcr~:llcd coatings are volume-re~l~-cing coatings.
In preferred embodiments the internal particle coating hinders the passage, and preferably ecsenti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa. and most preferably more than about 400 kDa; or of immune system colll~ollents such as Ig molecules or complement; or recipient-derived cells.
In preferred embodiments the super matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~t~- or agarose gel. In ~lcrcllcd embodiments the super matrix is other than a liquid.
The super matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g.. it can include polyethelyne oxide (PEO). polystyrene sulfonic acid (PSA), or polyornithine (PLO). A high molecular weight, molecule, e.g., a polymer, e.g., - PEO, with a molecular w eight of 1-8 million daltons. or more. can be added to the super matrix to confer immunoisolating ~lopcllies.
In ~lcrcllcd embo~limentc the super matrix hinders the passage. and preferably e~sçnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 CA 0221~039 1997-09-09 WO 96r28029 PCT/US96103135 kDa, and most preferably more than about 400 kDa; or of imml-n~ system components such as Ig molecules or complement, or recipient-derived cells.
In ~lcr~ ,d embo~liment~ the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this property being relegated to S other components of the microcapsule.
In ~l~r~llc;d embot1iment~ the outer coating (which is optional) is or incl~ltles: a poly~min--~cid, e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g., chitosan.
A particularly pler~..ed coating is polyamnino acid, e.g., polylysine or pol~o. ,.illl;...o, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly ~i~r~ ,d are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about S kDa to less than 15 IcDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also pl~fe~led are polyaminoacid, e.g., PLL or PLO. coatings in the range of 1 or 2- 10 kD, preferably in the range of 1 -2, 1 -3, or l -4 kD.
In preferred embo-liment~ the outer coating hinders the passage, and preferably ecs~nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement, or recipient-derived cells.
In preferred embo-liment~ the tli~meter of the internal particle, before application of a volume-reducing coating, is b~Lw~ell 50 and 700 microns, more preferably between 100 and 500 microns, more preferably between 200 and 400 microns. and most preferably about 300 microns in diameter. The t1i~meter of the internal particles. after application of a volume-re~ ring coating is preferably between 35 and 500 microns. more preferably between 75 and 400 microns, more preferably between 100 and 300 microns~ and most preferably about 200 microns in diameter.
In preferred embodiments the rli~mett-r ofthe composite microreactor is between 100 microns and 4 millimeters, between 300 and 1200 microns. between 300 and 1500 microns, between 400 and 1000 microns, or between 400 and 800 microns. More preferably the diameter is about 600 microns.
In ~.er~,led embo-liment~ the composite microreactor includes a plurality of internal particles, e.g., between 2 and 100, e.g., 2 and 10, internal particles.
In preferred embo-1imente the composite microreactor is a component of a higher order composite. e.g.. a double composite~ or a third order composite.
In p~r~ d embodiment one or more components of the composite is geometrically stabilized. For example: the first internal particle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days. e.g.. for between 12 hours and 4 or 5 CA 0221~039 1997-09-09 days, prior to coating it; the first int~rn~l particle is geometrically stabilized, e.g., by allowing it to age for b~;lwt;c;ll 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days prior to embedding it in the super matrix In plc;r~ d embo~1iment~ the int~rn~l particle coating has a lower molecular weight S exclusion number than does the super matrix, the outer coating (if present), or the combination of the super matrix and the outer coating, e.g., the intPrn~l particle coating excludes recipient immllne molecules, e.g., IgG or complement, and the super matrix, the outer coating (if present), or the combination of the super matrix and the outer coating, allows immtlne molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient 1 0 cells.
In plt;:rell~d embo-limt-nt~: the outer surface of the composite is biologicallycompatible, e.g., it is sufficiently smooth that it inhibits fibrotic encapsulation of the composite; the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic e~ pslll~tion of the composite but the surface of the internal particle is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic encapsulation.
In ~l~fell~d embo~liment~ at least one of the super matrix and the outer coatingprevent contact of fibrotic cells with the internal particle coating.
In ~-ef~.led embo-liment~ the composite microreactor further includes:
one. or a plurality, of a second intPrn~l particle which includes:
(i) a second source of a therapeutic substance, (ii) a second internal particle matrix which includes the second source, (iii) a second internal particle coating enclosing the second intern~l particle matrix, the second internal particle being embedded in the super matrix.
In preferred embo-liment~: super matrix prevents contact of fibrotic cells with the int~rn~l particle coating; the super matrix and the outer coating (if present) is free of defects which arise from the inclusion of non-geometrically stabilized components, e.g., non-geometrically stabilized int.-rn~l particles; at least two, or three, or four, components chosen from the group of the intern~l particle matrix, the internal particle coating, the super matrix, and the outer coating (if present), provides a molecular weight cutoff that prevents molecules larger than about 150,000 daltons from coming into contact with the sources; the internal particle molecular weight cutoff is provided by a pore structure of the internal particle matrix, and that pore structure results, e.g., from cross-linking of the internal particle gel; the molecular ~-eight cutoffthe super matrix is provided by a pore structure of the super matrix.
Preferred embo~lim~nt~ lack an outer coating.
In p-er~.led emborliment~ the outer surface of the composite microreactor is a gel.
e.g., an ~lginz~te gel. In more ~l~r~lled embo~liment~ the outer surface of the composite CA 0221~039 1997-09-09 WO 96/28029 PCr/US96/03135 microreactor is a gel, e.g., an ~lgin~te gel, the outer surface of which has been modified, e.g., by cross-linking, to produce a covalently modified gel s~ P, e.g., to form a coating.
In ~lcr~led embo~1imPnt~ the outer component of the composite mi-;lolca ;lol~ i.e., the component in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
S In pl~r~led embo-limPnt~ one or more col.. ponents of the composite microreactor is of sufficient diameter, or of sufficient thickness, such that it imposes a substantial ~1ict~nC e (or separation) between recipient cells, e.g., Iymphocytes, macrophages, or NK cells, and the source of a therapeutic substance. In more p.er~ d embo~limPnt~ the thickness (e.g., the distance between its inner surface and its outer surface) of a component, e.g., a matrix, e.g., a particle matrix or super matrix, is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more ~lefelled embodiments the distance between recipient cells and the source of a th~ uLic substance is: at least 5, 10, 20, 50, 75, 100, or 200 microns; sufficient such that exposure of the source of a therapeutic substance to small molecules (e.g., molecules which are not excluded by a component which excludes IgG, e.g., cytokines, nitric oxide (NO), and other 15 toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g., reduced at least 10. 20, 50, 75, or 90 %; sufficient such that the concentration of small molecules (e.g., molecules which are not excluded by the semipermeable barrier-components of the composite microreactor, e.g., cytokinPc, NO, and other toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, 20 or 90 % at the source of a therapeutic substance. In more p.er~ d emborlimPnt~: the distance is supplied by one or both of the particle matrix and the super matrix.In preferred embo~im~ntc one or more components of the composite microreactor isof sufficient diameter, or of sufficient thickness, such that it imposes a substantial distance (or separation) between recipient cells, e.g.. Iymphocytes, macrophages, or NK cells, and one or both of the source of a therapeutic substance or donor antigen (other than the therapeutic substance) released by the source (e.g., donor proteins, which could stim~ te a recipient response against donor tissue). In more pler~ d embodiments the thickness (e.g., the distance between its inner surface and its outer surface) of a component, e.g., a makix, e.g., a particle makix or super matrix. is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more p-~re-i cd embodiments the distance between recipient cells and the source of a thtila~u~ic substance is: at least 5, 10, 20, 50, 75, 100, or 200 microns; sufficient such that the amount, number, or concentration of a donor antigen, released into the recipient, or c~ nt~cting recipient cells, is substantially reduced (e.g., by diffusion, or by trapping in or exclusion by the component or components which supply the separation). e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient such that contact of cells of the recipient with donor antigens, e.g., proteins, which protrude from or extend through the jntPrn~l particle matrix or int~rn~l particle coating, or both, is substantially reduced (e.g., by diffusion, or by trapping in or CA 0221~039 1997-09-09 exclusion by the co~ ,onc--l or components which supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %, sufficient to inhibit acute release of donor antigens. In more ~lcrell~,d embo-liment~: the (1ict~nce or separation is supplied by one or both of the particle matrix and the super matrix.
S In another aspect. the invention fe~lu.es, a composite microreactor which includes:
(a) one, or a plurality, of an int~rn~l particle which includes:
(i) a source of a thel~l~e~-~ ic substance, e.g., an islet;
(ii) an internal particle matrix which contacts the source, and (iii) an internal particle coating enclosing the internal particle matrix, (b) a (non-liquid) gel super matrix in which the internal particle (or particles) is embedded, (c) (optionally) an outer coating enclosing the super matrix, the composite microreactor preferably providing a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from coming into contact with the sources.
In plcr~"led embo~liment~ the int~ l particle matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~t~ or agarose gel; a solid particle, e.g., a glass bead; a particle having pores or interstices. In ~lerellcd embo~liment~ the intPrn~l particle matrix is other than a liquid. The internal particle matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyo-l-ilhille (PLO).
In ~rerelled embo~1iment~ the int~rn~l particle matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa: or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In plerellcd embotliment~ the int~rn~l particle coatin~ is or includes: a polyaminoacid, e.g., polylysine (PLL) or PLO, a naturally occurring substance, e.g.. chitosan; a synthetic polymer e.g., PAN-PVC. A particularly p~r~ ed coating is polyamino acid. e.g., polylysine or polyol.li~lh~e, having a molecular weight of less than 15 kDa, more preferably of less than 30 10 kDa, more preferably of less than S kDa. Particularly plerelled are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa, about 1 kDa-less than 4 kDa.
e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g. 9.7 kDa. Also ~Jlerelled are polyzlmino~rid. e.g., PLL or PLO, coatings in the range of 1 or '~-10 kD, preferably in the range of 1-'~. 1-3, or 1-4 kD.
~ 35 Plere.,~,d coatings are volume-reducing coatings.
In ~ler~ d embodiments the int~ l particle coating hinders the passage, and preferably Pcsçnti~lly completely prevent the passage of: molecules having a molecular CA 022l~039 l997-09-09 WO 96/28029 PCI~/US96/03135 weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immlmP system components such as Ig molecules or complement; or recipient-derived cells.
In ~rer.,"ed embo~liment~ the super matrix is or includes: a gel, e.g., a hydrogel, e.g., S an ~lgin~t~ or agarose gel. The super matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), poly~lyl~ne sulfonic acid (PSA), or polyf, .,i~ (PLO). A high molecular weight, molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super matrix to confer immunoisolating properties.
In ~rc:r~;lled embo-liment~ the super matrix hinders the passage, and preferablyessentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa. preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~lef~;llc~d emborliment~ the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this ~ro~l Ly being relegated to other components of the microcapsule.
In ~ fellc~d embo~lim~nt~ the outer coating (which is optional) is or includes: a polyaminoacid, e.g.. polylysine (PLL) or PLO; a naturally occurring substance, e.g., chitosan.
20 A particularly plerelled coating is a polyamino acid, e.g.. polylysine or polyornithine~ having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly pl~r~ d are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about S kDa to less than 15 kDa, or about S kDa to less than about 10 kDa, e.g., 9 kDa-10 25 kDa, e.g., 9.7 kDa. Also plcr~ d are polyaminoacid. e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD. preferably in the range of 1-2, 1-3. or 1-4 kD.
In ~ r~ d embo~limt-nt~ the outer coating hinders the passage, and preferably e~enti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa preferably more than about 100 kDa. preferably more than about 150 30 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In pl~rtlled embo-1im~nt~ the diameter of the internal particle, before application of a volume-re~ cin~ coating, between 50 and 700 microns. more preferably between 100 and 500 microns. more preferably between 200 and 400 microns. and most preferably about 300 35 microns in ~ meter. The diameter of the internal particles~ after application of a volume-reducing coating, is preferably bet~,veen 35 and 500 microns. more preferably between 75 and .

CA 0221~039 1997-09-09 WO 96/28029 ~ 03135 400 microns, more preferably between 100 and 300 microns, and most preferably about 200 microns in flis3m~tPr.
- In ~ rt~ d embo~1iment~ the ~ m~tt?r of the composite microreactor is between 100 microns and 4 millimPt~rs, between 300 and 1200 microns, between 300 and 1500 microns, bGlW~t~ll 400 and 1000 microns, or bc;Lwe~;ll 400 and 800 microns. More preferably the met~r is about 600 microns.
In preferred embo-lim~nt~ the composite microreactor includes a plurality of int~
particles, e.g., between 2 and 100, e.g., 2 and 10, intt-rn~l particles.
In preferred embo-lim~ntc the composite microreactor is a con~ollent of a higherorder composite, e.g., a double composite, or a third order composite.
In preferred embodiment one or more components of the composite is geometricallystabilized. For example: the first internal particle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days, prior to coating it; the first internal particle is geometrically stabilized, e.g., by allowing 15 it to age for betueen 2 hours and 14 days, e.g.. for between 12 hours and 4 or 5 days prior to embedding it in the super matrix In preferred embo-1iment~ the internal particle coating has a lower molecular weight exclusion number than does the super matrix, the outer coating (if present), or the combination of the super matrix and the outer coating, e.g., the int~ l particle coating 20 excludes recipient immune molecules, e.g., IgG or complement, and the super matrix, the outer coating (if present). or the combination of the super matrix and the outer coating, allows immune molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In preferred embodiments: the outer surface of the composite is biologically 25 compatible~ e.git is sufficiently smooth that it inhibits fibrotic encapsulation of the composite, the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic encapsulation of the composite but the surface of the intern~l particle is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic encapsulation.
In ple~~ d embo(1iment~ at least one of the super matrix and the outer coating prevent contact of fibrotic cells with the internal particle coating.
In ~lefc.led embodiments the composite microreactor further includes:
- one, or a plurality. of a second internal particle comprising:
(i) a second source of a therapeutic substance.
(ii) a second internal particle matrix which includes the second source, (iii) (optionally) a second int~rn~l particle coating enclosing the second internal particle matrix, the second internal particle being CA 0221~039 1997-09-09 embedded in the super matrix.
In pleL~.led embodim~nt~: super matrix ~ contact of fibrotic cells with the intern~l particle coating; the super matrix and the outer coating (if present) is free of defects which arise from the inclusion of non-geometrically stabilized coll~ollellL~, e.g., non-S geometrically stabilized int~.rn~l particles, at least two, or three, or four, components chosenfrom the group of the internal particle matrix, the intern~l particle cs~ting, the super matri x, and the outer coating (if present), provides a molecular weight cutoff that pl~t;llL~ molecules larger than about 150,000 daltons from coming into contact with the sources, the int~rn~l particle molecular weight cutoff is provided by a pore structure of the internal particle matrix, 10 and that pore structure results, e.g., from cross-linking of the internal particle gel; the molecular weight cutoff the super matrix is provided by a pore structure of the super matrix.
Pl~r~l..,d emborliment~ lack an outer coating.
In ~ f~ d embo~liment~ the outer surface of the composite microreactor is a gel,e.g., an ~l~in~te gel. In more preferred embodiments the outer surface of the composite microreactor is a gel, e.g., an ~lgin~te gel, the outer surface of which has been modified, e.g., by cross-linking, to produce a covalently modified gel surface, e.g., to form a coating.
In ~l~f~ d embo~lim~ntc the outer component of the composite microreactor, i.e., the component in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
In plcf~ d embo~lim~-nt~ one or more components of the composite microreactor isof sufficient diarneter, or of sufficient thickness, such that it imposes a substantial rii~t~n~ e (or separation) between recipient cells, e.g., lymphocytes, macrophages, or NK cells, and the source of a therapeutic substance. In more yler~lled embodiments the thickness (e.g., the distance between its inner surface and its outer surface) of a component, e.g., a matrix, e.g., a particle matrix or super matrix, is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more l~ler~ d embodiments the distance between recipient cells and the source of a therapeutic substance is: at least 5. 10. 20, 50. 75, 100, or 200 microns; sufficient such that exposure of the source of a therapeutic substance to small molecules (e.g., molecules which are not excluded by a component which excludes IgG, e.g., cytokines, nitric oxide (NO), and other toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, or 90 %; sufficient such that the concentration of small molecules (e.g., molecules which are not excluded by the sellli~t:lllleable barrier-components of the composite microreactor, e.g.. cytokines, NO, and other toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, or 90 % at the source of a therapeutic substance. In more ~lere~ d emborliment~: the 35 ~ t~nee is supplied by one or both of the particle matrix and the super matrix.
In preferred embotliment~ one or more components of the composite microreactor is of sufficient diameter, or of sufficient thickness, such that it imposes a substantial ~ t~n~e (or CA 0221~039 1997-09-09 WO 96/28029 . PCTIU~ /t,313S

se~dldlion) bclwccll recipient cells, e.g., Iymphocytes, macrophages, or NK cells, and one or both of the source of a thrl~ ;c s~lbst~nce or donor antigen (other than the thc.d~culic ce) released by the source (e.g., donor proteins, which could stim~ te a recipient response against donor tissue). In more plcrcl.cd embo~limente the thickness (e.g., the S rliet~nre bcLwccll its inner surface and its outer surface) of a component, e.g., a matrix, e.g., a particle matrix or super matrix, is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more cr~"lcd embo-1imente the rii~t~nce between recipient cells and the source of a therapeutic sllhst~n~e is: at least 5, 10, 20, 50, 75, 100, or 200 microns; sufficient such that the amount, number, or conccllLldlion of a donor antigen, released into the recipient, or cnnt~cting 10 recipient cells, is substantially reduced (e.g., by diffusion, or by trapping in or exclusion by the component or components which supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient such that contact of cells of the recipient with donor antigens, e.g., ~teh~s, which protrude from or extend through the intern~l particle matrix or int~rn~l particle coating, or both. is substantially reduced (e.g., by diffusion. or by trapping in or 15 exclusion by the component or components which supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient to inhibit acute release of donor antigens. In more ~rcr~ ,d embodiments: the ~ t~n~e or separation is supplied by one or both of the particle matrix and the super matrix.
In another aspect. the invention features, a composite microreactor which includes:
(a) one, or a plurality~ of a geometrically stabilized internal particle which includes:
(i) an islet, (ii) an internal particle gel matrix in which the islet is embedded, (iii) an internal particle coating of polylysine enclosing the internal particle matrix; and (b) a gel super matrix in which the internal particle (or particles) is embedded, the composite microreactor providing a molecular weight cutoffthat prevents molecules larger than about 400~000 daltons from coming into contact with the sources.
In preferred embodiments the int~rn~l particle gel matrix is or includes: a hydrogel, e.g., an ~Igin~te or agarose gel. In preferred embodiments the internal particle matrix is other 30 than a liquid. The internal particle matrix can include sllbst~nces which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithin~ (PLO).
- In ~l~r~ d embodiments the int~rn~l particle coating is or includes: a polylysine (PLL) having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, 35 more preferably of less than 5 kDa. Particularly ~l~f~:lled are polylysines with a molecular weight of about 1 kDa-4 l;Da (or about 1 kDa-less than 4 kDa) e.g.. 3.7 kDa, or about S kDa to less than 15 kDa. or about 5 kDa to less than about 10 kDa. e.g.. 9 kDa-10 kDa, e.g., 9.7 CA 0221~039 1997-09-09 WO 96/2,8029 ~ /U~ 03135 kDa. Also ~ f.,~led are poly~min- iqc id, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Preferred coatings are volume-recl~ ing co~tings In ~lcr~ d emborlim~nt~ the internal particle coating hinders the passage, and preferably ees~nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immllne systemc.,lll~ol~ents such as Ig molecules or complement; or recipient-derived cells.
In ~lertlled embo~limentc the gel super matrix is or includes: a hydrogel, e.g., an 10 ~l~in~t~ or agarose gel. In ~lc;r~lled embo-1iment~ the super matrix is other than a liquid.
The super matrix can include sl~bst~n~ which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or poly~-rnithine (PLO). A high molecular weight. molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super 15 matrix to confer immunoisolating f~lopc~lLies.
In ~lerell~d embo-liment~ the super matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immlme system components such 20 as Ig molecules or complement; or recipient-derived cells.
In ~ler~ d embodiments the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this property being relegated to other components of the microcapsule.
In ~l~rt;lled embo-liment~ the diameter of the internal particle, before application of a 25 volume-re-lllcing coating. between 50 and 700 microns. more preferably between 100 and 500 microns, more preferably between 200 and 400 microns~ and most preferably about 300 microns in diameter. The diameter of the internal particles. after application of a volume-reducing coating. is preferably between 35 and 500 microns, more preferably between 75 and 400 microns, more preferably between 100 and 300 microns, and most preferably about 200 30 microns in diameter.
In ~l~rell~d embo-liment~ the diameter of the composite microreactor is between 100 microns and 4 millimeters, between 300 and 1200 microns. between 300 and 1500 microns, between 400 and 1000 microns, or between 400 and 800 microns. More preferably the mett~r is about 600 microns.
In ~ler~ll.d embodiments the composite microreactor includes a plurality of int~rnzil particles, e.g., bet~een 2 and 100, e.g., 2 and 10, internal particles.

CA 0221~039 1997-09-09 In ~lèr~ d embo-liment~ the composite microreactor is a co~ onent of a higher order composite, e.g., a double composite, or a third order composite.
In plerellèd embodiment one or more components of the composite is geometric~llystabilized. For example: the first intern~l pOEticle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours OEnd 4 or S
days, prior to coating it; the first intern~l pOEticle is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for beLweell 12 hours OEnd 4 or 5 days prior to embedding it in the super matrix In ~lere.l~,d embo~1imPrlt~ the intern~l particle coating has a lower moleculOE weight 10 exclusion number than does the super matrix; the intern~l particle coating excludes recipient immune molecules, e.g., IgG or complement, and the super matrix allows imml-ne molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In plcfclled embo-liment~: the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibits fibrotic encapsulation of the 15 composite; the outer surface ofthe composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic encapsulation of the composite but the surface of the internal pOEticle is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic encapsulation.
In ~lerel 1 ed embodiments: at least one of the super matrix and the outer coating 20 prevent contact of fibrotic cells with the internal pOEticle coating.
In plefellèd embo-liment~ super matrix prevents contact of fibrotic cells with the intern~l particle coating; the super matrix and the outer coating (if present) is free of defects which arise from the inclusion of non-geometrically stabilized components, e.g., non-geometrically stabilized internal particles, at least two, or three, or four. components chosen 25 from the group of the internal particle matrix~ the internal pOEticle coating, the super matrix, OEnd the outer coating (if present), provides a molecular weight cutoff that prevents molecules lOEger than about 150,000 daltons from coming into contact with the sources; the internal pOEticle molecular weight cutoff is provided by a pore structure of the int.orn~l pOEticle matrix, OEnd that pore structure results, e.g., from cross-linking of the intçm~l pOEticle gel; the 30 molecular weight cutoff the super matrix is provided by a pore structure of the super matrix.
Preferred embodiments lack an outer coating.
In plerelled embotliment~ the outer surface of the composite microreactor is a gel, e.g.. an ~lginz~te gel. In more ~lcrelled embotliment~ the outer surface of the composite microreactor is a gel, e.g.~ an ~l~in~te gel, the outer surface of which has been modified, e.g., 35 by cross-linking, to produce a covalently modified gel surface, e.g., to form a coating.
In ~refelled embo-lim~nt~ the outer collll)onent ofthe composite microreactor~ i.e.~ the component in contact with the recipient, is at least S0, 75, 90, 95, 97. or 98 %, water.

CA 0221~039 1997-09-09 WO 96/28029 PCTIUS96/0313~i In plcr~ ;d embo~liment~ one or more components of the composite microreactor isof sufficient diameter, or of sufficient thicknP~, such that it imposes a ~ . " i~ t~n~e (or sep~r~tion) between recipient cells, e.g., lymphocytes, macrophages, or NK cells, and the source of a th~ culic substance. In more p,ef~.,ed embo-liment~ the thickne~c (e.g., the 5 distance between its inner surface and its outer surface) of a co~ ollent, e.g., a matrix, e.g., a particle matrix or super matrix, is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more ,r~ ,d embo~ the tli~t~n~e between recipient cells and the source of a th~,~.,uLic s~bst~nce is: at least 5, 10, 20, 50, 75, 100, or 200 microns; sufficient such that exposure of the source of a therapeutic substance to small molecules (e.g., molecules which are not 10 excluded by a component which excludes IgG, e.g., cytokines, nitric oxide (NO), and other toxic moieties) released by recipient cells is subst~nti~lly reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, or 90 %; sufficient such that the concentration of small molecules (e.g., molecules which are not excluded by the semipermeable barrier-components of the composite microreactor, e.g., cytokines, NO, and other toxic moieties) released by recipient cells is subst~nti~lly reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75, or 90 % at the source of a therapeutic substance. In more ~ler~ ;d embo-liment~: the distance is supplied by one or both of the particle matrix and the super matrix.In preferred embot1iment~ one or more components of the composite microreactor is of sufficient diameter, or of sufficient thickness, such that it imposes a substantial distance (or separation) between recipient cells~ e.g., lymphocytes, macrophages, or NK cells, and one or both of the source of a therapeutic s~lbst~nre or donor antigen (other than the tht~ uLic substance) released by the source (e.g., donor proteins, which could stim~ te a recipient response against donor tissue). In more ~ult;r~ ,d embo-liment~ the thickness (e.g., the distance between its inner surface and its outer surface) of a component, e.g., a matrix, e.g., a particle matrix or super matrix, is: at least 5, 10~ 20, 50, 75, 100, or 200 microns. In more p-efe..~d embodiments the distance between recipient cells and the source of a tht.~c;ulic substance is: at least 5~ 10, 20, 50~ 75, 100, or 200 microns; sufficient such that the amount, number, or concentration of a donor antigen, released into the recipient, or contacting recipient cells, is s~lbst~nti~lly reduced (e.g., by diffusion, or by trapping in or exclusion by the component or components which supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient such that contact of cells of the recipient with donor antigens, e.g., proteins, which protrude from or extend through the internal particle matrix or intern~l particle co~ting, or both, is substantially reduced (e.g., by diffusion, or by trapping in or exclusion by the component or components which supply the separation), e.g., reduced by at least 10? 20, 50, 75, or 90 %: sufficient to inhibit acute release of donor antigens. In more ~-~r~ d embo~limentc: the distance or separation is supplied by one or both of the particle matrix and the super matrix.

CA 0221~039 1997-09-09 WO 96/28029 ~ 03135 The iLvt;llLol~ have discovered that a single or simple composite microreactor, e.g., one which in~ d~s one or more microç~psl-lPs cont~inp~l in a larger particle, can be used to immlmc)isolate donor tissue. They have further discovered that higher order composites, e.g., double composites, which include one or more single composites contained in a larger S particle, are also effective for immunoisolating donor tissue.
Accordingly, the invention features, a double composite microreactor which includes:
(1) one, or a plurality, of an intern~l particle which includes:
(a) a source of a therapeutic substance, e.g., an islet, (b) an internal particle matrix which contacts the source; and (c) (optionally) an intern~l particle coating enclosing the first intPrn~l particle matrix;

(2) one, or a plurality, of a particle which includes:
(a) the internal particle or particles of ( 1 ) (b) a particle matrix in which the intern~l particle (or intern~l particles) is embedded; and (c) (optionally) a particle coating enclosing the particle;

(3) a super matrix in which the particle (or particles) of (2) is embedded, and (4) (optionally) a super matrix or outer coating, e.g., of polylysine enclosing the super matrix.
In ~,~ere,led embolliment~ the int.?rn~l particle matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~te or agarose gel, a solid particle, e.g., a glass bead, a particle having pores or interstices. In pl~rt;:lled embo-liment~ the internal particle matrix is other than a liquid. The internal particle matrix can include subst~ncc~ which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), 25 polystyrene sulfonic acid (PSA), or polyornithine (PLO).
In preferred embodiments the intern~l particle matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of imm~ne system 30 components such as Ig molecules or complement; or recipient-derived cells.
In ~l~r~ d embo-1iment~ the intern~l particle coating is or includes: a polyaminoacid, e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g.. chitosan, a synthetic polymer e.g., PAN-PVC. A particularly plef~ d coating is polyamino acid. e.g., polylysine or polyc ll,iLhille~ having a molecular weight of less than 15 kDa~ more preferably of less than 35 10 kDa, more preferably of less than 5 kDa. Particularly ~l~f~lled are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa, about 1 kDa-less than 4 kDa.
e.g., 3.7 kDa, or about S kDa to less than 15 kDa. or about S kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~,ef~ d are polyaminoacid, e.g.. PLL or PLO, CA 0221~039 1997-09-09 WO 96J28029 PCI'IUS96/03135 co~tin~.~ in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
Preferred co~tings are volume-rec~ in~ CQ~tin~.c In ~lcr.,.~ed embo-liment~ the intt-rn~l particle coating hinders the passage, and preferably ~o~s~nti~lly completely prevent the passage of: molecules having a molecular S weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immllne systemcomponents such as Ig molecules or complement; or recipient-derived cells.
In ~r~:r~ d embo-lim~nt~ the rli~m~er of the internal particle, before application of a volume-reducing coating, between 50 and 700 microns, more preferably between 100 and 10 500 microns, more ~ler~lably between 200 and 400 microns, and most preferably about 300 microns in diameter. The diameter of the intPrn~l particles, after application of a volume-retlucin~ coating, is preferably between 35 and 500 microns, more preferably bet~,veen 75 and 400 microns, more preferably between 100 and 300 microns, and most preferably about 200 microns in diameter.
In preferred embodiments the particle matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~te or agarose gel. In ~lcr~ d embo-1imPnt~ the particle matrix is other than a liquid. The particle matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), poly ,Ly.e..e sulfonic acid (PSA). or polyornithine (PLO).
In p.er~.lcd embodiments the particle matrix hinders the passage, and preferablyç~çnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa. preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immlme system components such as Ig molecules or complement, or recipient-derived cells.
In ~-er~ d embodiments the particle coating is or includes: a polyaminoacid, e.g., polylysine (PLL) or PLO, a naturally occurring ~llbst~nce, e.g., chitosan, a synthetic polymer e.g., PAN-PVC. A particularly ~lert;llc:d coating is polyamino acid. e.g., polylysine or polyornithine, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly ~left;:llcd are polyamino acids, e.g., polylysines, with a molecular weight of about I kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about S kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa. e.g., 9.7 kDa. Also ~lef~ d are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3. or 1-4 kD.
Preferred coatings are volume-reducing co~ting~
In ~l~r~ d embo-lim~nt~ the particle coating hinders the passage, and preferablyç~enti~lly completelv prevent the passage of: molecules having a molecular weight of more than about S0 kDa. preferably more than about 100 kDa, preferably more than about 150 CA 0221~039 1997-09-09 WO 96128029 PCI~/U~ 3135 kDa, and most preferably more than about 400 kDa; or of immllne system components such as Ig molecules or complement; or recipient-derived cells.
In ~crcl~,d embo-lim~nt~ the diameter of the particle, before application of a volume-reducing co~ting, is between 200 and 1,000 microns, more preferably between 400 and 800 5 microns, more preferably between 500 and 700 microns, and most preferably about 600 microns in ~ m~t~r. The ~ met~r of the particles, after application of a volume-re~ in~
co~ting, is preferably bc;Lweell 100 and 700 microns, more preferably between 250 and 550 microns, more preferably between 300 and 500 microns, and most preferably about 400 microns in t1i~m~ter.
In ~l~r~.lled embo-liment~ the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this ~lo~ Ly being relegated to other components of the microc~psllle.
In plert;llc~d embotliment~ the gel super matrix is or includes: a hydrogel, e.g., an ~lgin~te or agarose gel. In ~l~r~lled embodiments the super matrix is other than a liquid.
15 The super matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyolllilhine (PLO). A high molecular weight, molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super matrix to confer immunoisolating plvp~l~ies.
In l~lercllc~d embo-liment~ the super matrix hinders the passage, and preferably~ossenti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immllne system components such as Ig molecules or complement; or recipient-derived cells.
In plcfcllc:d embo-liment~ the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement. with this property being relegated to other components of the microcapsule.
In plefelled embo-limentc the super matrix coating (which is optional) is or includes:
a polyaminoacid, e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g.~
30 chitosan. Particularly plc:rtlled coating is polyamino acid. e.g., polylysine or polyolllilhille, having a molecular weight of less than 15 kDa. more preferably of less than 10 kDa. more preferably of less than S kDa. Particularly ~lcfclled are polyamino acids. e.g., polylysines, - with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g.. 3.7 kDa, or about S kDa to less than 15 kDa, or about S kDa to less than about 10 kDa, e.g., 9 kDa-10 35 kDa, e.g.~ 9.7 kDa. Also ~ rell~id are poly~min- ~cid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2. 1-3, or 1-4 kD.

CA 0221~039 1997-09-09 WO 96/28029 PCT/U' ,5/0~135 In pl~;f~,.,ed emborliment~ the particle coating hinders the passage, and preferably ess~nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 lcDa, and most preferably more than about 400 kDa; or of immlm.? system components such 5 as Ig molecules or complement, or recipient-derived cells.
In pl~r~,.,d emborliment~ the fli~met~r of the double composite microreactor is,before application of a volume-reducing coating, between 400 and 1,500 microns, more preferably between 500 and 1,300 microns, more preferably between 600 and 1,100 microns, and most preferably about 900 microns in diameter. The diameter of the double composite 10 microreactor is, after application of a volume-reducing coating, is preferably between 300 and 1,300 microns, more preferably between 400 and 1,200 microns, more preferably between 500 and 1,000 microns, and most preferably about 800 microns in ~ meter.In preferred embo-liment~ the composite microreactor includes a plurality of int~
particles, e.g., between 2 and 100, e.g., between 2 and 10, internal particles.
In preferred embodiments the composite microreactor includes a plurality of particles, e.g., between 2 and 100, e.g., 2 and 10, particles.
In ~,~rel.cd embodiments the double composite microreactor is a component of a higher order composite, e.g., a third or fourth order composite.
In p.cfc~.~d embodiment one or more components of the composite is geometricallystabilized. For example: the first internal particle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days, prior to coating it; the first internal particle is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days prior to embedding it in the particle matrix; the first particle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days, prior to coating it~ the first particle is geometrically stabilized, e.g.. by allowing it to age for between 2 hours and 14 days, e.g~ for between 12 hours and 4 or 5 days prior to embedding it in the super matrix; the super matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days 30 prior to coating it; the coated super matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days.In p~cre.~d embo~liment~ the internal particle coating has a lower molecular weight exclusion number than does the particle matrix, the super matrix, the outer coating (if present), or a combination of one or more of these, e.g., the internal particle coating excludes 35 recipient immune molecules, e.g., IgG or complement, and the particle matrix, super matrix, the outer coating (if present), or a combination of these, allows immlme molecules~ e.g., IgG, or complement, to pass but excludes the passage of recipient cells.

CA 0221~039 1997-09-09 W0 96/28029 PCT/u~ v3135 In ~r~rcllcd embo~liment~: the outer surface of the composite is biologically co. ~ ~p~l ;hle, e.g., it is sufficiently smooth that it inhibits fibrotic ~ til~n of the composite, the outer surface of the composite is biologically cnmr~tible, e.g., it is sufficiently smooth that it inhibit fibrotic e~ps~ tion of the composite but the surface of the intPrns~l S particle (or of the particle) is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic ~ne~rs~ tion In plcfe,l~d embo-1im~nt~: the first source is an islet; the second source is an islet; the third source is an islet; the fourth source is an islet; one source is an islet and another source is other than an islet, e.g. an erythrocyte, an acinar cell, or an adrenal cell.In ~lcfellcd emborliment~: an intern~l particle coating is a low molecular weight polyamino acid e.g., 1 kDa-4 kDa, about 1 kDa-less than 4 kDa and a particle coating is a low molecular weight polyamino acid e.g., 5 kDa to less than about 10 kDa, S kDa to less than about 15 kDa, e.g., about 9 kDa-10 kDa. Also plcrcllcd are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In ~lcf~;llcd embodiment~: an internal particle coating has an exclusion limit of about 150 kDa and the first particle coating has an exclusion limit of about 400 kDa.
In ~lcfe~l~d embo~lim~nt~ an int~m~l particle coating has an exclusion limit of about 150 kDa and the particle matrix, first particle coating, super matrix, outer coating, or a combination thereof. has an exclusion limit of about 400 kDa.
In plef~:llcd embotliment~ an internal particle coating has an exclusion limit which is lower than that of the particle coating, e.g., the int~rnzll particle coating has an exclusion limit which will exclude molecules which are the size of IgG. or C 1 q, or larger, and the particle coating has an exclusion limit which, though permeable to IgG, or C 1 q, will exclude objects, e.g., cells, having a molecular weight of one million or more.
In ~lcrellcd embo~iiment~ an internal particle coating has an exclusion limit which is lower than that of the particle co~ting~ e.g., the internal particle coating has an exclusion limit which will exclude molecules which are the size of IgG, or C 1 q, or larger, and the particle matrix, first particle coating, super matrix, outer coating. or a combination thereof, has an exclusion limit which~ though permeable to IgG, or Clq, will exclude objects, e.g., cells, having a molecular weight of one million or more.
In ~rcfellcd embo~liment~ there is a buffer-zone component, e.g., the particle matrix~
which is disposed between a component which is not biocompatible, e.g., which is not anti - fibrotic, e.g., the internal particle co~ting~ and a component which has an exclusion limit which excludes the passage of recipient cells, e.g.. the super matrix or outer coating.
In preferred emborliment~ an internal particle coating has an exclusion limit which will exclude molecules which are the size of IgG, or Cl q, or larger. the particle matrix is not free of defects which arise from the use of non-geometrically stabilized components, and the CA 0221~039 1997-09-09 WO 96/28029 PCI~/US96/03135 super matrix, outer co~ting, or a combination thereof, has an exclusion limit which, though p~rmt~hle to IgG, or Clq, will exclude objects, e.g., cells, having a molecular weight of one million or more.
In ~l~rtl-~d embo~liment~, the first particle of the double composite microreactor S further includes:
one, or a plurality, of a second int~rn~l particle which includes:
(i~ a second source of a therapeutic substance, e.g., an islet;
(ii) a second intt-rn~l particle matrix which contacts the second source, (iii) a second internal particle coating enclosing the second int~rn~l particle 1 0 matrix;
In plt:r~lled emborliment~, the double composite microreactor further includes:
one, or a plurality, of a second particle which includes:
(a) a third internal particle which includes:
(i) a third source of a theld~;uLic s~lbst~nce, e.g., an islet, (ii) a third internal particle matrix which contacts the third source, (iii) (optionally) a third internal particle coating enclosing the third int~rn~l particle matrix; and (b) (optionally) a fourth internal particle which includes:
(i) a fourth source of a therapeutic substance, e.g., an islet, (ii) a fourth internal particle matrix which contacts the fourth source, (iii) a fourth internal particle coating enclosing the fourth int~rn~l particle matrix.
In preferred embo~limPnt~: one or more of the particle matrix, particle covering, or super matrix, prevents contact of fibrotic cells with the internal particle coating; the particle matrix, super matrix or the outer coating (if present) is free of defects which arise from the inclusion of non-geometrically stabilized components. e.g., non-geometrically stabilized intt~rn~l particles; at least two, or three, or four, components chosen from the group of the particle matrix. the particle coating, the super matrix. and the outer coating (if present), provides a molecular weight cutoffthat prevents molecules larger than about 150,000 daltons from coming into contact with the internal particle coating; the internal particle molecular weight cutoff is provided by a pore structure of the internal particle matrix. and that pore structure results. e.g.. from cross-linking of the internal particle gel; the molecular weight cutoff the super matrix is provided by a pore structure of the super matrix.
Preferred embodiments lack an outer coating.
In ~l~rt.. ~d embo~liment~ the outer surface of the double (or higher order) composite microreactor is a gel. e.g., an ~l~in~t~ gel. In more preferred embo-liment~ the outer surface of the composite microreactor is a gel, e.g., an ~lgin~tt~ gel~ the outer surface of which has CA 0221~039 1997-09-09 WO 96/28029 PCT/U~,~ 'l.3135 been modified, e.g., by cross-linking, to produce a covalently modified gel surface, e.g., to form a coating.
In plcr~llcd embo~liment~ the intern~l particle coating is polylysine of about 5 kDa to less than about 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, and the 5 particle coating is polylysine of about 5 kDa to less than about 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., about 9 kDa-10 kDa. Also ~l~,r~,.lcd are poly~min--~cid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In pLere.lcd embo~limentc the int~rn~l particle coating is polylysine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa, and the particle coating is 10 polylysine of about 5 kDa to less than about 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., about 9 kDa-10 kDa. Also ~lef~llcd are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In pler~llcd embodiments the int.orn~l particle coating is polylysine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa, and the particle coating is 15 polylysine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa.
Also E~rcrellcd are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In plercll~d embodiments the outer component of the double composite microreactor, i.e., the component in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
In preferred embo~1imentc one or more components of the double composite microreactor is of sufficient diameter, or of sufficient thickness, such that it imposes a substantial distance (or separation) between recipient cells, e.g., lymphocytes, macrophages, or NK cells. and the source of a therapeutic substance. In more ~.er~ d embo-liment~ the thickness (e.g., the distance between its inner surface and its outer surface) of a component, 25 e.g., a matrix, e.g., a particle makix or super matrix. is: at least 5, 10~ 20, 50, 75, 100, or 200 microns. In more ~.~r~ d embodiments the distance between recipient cells and the source of a therapeutic substance is: at least 5, 10, 20. 50, 75, 100, or 200 microns; sufficient such that exposure of the source of a therapeutic substance to small molecules (e.g., molecules which are not excluded by a component which excludes IgG, e.g., cytokines, nitric oxide 30 (NO). and other toxic moieties) released by recipient cells is subst?nti~lly reduced (e.g., by diffusion), e.g., reduced at least 10, 20, 50, 75. or 90 %, sufficient such that the concentration of small molecules (e.g., molecules which are not excluded by the semipermeable balrier-- components of the double composite microreactor, e.g., cytokines, NO, and other toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g.. reduced ~ 35 at least 10, 20, 50, 75, or 90 % at the source of a therapeutic substance. In more plefelled embo~liment~: the distance is supplied by one or both of the particle makix and the super matrix.

CA 0221~039 1997-09-09 In ~c;r~,~lcd emboAiment~ one or more eomponents of the double composite mieroreaetor is of suffieient Ai~m~t~or~ or of suffieient thiekness, sueh that it imposes a substantial Ai~t~nce (or separation) between reeipient eells, e.g., lymphoeytes, maerophages, or NK eells, and one or both of the souree of a th~.dlJcuLie sllbst~nre or donor antigen (other 5 than the Lh~,~d~c~ulie sllhst~nee) released by the souree (e.g., donor proteins, whieh eould stim~ te a reeipient response against donor tissue). In more p~cr~ cd emboAim,ont~ the thieknee~ (e.g., the Ai~t:~nre between its inner surfaee and its outer surfaee) of a eomponent, e.g., a matrix, e.g., a partiele matrix or super matrix~ is: at least 5, 10, 20, 50, 75, 100, or 200 mierons. In more ~.crc..ed embo~lim~-nt~ the Ai~t~nre between reeipient eells and the souree of a therapeutie substanee is: at least 5, 10, 20, 50, 75, 100, or 200 mierons; suffieient sueh that the amount, number, or eoncentration of a donor antigen, released into the reeipient, or eontacting recipient cells, is substantially redueed (e.g., by diffusion, or by trapping in or exelusion by the eomponent or eomponents whieh supply the separation), e.g., redueed by at least 10, 20, 50, 75, or 90 %; sufficient sueh that eontaet of eells of the reeipient with donor antigens, e.g., proteins, whieh protrude from or extend through the internal partiele matrix or internal partiele coating, or both, is subst~nti~lly reduced (e.g., by diffusion, or by trapping in or exclusion by the component or compo.lc.lL:j whieh supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient to inhibit acute release of donor antigens. In more preferred embo~limentc the ~ t~nce or separation is supplied by one or both of the partiele matrix and the super matrix.
In yet another aspeet, the invention features, a double eomposite microreactor whieh ineludes:
(1) one, or a plurality~ of an internal particle whieh ineludes:
(a) an islet;
(b) an internal particle matrix of ~l~in~te in which the islet is embedded;
(c) an internal particle eoating of polylysine enclosing the first interns~
particle matrix;
(2) one, or a plurality of. a geometrically stabilized partiele which ineludes:
(a) the internal particle (or internal particles) of ( 1 );
(b) a particle matrix of ~lgin~te in which the internal particle (or intern~l particles) is embedded; and (c) a particle coating of polylysine enelosing the particle; and (3) a gel super matrix of agarose in whieh the particle (or particles) of (2) is embedded.
In ~lt;r~ d emboAiment~ the internal particle coating is polylysine of about S kDa to less than about 15 kDa. or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, and the partiele eoating is polylysine of about 5 kDa to less than about 15 kDa. or about 5 kDa to less CA 022l~039 l997-09-09 W O g6/28029 P~~ '03135 than about 10 kDa, e.g., about 9 kDa-10 kDa. Also plcrcllcd are poly~n~ino~icl e.g., PLL or PLO, coiqtin~S in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In ~le~.lcd embo-lim~nt~i the int~rn~l particle coating is polylysine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa, and the particle coating is polylysine of about 5 kDa to less than about 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., about 9 kDa-10 kDa. Also ~lcrcll~,d are polyaminoacid, e.g., PLL or PLO, co~tin~s in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In ~.lcrclled emborliment~ the intern~1 particle coating is polylysine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa, and the particle coating is polylysine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa.
Also ~lcr~ d are poly~nnino~id, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In plcrcllcd embo~liment~ the intern~l particle coating is polylysine of about S kDa to less than about 15 kDa. or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, and the particle coating is polylvsine of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., about 2 kDa- 3 kDa. Also pl~f~llcd are polyaminoacid, e.g.. PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In pl~r~led embodiments the internal particle matrix is other than a liquid. Theintern~l particle matrix can include substances which impede the passage of recipient-derived 20 molecules or cells, e.git can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO).
In pl~r~ d embo~liment~ the internal particle matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa: or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~l~r~ d embotliment~ the intern~l particle coating is or includes: a polylysine having a molecular weight of less than 15 kDa, more preferablv of less than 10 kDa, more preferably of less than 5 kDa. Particularly preferred are polylysines. with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa. or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 l;Da-l O kDa, e.g., 9.7 kDa.
Also pl~;f.,Ll~,d are polvaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, - preferably in the range of 1-2, 1-3, or 1-4 kD. Preferred coatings are volume-re~lllcing coatings.
In ~lefell~d embo~1iment~ the internsll particle coating hinders the passage, and preferably ess.?nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa. preferably more than CA 0221~039 1997-09-09 about 150 kDa, and most preferably more than about 400 kDa; or of immlme system CO~ ull~ such as Ig molecules or complement; or recipient-derived cells.
In ~.er~ l.ed embo-liment~ the ~ met~-r of the internal particle, before application of a volume-red~cin~ co~ting~ between 50 and 700 microns, more preferably between 100 and S 500 microns, more preferably b~w~ 200 and 400 microns, and most preferably about 300 microns in fli~m~t~r. The ~ m~ter of the int~rn~l particles, after application of a volume-reducing coating is preferably between 35 and S00 microns, more preferably between 75 and 400 microns, more preferably between 100 and 300 microns, and most preferably about 200 microns in diameter.
In preferred embo~1im.ont~ the particle matrix is other than a liquid. The particle matrix can include s~lbst~nces which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO).
In preferred embo-liment~ the particle matrix hinders the passage, and preferably 15 e~enti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa. preferably more than about 100 kDa. preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immllne system components such as Ig molecules or complement; or recipient-derived cells.
In preferred embo~liment~ the particle coating is polylysine having a molecular weight 20 of less than 15 kDa. more preferably of less than 10 kDa. more preferably of less than S kDa.
Particularly preferred are polylysines with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g.. 9.7 kDa. Also ~Jlef.,~lcd are polyaminoacid. e.g.. PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2. 1-3. or 1-4 kD. Preferred coatings are volume-reducing coatings.
In preferred embo~liment~ the particle coating hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 l;Da. preferably more than about 100 kDa~ preferably more than about 150 kDa, and most preferably more than about 400 kDa~ or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In preferred embodiments the diameter of the particle, before application of a volume-re~ln~ing coating. is between 200 and 1,000 microns. more preferably between 400 and 800 microns. more preferably between 500 and 700 microns. and most preferably about 600 microns in diameter. The diameter of the particles. after application of a volume-reducing coating. is preferablv between 100 and 700 microns. more preferably between 250 and 550 microns~ more preferably between 300 and 500 microns. and most preferably about 400 microns in ~ m~t~r.

CA 0221~039 1997-09-09 WO 96128029 PCI'tUS96/03135 In plefe,l~d embo-1imt-nte the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this ~lopclly being relegated to other COlll~ "ents of the microcapsule.
In l~ert;~cd embo-lim~nt~ the super matrix is other than a liquid. The super matrix 5 can include substances which impede the passage of reçipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyc . .,;ll.;.~t? (PLO). A high molecular weight, molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super matrix to confer immunoisolating prol ~;,lies.
In ~l~f~led embo-1imente the super matrix hinders the passage, and preferably eee~nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In plcrcllcd embo-limente the super matrix has little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this property being relegated to other components of the microcapsule.
In preferred emborlimPnte the super matrix coating (which is optional) is or includes:
a polyaminoacid, e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g., 20 chitosan. Particularly ~lcrellcd coating is polyamino acid. e.g., polylysine or polyolllilhi,le, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly plcfellcd are polylysines with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa.
25 Also ~l~fclled are polyaminoacid, e.g., PLL or PLO. coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In ~lc-fc~lcd embodiments the particle coating hinders the passage, and preferably ese~nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 30 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~ler~llcd emborlimente the ~ m~ter of the double composite microreactor is.
before application of a volume-redtlcinE coating. between 400 and 1,500 microns, more preferably between 500 and 1,300 microns, more preferably between 600 and 1,100 microns, 35 and most preferably about 900 microns in diameter. The diameter of the double composite microreactor is. after application of a volume-reducing coating, is preferably between 300 ~9 CA 022l~039 l997-09-09 and 1,300 microns, more preferably between 400 and 1,200 microns, more preferably between 500 and 1,000 microns, and most preferably about 800 microns in rliz3m~ter.
In ~lerGlled embot1imentc the composite microreactor includes a plurality of intern~l particles, e.g., bGLWGGll 2 and 100, e.g., b~LWGGll 2 and 10, intern~l particles.
S In ~rGrGllGd embotlimentc the composite mi-,lolGacLor includes a plurality of particles, e.g., between 2 and 100, e.g., 2 and 10, particles.
In ~rcllGd embotiimentc the double composite miclorGa~;Loi is a component of a higher order composite, e.g., a third or fourth order composite.
In ~lGrGlled embodiment one or more components of the composite is geometrically10 stabilized. For ex~mrle: the first internal particle matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days, prior to coating it; the first internal particle is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or S days prior to embedding it in the particle matrix: the first particle matrix is geometrically stabilized, e.g., lS by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or S
days, prior to coating it; the first particle is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or S days prior to embedding it in the super matrix; the super matrix is geometrically stabilized, e.g., by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or S days 20 prior to coating it; the coated super matrix is geometrically stabilized, e.g.~ by allowing it to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or S days.In plerGllGd embo-limentc the internal particle coating has a lower molecular weight exclusion number than does the particle matrix, the super matrix, the outer coating (if present), or a combination of one or more of these, e.g., the internal particle coating excludes 25 recipient immllne molecules, e.g., IgG or complement, and the particle matrix, super matrix, the outer coating (if present). or a combination of these, allows immlme molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In plerGll~d embodiments: the outer surface of the composite is biologically colllp~Lible, e.g., it is sufficiently smooth that it inhibits fibrotic encapsulation of the 30 composite; the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic çnc~r5nl~tion of the composite but the surface of the intern~l particle (or of the particle) is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic enc~rsulation.
In preferred embo~lim~ontc the first source is an islet; the second source is an islet; the 35 third source is an islet: the fourth source is an islet; one source is an islet and another source is other than an islet, e.g. an erythrocyte, an acinar cell. or an adrenal cell.

CA 0221~039 1997-09-09 WO g6~28029 PCT/US96/03135 In ~rer~ d embo~liment~ an intern~l particle coating is a low molecular weight polyamino acid e.g., 1 kDa-4 kDa, about 1 kDa-less than 4 kDa and a particle coating is a low molecular weight polyamino acid e.g., S kDa to less than about 10 kDa, S kDa to less than about 15 kDa, e.g., about 9 kDa-10 kDa. Also ~r~;r.,lled are poly~minc-~cid, e.g., PLL or S PLO, co~tin~ in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In pler~ ,d embot1iment~ an intern~l particle coating has an exclusion limit of about 150 kDa and the first particle coating has an exclusion limit of about 400 kDa.
In ~lerel.cd embor1iments an internal particle coating has an exclusion limit of about 150 kDa and the particle matrix, first particle coating, super matrix, outer coating, or a 10 combination thereof, has an exclusion limit of about 400 kDa.
In preferred embo~liment~: an internal particle coating has an exclusion limit which is lower than that of the particle coating, e.g., the intern~l particle coating has an exclusion limit which v~ ill exclude molecules which are the size of IgG, or C 1 q, or larger, and the particle coating has an exclusion limit which, though permeable to IgG, or Cl q, will exclude 15 objects, e.gcells. having a molecular weight of one million or more.
In preferred embodiments: an internal particle coating has an exclusion limit which is lower than that of the particle coating, e.g., the intern~l particle coating has an exclusion limit which will exclude molecules which are the size of IgG, or C 1 q, or larger, and the particle matrix. first particle coating, super matrix, outer coating, or a combination thereof, 20 has an exclusion limit which~ though permeable to IgG, or Clq, will exclude objects, e.g., cells, having a molecular weight of one million or more.
In preferred embodiments: there is a buffer-zone component, e.g., the particle matrix, which is disposed between a component which is not biocompatible, e.g., which is not anti fibrotic, e.g., the internal particle coating, and a component which has an exclusion limit 25 which excludes the passa_e of recipient cells, e.g., the super matrix or outer coating.
In preferred embo~liment~: an intern~l particle coating has an exclusion limit which will exclude molecules which are the size of IgG, or Clq, or larger. the particle matrix is not free of defects which arise from the use of non-geometrically stabilized components, and the super matrix, outer coating. or a combination thereof, has an exclusion limit which, though 30 permeable to IgG, or C l q. will exclude objects, e.g.~ cells, having a molecular weight of one million or more.
In IJler.,.led emborliment~ one or more of the particle matrix, particle covering, or super matrix, prevents contact of fibrotic cells with the internal particle coating, the particle matrix, super matrix or the outer coating (if present) is free of defects which arise from the 35 inclusion of non-geometrically stabilized culllpo~lents, e.g.~ non-geometrically stabilized intern~l particles, at least two, or three, or four, components chosen from the group of the particle matrix, the particle coating, the super matrix, and the outer coating (if present), CA 0221~039 1997-09-09 provides a molecular weight cutoff that prevents molecules larger than about 150,000 daltons from coming into contact with the internal particle coating; the int~rn~l particle molecular weight cutoff is provided by a pore structure of the internal particle matrix, and that pore structure results, e.g., from cross-linking of the internal particle gel; the molecular weight 5 cutoffthe super matrix is provided by a pore structure of the super matrix.
Preferred embo-1iment~ lack an outer coating.
In plc;r~"~ed embo-liment~ the outer surface of the composite microreactor is a gel, e.g., an ~lgin~te gel. In more ~lc;rel~ed embodiments the outer surface of the composite microreactor is a gel, e.g., an ~l~in~te gel, the outer surface of which has been modified, e.g., 10 by cross-linking, to produce a covalently modified gel surface, e.g., to form a coating.
In ~ r~l,ed embodiments the outer component of the double composite microreactor, i.e., the component in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
In ~l~rc~ d embolliment~ one or more components of the double composite microreactor is of sufficient diameter, or of sufficient thickness, such that it imposes a 15 substantial distance (or separation) between recipient cells, e.g., lymphocytes. macrophages, or NK cells. and the source of a therapeutic substance. In more preferred embo~liment~ the thickness (e.g.~ the distance between its inner surface and its outer surface) of a component, e.g., a matrix, e.g.. a particle matrix or super matrix. is: at least 5, 10, 20, 50, 75, 100, or 200 microns. In more preferred embo~liment~ the distance between recipient cells and the source of a therapeutic substance is: at least 5, 10, 20, 50, 75, 100, or 200 microns; sufficient such that exposure of the source of a the,al,~ulic substance to small molecules (e.g., molecules which are not e~;cluded by a component which excludes IgG. e.g., cytokines, nitric oxide (NO), and other toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g.. reduced at least 10, 20, 50, 75. or 90 %; sufficient such that the concentration 25 of small molecules (e.g.. molecules which are not excluded by the s~ "lleable barrier-components of the double composite microreactor. e.g.. cytokines, NO, and other toxic moieties) released by recipient cells is substantially reduced (e.g., by diffusion), e.g., reduced at least 10, 20. 50. 75, or 90 % at the source of a therapeutic substance. In more ",ef~"~d embodiments: the distance is supplied by one or both of the particle matrix and the super 30 matrix.
In ~ r~lled embodiments one or more components of the double composite microreactor is of sufficient diameter, or of sufficient thickness, such that it imposes a substantial distance (or separation) between recipient cells. e.g., lymphocytes, macrophages, or NK cells. and one or both of the source of a therapeutic substance or donor antigen (other 35 than the therapeutic substance) released by the source ~e.g.. donor proteins, which could stim~ te a recipient response against donor tissue). In more ~ d embo-lim~ntc the thickness (e.g.. the ~ t~nce between its inner surface and its outer surface) of a component, CA 0221~039 1997-09-09 e.g., a matrix, e.g., a particle matrix or super matrix, is: at least S, 10, 20, 50, 75, 100, or 200 microns. In more p,er~ ;d embo~ ; the ~ t~nre between recipient cells and the source of a the~a~culic sllhst~nl e is: at least S, 10, 20, S0, 75, 100, or 200 microns; sufficient such that the amount, number, or concentration of a donor antigen, released into the recipient, or S cont~-ting recipient cells, is subst~nti~lly reduced (e.g., by diffusion, or by trapping in or exclusion by the cc,l,l~,o~,ent or components which supply the separation), e.g., reduced by at least 10, 20, S0, 75, or 90 %; sufficient such that contact of cells of the recipient with donor antigens, e.g., proteins, which protrude from or extend through the int~rn~l particle matrix or int~rn~l particle coating, or both, is substantially reduced (e.g., by diffusion, or by trapping in 10 or exclusion by the component or components which supply the separation), e.g., reduced by at least 10, 20, 50, 75, or 90 %; sufficient to inhibit acute release of donor antigens. In more ~,cre"ed embo~limPntc: the distance or separation is supplied by one or both ofthe particle matrix and the super matrix.
The inventors have also discovered a method of improving the performance of a 15 microcapsule, e.g., a composite microreactor which includes "geometrically stabilizing"
one or more components of the microcapsule.
Microc~ps-llPs are generally formed in a sequence of steps wherein a first component is combined with a second component, e.g., wherein an internal component is formed and a more exterior, surrounding culll~o~ , is subsequently formed around the intern~l20 component. For example, a simple microcapsule (i.e., a non-composite microcapsule) can be formed as follows: a core co..l;1;..il-F living cells, e.g., a gel core, is formed, and a semipermeable coating is subsequently applied to the core. A simple or single composite microreactor can be formed as follows: internal particles co.~l~;.l;..P living cells are formed, the internal particles are embedded in a surrounding super matrix, and (optionally) a semipermeable coating is subsequently applied to the internal particle-cont~ininP; super matrix.
The inventors have discovered that the pt;~r~"~ ce of a microcapsule can be improved if one or more of the components is geometrically stabilized, e.g., if an internal component is geometrically stabilized prior to the application of a more exterior, surrounding component. For example, the p~ ~ro""ance of a simple or single microcapsule can be improved by~ prior to the application of a semipermeable membrane, allowing the gel core to become geometrically stabilized. The performance of a composite microcapsule can be improved. e.g., by, prior to embedding the internal particles in the super matrix, allowing the intern~l particles to become geometrically stabilized.
A geomPtric~lly stabilized component. as used herein, refers to a component that is treated such that it undergoes no substantial change in shape or volume when incorporated ~ into a larger structure. In typical embotliment~ of the invention, a component, e.g.. an interior CA 0221~039 1997-09-09 WO 96/28029 ~ 03135 or intPrn~l coll~ol,ent, is geometrically stabilized prior to its incol~vldLion into a larger ~Llu,lult;. Generally, a component can be geom~fric~lly stabilized by allowing it to age ffir a period of time. For ~mrle, in embo-liment~ of the invention a con,l~o.~ent is allowed to age prior to inc~ ,~ur~Li~lg it into a larger structure or prior to depositing or otherwise forrning a S structure on its exterior surface. For example, a gel core can be geom~trir~lly stabilized, e.g., by aging, prior to depositing or forming a selectively permeable membrane on its outer surface; an int~rn~l particle can be geometrically stabilized, e.g., by aging, prior to its hlcol~oldlion into a composite microreactor; and the super matrix of a composite can be geometrically stabilized, e.g., by aging, prior to depositing or forming a selectively p~rme~hle 10 membrane on its outer surface. When a coll~vnent, e.g., a gel, e.g., an ~lgin~t~ particle, is geometrically stabilized by aging, the aging period can be equal to or longer than 2 hours, equal to or longer than 12 hours, equal to or longer than 24 hours, equal to or longer than 36 hours, equal to or longer than 48 hours, equal to or longer than 3 days, or equal to or longer than S days. The geometric stabilization tre~tm~-nt should be such that viability or activity of 15 the enr~ps~ tecl material is not unacceptably altered and that the desirable ~JlVpC~l lies of the component itself are not unacceptably col~ vlllised.
Although aging is the ~ Ç~ d method of geometric stabilization other methods canbe used. For example, an ~lFin~te gel which has been treated with CaC12 can be further treated with a metal ion, e.g., a Ba or Fe ion, to geometrically stabilize the gel. A gel core 20 can also be stabilized by cross-linking its surface.
A component is geometrically stabilized when any of the following conditions is met:
(1) it will undergo no s~lbst~nti~l change (a sllhst~nti~l change is one which co~ olllises the perf~ rm~n~P, e.g., the ability to exclude host cells) in shape or volume when the component is subjected to the next stage of the manufacturing process;
(2) when it is incorporated into a larger structure it induces fewer structural faults, or reduces the performance (e.g.. the ability to immunoisolate) of the structure to a lessor degree, than would be the case if a non-geometrically stabilized, but otherwise similar, component were used (the presence of structural faults can be determined by microscopic e~c~min~tion or by the ability to inhibit passage of a test component, e.g., a molecule or cell;
the ability to immllnoisolate can be determined, in vivo, by implar~ting the structure in question in an animal and ~lptermining if the structure is capable of immlm~lisolation and resisting fibrotic inactivation. or in vitro, by incubating the structure with molecules of the immune system of a test animal);
(3) the component appears stable, in terms of shape and size. for at least 12 hours.
The method described above can be used to determine if a treatment is suitable for geometrically stabilizing a component.

CA 0221~039 1997-09-09 WO 96/28029 PCI~/US96/03135 Aecordingly, in another aspect, the invention features a method of improving thep~ . r~ nl~e of a microe~rslllP The method includes:
(a) forming a first conll onent of a mieroeapsule, (e.g., a eore, e.g., a gel eore);
(b) treating the first eollll,~,llent so as to geometrieally stabilize the first eomponent S (e.g., by allowing the first eomponent to age for at least 12 hours); and (e) eombining the geometrieally stabilized first eomponent with a seeond eomponent of a mieroeapsule, (e.g., forming an exterior eomponent, e.g., a semipPrm~ble eoating, around a geometrieally stabilized gel eore). In pl~r~ d embo-lim~ntc step e is performed after step b.
In ~l~;ftl.~d emborlimentc the first eomponent is: a core, e.g., a gel eore, e.g., an ~lgin~te eore; an internal particle matrix; an intern~l particle; a super matrix; a particle; a partiele matrix.
In ~ler~ d embodiments the second component is: a coating; a matrix, e.g., a partiele matrix or a super matrix; a component of a higher order composite.
In pl~:ftlled emborlimentc: the first component is a gel core or matrix and the second eomponent is a coating; the first eomponent is a particle or an internal particle and the second eomponent is a eoating; the first eomponent is a particle or intern~l partiele and the seeond eomponent is a matrix, particle matrix, or super matrix.
In preferred embo-limentc one of the eomponents, e.g.. the first eomponent, eonL~ills a 20 souree of a therapeutic snkst~nre In ~ rell~d embodiments the first component is a gel matrix, e.g.,: a gel, e.g., a hydrogel, e.g.. an ~Igin~te or agarose gel. In plef~ d embo-limentc the gel matrix is other than a liquid. The gel matrix can include substances which impede the passage of recipient-derived molecules or cells. e.g., it can include polyethelyne oxide (PEO), polystyrene 25 sulfonic aeid (PSA). or polyornithine (PLO).
In preferred embodiments the gel matrix hinders the passage. and preferably essentially eompletely prevent the passage of: moleeules having a moleeular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immnne system eomponents sueh 30 as Ig molecules or complement; or recipient-derived cells.
In plertllc~d embodiments the first eomponent is a gel matrix having a eoating. In more ~lefelled the coating is or ineludes: a polyaminoaeid, e.g.. polylysine (PLL) or PLO; a - naturally oecurring substance, e.g., chitosan; a synthetie polymer e.g.. PAN-PVC. A
partieularly plefelled coating is polyamino acid, e.g., polylysine or pol~/olllilhille~ having a 35 moleeular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly pl~efellc;d are polyamino acids. e.g.. polylysines, with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or - ' ~
CA 0221~039 1997-09-09 about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also plerelled are poly~mino~id, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Plefelled coatings are volume-recl~lcin~ co~ting~
In ~lèrelled emborlim~nt~ the coating hinders the passage, and preferably ess~nti~lly complet~ly prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immllne system components such as Ig molecules or complement; or recipient-derived cells.
In ~lcrelled embo~liment~ the second component is a matrix. In more ~lerellcd embo-limçnt~ the matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~te or agarose gel.
In p~ere~ed embodiments the super matrix is other than a liquid. The matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), poly~ly-clle sulfonic acid (PSA), or polyornithine (PLO).
A high molecular weight, molecule, e.g., a polymer, e.g.~ PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super matrix to confer immunoisolating properties.
In ~lerelled embodiments the matrix hinders the passage, and preferably essf nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~lefelled embodiments the second component is a coating. In more ~efelled embodiments the coating is or includes: a polyaminoacid. e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g., chitosan; a synthetic polymer e.g., PAN-PVC. A
particularly preferred coating is polyamino acid. e.g., polylysine or polyornithine, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly plerelled are polyamino acids, e.g., polylysines, with a molecular ueight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than abou~ 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also plefell~,d are poly~rnino~t~id~ e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD. preferably in the range of 1-27 1-3. or 1-4 kD. Preferred coatings are volume-reducing coatings.
In ~lefe~led embodiments the coating hinders the passage. and preferably e~senti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa. and most CA 0221~039 1997-09-09 WO 96128029 PCTtUS96103135 preferably more than about 400 kDa; or of immlme system components such as Ig molecules or comrlennent; or recipient-derived cells.
In pler~ d embodiment the first component is geometrically stabilized by aging. In ~l~r~ d embo-limente the aging period is: equal to or greater than 2 hours, 6 hours, 12 S hours, 24 hours, 48 hours, 4 days, 5 days, 7 days, 10 days, and 14 days. In more plcr~ ,d embo~limente the first colllpo"ent is allowed to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days.
In plert;"ed emboflimente the first component has a lower molecular weight exclusion number than does the second component, e.g., the first component excludes recipient immune 10 molecules, e.g., IgG or complement, and the second component, allows immune molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In ~l~relled embo-1imente: the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibits fibrotic encapsulation of the composite; the outer surface of the composite is biologically compatible, e.g., it is sufficiently 15 smooth that it inhibit fibrotic ent~pslll~tion of the composite but the surface of the internal particle is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic encapsulation.
In ~l~r~lled embo-1imente the method includes (a) forming a gel core co.~ g a living cell, e.g., an islet cell;
(b) aging the gel core for between 2 hours and 5 days, and (c) forming a semipermeable coating around the geometrically stabilized gel core), wherein step c is ~ ro.l.~ed after step b.
In preferred embo-limente the microcapsule is a double composite microreactor and:
the first component is an internal particle and the second component is an intern~l particle 25 coating: the first component is an internal patticle and the second component is a particle matrix: first component is an internal particle coating and the second component is a particle matrix first component is a particle and the second colllpollent is a particle coating; first component is a particle and the second component is a super matrix; first component is a particle coating and the second component is a super matrix; first component is a super 30 matrix and the second component is an outer coating.
In ~ r~:llcd embodiments the outer component of the microcapsule, i.e., the component in contact with the recipient, is at least 50, 75, 90, 95, 97. or 98 %. water.
In another aspect, the invention features. a method of improving the performance of a composite microreactor. The method includes:
~ 35 (a) forming a first component of a composite microreactor, (e.g., a coated or uncoated intern~l core, e.g., an int~rn~l gel core);

CA 0221~039 1997-09-09 WO 96/28029 ~: ~_l/U~ 03135 (b) combining the first eomponent with a second component of a composite microc~rs~ , (e.g., ennbell~ling the internal core in a super matrix, e.g., a gel super matrix);
and ~c,r~. ~l"illg one or both of the following steps (c) and (d):
S (c) prior to combining the first and the second component, geometrically stabilizing the first component, (e.g., prior to embedding an int~rn~l core in a super matrix, treating the int~rn~l core so as to geometrically stabilize the int~ l core); and/or (d) prior to combining the first and the second component with a third component, geometrically stabilizing the second component, (e.g., geometrically stabilizing a super 10 matrix prior to forming an exterior component around the stabilized super matrix).
In preferred embodiments the first component is: a core, e.g., a gel core, e.g., an ~lgin~t~ core; an internal particle matrix; an internal particle; a super matrix; a particle; a particle matrix.
In preferred embo-limentc the second component is: a coating: a matrix, e.g., a 15 particle matrix or a super matrix; a component of a higher order composite.
In preferred embo-liment~ the first component is a gel core or matrix and the second component is a coating; the first component is a particle or an internal particle and the second component is a coating; the first component is a particle or internal particle and the second component is a matrix, particle matrix, or super matrix.
In preferred embo-liment~ one of the components, e.g.. the first component, contains a source of a therapeutic substance.
In preferred embodiments the first component is a gel matrix, e.g.: a gel, e.g., a hydrogel, e.g., an ~ in~te or agarose gel. In plcrellcd embo-limentc the gel matrix is other than a liquid. The gel matrix can include substances which impede the passage of recipient-25 derived molecules or cells, e.g., it can include polyethelyne oxide (PEO)~ polystyrenesulfonic acid (PSA), or polyornithine (PLO).
In ~lcrcll~d embo-1im~nt~ the gel matrix hinders the passage, and preferably t-c~enti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 30 kDa, and most preferably more than about 400 kDa; or of immnne system components such as Ig molecules or complement; or recipient-derived cells.
In ~lcrel~ed embodiments the first component is a gel matrix having a coating. In more ~ulcrcllcd the coating is or includes: a poly~mino~cid, e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g.. chitosan; a synthetic polymer e.g.~ PAN-PVC. A
35 particularly ~lcrcllcd coating is polyamino acid. e.g., polylysine or polvornithine, having a molecular weight of less than 15 kDa. more preferably of less than l O kDa, more preferably of less than 5 kDa. Particularly plercllcd are polyamino acids, e.g., polvlysines~ with a CA 0221~039 1997-09-09 WO 96/28029 PCr/US96/03135 molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also plGrG~ d are poly~mino~cid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Plefel,Gd co~tin~c are S volume-re~ c.ing co~tin~s In "l-,r~ d embo~1iment~ the coating hinders the passage, and preferably essPnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immnne system components such as Ig molecules 10 or complement, or recipient-derived cells.
In ~lGrGlled embo~liment~ the second component is a matrix. In more plGr~,llGd embo-liment~ the matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~te or agarose gel.
In plGfGllGd embodiments the matrix is other than a liquid. The matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can 15 include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO).
A high molecular weight. molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super matrix to confer immunoisolating o~.,l Lies.
In ~lefGllGd embo-liment~ the matrix hinders the passage. and preferably ~-ssenti~lly 20 completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In preferred embodiments the second component is a coating. In more plGrGllGd 25 embodiments the coating is or includes: a poly~minoiqcid. e.g.t polylysine (PLL) or PLO; a naturally occurring substance, e.g., chitosan; a synthetic polymer e.g., PAN-PVC. A
particularly preferred coating is polyamino acid. e.g., polylysine or polyornithine, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly prGfG.lGd are polyamino acids. e.g., polylysines, with a 30 molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDat e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also preferred are polyaminoacid, e.g., PLL or PLO. coatings in the range of 1 or 2-10 kD. preferably in the range of 1-2, 1 -3t or 1-4 kD. Preferred coatings are volume-re~lllcin~ coatings.
In preferred embolliment~ the coating hinders the passage. and preferably ~çnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most - .
CA 0221~039 1997-09-09 WO 96/28029 PCrtlJS96/03135 preferably more than about 400 kDa, or of immlme system components such as Ig molecules or complement; or recipient-derived cells.
In ~lcrtl.cd embodiment the first component is geometrically stabilized by aging. In ~crtl~,d embo~liment~ the aging period is: equal to or greater than 2 hours, 6 hours, 12 S hours, 24 hours, 48 hours, 4 days, 7 days, 10 days, and 14 days. In more plcr~,d embo-lim~nt~ the first component is allowed to age for bCLWeC11 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days.
In preferred embo-limentc the first component has a lower molecular weight exclusion number than does the second component, e.g., the first component excludes recipient immlme molecules, e.g., IgG or complement, and the second component, allows immune molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In preferred embot1iment~: the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibits fibrotic encapsulation of the composite; the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic encapsulation of the composite but the surface of the int~-rn~l particle is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic encapsulation.
In preferred embodiment the second component is geometrically stabilized by aging.
In preferred embo-liment~ the aging period is: equal to or greater than 2 hours, 6 hours, 12 hours, 24 hours. 48 hours, 4 days, 7 days, 10 days, and 14 days, In more ~.cfcllcd embodiments the first component is allowed to age for between 2 hours and 14 days, e.g., for between 12 hours and 4 or 5 days.
In ~icf~ d embo-liment~ the third component is a matrix. In more prer~i..cd embodiments the matrix is or includes: a gel, e.g.. a hydrogel~ e.g.. an ~l~in~te or agarose gel.
~5 In preferred embodiments the matrix is other than a liquid. The matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyo., .ill.i ..e (PLO).
A high molecular ~-eight, molecule, e.g., a polymer~ e.g.. PEO, with a molecular weight of 1-8 million daltons. or more, can be added to the super matrix to confer immunoisolating 30 plopc.~ies.
In ~cr~cd embo-lim.?nt~ the matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa, or of immune system components such as Ig molecules 35 or complement: or recipient-derived cells.
In p.~rt:..ed embodiments the third component is a coating. In more ~cr~ Gd embo-liment~ the coating is or includes: a polyaminoacid. e.g., polylysine (PLL) or PLO, a CA 0221~039 1997-09-09 WO g6/28029 ~ 03135 n~hlr~lly occllrring substance, e.g., chitosan; a synthetic polymer e.g., PAN-PVC. A
particularly ~ler~,.led coating is polyamino acid. e.g., polylysine or polyo-.-ilhil-e, having a molecular weight of less than l S kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly ~lertlled are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about S kDa to less than l S kDa, or about S kDa to less than about 10 kDa, or about S kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~l~,rclled are poly~mino~ci~l, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Plcfellcd coatings are volume-recl~lc-in~ coatings.
In preferred embotliment~ the coating hinders the passage, and preferably e~nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about l S0 kDa, and most preferably more than about 400 kDa, or of immune system components such as Ig molecules or complement: or recipient-derived cells.
In preferred embo-liment~ the second component has a lower molecular weight exclusion number than does the third component, e.g., the second component excludes recipient immune molecules, e.g., IgG or complement, and the third component, allows imml-ne molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In preferred embodiments the composite microreactor is a component of a higher order composite~ e.g., a double composite, or a third order composite.
In a preferred embodiment, the method further includes incorporating the composite into a higher order composite, e.g., a double composite. In more preferred embo-liment~, the composite is geometrically stabilized prior to incorporating it into a higher order composite, e.g., a double composite. For example, an internal z~lgin~t~ particle coated with polylysine of between about 1 kDa-4 kD and co~ g a living cell, e.g., an islet, is formed. The intçrnzll particle is aged for between about 3-5 days prior to incorporating it into an ~lgin~te particle matrix. The aged internal particle is then incorporated into a particle matrix. The particle matrix is then coated with polylysine of between about S kDa to less than about 15 kD, and is aged for between about 2-24 hours. Finally, the aged coated particle is embedded into an :~lgin~te matrix to form a double composite.
In preferred embotliment~ the composite microreactor is a double composite microreactor and: the first component is an internal particle and the second component is an intt?rn~l particle coating, the first component is an intern~l particle and the second component ~ 35 is a particle matrix: first component is an internal particle coating and the second component is a particle matrix; first component is a particle and the second component is a particle coating: first component is a particle and the second component is a super matrix; first CA 0221~039 1997-09-09 ~ WO 96/28029 ~. ~ , ~ /03135 colllpollent is a particle coating and the second component is a super matrix, first component is a super matrix and the second component is an outer coating.
In ~ref~ ;d embo-lim~nte the outer colllpolle~lL of the composite microreactor, i.e., the Colllpr;)n~ in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
S The inventors have also discovered that application of a coating of a relatively low molecular weight polymer, e.g., a relatively low molecular weight polyamino acid, to a microcapsule component, e.g., to a core, e.g., a gel core or a super matrix, can result in a decrease in the diameter and volume of the re~sllltin~r coated component.
Accordingly, in another aspect, the invention features, a method of making a microcapsule having reduced volume. The method includes:
(a) forming a component of a microcapsule, (e.g., a core particle, e.g., a gel particle); and (b) applying a volume re(lllcing coating to the component, thereby producing a microcapsule including a component and a coating and having a volume less than the volume of the uncoated component.
In preferred embodiments the volume reducing coating is or includes: a polyarnino acid, e.g., polylysine, or polyornithine, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly l~lcrt;lled are polyamino acids with a molecular ~eight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-l 0 kDa, e.g.. 9.7 kDa. Also pler~lled are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or ~-10 kD. preferably in the range of 1-2, 1-3, or 1-4 kD.
In preferred embodiments the component is a gel matrix, e.g.: a gel, e.g., a hydrogel, e.g., an algin~te or agarose gel. In preferred embodiments the gel matrix is other than a 25 liquid. The gel matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g.. it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO).
In pler~ d embodiments the gel matrix hinders the passage, and preferably ~eeerlti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa? and most preferably more than about 400 kDa: or of immune system colllpollents such as Ig molecules or complement; or recipient-derived cells.
In plc:r~ d embo-limente the component is a gel matrix having a coating. In morepreferred the coating is or includes: a poly~mino~cid, e.g., polylysine (PLL) or PLO, a 35 naturally occllrring substance. e.grhitoe~n~ a synthetic polymer e.g., PAN-PVC. A
particularly plef~ d coating is polvamino acid. e.g., polylysine or polyo.,.;lh;l.~ having a molecular weight of less than 15 kDa. more preferably of less than 10 kDa, more preferably CA 0221~039 1997-09-09 WO 96/28029 ~ ~_l/U~ 3135 of less than S kDa. Particularly plertil-ed are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa ~or about i kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than lS kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~leftlled are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. P~re.l~d coatings are volume-re~ ing coatings.
In l)lc;rt;lled embo~1iment~ the coating hinders the passage, and preferably essçnti~lly eompletely prevent the passage of: molecules having a moleeular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most 10 preferably more than about 400 kDa; or of immune system colllpollents such as Ig molecules or complement; or recipient-derived cells.
In pl~r~;lled embo-liment~ the component contains a source of a therapeutic substance.
In l)ler~ d embo-liment~ the microcapsule is incorporated into a composite, e.g. a double, or higher order composite.
In ~lc;fc.led embo-lim~nt~ the coating is geometrically stabilized prior to being implanted in a recipient.
In pler~lled embo-limentc the method includes (a) forming a gel core cont~ining a living cell, e.g., an islet cell;
(b) forming a relatively low molecular weight polyamino acid eoating on the eore.
In pler~ d embo~limentc the outer component of the microcapsule, i.e., the component in contact with the recipient, is at least S0, 75, 90, 95, 97, or 98 %, water.
In another aspect. the invention features, a method of making a composite microreactor having reduced volume. The method includes:
(a) forming a first component of a composite microreactor. (e.g., forming an intern~l 25 core particle, e.g., an internal gel particle);
(b) combining the first component with a second component of a composite microreactor, (e.g., embedding an internal core particle in a super matrix, e.g., a gel super matrix); and performing one or both of the following steps (c) and (d):
(c) prior to combining the first component with the second component, applying avolume reducing coating to the first component, (e.g., prior to embedding an intern~l core particle in a super matrix. applying a volume re~ cing coating of low molecular weight ~ polylysine to the core particle); and/or (d) applying a volume reducing coating to the second component, (e.g., applying a - 35 volume reducing coating to the super matrix), thereby producing a composite microreactor having a reduccd volume.

CA 0221~039 1997-09-09 WO 96128029 PCI~/US96/03135 In ~Jlcrelled embo~lim~ntc the volume re~ cing coating is or inclll~e~ a polyamino acid, e.g., polylysine, or polyo. . ,i~ , having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly ~lc;r~,~led are poly amino acids with a molecular weight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa. or about 5 kDa to less than 15 kDa, or about S kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~ler~lled are poly~minc~ l e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD.
In pler~ ,d embo-liment~ the first component is: a core, e.g., a gel core, e.g., an ~lginzlte core; an internal particle matrix; an internal particle; a super matrix; a particle, a particle matrix.
In ~lerell~d embodiments the second component is: a coating; a matrix, e.g., a particle matrix or a super matrix; a component of a higher order composite.
In plefe~lcd embo-liment~ the first component is a gel core or matrix and the second component is a coating; the first component is a particle or an internal particle and the second component is a coating; the first component is a particle or int~rn~l particle and the second component is a matrix, particle matrix, or super matrix.
In plcr~llcd embo(1iment~ one of the components contains a source of a therapeutic ~lbst~nt~e In ~lerclled embodiments the first component is a gel matrix, e.g.: a gel, e.g., a 20 hydrogel, e.gan ~lgin~te or agarose gel. In ~lerelled embo-1iment~ the gel matrix is other than a liquid. The gel matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA). or polyornithine (PLO).
In preferred embodiments the gel matrix hinders the passage, and preferably esc~ nti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa. preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ple~elled embodiments the first component is a gel matrix having a coating. In more prer~ ,d the coating is or incl~lclec: a polyaminoacid, e.g., pQlylysine (PLL) or PLO; a naturally occurring cnbst~n~e, e.g., çhitos~n, a synthetic polymer e.g., PAN-PVC. A
particularly ~lefelled coating is polyamino acid. e.g., polylysine or polyornithine, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly plerelled are polyamino acids, e.g., polylysines, with a molecular weight of about 1 kDa-4 kDa (or about I kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 - kDa, e.g., 9.7 kDa. Also ~lerc~led are polyaminc ~cid. e.g.. PLL or PLO, coatings in the CA 0221~039 1997-09-09 WO 96/28029 PCI~/US96103135 range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Preferred coatings are volume-re~lcinp co~tin~s In ~ler~,.led emborliment~ the coating hinders the passage, and preferably ec~enti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immllne system components such as Ig molecules or complement, or recipient-derived cells.
In ~lèrelled embo~liment~ the second component is a matrix. In more ~ler~lled embo-1iment~ the matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lpin~te or agarose gel.
10 In ~Jlef~ d embofliment~ the matrix is other than a liquid. The matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO).
A high molecular weight, molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons. or more, can be added to the super matrix to confer immunoisolating 15 ~lol.~,lLies.
In preferred embodiments the matrix hinders the passage, and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules 20 or complement; or recipient-derived cells.
In preferred embodiments the second component is a coating. In more plef~:lled embodiments the coating is or includes: a polyaminoacid, e.g., polylysine (PLL) or PLO; a naturally occurring substance, e.g., chitosan; a synthetic polymer e.g., PAN-PVC. A
particularly preferred coating is polyamino acid. e.g., polylysine or polyollliLlline, having a 25 molecular ~-eight of less than 15 kDa. more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly preferred are polyamino acids, e.g., polylysines, with a molecular w eight of about 1 kDa-4 kDa (or about 1 kDa-less than 4 kDa) e.g., 3.7 kDa, or about 5 kDa to less than 15 kDa, or about 5 kDa to less than about 10 kDa, e.g., 9 kDa-10 kDa, e.g., 9.7 kDa. Also ~lerell~d are polyaminoacid, e.g., PLL or PLO, coatings in the range of 1 or 2-10 kD, preferably in the range of 1-2, 1-3, or 1-4 kD. Preferred coatings are volume-reducing coatings.
In preferred embodiments the coating hinders the passage, and preferably ç~enti~lly ~ completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa. preferably more than about 100 kDa. preferably more than about 150 kDa, and most - 35 preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.

CA 0221~039 1997-09-09 WO 96/28029 PCI~/US96/03135 In ~crtll~,d embodiment the first component is geometrically stabilized by aging. In p-~rt:lred embo-1im~nt~ the aging period is: equal to or greater than 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 4 days, 7 days, 10 days, and 14 days; In more ~.~;r~ t;d embo-limt-nte the first component is allowed to age for between 2 hours and 14 days, e.g., for 5 between 12 hours and 4 or 5 days.
In ~l~r~"~d embo-lim~nt~ the first component has a lower molecular weight exclusion number than does the second Cu~ o~ent, e.g., the first component excludes recipient immlm~?
molecules, e.g., IgG or complement, and the second component, allows immlme molecules, e.g., IgG, or complement, to pass but excludes the passage of recipient cells.
In ~.ert.. ~,d embo-lim~ntc the outer surface of the composite is biologically con~ ible, e.g., it is sufficiently smooth that it inhibits fibrotic encapsulation of the composite; the outer surface of the composite is biologically compatible, e.g., it is sufficiently smooth that it inhibit fibrotic ens~pslll~tion of the composite but the surface of the int~
particle is not biologically compatible, e.g., it is not sufficiently smooth to inhibit fibrotic 1 5 encapsulation.
In ~rer~ d embodiment the second component is geometrically stabilized by aging.In ~.~f~ ;d embo-liment~ the aging period is: equal to or greater than 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 4 days, 7 days, 10 days, and 14 days; In more p.~r~ dembodiments the first component is allowed to age for between 2 hours and 14 days, e.g., for 20 between 12 hours and 4 or 5 days.
In ~cr~ed embo-limt?nt~ the composite microreactor is a component of a higher order composite, e.g., a double composite, or a third order composite.
In a pl~:f~lled embodiment. the method further includes incorporating the composite into a higher order composite, e.g.. a double composite. In more preferred embo-liment~, a 25 volume-reducing coating is applied to the composite (or one of its components) prior to incorporating it into a higher order composite, e.g., a double composite. For example, an intf-rn~l agarose particle coated with polylysine of between about I kDa-4 kD and co,-l;-;";,~g a living cell, e.g., an islet, is formed. The internal particle is then incorporated into the particle matrix. The particle matrix is then coated with polylysine of between about S kDa to 30 less than about 15 kD. Finally, the coated composite is embedded into an agarose matrix to form a double composite.
In plc;rt;lled embodiments the composite microreactor is a double composite microreactor and: the first component is an internal particle and the second component is an internal particle coating; the first component is an internal particle and the second component 35 is a particle matrix; first component is an internal particle coating and the second component is a particle matrix; first component is a particle and the second component is a particle coating, first component is a particle and the second component is a super matrix; first CA 0221~039 1997-09-09 WO 96/28029 PCI'IUS96103135 conl~ollent is a particle coating and the second component is a super matrix; first component is a super matrix and the second colll~,ollent is an outer co~ting In ~lcÇ~ ,d embo-liment~ the outer component of the composite microreactor, i.e., the colll~ollelll in contact with the recipient, is at least 50, 75, 90, 95, 97, or 98 %, water.
The inventors have also discovered that a high molecular weight molecule, e.g., a polymer, e.g., PEO, can be added to a microcapsule to improve its immllnc isolating p~ lies. Because of its high molecular weight, the molecule does not need an outer coating to prevent it from leaking out of the matrix, yet the molecule does not result in unacceptable viscosity.
Accordingly, in another aspect, the invention features, a microcapsule, e.g., a composite microreactor as described herein, which includes:
a source of a therapeutic substance, a molecule, e.g., a polymer, e.g., PEO, of at least 1-8 million Da in molecular weight, and a matrix, the source and the molecule of at least 1-8 million Da embedded in the matrix.
In plcfcllcd embo~1iment~ the matrix does not have an outer coating.
In ~lefcll~d embodiments the matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~lgin~te or agarose gel. In p,crcllcd embo~liment~ the matrix is other than a liquid. The 20 matrix can include substances which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO). A high molecular weight, molecule, e.g.~ a polymer, e.g., PEO, with a molecular weight of 1-8 million daltons, or more, can be added to the super matrix to confer immunoisolating properties.
In preferred embo-liment~ the matrix hinders the passage. and preferably essentially completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells.
In ~lcrclled embo~liment~ the outer component of the microcapsule, i.e., the component in contact u-ith the recipient, is at least 50, 75, 90, 95. 97, or 98 %, water.
The inventors have also discovered that it is not necess~ry to reliquify the gel matrix of a microcapsule, e.g.. the internal matrix, particle matrix, or super matrix of a composite microreactor as described herein. Allowing the matrix to remain a gel elimin~tes the 35 liquefaction step, preserves the immunoisolating ~ ;>p~_L Lies of the matrix, and does not unacceptably hinder diffusion or cell viability.

CA 0221~039 1997-09-09 Accordingly, in another aspect, the invention realules, a microcapsule, e.g., a composite microreactor as described herein, which in-~ln~ s:
a source of a th~ld~ Lic substance, and a matrix which is not a liquid.
In ~l~r.,.led embo~liment~ the matrix is or includes: a gel, e.g., a hydrogel, e.g., an ~l~in~tc or agarose gel. The matrix can include s~lhst~nce~ which impede the passage of recipient-derived molecules or cells, e.g., it can include polyethelyne oxide (PEO), poly~Lylelle sulfonic acid (PSA), or poly-,., .; L~ e (PLO). A high molecular weight, molecule, e.g., a polymer, e.g., PEO, with a molecular weight of 1-8 million rl~ltQn~, or more, 10 can be added to the super matrix to confer immnnnisolating ~lop~;lLies.
In pr~r~;lled embo-lim~nt~ the matrix hinders the passage, and preferably ec~enti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immune system components such as Ig molecules IS or complement; or recipient-derived cells.
In plefc.led embo~liment~ the outer component of the microcapsule, i.e., the component in contact with the recipient, is at least S0, 75, 90, 9S, 97, or 98 %, water.
In yet another aspect, the invention features~ a single composite microreactor which includes:
(a) one, or a plurality, of an internal particle (preferably cont~ining a living cell);
(b) optionally, an internal particle coating covering the internal particle;
(c) a matrix component in which the internal particle is embedded;
(d) optionally, an outer coating covering the matrix component, 25 wherein, at least one component of the single composite microreactor is free of defects which arise from the inclusion of non-geometrically stabilized components.
In yet another aspect, the invention features. a double composite microreactor which includes:
(a) one, or a plurality, of an internal particle (preferably co.,~ g a living cell);
(b) optionally. an internal particle coating covering the internal particle, (c) one, or a plurality, of a matrix a component in which the intern~l particle is embedded;
(d) optionally. a particle or matrix coating covering the matrix component;
(e) a super matrix in which the matrix component is embedded;
(f) optionally. an outer coating covering the double composite, wherein, at least one component of the double composite microreactor is free of defects which arise from the inclusion of non-geometricallv stabilized components. In ~lert;lled CA 0221~039 1997-09-09 WO 96/28029 PCI~/US96/03135 embo~liment~ one co.,l~ollent of the double composite microreactor, e.g., the matrix ~ cGl"l)o,lent or its co~tin~, is not free of defects which arise from the inclusion of non-geometrically stabilized con~onents, but the super matrix is free of such defects.
In yet another aspect, the invention r~Lul~;s, a method of impl~nting a living donor S cell into a recipient. The method includes the steps of:
providing a microcapsule, e.g., a composite microreactor, of the invention whichco"l~ins the living donor cell; and implanting the microcapsule into the recipient animal.
In ~ r~ d embofliment~ the method further includes testing the recipient for antibodies to the living cell. The test can be performed before or after the microcapsule is 1 0 implanted.
In ~l~Ç~ d embo-liment~ no adjunctive imm-m~ ,lession is ~11mini~tered to the recipient.
In pler~ d embo-liment~ the method further includes: ~imini~tering to the recipient adjunctive immuno 7U~ S ,ion for less than 30 days; the step of ~imini~tering a drug to the host animal at a dosage effective to inhibit fibrosis and infl~mm~tion around the uncoated particle, but at a dosage lower than that required to achieve immlmf~u~pl~ion when the donor cell is implanted into the host animal without encapsulation, e.g., wherein the drug is cyclosporin A and is ~imini~tered at a dosage that achieves a whole blood trough level of less than about 100 ng/ml in the host animal.
In ~lefelled embo-liment~ the cell is: a living cell; a eukaryotic cell, e.g., a rodent, canine, porcine, or human cell; a prokaryotic cell, e.g., a bacterial cell; a fungal or plant cell;
a fungal or a plant cell; a cell which is genetically engineered, e.g., a cell which is genetically engineered to produce a protein, e.g., a human protein.
In pler~ d embo-iiment~ the cell is an autologous, an allogeneic, or a xenogeneic cell. For example, the cell is: an autologous cell, i.e., a cell which is taken from the individual recipient into which the cell will be implanted; an allogeneic cell, i.e., a cell which is taken from a different individual of the same species as the recipient into which the cell will be implanted; a xenogeneic cell, i.e., a cell which is from a ~lirr~l~llL species than the recipient into which the cell will be implanted. In the case of an allogeneic cell, the cell can be fully m~tched or partially m~tched for MHC class I loci, fully m~tch~(l or partially matched for MHC class II loci, and fully matched or partially matched for minor loci.
In preferred embo~liment~ the recipient animal is a dog, a pig, or a human.
- In ~ fclled embotliment~ the donor cell is a pancreatic islet cell.
In plefelled embo-liment~ the composite microreactor contains pancreatic islets. e.g., at e.g., a density of 5,000 to 100,000 islets per milliliter of medium; the composite microreactor contains living cells at a density of about 105 to 105 cells per milliliter of medium.

-CA 0221~039 1997-09-09 WO 96/28029 PcT/u:~5~ 3l35 In plert;;lled embol1imenti the recipient is s11ff~ring from a disorder, e.g., diabetes, caused by a deficient production of a substance, e.g., insulin, in the recipient and the donor cell provides a substance which treats the disorder.
In ~lcrelled embo-limt-nt~ the donor cell secretes Factor IX, Factor VIII, an intrr1e11kin, e.g., intPr1enkin 2, an hl~;lrt;loll, an endocrine hormone, a nerve growth factor, tumor necrosis factor alpha, a n~;uloLlu~ic factor, or a n~uroL~ mittrr.
In ~lt;Çell~id embo-lim~nt~ the disorder is diabetes mellitus, hepatic ~ e~e~
amyotrophic lateral sclerosis, hemophilia, hypothyroidism, Parkinson's ~ e~ee, acquired immune deficiency syndrome, Dllrl~pnnr~s muscular dy~Llu~hy, infertility, epilepsy, Huntington's rli~e~ce~ hy~,op~dLllyl~,idism, a mood disorder, a motor neuron tli~e~e~
osteoporosis, or ~17heim~r's disease.
In lJle~lled embo-1iment~ the composite microreactors are implanted into an privileged site in the patient; the composite microreactors are implanted by injection.
In yet another aspect, the invention features, an artificial organ suitable for l 5 implantation into a m~mm~1, which includes one or a plurality of an effective number of the composite microreactors of the invention.
In yet another aspect, the invention features, an insulin producing system, including a composite microreactor (including a living cell) in a culture medium, wherein the living cell is a m~mm~ n islet of Langerhans cell, and wherein the culture medium comprises nutrients and amino acids sufficient to m~int~in the cell and allow the cell to synthesize insulin.
In yet another aspect, the invention redlules, an in vivo method of culturing a living cell, the method including the steps of: çnr~ps~ ting the living cell in a microreactor of the invention, inserting the microreactor into an animal, e.g., a non human animal, and culturing the cell in the animal.
In yet another aspect, the invention features, a preparation of: a microcapsule which includes a living cell, e.g., a composite microreactor, of the invention, and a carrier suitable for implantation into a human. In ~lef~lled embo-liment~ the pl~dLion is contained in a device suitable to deliver the microcapsules to a recipient, e.g., a device for injection the ~lepaldLion into a recipient.
In yet another aspect, the invention features, a microcapsule which includes a living cell, e.g., a composite microreactor, of the invention, in a culture medium which will support the living cell.
In yet another aspect, the invention features, a microcapsule which includes a living cell, e.g., a composite microreactor. made by a method described herein.
Microcapsules of the invention. e.g., single or double composites, are preferably less than 4,000, more preferably less than 3,500. 3,000, 2,500, 2,000, l,500, or l.000 microns in meter.

CA 0221~039 1997-09-09 In single composite microreactors of the invention, the thickness of the super matrix (i.e., the ~liet~n~ e bcLwc;en the inner and outer surfaces of the super matrix) is preferably at least 10, 25, 50, 100, 150, 200, or 250 microns.
In double composite mi~ ea~;k~l~ of the invention, the thickness of the particleS matrix (i.e., the ~ t~n~ e b~;lwt;~n the inner and outer surfaces of the particle matrix) is preferably at least 10, 25, 50, 100, 150, 200, or 250 microns; the thickness of the super matrix (i.e., the ~ t~nre bc;Lwe~ll the inner and outer surfaces of the super matrix) is preferably at least 10, 25, 50, 100, 150, 200, or 250 microns.
Microcapsules of the invention, e.g., single or double composites, are preferably more 10 than 80, more preferably more than 100, 200, 400, 600, 800, 1.000, 1,200, 1,400, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 microns in diameter.
In single composite microreactors of the invention, the diameter of the int-orn~l particle is preferably more than 80, more preferably more than 100, 200, 400, 600, 800, 1,000, 1,500, or 2,000 microns. The diameter of the single composite microreactor is 15 preferably more than 200, more preferably more than 400, 600. 800, 1,000, 1,200, 1,400, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 microns.
In double composite microreactors of the invention, the diameter of the internz~l particle is preferably more than 80, more preferably more than 100, 200, 400, 600, 800, 1,000, 1,500, or 2,000 microns. The diameter of the particle is preferably more than 100, 20 more preferably more than 200, 400, 600, 800, 1,000, 1,500, 2.000. or 2,500 microns. The diameter of the double composite microreactor is preferably more than 200, more preferably more than 400, 600. 800~ 900, 1,000, 1,200 1,400, 2,000, 2,500. 3,000, 3,500, 4,000, 4,500, or 5,000 microns.
As used herein, microcapsule, refers to a source of a therapeutic substance enclosed in 25 an applied semipermeable component, e.g., a matrix and/or a coatin~. The component is one which excludes one or more of IgG, complement, or cells.
As used herein. a source of a therapeutic substance can include a composition ofmatter which produces or releases a therapeutic substance, e.g.. a protein, e.g., an enzyme, hormone, antibody, or cytokine, a sense or anti-sense nucleic acid, e.g., DNA or RNA, or 30 other substance which can exert a desired effect on a recipient. The therapeutic substance can also be a composition of matter which absorbs or modifies or detoxifies a substance produced by the recipient. The source of a therapeutic substance can be a tissue or a living cell; a eukaryotic cell, e.g.. a rodent. canine, porcine, or human cell: a prokaryotic cell, e.g., a b~ct~ri~l cell; a fungal or plant cell; a cell which is genetically engineered, e.g., a cell which 35 is genetically engineered to produce a protein, e.g., a human protein. The source of a therapeutic substance can be or include an autologous, an allogeneic. or a xenogeneic cell.
For example, the cell is: an autologous cell, i.e., a cell which is taken from the individual CA 0221~039 1997-09-09 WO 96/28029 ~,llU' _ -~0313~

recipient into which the cell will be implanted; an allogeneic cell, i.e., a cell which is taken from a ~lirr~,~e.,l individual of the same species as the recirient into which the cell will be implanted; a xenogeneic cell, i.e., a cell which is from a dirrtle l~ species than the recipient into which the cell will be implanted. In the case of an allogeneic cell, the cell can be fully S m~trhPrl or partially m~trhPd for MHC class I loci, fully m~trhP-l or partially m~tchPcl for MHC class II loci, and fully m~tc hPcl or partially m~trhPcl for minor loci.
Many clinically useful cells, e.g., normal pancreatic islets, cannot presently be efficiently propagated in culture. This, together with a relative shortage of human donor tissue, means that widespread clinical transplantation of such cells, e.g., to treat diabetes, will 10 likely require the use of discordant xenografts. The invention allows transpl~nt~ti~ n of microellr~ps~ t~cl allogeneic or xenogeneic donor tissue, even discordant xenogeneic tissue, with little or no adjunctive immuno~u~,~;s:jive therapy.
Composite microreactors of the invention allow the use of internal particle coatings which are not biocompatible, e.g., polyacrylate or plastics. Methods of the invention allow 15 the use of virtually any material which can be used to form permselective barriers, e.g., materials such as, chitosan, polyacrylate, or synthetic polymers, which alone might be digested by enzymes. or which might trigger a fibrotic response when they come into direct contact with host tissues. Furthermore, microcapsules of the invention having an outer hydrogel coating inhibit the scarring of organs which often accompanies the long-terrn use 20 of hard plastic shells.
The ,~eometrically stabilized microcapsules of the invention have improved performance characteristics. For example, they are more resistant to invasion by components of the host immune system. Geometrically stabilized microcapsules are also more effective in preventing release of implant antigens, and thereby help to ~ "i,P the host's anti-25 implant response.
Embo~limPnt~ of the invention feature the use of coatings, which, result in a decreasein the volume of the coated particle. These coatings are referred to herein as volume-reducing coatings. The use of volume-reducing coating confers a number of advantages on a microcapsule, e.g.. a composite microreactor. With many prior art encapsulation methods, 30 the encapsulation of a number of cells which is sufficient to provide a sustainable biological effect in the host results in an undesirably large volume. Use of the volume-reclnring methods and materials of the invention allows a useful nurnber of cells to be delivered in a smaller volume. The use of volume-re~ cing coating. e.g.~ low molecular weight polylysine, can result in a 30% decrease in diameter of the internal particle (with an even greater percent 35 reduction in v olume of the particle). Without the use of volume-recltlr,in~; methods, the volume of the enr~pslll~tP~l beads can give a greater than 300% increase in the volume of the particles. For example. a 30% increase in diameter gives a 3-fold increase in volume.

CA 0221~039 1997-09-09 The use of smaller microcapsules can also reduce the risk of producing a fibrotic response. There are two types of fibrotic responses, an acute response and a chronic response. The acute response is gt?ner~t~cl in large part by the degree of smoothness of the exterior surface of the mi~;locdl,sule. The introduction of a microcapsule having a rough S surface will trigger a relatively rapid fibrotic response. An acute response can usually be avoided by the use of a microcapsule having a smooth exterior surface. The size of a microcapsule plays a large part in the generation of a chronic fibrotic response. A smooth surface mic.oca~sule, if it has a diameter of about 2 mm or more, will, after few weeks-months, often elicit a chronic response. By using a volume-re~lucin~ coating, relatively 10 small, preferably smaller than or about equal to 1 mm in diameter, single or even double composite microreactors can be produced. Furthermore, the volume reducing coating will allow for easier ~le~a,dlion of microcapsules, e.g., composite microreactors, as smaller the microcapsules are generally easier to produce.
The reduced size microcapsules of the invention can contribute to sustaining the15 viability of encapsulated cell. Microcapsules having a relatively large diameter, e.ga radius of greater than 0.5 mrn generally make it more difficult for the encapsulated cells to exchange necessary nutrients and oxygen with the outside environment. This can result in premature cell death. Again, by use of a volume re~ ing coating, the diameter of single, or double composite microreactors can be minimi7~-1 and thereby prolong the period for which an encapsulated cells is viable.
The composite microreactors of the invention allow important properties of the composite microreactors to be allocated or partitioned to different components of the composite microreactor, thereby allowing the properties to be optimi7~ ~1 For example, it has been found that although the use of lower weight polyamino acids as coatings results in a more effective barrier to molecules of the host's immune system, e.g., IgG~ these coating are sometimes associated with undesirable l,~u~ ies. For example, they can have surfaces which do not effectively inhibit fibrosis and they are often not geometrically stable just after m~nuf~cture and can thus introduce faults in other components of the composite microreactor. As is discussed herein, the inventors have found that geometrically stabilizing the intern~l particles can minimi7~ the induction of faults and that the inability to inhibit fibrosis can be overcome by using such surfaces as an internal component of a composite microreactor. but not on the surface which faces the body of the recipient.
- The inventors have also discovered that the tendency of certain components. e.g.~ low molecular weight polyamino acids to induce faults~ or to otherwise co~ .o..lise performance, - 35 can be countered not only by geometric stabilization~ but by using such coatings on the most int~rn~l components of a microcapsule. For example~ in the case of a double composite, these materials can be used as coatings on the internal particles. The intPrn~l particle coating CA 0221~039 1997-09-09 WO 96/28029 1 ~ u~g6lo3l35 is se~ Led from the host by the particle matrix, the particle coating (if present), the super matrix, and, the outer coating (if present). Even if the int~rn~l particle coating in~ ces a fault in the particle matrix, the particle coating (if present), the super matrix, and the outer coating (if present) still hinder the passage of host components, e.g., host cells.
S This approach is particularly advantageous when it is desirable to m;.l;lll;~t~ the amount of time taken by geometric stabili_ation. Prolonged cell culture is not compatible with viability in all cell types. Some cells, e.g., some l~lh,.~ cells, can survive in the host but cannot survive for exten~ periods in culture. Stabili_ation generally takes longer the lower the molecular weight of the coating. For example, in the contlition~ described herein, 10 ~ ullial stabilization of 5 kDa to less that 15 kDa polylysine coatings takes about a day, while substantial stabilization of 1 kDa-4 kDa, about 1 kDa-less than 4 kDa coatings takes 3-5 days) Thus, in some applications it is desirable both to use a geometrically "unstable-' component (or at least to minimi7e the amount of stabilization time) and to minimi7~ the "culture" time of the source, e.g., a cell, to be delivered. In embo-liment~ of the invention, 15 performance is hl.~,lov~d by using a double composite in which the internal particle coating provides protection from smaller species, e.g., molecular as opposed to cells, e.g., IgG
molecules, while other components e.g., the particle coating and/or super matrix, while not providing a barrier to smaller species, provide a barrier to large species, e.g., cells. Thus, in embo~limcnt~ of the invention, the component which provides protection from the smaller 20 species, and which may induce faults in an adjacent component, is separated from the component(s) (e.g., the particle coating and super matrix) which provide protection from invading cells, by an intervening, or ":buffer" component, e.g., the particle matrix. Thus even if the internal particle coating induces faults in the particle matrix, the particle coating and/or super matrix prevent contact of cells with the internal particle coating ~which is often not 25 anti-fibrotic).
If polylysine of about 1 kDa-4 kDa, about 1 kDa-less than 4 kDa is used on the int~qrn~l particles, and a less unstable coating, e.g., a coating of about S kDa to less than 15 kDa, or of about 5 kDa to less than about 10 kDa, is used on the particle. a geomet-ric stabilization of as little as about 24 hours can be used with the internal particle. A
30 particularly effective composite uses a polyamino acid, e.g., polylysine~ of less than 5 kDa as the intern~l particle coating and a polylysine of about 9 kDa-10 kDa as the particle coating.
The diameter of the internal particle is about 300 microns, the particle about 600 microns, and the entire composite about 900 microns.
Composite microreactors of the invention have reduced size, and can therefore have 35 reduced size and allow delivery by injection. For example, composite microreactors of about 1,000 microns or less in ~ met~?r can be delivered with a 16 gauge needle. Larger microreactors, e.g., those of 1, 500 microns in diameter, generally require larger needles.

CA 0221~039 1997-09-09 The release of small components, e.g., cytokines, NO, and other toxic moieties by recipient immlmP cells plays an i~ olL~-l role in the inactivation of implanted donor tissue.
Microc~psllles of the art often possess only a thin barrier, e.g., a polylysine coating and/or a thin electrostatic exterior coating of agarose, between the en~rslll~tetl cells and reciri~ont 5 cells. Even though recipient cells may be denied direct contact with the enf ~rslll~t~-l cells, the recipient cells can come into close enough ploxillliLy such that small molecules (which pass through any se,.li~....eable membrane present on the microcapsule) they release are present in sufficient con~çntrqtion inside the microcapsule to damage the en~rslll~t~cl cells.
Composite microreactors of the invention can include one or more components which, 10 s~,~dL~ly or togetherr are of sllfficient tli~m~ter~ or of sufficient thickness, such that they provide a substantial ~ t~nre between recipient cells, e.g., lymphocytes, macrophages, or NK
cells, and the source of a therapeutic subst~nre The distance is sufficient such that exposure of the source to small molecules e.g., cytokin~, NO, and other toxic moieties, released by recipient cells, is suhst~nti~lly reduced by diffusion. Composite microreactors of the 15 invention can thus minimi7~ the concentration of recipient cell-released small molecules and thereby promote the viability or function of the encapsulated cells.
The release of donor antigens, e.g., proteins, into the recipient, or into outercomponents of the composite which might contain recipient cells. can stim~ te a recipient immune response to donor antigens. Composite microreactors of the invention can include 20 one or more components which, separately or together, are of sufficient diameter, or of sufficient thickness, such that they provide a substantial distance or s~dlion between antigens released by the source and recipient cells, e.g., lymphocvtes, macrophages, or NK
cells. The ~ t~n~e is sufficient such that diffusion, or trapping in the matrix of the component or components which provide the separation, substantially reduces exposure of 25 recipient cells to released antigens. This is particularly important in inhibiting recipient cell:donor antigen contact in the case of acute release of donor antigen, e.g., the release of comparatively large amounts of antigen over a short time as a result of cell death. The matrix of the component or components which supply the separation can also act to inhibit diffusion of larger protein antigens which are efficiently processed into antigenic species by the 30 recipient. The ~ t~n~e also minimi7~-s the contact of cells of the recipient with donor antigen, e.g., proteins. w hich protrude from or extend through one or both of the int~rnsll particle matrix or internal particle coating. Composite microreactors of the invention can - thus minimi7~ the recipient response to donor antigens. This is particularly important because once the recipient is sensitized to a particular donor species it is much more difficult 35 to provide s~lcces~ful encapsulation is subsequent implantations.

CA 0221~039 1997-09-09 WO 96/28029 PCT/Ui~ 3135 Composite reactors of the invention, because they allow sources to be sequestered within int~rn~l particles, can prevent the protrusion of sources through the outer surface of the composite.
Multiple barrier devices of the art can have unacceptable ~ m.otPrs. The methods of S the invention disclosed herein allow production of multiple barrier devices of 1i~-;, diameter.
The inventors have discovered that ~i~nific~nt advantages flow from the use of low molecular weight polyamino acids (e.g., in the range of 1 or 2-10 kD, preferably in the range of 1-3 or 1-4 kD) as s~,l,il,c;""eable co~ting~ The inventors have discovered that a low molecular weight poly amino acid, e.g., polylysine, covering "shrink wraps" the enr~pslll~tr(l particle and actually reduces the diameter and volume of the encapsulated particle. A
reduction in size confers a number of advantages.
The shrinl;age intlllred by the low molecular weight poly amino acid, e.g., polylysine, allows for a relati~ely greater part of the radius of a reactor to be devoted to the particle matrix and super matrix components. In other words. we can devote more of the rli~me,ter to, e.g., the particle matrix or super matrix. This means that the physical ~ t~nre between the islet and the outer surface of the microreactor can be m~imi7~rl This results in more separation from toxic substances, e.g., NO, produced by the host. It also increases the amount of matrix through which graft antigens must sieve to reach the outer surface. The particle and super matrix are relatively thick layers and provide much greater protection than the thin electrostatic layers of ~lgin~tr seen in multi-layer capsules in the art.
The shrin~;age in~ cetl by the low molecular weight poly amino acid, e.g., polylysine, results in a compression of the encapsulated :~lgin~tr and results in a higher ~lgin~te concentration at the poly amino acid gel, e.g., ~lgin~tr intrrf~re This "çnh~nr,e-l" intrrf~re has a high positive charge density on one side and a high negative charge density on the other side. The gel concentration at the interface is ~ignific~ntly greater than that prior to shrink~ge or that found in the center of the particle. Thus, the invention includes a microreactor having one or more components with an enhanced interface. This unique interfacial structure is believed to contribute to improved isolation and may modulate release of implant antigen.
The shrinkage indllred by the low molecular weight poly amino acid, e.g., polylysine, provides a semi-conformal covering. A semi-conformal coating is one which reduces the amount of volume around the enclosed entity. A shrink wrapped islet is pedestal in shape and allows better diffusion . It also allows increased density of encapsulated tissue, i.e., it allows a greater amount of encapsulated tissue per unit of surface area or increased metabolic activity per unit of volume of the microreactor. The pedestal greatly increases the surface to volume ratio. Thus. the invention includes a microreactor having one or more components with a semi-conformal coating.

CA 0221~039 1997-09-09 WO 96/28029 P~;l/u~ -'03135 The chrink~ge in-1ucecl by the low molecular weight poly amino acid, e.g., polylysine, h,~ es the volume to islet ratio and allows tre~tmtont with a smaller ~t1mini~t~red volume.
This is hllL,ol L~lt because it allows for a safe and useful volume. This is particularly hll~ when booster injections are con~i(lered. Xenografts also require more islets as the insulin output is lower, so volume is even more important.
The invention inc lll-lç~ the use of polyamino acids, e.g., polylysine, of low molecular weight. Low molecular weight co~tings are less immunogenic than are larger polymers.
The low molecular weight polyamino acid, e.g., polylysine, coatings results in a 3-~limen~ional and cross-linked co~ting- The coating penetrates more deeply into the surface of 10 the gel particle than would a coating of higher molecular weight.
Geometric stabilization is crucial for permselectivity of the poly arnino acid, e.g., polylysine coating. If poly amino acid, e.g., polylysine, is applied to early Igg protection will be col"p,~ ised. If applied too late there is le~c-hing and co"~lol"ised coverage with PLL.
The ~.~rel.ed ranges for islet density in the reactor is 10-100, preferably 20-50 15 islets/mm3.
Small size is also preferably because it allows the reactors to be delivered by injection.
Unless otherwise clefin~-l all technical and scientific terms used herein have the same me~ning as commonly understood by one of oldi--~-y skill in the art to which this invention 20 belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the ~-ert;:l,ed methods and m~tf ri~l~
are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In addition, the materials, methods, and examples are illustrative only and not intt-n~lP~l to be limiting.
Other features and advantages of the invention will be a~ale~L from the following detailed description and from the claims.
Detailed Description Brief Description of the Drawings Fig. 1 is a sch~m~tic diagram of a composite microreactor.
Fig. 2 is a srhPm~tic diagram of a double composite microreactor.
Composite microreactors A microcapsule should possess a variety of diverse p-op~ ies if it is to preserve the viability of the encapsulated material~ allow rapid~ timely and adequate release of donor tissue produced substances, and satisfy other clinical requirements. The microcapsule should 35 possess the following plop~,.Lies:
( 1 ) it should allow relatively efficient diffusion of critical nutrients from the recipient environment into the microcapsule;

CA 0221~039 1997-09-09 WO 96/28029 ~ 96/0313~

(2) it should allow relatively efficient diffusion of donor cell waste products out of the rnicrocapsule;
(3) it should allow eff1cient diffusion of recipient signal molecules, e.g., in the case of the tre~trnent of diabetes, glucose, into the microcapsule, (4) it should allow diffusion of the critical s~lbst~nce supplied by the en~ps-ll~te~
cells, e.g., in the case of the tre~tm~ont of diabetes, insulin, into the recipient milieu, (5) it should ,..;.,i.";~ non-immnne inactivation, e.g., by fibrotic ellc~ulation, ofthe microcapsule;

(6) it should minimi7.o contact of the recipient's immllne system with the ~ne~rslll~tecl 1 0 cells;

(7) it should have a life time of about the same length as the encapsulated cells;

(8) it should use, to the extent possible, biocompatible m~teri~

(9) it should be biocompatible with respect to the graft or islet tissue, and (10) it should minimi7~ release, particularly acute release, of antigens which might 5tim~ te a recipient immune response.
Although some of these plo~ ies, e.g., 4, 5, and 6, must be possessed by a microcapsule, the properties need not be po~e~e~l by all components of the microcapsule.
E.g., internal components need not be anti-fibrotic and not everv element of the device needs to be effective in inhibiting the passage of recipient immune components, e.g., Ig molecules or complement; or recipient-derived cells. As described in more detail below, the multi-c.,llll,onent structure of the composite microreactors of the invention allow segregation of functions and properties and thus allow greater freedom in the choice of m~teri~lc and greater efficacy of the microcapsule.
Structural Components Fig. 1 is a schematic diagram of a simple or single composite microreactor (10). The composite microreactor (10) contains at least one, and preferably a plurality of intern~l particles (20). An internal particle (20) in~hlc~ one or a plurality of sources (30) of a therapeutic or otherwise desirable s~lhst~nre The sources (30) are embedded carried on, adhered to, or in an internal particle matrix (40). The internal particle (20) can optionally include an internal particle coating (50). The internal particles can be embedded in a super matrix 60. The composite microreactor 10 can (optionally) include an outer coating 70.
The ~ met~r of the composite microreactor ( 10) is preferably between 100 microns and 4 millimeters, between 300 and 1200 microns, between 300 and 1500 microns, between 400 and 1000 microns. or between 400 and 800 microns. More preferably the diameter is about 600 microns.
The diameter of the internal particles (20) before application of a volume-reducing - coating (described below) is preferably between 50 and 700 microns. more preferably CA 0221~039 1997-09-09 .

bclwcen 100 and 500 microns, more preferably between 200 and 400 microns, and most preferably about 300 microns in ~ m~ter. The ~ metpr of the intPm~l particles (20) after application of a volume-re(lnc ing coating (described below) is ~lcre~bly between 35 and 500 microns, more preferably between 75 and 400 microns, more preferably between 100 and 300 microns, and most preferably about 200 microns in ~ mptpr.
As is discussed in more detail elsewhere herein, the source 30 can be a cell, or a group of cells, e.g., an islet. The sources of an intPrn~l particle can all be of one type or more than one type of source can be included in an intPrn~l particle. Furthermore, the composite microreactor 10 can include more than one type of intern~l particle (20), e.g., the co",~o ,iLc mi-,lorca.ilor 10 can include a first type of intPrn~l particle (20) having within it a first source, e.g., a first type of cell, and a second type of intPrn~l particle (20) having within it a second source, e.g., a second type of cell.
The intemal particle matrix (40) can be a gel, e.g., a hydrogel, e.g., ~lgin~te or agarose. The intemal particle matrix can also be a solid particle, e.g., a glass bead, or a porous structure, on which anchorage dependent cells can be seeded. The intern~l matrix (40) can have immnnc isolative properties. In some embo-liment~ it has little or no ability to exclude low molecular ueight species, e.g., IgG or complement. with this plopCl~y being relegated to other components ofthe composite microreactor (10). The intPrn~l particle matrix (40) can be rendered imml~noisolating by controlling its porosity, e.g., such that it hinders the passage of molecules of relatively large molecular weight, or by adding to it components, e.g., polyethylene oxide (PEO), polystyrene sulfonic acid (PSA), or polyornithine (PLO) which hinder the passage of molecules of relatively large molecular weight. Regardless of the method of controlling its permeability. the intPrn~l matrix (40) will, in preferred embodiments will hinder the passage, and preferably~ ec~Pnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa~ preferably more than about 150 kDa, and mostpreferably more than about 400 kDa; or of immune system components such as Ig molecules or complement; or recipient-derived cells. The intem~l matrix (40) need not be anti-fibrotic and need not be biocompatible. The composite microreactor (10) can include more than one type of intPrn~l particle (~0). e.g., the composite microreactor ( l O) can include a first type of intPrn~l particle (20) having within it a first type of intPrn~l matrix (40) and a second type of intPrn~l particle (20) ha~-ing within it a second type of internal matrix (40).
The intern~l particle coating (50) is optional. It can be made of a poly:~mino~cid, e.g., polylysine (PLL), PLO chitosan, or PAN-PVC, or any coating used to coat non-composite microreactors. In addition. the coating can be a formed by modifying the structure of the matrix, e.g., the matrix can be cross-linked, e.g., with metal ions. e.g.. Ba or Fe ions, or by photo-cross-linking to form an coating. A ~ r~llcd coating is polylysine having a CA 0221~039 1997-09-09 W O 96/28029 PC~rlU~ 3135 molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than S kDa. Particularly I~lcr~ d are polylysines with a molecular weight of about 3-4 kDa, e.g., 3.7 kDa, or about 9 kDa-10 kDa, e.g., 9.7 kDa. Pl~r~llcd coatings are volume-re~1n~ ing co,.ting~ Furthermore, the composite microreactor 10 can include more than one 5 type of internal particle (20), e.g., the composite microreactor 10 can include a first type of int~ n~l particle (20) having a first type of internal particle coating and a second type of int~rn~l particle (20) having a second type of internal particle coating (50). Because, in some embo~1iment~ the internal particles coating (50) need not be biocolll~Lible and need not be anti-fibrotic, other properties of the internal coating (50). e.g., its ability to immllnnisolate~
10 can be optimized without the nece~city of any colllplulnise to allow confer biocompatibility or anti-fibrotic activity.
Super matrix (60) can be a gel, e.g., a hydrogel. e.g., ~lgin~te or agarose. The super matrix (60) can have immunoisolative properties. In some embodiments it can have little or no ability to exclude low molecular weight species, e.g.. IgG or complement, with this 15 pl~ y being relegated to other components ofthe composite microreactor (10). The super matrix (60) can be rendered immunoisolating by controlling its porosity, e.g., such that it hinders the passage of molecules of relatively large molecular weight or by adding to it components, e.g.. PEO, PSA, or PLO which hinder the passage of relatively large molecules.
Regardless of the method of controlling its permeability. the super matrix (60) will in 20 ~l~r~lled embodiments, will hinder the passage, and preferably e~sPnti~lly completely prevent the passage of: molecules having a molecular weight of more than about 50 kDa, preferably more than about 100 kDa, preferably more than about 150 kDa, and mostpreferably more than about 400 kDa; or of immnne system components such as Ig molecules or complement: or recipient-derived cells. The super matrix (60) need not be anti-fibrotic and 25 need not be biocompatible if a more proximal or more exterior component supplies these functions.
Outer coating (70) is optional. It can be made of a polyaminoacid, e.g., PLL or PLO, chitosan, or PAN-PVC. or any coating used to coat non-composite microreactors.
~ltern~tively, the coating can be a formed by modifying the structure of the matrix, e.g., the 30 matrix can be cross-linked, e.g., with metal ions, e.g.~ Ba or Fe ions, or by photo-cross-linking, to form an coating. A plc~rtl,c~d coating is polylvsine having a molecular weight of less than 15 kDa. more preferably of less than 10 kDa. more preferably of less than S kDa.
Particularly p~eftlled are polylysines with a molecular ~ eight of about 3-4 kDa, e.g., 3.7 kDa, or about 9 kDa-10 kDa, e.g., 9.7 kDa. Preferred coatings are volume-re-lllcing co~ting~
35 The outer coating (70) need not be immunoisolating if other components supply this function.
The multi-component structure of the composite microreactor allows selection of m~t~ris~l~ which can optimize perform~n~ e Coating materials which are highly CA 0221~039 1997-09-09 WO 96/28029 PCI'/US96/03135 immlmoisolating, but less desirable in terms of their bioconl~libility or anti-fibrotic activities, can be used in the int~rn~l particle coating. The multi-co~ ullent structure also allows for mtlltiple lines of defense against invasion by recipient immnne system CU111~One11l~. E.g., the use of an outer coating which passes 1 in 1 o2 recipient IgG molecules, a super matrix which passes 1 in 102 recipient IgG molecules, and an int~rn~l particle coating which passes 1 in 1 o2 recipient IgG molecules, results in a composite pass rate of only about 1 in 106-The ability to segregate functions also allows construction of composite microreactors the life of which are roughly commencllr~te with the useful life of the enclosed sources. For example, gelatin, which weakens the matrix, could be added. If it is nPcesc~ry to strengthen the matrix, fibers can be added.
A ~lcr~lled composite microreactor is one in which: the composite microreactor contains at least two internal particles; the source of a therapeutic or otherwise desirable substance is a cell, e.g., an islet cell; the internal particle matrix is ~lgin~te; the intern:~l particle includes an internal particle coating of polylysine; the intern~l particles are embedded in a super matrix of ~lgin~te, and the composite microreactor includes an outer coating of polylysine; the polylysine of the internal particle coating is of a molecular weight of b~lw~c;
2 and 10 kDa; the polylysine of the outer coating is of a molecular weight of between 2 and 10 kDa; the int~rn~l particles are geometrically stabilized, as is described below; the composite microreactor is generally stabilized, as is discussed below; the super matrix is free of fissures or other defects which arise form the use of int~rn~l particles which have not been geometrically stabilized; the diameter of an internal particle, is between 100 and 400 microns, preferably about 200 microns; the diameter of the composite microreactor is between 400 and 800 microns, preferably about 600 microns.
As described above, the internal particle (20) can consist of sources embedded in a matrix. the matrix being enclosed in an internal particle co~ting The int~rn~l particle (20) can also have other structures. For example, the inner particle can consist of a solid bead, e.g., a plastic bead, a sepharose bead, or a glass bead, on which cells, e.g., anchorage dependent cells, are allowed to grow. Cells can be allowed to grow on a surface of the solid bead or. if they are present, within i.lL~l~LiLial spaces of the bead. Such an intern~l particle can be coated as described herein, or left uncoated. The internal particle can be coated or left uncoated. The internal particle, can be embedded in a supper matrix, the super matrix being - enclosed by coating.
Composite microreactors can also contain fibers or materials to enhance the - 35 m~-c h~nical strength of the sphere. Similarly, the composite material can contain substances such as PEO which may act to repel protein and to hinder the fibrotic response. Other - mz-tt~ri~lc such as gelatin or collagen can also be added to either increase or decrease the CA 0221~039 1997-09-09 WO 96/28029 PCI/~JS96/03135 porosity so as to influence the transport ~ ies (permeability/and or molecular weight cutoff).
In addition to advantages, such as ease of retrieval, the embo-1iment~ of the invention permit~ the use of immlm~roL~;~;L~ulL~ which are not biocompatible. ~t~ri~l~ which alone 5 might be digested by el~y~lles, or which might trigger a fibrotic response when they come into direct contact with host tissues can be used to form permselective barriers. Methods of the hl~,ellLol can also be used to ~lgin~t~-coat particles made of neutral or positively-charged substances. More hllpulL~lLly, the ~lgin~te coating filrni~h~s the composite structure with a physical barrier of substantial thickness versus the "coating" formed by mere electrostatic 10 interactions. The composite structure (ranging in diameter from ~50~Lm to ~Smm) can be made of any m~teri~l Tntern~l particles can be of any shape, including, for example, planar, cubical, tubular, and disk-shaped particles and chambers, or other shapes which might otherwise become fibroencapsulated.
The ratios of the volume of the internal particles to the volume of the composite microreactor can be tailored to particular applications, but ~rerel,ed ratios are 0.5:5.0, preferably 1.0:3.5, more preferably 1.0:2.5, or 1.0:3Ø
Hi~her Order Composites Embo-liment~ of the invention include higher order composite microreactors, e.g., a double composite in which single composite microreactors (10) are embedded in a matrix which is (optionally) coated with an outer coating. Accordingly, Fig. ~, shows a second order, or double composite microreactor (100).
The double composite microreactor (100) contains one or a plurality of compositemicroreactors (10) (as described above and elsewhere herein) embedded in a double composite microreactor matrix or super matrix (110) which is (optionally) enclosed in a double composite microreactor outer coating (120).
The diameter of the double composite microreactor ( 100) is preferably between 100 microns and 4 millimeters, between 300 and 1500 microns, between 400 and 1000, or between 500 and 900 microns. More preferably the ~ metPr is about 600-800 microns.
Double composite microreactor matrix or super matrix (110) can be a gel, e.g., ahydrogel, e.g., ~l~in~te or agarose. The double composite microreactor matrix or super matrix (1 10) can have immunoisolative properties. In some embo~liments it can have little or no ability to exclude low molecular weight species, e.g., IgG or complement, with this property being releg~ted to other components ofthe composite microreactor (10). The double composite microreactor matrix or super matrix (110) can be rendered immlln~isolating by controlling its porosity, e.g., such that it hinders the passage of molecules of relatively large molecular weight or by adding to it components, e.g., PEO, CA 0221~039 1997-09-09 PSA, or PLO which hinder the passage of relatively large molecules. Regardless of the method of controlling its permeability, the matrix or super matrix (110) will, in plc;r~ ,d embo~1iment~ will hinder the passage, and preferably, ~ssenti~lly completely prevent the passage of molecules having a molecular weight of more than about 50 kDa, preferably more 5 than about 100 kDa, preferably more than about 150 kDa, and most preferably more than about 400 kDa; or of immlme system components such as Ig molecules or complement; or recipient-derived cells. The double co...~osile mi~ a;lor matrix (110) need not be anti-fibrotic and need not be bioco~ dlible if a more proximal or more exterior component supplies these functions. In double composite, the matrix of the inner most particle is usually referred to as the internal particle matrix. The matrix in which the int~rn~l particles are embedded is usually referred to as the particle matrix, and the matrix in which the single composite particles are embedded is usually referred to as the super matrix.
Double composite microreactor outer coating ( 120) is optional. It can be made of a poly~mino~eid, e.g., PLL. PLO, chitosan, or PAN-PVC, or any coating used to coat non-composite microreactors. A ~cf~lled coating is a poly amino acid, e.g., polylysine, having a molecular weight of less than 15 kDa, more preferably of less than 10 kDa, more preferably of less than 5 kDa. Particularly ~l~r~ d are polylysines with a molecular weight of about 3-4 kDa, e.g., 3.7 kDa. or about 9 kDa-10 kDa, e.g., 9.7 kDa. Plef~llc;d coatings are volume-reducing coatings. The double composite microreactor outer coating (120) need not be immllnnisolating if other components supply this function.
Other embodiments of the invention include higher order composite microreactors,e.g., third order composites which include double composite microreactors embedded in a matrix and (optionally) enclosed in an outer coating, and forth order composites, which includes third order composites embedded in a matrix and (optionally) enclosed in an outer coating. The materials and methods ~ cl-~se~1 for use in simple and double composites can be used for the matrices and coatings of higher order composites.
Isolation of Cells Living cells can be isolated from surrounding tissues or grown in culture by procedures known to the art, and then suspended in a liquid medium prior to enc~pslll~tjon.
The living cells can provide biological substances, e.g., enzymes or co-factors, hormones, clotting factors, or gro~th factors. Cells, e.g., pancreatic cells. can provide enzymatic or hormonal functions. Cells such as hepatic cells can provide a detoxification function.
- As an example. pancreatic islet cells were prepared from either adult mongrel dogs, pigs, or bovine calves (0-2 weeks old) by a modification of the methods of Warnock and - 35 Rajotte, Diabetes~ 37:467 (1988), as previously described in Lanza et al., P.N.A.S. USA.
88:1 1 100-1 1 104 (1991).

CA 0221~039 1997-09-09 Briefly, aseptic, viable porcine pdllCledLd were obtained under aseptic operating room procedures. After resection (warm i.~ emi~ for less than about 15 min~ltes), the glands were c~nmll~te(l and infused with cold (4~C) University of Wisconsin (UW) organ preservation solution. Pancreatic tissues were dissociated using an intraductal collagenase digestion 5 procedure. The collagenase is delivered by peristaltic pump, and the digested pancreas is mechanically disrupted in a polypropylene dissociation chamber cu..~ g 2-6 mm glass beads. The islets were separated from the exocrine tissue by disculllhluous density gradient ce--l. ;r~lg~tion (27%, 20.5%, and 11% (w/v) FICOLLt~) (Sigma, F 9378) in Eurocollins solution).
10Isolated islets were then cultured for one day either in M199/Earle's merlillmsupplementerl with 10% (vol/vol) fetal bovine serum, 30 mM HEPES, 100 mg/dl glucose, and 400 IU/ml penicillin (canine), or in a-MEM plus 10% heat-inactivated horse serum (bovine and porcine) in a hllmi~lified atmosphere of 5% CO7/95% air at 37~C. A typical yield of islets should be in the range of 0.5-1.8 x 1 o6 islets for adult pancreas (400 gm wet weight, 15islet diameter 80-125 ~um, purity 85-95%, viability greater than 90% (see below). The cells may also be isolated by other procedures and cultured under other suitable conditions.
T~l~hernic deterioration of the islet cells is minimi7ecl by using tissue fr~gment~ of a suitable size, e.g., islet fr~gment~ should be less than about 150 microns, and preferably 50 to 125 microns, in diameter. Viability, growth, longevity, and/or function of the islet cells can 20 be enhanced by co-culturing, i.e., by mixing other cell types in the liquid medium prior to enc~ps~ tion. Useful cell types include cells which secrete growth hormone, e.g., GH-3 cells, or cells which secrete connective tissue and/or extracellular matrix components, e.g., fibroblasts and endothelial cells. In addition, cells, e.g.~ islets, can be co-cultured with red blood cells, or hemoglobin, or other oxygen carrying agents can be added, to enhance oxygen 25 availability. Red blood cells can also be used to scavenge nitrous oxide.
Islet quality control procedures are used to enable comparison of dirr~ lcnt lots of islets prepared at different times. Purity (amount of islet tissue compared to exocrine tissue co.~l;.~..;..~tion) can be ~l.?termin~i by ability of pancreatic islets to rapidly take up diphenyl thioc~ba~olle (dithizone) Islets can be incubated for five to ten min~lte~ with 50 ~lg/ml of 30 dithizone (D5130, Sigma) to stain them red. The ple~dldlion is then ex~mine-l under light microscopy for a qualitative estimate of purity. Quantification of purity is effected by islet dispersion and counting of stained and unstained cells! or with a spectrophotometric assay of dithizone uptake/~g DNA.
Viability can be ~ terminecl by any one of several assays that depend on the capability 35 of viable cells to exclude certain dyes. For example~ one assay uses a combination of the fluol~esct:llL stains acridine orange, which stains only viable cells green, and propidium iodide~
which stains only the nuclei of dead cells red. The islets are inc~b~t~l with the dyes (acridine CA 0221~039 1997-09-09 orange, Sigma A6014, 50~g/ml, and propidium iodide, Sigma P4170, 2.5~g/ml) in a PBS
solution for 10 to 15 ~ es and then dispersed into single cells. Counts of red and green fluorescing cells are used to calculate % viability.
Insulin se~ Lol y activity of the islets is ~letermin~d both in static culture, e.g., 5 expressed as units of insulin per islet volume, and based on the capability of the islets to respond to graded con~ ~ntr~tions of glucose. These values are 4~ ely established by measuring the insulin secreted by islets exposed to a range of glucose concentrations exterl~ling from 2.8 to 28 mM glucose.
Form~tion of Mi-;J~y~ules Living cells, e.g., islet cells, can be encapsulated in a variety of gels, e.g., ~l~in~t~, to form microparticles, e.g., microbeads or microspheres to physically isolate the cells once implanted into a host. To prevent entry of smaller molecular weight substances such as antibodies and complement (with a molecular weight of about 150 kDa) into these microparticles, they can be coated with a material such as poly-L-lysine, chitosan, or PAN-15 PVC, which provides an outer shell with a controlled pore size, or they can be treated by, e.g., cross-linking, to control their internal porosity. Alternatively, their porosity can be controlled by mixing various substances such as polyethylene oxide (PEO) directly into the gel mixture. The use of a high molecular weight molecule, e.g., a high molecular weight PEO, e.g., of about 1-8 million Da, will .. ,i"i.. ,i~ the escape of the porosity controlling substance. Molecules of this size range can be used with out an outer coating.
Enca~sulation Once the cells are isolated and suspended in liquid medium, they can be encapsulated by a supporting matrix, e.g., a hydrogel matrix to form a microbead, which serves as a core of a microcapsule, e.g., or int~rn~l particle. The core m~int~in~ a proper cell distribution, provides strength, and enhances cell viability. longevity, and function. The core can also contribute to immlln~isolation. For example, the physical rli~t~n~ e that is created by embedding the internal particle in a supporting matrix, can provide protection from, e.g., nitric oxide and cytokines. It also protects the internal particle from direct cell-cell interactions which can elicit an undesirable host response.
Using standard techniques, a gel matrix is formed by adding cells, e.g., pancreatic islets. to a solution of nutrient medium and liquefied gel, e.g., sodium ~Igin~t~, to form a suspension. and then cro~linking the gel. The gel matrix can be any one or a combination of - a variet~ of substances, preferably substances which are biocompatible with the host animal, and are capable of m~int~ining cellular viability and physically supporting the tissue or cells in suspension.
The gels can be gelled or cros~linke~l e.g., by the addition of ions such as calcium, potassium. or barium, or by a change in le~ dLul~. Though. if temperature change is used CA 0221~039 1997-09-09 ~VO 96/28029 PCTIUS96103135 care should be taken so that the L~ lp~ldLul~; changes required for gelation are not harmful or fatal to the living cells to be ~ terl Te~ ldLule-independent gels include ~l~in~tes, carrageenans, and gums such as xanthin gum. As used herein, the term ~l~in~te includes ~lpin~t~ derivatives. These gels should be treated to remove polyphenols, S lipopolysacrh~ri~e~, endotoxins, and other hll~uliLies using standard techniques.
Alginate is composed of blocks of 1,4 linked ~-D-rnal.llulollic acid (M) and a-1-guluronic acid (G) linked together, e.g., in ~lt~rn~ting MG blocks. The ~l~;r~ ;d ~lgin~te is one formnl~ted with a high G block content, e.g., at least about 60 percent. The higher the pelc~:llLage of G blocks, the greater the pore size and the strength of the gel matrix. In 10 addition, zllgin~t~ gels with a high M block content appear to be more immnn()genic than gels with a high G block content. See, e.g., Soon-Shiong et al., Transplant. Proc.. 23:758-759 (1991), and Soon-Shiong et al., Transplantation. 54:769-774 (1992).
The gel matrix should be sufficiently viscous to m~ints~in the cells in a dispersed state.
When ~lgin~te is used as the gel matrix, it is added up to about 3%, preferably to about 1 to 2%, of the liquid medium, and the solution is cross-linked to form a semisolid gel in which the cells are suspended. These percentages provide a matrix that m~int~in~ its shape and has sufficient mechanical strength to remain intact in vivo for several months.
Alginate hydrogels are plt:ftlled for the microbead cores of the internal particles for a number of reasons. Alginate allows rapid polymerization and immobilization of cells at room temperature using relatively benign CaCl2, provides consistent gel rheology that can be conveniently varied by increasing ~1, in~t~ concentration, and produces microbeads with good mechanical strength.
In contrast, although agar/agarose has been found to be an excellent medium for embedding isolated porcine, canine. and bovine islets, significant problems were encountered in preliminary studies to embed isolated islets in agar microspheres. Requirements for elevated temperatures and the heterogeneous rheology of agar significantly complicated the procedure. For example, temperatures above the physiologic range irreversibly damaged the islets.
A preferred method for making hydrogel microbeads is with an air jet.
Other methods for making hydrogel microbeads including em~ ification~ dripping, and the Rayleigh jet.
Emulsification Fmlll~ification depends upon shear forces in an immiscible liquid to break up the pre-gel liquid. The shear forces are usually produced by agitation although they can be produced by wall shear in a lumen or by sonification. Well controlled agitation can produce droplets which are uniform to about +15% in diameter, and it is an acceptable technique often used to enr~pslll~t~o living cells. See, e.g.. Lencki et al., U.S. Patent No. 4,822,534.

CA 0221~039 1997-09-09 Dripp;n~
In this group of related techniques, a force is applied to pre-gel liquid in which living cells are suspended, which overcollles the surface tension force between the forming droplet and the extruding orifice. The orifice may take many forms; frequently a blunt-tipped S hypodermic needle is used.
Various forces can be applied to the droplets, e.g., centrifugal, electrostatic, or inertial.
A c~ ;rugal force of about 536 g is suitable for use with an extrusion orifice large enough to pass 100,um islets to produce 200 ~lm droplets. A suitable rotor to produce microspheres with ce~ g~l force is a multiorifice c~ irugal head, e.g., as described in Deasy (ed.), 10 "Micro~ tion and Related Drug Processes," Chapter 13 (Marcel Dekker, Inc., NY &
Basel, 1984) (Suu~hwe~L Research Institute, San Antonio, TX). Another suitable rotor is described in U.S. Patent No. 4,386,895.
Axial inertial forces, as employed in ink jets which "spit" a single droplet from an orifice by pulsing a pumping chamber with a piezoelectric crystal. or in so-called "bubble"
15 jets which boil microvolumes of fluid to create a pl-,S:iUle pulse. are also suitable for the present invention.
Fluid flow drag is another common method used to form microdroplets in the 200 to 1000 ~m size range. In this method, a liquid is extruded steadily from an orifice (usually a hypodermic needle) which is arranged within a coaxial jet of gas. The drag force "shear" of 20 the gas flow, with a velocity of up to 400 m/sec, pulls the forming droplet away from the needle against the ret~ining force of surface tension. The droplets are typically twice the diameter of the orifice. which makes 400 ~m diameter droplets. with less than or equal to 100 m diameter islets, the smallest practical size using this method.
Rayleigh Jet The Rayleigh jet can be used to manufacture very uniform (+ 1%) microdroplets and even encapsulated microdroplets by means of two coaxial jets. This technique is based upon the principle, first illustrated by Lord Rayleigh in 1873, that a liquid jet is inherently unstable against surface tension. Rayleigh demonstrated that there is a particular geometry that produces a characteristic frequency (wheref= frequency; V = jet velocity, D = diameter of 30 microdroplet): f= 0.419 V/D.
Although the drop-passing frequency is completely cletermined by the jet velocity, the droplet-forming process is completely independent of the jet velocity. This phenomenon has been used to m~nllf~rtnre microdroplets from 20 ~lm to lOOO ,um diameter from liquids ranging in viscosity from 1 to 100,000 cps. The jet is disturbed at the Rayleigh frequency by 35 either natural turbulence or. usually, by driving the plenum supplying the jet with an oscillator, typically a piezoelectric crystal, or by inertial forces arising from vibrating the CA 022lS039 l997-09-09 WO 96128029 PCI'IUS96/03135 noz71e transversely or axially. Eleetrostatic or aeoustie exeitation of the jet ean also be employed.
By employing a eoaxial flow of an ~nr~ps~ ting liquid and a eoating liquid, eoated droplets ean be formed. Coextrusion is very attraetive as long as the eore m~teri~l does not S eause a rapid eoagulation of the shell m~t~ri~l When the droplets are to be eoated ~,vith a rapidly eoagulating m~teri~l sueh as an aerylie eopolymer, a temporary barrier liquid (e.g., vegetable oil or a polymer solvent) should be interposed between the eore and eoating materials.
Controllin~ Pore Size of Mieropartieles The pore size of the mieropartieles ean be eontrolled either by applying a semipermeable shell having a partieular molecular weight cutoff. This can be effeeted by applying an "eleetrostatic" coating, e.g., a coating of a polyamino aeid, e.g., polylysine. Pore size ean also be controlled by treating the gel matrix of the microparticles th~mcelves to change the pore size of the matrix without any subsequent coating. E.g., the surface of the eore ean be altered by, e.g., eross-linking, to produce covalently modified gel matrix surface.
A coating can be a formed by modifying the structure of the matrix, e.g., the matrix can be cross-linked, e.g., with metal ions, e.g., Ba or Fe ions, or by photo-cross-linkin~ to form an coating.
As used herein, "molecular weight cutoff" refers to the size of the largest moleeule that is not sllbst~nti~lly blocked, e.g., by a semipermeable shell or coating surrounding a mierosphere or by the gel matrix itself or both. Moleeules with a molecular weight above the cutoff are substantially prevented from ent~rin~ or leaving the particle. The composite microreactor should generally provide a molecular weight cutoff of about 50,000, more preferably about lO0.000. more preferably about lS0,000. and most preferably about 400,000 daltons. In preferred embotlimçntc, the molecular weight cutoff is sufficient to prevent Ig molecules, e. g., IgG, and complement, from entering and coming into contact with the enc~ps~ t~l m~tPri~l Alterin~ the Pore Size of the ~el Matrix The pore size of the gel matrix can be altered in several ways. For example, the gel 30 matrix can be altered, e.gthe porosity can be either increased or decreased so as to influence the transport properties, e.g., permeability and/or molecular weight cutoff, by adding, e.g., gelatin, or collagen or barium, or other ions with the same valance as Ca++ ions. Changes in the t~lllp~ ule will also affect the pore size. An increase in the temperature will result in shrinkage of the gel matrix. The addition of eompound. e.g., PEO, to the gel matrix ean also result in altered pore size. PLO may act to repel protein and to hinder fibrotie response. In preferred embo-lim~ntc, PEO of moleeular weight greater than 1,000,000 Da. more preferably greater than 4.000.000 Da. and most preferably greater than 8,000~000 Da, is mixed with the CA 0221~039 1997-09-09 WO 96/28029 PCr/US9610313 gel matrix. PEO of relatively high molecular weight will not diffuse out and thus does not require cros~linkin~
Co5lti~ca Microc~ps~ , e.g., the intt~ l particles of a composite microlea~;lor, can be coated S or uncoated. The inttorn~l particles of a composite microreactor can be coated with any coating used to coat non-composite microreactors. Because the int~rn~l particles of a composite microreactor do not generally come into contact with recipient tissue, the outer surface of an internal particle, whether or not coated, need not be anti-fibrotic.
Microbeads, or pre-gel microdroplets, can be coated with a coating of a polymer, e.g., 10 a biocompatible polymer, e.g., an electrostatic coating, e.g., a polyaminoacid, e.g., PLL, or PLO, chitosan, "PEO", polyvinyl acetate ("PVA"), or an acrylic copolymer such as PAN-PVC, which is then solidified to form a shell. Known coating methods can be used for coating materials such as poly-L-lysine or chitosan, but polymers which can have undesirable effects on the encapsulated cells, such as PAN-PVC, present special coating problems 15 described below.
A suitable copolymer is PAN-PVC, which, when solubilized in an organic, polar solvent such as n-methyl-2-pyrrolidone (NMP), dimethylsulfoxide (DMSO), dimethylformamide (DMF), or dimethyl <qcetz~mide (DMAC)(all available from Sigma Chemicals Co., St. Louis, MO), is coated onto the microspheres and then precipitated by solvent exchange 20 with water to form a porous membrane with pores that exclude molecules larger than about 80,000-150,000 MW.
The coating thickness is preferably greater than 20 ,um, but less than 100 ,um so as not to compromise the microbead core volume. A plefell~d geometry is a 300 ~m diameter microbead containing two or three islets encapsulated in a 25 ~m thick acrylic copolymer 25 coating to give an outer diameter ("OD") of 350 ~um. If the OD is increased to 500 ,um, a coating thickness up to 100 ~Lm can be employed, but a thicker coating generally increases the mass-transfer reci~t~nce and decreases the number of cells that can be supported by the surface area of the coating. Therefore, the coating is preferably at least 25 ~Lm, to give sufficient strength, but as thin as possible to allow rapid mass transfer.
Initial attempts to coat batches of microbeads by dipping them in a PAN-PVC coating solution were less than entirely ~lccecsful because the solvents required to dissolve PAN-PVC are toxic to cells and the solvent quickly diffuses throughout the microbeads when they are dropped into the coating solution. In addition, the PAN-PVC precipitates and solidifies almost inct~nt~n~ously in the presence of water~ making it almost impossible to prevent the - 35 microbeads submerged in the coating solution from sticking to each other.The problem is that the outer surface of the coating around the microbeads is sticky, because at first only the int~rn~l surface of the coating that contacts the microbead~ and thus CA 0221~039 1997-09-09 WO 96/28029 1 ~ 03135 the water in the bead, is solidified. As a result, the beads stick together. The outer surface of the coating will solidify only when exposed to an aqueous solution; but by then all the beads are already stuck together. This is especially a problem with large batches of microbeads.
Thus, PAN-PVC, and other polymers that require the use of toxic solvents, must be 5 coated onto microbeads with methods that generally include the following commc-n r~LuL~s:
(1 ) a very brief contact of the cell-co, .~ p pre-gel microdroplets or gelled microbeads with the coating solution to avoid toxic effects, (2) the elimin~tion of any h~n-lling of or contact bc:Lw~n the microbeads before the coating is solidified, and (3) the coating of large numbers of microbeads in a short period of time and on an individual basis.
In each of the methods described below, the polymer solution, e.g., PAN-PVC, is dissolved in a water-soluble, organic, polar solvent, e.g., NMP, and a small amount of water.
The solution is solidified by exposure to water, which dilutes the solvent by counter diffusion of the t~,vo liquids, causing a fibrous network to form a porous coating on the surface of the microbeads. By varying the composition of the polymer solution, casting tt;m~el~Lul~, pre-15 evaporation of solvent, etc., the molecular exclusion size of the copolymer coating can be varied.
In one method of coating microdroplets or gelled microbeads with PAN-PVC, the microbeads are shot through a free-standing thin film "curtain" of a coating solution created by flowing the solution through a narrow slit in the wall of a tube, over a straight edge, i.e., 20 weir, to form a "waterfall" curtain. or out of a rotating centrifugal cup-like device as shown in Fig. 5-15A. Pre-gel microdroplets co~ lg immobilized cells are generated, e.g., by Rayleigh jet as described above. and are shot on a ballistic trajectory to penetrate through the free-st~n~ling coating film (Fig. S-lSA) at velocities of 1 to 40 m/sec. During the penetration they are coated with a thin layer of the polymer solution (Fig. 5-1 5B). In order to enhance 25 complete coating of difficult shell liquids (i.e., liquids that are very viscous and/or have a high surface tension), the microdroplets can be given a spin by injecting vorticity, i.e., a swirl, upstream of the droplet-forming orifice. The coated microbeads are then dropped into an aqueous biocompatible collection medium in which the coating, e.g.~ PAN-PVC, solidifies by contact with water and the coating solvent diffuses away from the gel core into the 30 surrounding aqueous bath. The excess coating solution is collected, e.g.. in a funnel, and recycled.
Since the plef~l,ed Rayleigh jet can produce large numbers of microdroplets in a short period of time. The microbeads pass through the film one-by-one, so they are all coated on an individual basis. Since the total "in-flight" time before they drop in the collection medium 35 is only a few milli~econds, toxicity of the organic solvents does not play a role. All microbeads remain separate at all times until the outside of the coating is solidified and thus CA 0221~039 1997-09-09 no longer sticky, and are not h~n-ll.o~l physically. As a result, the sticking/agglomeration ph~nnmen-)n described above is avoided.
After penetration, coating, and time to regain a spherical shape, the coated microcapsules fall into an aqueous solution that solidifies the shell co~ting Ions, e.g., Ca~, S for cros~linking the ionic gel core, e.g., sodium ~lgin~te7 diffuse through the shell during and after solvent ç~ n~e. The coating film, e.g., from a ct;:llLI;rugal cup, can be S to 50 llm thick depending upon the feed rate, the centrifugal acceleration at the cup lip, and fluid pl~ ies, i.e., viscosityr density, and surface tension. If the shell polymer solidifies too rapidly on the gel microbeads, they may be frozen at their s~ e~ by liquid nitrogen or its 1 0 vapor.
This method of curtain coating can also be used for other applications in which only the surface of a small particle, such as a gel microbead or pre-gel droplet, is exposed to a solution that is used to change the surface characteristics of the microbead or droplet. For example, a curtain solution of BaCl7 can be used to selectively gel only the surface of sodium ~l~in~te microdroplets to form a gel surface layer that is denser than the gel formed inside the microdroplet when the droplet is subsequently immersed in CaC12. If the microdroplets were immersed in a BaCl2 solution, the entire microdroplet would be gelled to the same density which probably would harm the living cells because the pore size could be too small to give them adequate space.
Coating with Polvlysine To coat an ~lgin~te core with polylysine the ~l~in~te core is dropped into a solution of 0.05% polylysine in serum free culture medium. The thickness of the polylysine coating can be increased by increasing the time the ~l~inslt~ core is left in the solution, or alternatively, by increasing the concentration of the solution. The volume of beads to solution can be, e.g., 1:5, 1:10, or 1:20. For smaller beads a greater proportion of solution is desirable.
Coatin~s which minimi7e particle volume Embo~liment~ of the invention use coatings which reduce the volume of a component, e.g., a core, to which they are applied. For example, a poly~min- ~id coating, e.g., a polylysine, or polyornithine made from a polyaminoacid of a relatively low molecular weight, can result in a significant reduction in the volume of a gel core, e.g., an ~lgin~te core, to which it is applied. In many cases the reduction in volume is as much as about 50%, or even 60-70%, or more.
- Relatively low molecular weight, as used herein, means about 30, 000 Da or less, more preferably about 15. 000 Da or less, more preferably about 10. 000 Da or less, more ~ 35 preferably about 8, 000 Da or less, more preferably about 7, 000 Da or less. more preferably about 5, 000 Da or less. more preferably about 4, 000 Da or less. more preferably about 3, 000 Da or less, and most preferably about 1,500 Da or less.

-CA 0221~039 1997-09-09 W O 96/28029 PC~rnUS96/03135 For example, the use of polylysine of a relatively low molecular weight, e.g., 3, 7, or 9.6 kDa, can result in a ~ignifir~nt reduction, (approximately 30% in some cases) in the diarneter, of the core to which it is applied. In addition to the decrease in volume, the use of a low molecular weight poly~minoqcid will result in a coat having superior permselective 5 ~JlU~J~,. Lies. However, the use of a low molecular weight poly~mino~cid often results in a surface which is "pruned", i.e., relatively convoluted or rough, and which can elicit a fibrotic response. The composite microreactor of the invention, by using this coating on the intt-.rn~l particle, and a smooth surface, e.g., of ~l~in~t~, on the exterior of the composite microreactor, can obtain the benefits of a coating of relatively low molecular weight and also inhibit 1 0 fibrosis.
The pcrm~electivity properties of a poly amino acid, e.g., a polylysine, coatingimprove after the coating has been aged 2 or more hours. Thus, for best results, particles coated with these coatings should not be impl~nt~-l in recipients until the coating has aged.
Geometric Stabilization Some particles or components are not geometrically stable immediately after m~mlf~ctllre, e.g.. the particle or component can change size or shape. If intern~l particles which are incorporated into a composite microreactor change geometry, the components of the composite microreactor, e.g., the super matrix or outer coating, can be damaged and the integrity of the composite microreactor can be colllprulllised. Although not wishing to be 20 bound by theory. the hlv~llLol:j believe that rh~n~es in the geometry may ~l~m~ge the super matrix or the outer coating, e.g., by in~ cin~ fissures or discoll~hllliLies. Damaged particles can allow the fibrotic proliferation of recipient cells on the inner particles when implanted into a recipient. Therefore, it is often desirable to geometrically stabilize int.om~l particles, preferably prior to incorporating them into composite microreactors. Stabilization can 25 generally be accomplished by allowing the particles to "age" for a short time before incorporation into larger structures. The aging should be done under condition which m~imi7~ the viability of encapsulated cells. Geometric stabilization is particularly important when the particles are coated with a relatively low molecular weight poly-amino acid.
Polylysine-coated ~lgin~te particles, especially those coated with relatively low molecular weight polylysine, should be geometrically stabilized. The polylysine coated zllgin~te particles should be placed in a culture medium. suitable for the cell being used, and allowed to stabilize overnight.
Form~tion of Composite microreactors Composite microreactors can be made by materials which are analogous with the methods used to make internal particles: individual or small numbers of int~m~l particles .

CA 0221~039 1997-09-09 (rather than cells) are embedded in a matrix (referred to herein as a super matrix to distinguish it from the intern~l particles matrix) and an (optional) outer coating applied.
For ex~mrle, after the intern~l particles are prepared and, e.g., either coated or otherwise treated, e.g., cross-linked, they should be washed in medium to prevent the exictinp S microparticles from stiel~ing to each other (particles that have not been coated should be washed in calcium and m~gneeillm free medium), mixed with a liquid hydrogel such as ~lgin~te~ and formed into a composite microparticle with a ~ meter from less than 50 ~Lm up to more than 5 mm. For example, in a method which is analogous to that described above for the creation of the internal particles, a mixture of intern~l particles in a liquid gel can be 10 extruded through an 18 gauge catheter to form composite microreactors.
As is ~ cu~ce~1 elsewhere herein, it may be desirable to geometrically stabilize the int~rn~l particles before incol~uldlhlg them into a composite microreactor.
The super matrix of a composite microreactor can provide a sclllipclllleable shell of a hydrogel material around all of the encomp~c~ecl intern~l particles can provide a physical 15 barrier of substantial thickness compared to the individual coatings on each of the microcapsules. Electrostatic interactions in the super matrix can contribute to immunoisolation.
The super matrix can be made of the same material as the intern~l particle matrix or it can differ from the matrices of some or all of the internal particle matrices.
A composite microreactor can contain internal particles of any shape, including, for example, planar~ cubical, tubular, and disk-shaped particles and chambers, which might otherwise become fibroencapsulated.
A composite microreactor can also contain other substances to modify the l,lopcllies of the composite microreactor and can, e.g., include fibers or materials in addition to the 25 hydrogel matrix and internal particles to enh~nre the mechanical strength of the composite microreactor. Similarly, the composite microreactors and particularly the super matrix, can include substances such as PEO which act to repel proteins and to hinder the fibrotic response. Other m~t~ri~l~ such as gelatin or collagen can also be added to the super matrix to either increase or decrease the porosity so as to influence the transport properties 30 (permeability and/or molecular weight cutoff) of the composite microreactors. Higher order composites can be made by analogous methods.
Outer Coatin~
Composite microreactors can (optionally) be provided with an outer coating.
Although any coating used with non-composite microreactors or for internal particles can be - 35 used for the outer coating, other coatings, or no coating, can be used as well. Because the various p~ ies need by the implanted device. e.g., biocompatibility, the ability to resist fibrotic en~ps~ tion, the ability to prevent recipient imm~me inactivation of the implanted CA 0221~039 1997-09-09 WO 96128029 . P~:1J~ 03135 donor tissue, can be distributed among the various components of the composite microl~a ;lul, the outer coating need not supply all of these ~ll U~ l Lies. It may be desirable to geometric~lly stabilize the :iu~r~ l ;x prior to application of a coating.
Impl~nt~tion The composite microreactors can be implanted into a host by injection with a standard ez~thPtPr or syringe, e.g., with a 16 gauge needle for beads less than 1000 ~lm in ~ m~ter.
Larger composite microreactors can be inserted via a small incision, e.g., with a catheter or funnel-like device. The beads are preferably implanted into the host intraperitoneally. The beads can also be implanted intr~mllcclll~rly or ~ullcul~leously. ~ltern~tively, the beads can 10 also be implanted into immunnprivileged sites such as the brain, testes, or thymus, where the host's immunto response is least vigorous, as described in Chapter 7 of Lanza et al. (eds.), Immunomodulation of Pancreatic Islets (RG T.zln-1Ps7 Texas, 1994). Composite microreactors can also be introduced at a site where the ~llbst~n~ e provided by the composite microreactor is needed locally. E.g., a microcapsule which provides a-interferon could be implanted in 15 tumors. The microreactors of the invention can be delivered to a subcutaneous site. The composite microreactors can be inserted through a small surgically created opening using a gun/trocar type device that slips the beads under the skin.
Fxamplç ]: FJncapsulation of Pancreatic Islets Pancreatic islets were encapsulated as follows. After preculturing ov~rni~ht islet 20 cells were suspended uniformly at a density of 30,000 isletstml, which is 30 islets/mm3, in a solution of 1.5% (wt/vol) Pronova LVG sodium ~lgin~te (Protan, Drammen, Norway) in culture medium plus additives (a-MEM, lOmM HEPES pH 7.1, penicillin, 2 mM ~ ",;"e for porcine islets; and M199 with the same additives for canine islets). A syringe pump was used to pump the suspension through an air jet ~aldlu~ (co..~ ,g a straight-edged 22 25 gauge needle) at a speed of 3 ml/min. Droplets formed at the tip of the needle were stripped offby means of a concentric flow of air at an air speed of 7 to 8 m/sec. The resulting droplets fell a rii~t~n~e of 4 cm and were collected in a solution of 1.5% CaCl~ in 10 mM HEPES (pH
7.1 ) to form gelled beads. These beads can be made in various sizes ranging from about 100 ~Lm to 400 ~m in diameter by altering the air flow speed, the faster the flow rate the smaller 30 the beads.
Each bead contains appr~-xim~tely 1 to 2 islets. After gelation, the beads were washed three times with culture medium (~plupliate for the species of islets in use), and were then cultured in a tissue culture incubator at 37~C and 5% CO~ until implantation.
Larger beads up to 3 mm in diameter can be made in a similar manner, or can be 35 extruded through a syringe with a 18 gauge ç~theter.
F~mrle 2: ~ormation comrosite microreactors Composite microreactors are made as follows:

CA 0221~039 1997-09-09 WO 96/28029 PCI'/u~ .3135 Step: 1: Day 0. A ~ Lul~ of islets and sodium ~lgin~t~ (30,000 islets/ml) are extruded with a droplet generation device (an airjet with a 22 gauge needle) into a 1.5%
CaC12/lOmM HEPES solution. The al~p~dLus is run on ~Lol"i;~Lion mode to generatemini~ph.ores a~ploxin~ately 300,um in ~ m~ter The gelated spheres are collected and 5 washed in culture media several times. Generally the gelated spheres are aged for about 24 hours before procee~ling to Step 2.
Step: 2: Day 1. The mini~ph~res are washed in serum free media several times andcoated with polylysine by imm~r~ion in a solution of 0.2% 9.6K (or 5-less than 15 kDa) polylysine (or 3.7K (or 1 kDa-4 kDa polylysine)) for 2 minllte~ The spheres are then washed 10 in complete culture media (i.e. media co~ serum).
Step: 3: Day 2. There is usually a 3-5 day aging period between Step 2 and the be~inninp of Step 3 when 1 kDa-4 kDa polylysine is used. Otherwise the outer composite matrix can develop cracks which could allow host cell penetration. There is usually a 24 hour aging period between Step 2 and the beginning of Step 3 when 5 kDa-less than about 15 15 kDa polylysine is used. The polylysine-coated mini~pheres are mixed with sodium ~qlgin~te (the ratio of settled bead volume to ~l~in~te is 1:3). The mixture is run through the airjet using a 20 gauge needle to generate spheres approximately 600 ~lm in diameter if single composites are to be made. The ",i~Lu,e is run through the airjet using an 18 gauge needle to generate spheres approximately 900 ~:Lm (or larger) in diameter if double composites are to be 20 made.
The above recited protocol results in composite microreactors. these can be used as is to deliver islets to a host. coated with an outer coating and used to deliver islets to a host, or used to make higher order composites to deliver islets to a host.
Example 3: Forrnation of double composite microreactors Double composites are made as follows:
Perform steps 1-3 as described in Example 2 above.
Step 4: Day 3. There is usually an aging period of about 24 hours between Step 3and Step 4. Wash and coat composite spheres with polylysine as described in step 2 in Example 2.
Step 5: Day 4. There is usually an aging period of about 24 hours between Step 4 and Step 5. The polylysine-coated composite spheres are mixed with sodium ~l~in~t~ (ratio 1:3 as above). The ~ Lule is run through the airjet using an 18 g needle to generate spheres approximately 900 ,um in ~ met~r.
The above recited protocol results in double composite microreactors. these can be 35 used as is to deliver islets to a host, coated with an outer coating and used to deliver islets to a host, or used to make hi_her order composites to deliver islets to a host.
~ - Fx~mrle 4: In Vitro Tes~in~ of Composite microreactors CA 0221~039 1997-09-09 WO 96J28029 PCT/lJS96/03135 Figure 3 shows the in .vitro insulin output of bovine islets cn~ ulated using composite miclvr~a.;lol:i (made as described in Example 2). The microreactors ~H;IIIH;~lod excellent secretory ffinrtion, averaging 144 + 1 luU/ElN/day during the first six weeks. The ability of these microreactors to ~ ~ IH;~IIH; 1- activity during long-term culture show they can S provide an in vitro implant with r~ nHble longevity.
In addition to d~nnc" ,~ long-term islet viability and function, in ~Q studies were also carried out to test the insulin se.ilcl~ly activity from the islets seeded in the microreactors and to evaluate the kinetic ~clr~ .l " ,~nre of the composite microreactors. An a~pl~,~i.l.ately four- to fivefold increase from the basal insulin secretion was observed. The 10 secretory response of the encHpslll~te~l islets was sustained for one hour of glucose stimulation (300 mg/dl) and returned to basal levels after perfusion with the low-glucose solution (50 mg/dl). A second glucose challenge resulted in a similar insulin secretory response with virtually no delay before the insulin concc~Ll~Lion in the perifusate began to increase. These finding show that islets encapsulated by this procedure can respond in a 15 physiological fashion to fluctuations in concentrations of glucose.
Exam~ple 5: Introduction of Composite Microreactors into niabetic Rats Experiments in streptozotocin-in~luc ed diabetic rats clearly demonstrate the advantages of composite microreactors of the invention. When non-immlmo~u~l..cs~,ed diabetic rats received either bovine or porcine islets enc~rs -l~ted using the standard PLL-20 HlginHte procedure cited in the liLclclLulc (see e.g., Immunomodulation of Pancreatic Islets(RG T Hn~les Texas, 1994) the Hnim~lc became hyperglycemic in <4 days. The addition of CsA therapy (20-30 mg/kg/day s.c.) prevented this primary nonfunction. However, by 12 to 16 days postimplantation the implants failed despite the immlmc)suppressive therapy. In contrast, when STZ-in~l~-cecl diabetic rats received bovine islets encapsulated using composite 25 microreactors (made as described in Example 2)the implants continued to mHintHin prolonged function without the use of any immuno~iupplcs:iive therapy. In one set of experiments, seven rats received bovine islet grafts (2 X 105 islet equivalents [EIN]) encapsulated in composite microreactors. No~-rH~ g plasma glucose concentrations plo~ Lly dropped from a preimplantation value of 3 75~25 to 99+21 mg/dl (mean ~SEM) during the first week.
30 Three of the animals were sacrificed with functioning grafts 3 to 4 weeks postimplantation.
The remHinin~ animals snctHin~d normoglycemia for >8 weeks. Immunohistoch~mic~l stHining ofthe explanted microreactors revealed multiple. viable, insulin-co~lH;,~ g ~3-cells conci~tent with functionally active hormone synthesis and secretion; the external surfaces of the microreactors were free of fibrotic overgrowth and exhibited only occasional host cell 35 adherence. To test further the secretory fimction of the bovine islets, microreactors recovered two months after implantation were inc~-b~t~d in medium cu~lH;~;I)g either basal (2.8 mM
[50 mg/dl] or stimul~trry (16.8 mM [300 mg/dl]) concentrations of glucose for 24 hours. In CA 0221~039 1997-09-09 three separate ~x~.;.l.ent~ in which this test was performed, the islets responded with an approxim~tely one-to three-fold increase above basal insulin secretion. In contrast, few or no bovine islets survived when immobilized in uncoated ~lgin~te microspheres and implanted into the peritoneal cavity of five non-immlmo~u~ ed rats for 9 to 14 days. In other S studies, loss of blood glucose control and histology confirmerl that the islets immobilized inside these nn-~o~tecl ~lpin~te microspheres were rejected within dl proxilllately one to two weeks after implantation.
Fx~mr~le 6: Tntroduction of ComI osite Microreactors into Norm~l Do~
Composite microreactors have also been used to prevent immnne rejection of 10 discordant bovine islet xenografts in dogs (made as described in Example 3). The islets were immobilized in composite microreactors and implanted into the peritoneal cavity of adult mongrel dogs both with and without immnnc :iu~lc~ion. In a preliminary set of . ;...ent~ in dogs (n=S), bovine islets were implanted in either uncoated, IgG- and complement-permeable, ~l~in~te spheres or in composite microreactors for 3-4 weeks. No 15 islets survived in the uncoated ~Igin~te microspheres--even with the use of immnnn~ ressi~e therapy (CsA, 10 mg/kg/day). However, when the islets were immobilized within the composite microreactors, viable tissue was observed both with and without immuno~u~ple~:iion. Tmmllnohistochemical st~ining revealed well-gr~nnl~te~l J3-cells and indicated that the microreactors excluded canine IgG. Explanted islets responded to 20 glucose stimulation with an approximately three to six fold average increase above basal insulin secretion. These results indicate that survival of discordant xenografts can be achieved in both rodents and dogs without immuno~u~p~ ion.
Other Fmbodiments The composite microreactors of the invention can be used to treat a variety of 25 disorders. These include disorders that result from the defective or insufficient production of a particular substance, e.g., enzyme or hormone, and other disorders, e.g., trauma-related disorders, such as spinal cord injury.
A number of well-characterized disorders caused by the loss or malfunction of specific cells in the body are amenable to composite microreactor-medicated replacement 30 therapv. For example, in addition to the use of islets of Langerhans, which can be used for the tre~tment of diabetes as is described above, hepatocytes can be used for the tre~tment of hepatic failure, adrenal gland cells can be used for the treatment of Parkinson's ~ e~e7 cells that produce nerve growth factor (NGF) can be used for the trç~fm~nt of ~l7heimer's r1i~ç~e, cells that produce factors VIII and IX can be used for the trÇ~tm~-nt of hemophilia, and 35 endocrine cells can be used for the tre~tment of disorders resulting from hormone deficiency, e.g., hvpo~d~lyluidism.

CA 0221~039 1997-09-09 Moreover, by using recombinant DNA methods to supply a cell which produces a disease product, or encapsulating other tissues, composite microreactors can be used to treat p:~ti~ntc j,.rr~, ;.lf~ from chronic pain, cancer (e.g., hairy cell lel-k~mi~ melanoma, and renal carcinoma), AIDS (treated by immlmological augmentation), Kaposi's Sarcoma (treated by S ~timinictration of interferon, IL-2, or TNF-a), primary hematologic disorders, patients with long-lasting aplasia, and patients who are myelo~u~ ssed (treated by bone marrowtransplantation and aggressive chemotherapy). Composite microreactors should also be useful in the trç~tment of affective disorders, e.g., Huntington's Disease, D~ h-onne's Muscular Dystrophy, epilepsy, infertilit,v. Composite microreactors can also be used to 10 promote wound healing and to treat traumatic, mechanical, chemical, or thermal injuries, e.g., spinal cord injuries, and in wound he~lin~
Implantation of specific cells can also serve to detoxify, modify, or remove substances from the circulation, e.g., drugs, poisons, or toxins. For example, the implantation of ~lol)liate living cells restores normal physiologic function by providing replacement for the 15 ~ e~cecl cells, tissues, or organs, e.g., in hepatic encephalopathy (produced by liver disease) or uremia (produced by kidney failure).
In embodiments of the invention, the encapsulated cells can release fairly largemolecules, e.g., IgG molecules. In many applications the critical host component which must be excluded is C 1 q, which has a molecular weight of about 41 OkDa. Thus, the molecular 20 weight cutoff will be about 400kDa and molecules of up to this size can be released.
Genetically engineered cells can also be used in the methods of the invention. For example, cells can be engineered to release larger products. e.g., IgG.
In each application, a sufficient number of composite microreactors, cont~ining the desired living cells, can be impl~nte~l into the patient, e.g., surgically or with a syringe. The 25 composite microreactors are implanted, e.g., intraperitoneally, for a systemic effect, or into a particular location, e.g.. the brain to treat Parkinson's disease. or the spinal cord to chronic pain or treat spinal cord injuries, for a local effect.
The dose of composite microreactors to be used is determined initially from results of in vitro studies. In addition, in vivo results in, e.g., mice, rats. or dogs will facilitate more 30 accurate ~cct~ccment of required doses, as these tests are generally predictive of efficacy in human patients. For example, canine insulin dependent diabetes represents an excellent model of cellular and humoral autoil,--llullity (Nelson, Diabetes Spectrum 5:324-371 (1992)) The composite microreactors are int~n~1ed to remain in the patient with viable donor cells for ext~nrie-l periods of time up to several months or years. However. if it is determined 35 that the donor cells are no longer viable, e.g., by monitoring the patient's blood for a certain level of the protein secreted by the donor cells, it is a simple task to remove the composite microreactors and renew the supplv of beads in the patient.

CA 0221~039 1997-09-09 WO 96/28029 PCT/US96/0313~i Diabetes Mellitus To treat ~ hete~, e.g., in a dog or human patient, the impl~nt~hle beads preferably p~ t~ isolated canine or porcine islets or other cells that produce insulin or insulin-like growth factor 1 (IGF-l). Islets are prepared and encapsulated using procedures described S above. Insulin secretory activity of the en~ ~ps~ te~l cells or islets is ~et~rrninto~l both in static culture, e.g., expressed per islet volume, and based on the capability of the islets to respond to graded concentrations of glucose. These values are established as described above. Once the insulin secretion activity of a particular batch of en~pslll~tt?~l islets is .l~L~ erl, the proper number of beads can be ~1~1r~ ?(l and implanted into a diabetic patient. For exarnple, to 10 treat a human patient that requires 20 to 50 units of insulin per day, the total number of beads should be selected to contain a total of about 1.0 to 2.5 million porcine islets. For beads lle~ign.?~l to contain, on average, 30,000 islets/ml of gel, the proper dosage would be beads made from 30 to 85 ml of gel.
Hemollhilia Hemophilia is an X-linked hereditary bleeding disorder caused by Factor VIII or Factor IX deficiency. Recombinant methods have now been successfully used to create Factor VIII- and Factor IX-producing cells as described above. Encapsulation in composite microreactors and implantation of such cells according to the present invention can thus be used for an improved tre~tment for hemophilia.
Hepatic Diseases Hepatocyte transplantation is useful not only for irreversible hepatic failure, but for several disease processes including hereditary enzyme abnormalities, acute hepatic failure, where the ability of the liver to regenerate may still exist, and as a bridge to whole liver transplantation in patients who develop sudden hepatic failure~ either because of medical 25 progression or because of rejection-related complications.
Wong and Chang, Biomat. Art. Cells Art. Or~.~ 16:731 (1988), have demonstratedthe viability and regeneration of microencapsulated rat hepatocytes implanted into mice. Viable hepatocytes were microencapsulated in z~lp;in~te-poly-(L-lysine) and implanted h~ oneally into normal and galactosamine-in~ çe-l liver failure mice. Eight days after 30 implantation in the mice with in~ çecl liver failure, the viabilit~ of the encapsulated rat hepatocytes increased from 42% to nearly 100%. After 29 days. the viability of the encapsulated hepatocvtes implanted in normal mice also increased from 42% to nearly 100%.
By contrast, free rat hepatocytes implanted into mice all died within four or five days after xenotransplantation. The composite microreactors of the invention are well-suited to treat 35 hepatic failure.
Other investigators have shown that microencapsulated hepatocytes continue the synthesis and secretion of many specific proteins and enzymes. Cai et al., H~patolo~rv.

CA 0221~039 1997-09-09 10:855 (1989), developed and evaluated a system of microenr,~ps~ tinn of primary rat hepatocytes. Urea formation, ~lvlluulllbin and cholinesterase activity, the incol~vldLion of triti~t~l leucine into intracellular proteins, and the immunnlocation of synthr~i7P~l albumin were monitored in culture. Despite gradual decreases in some of these activities, the S enr~pslll~t~l hepatocytes contimlrd to function throughout the 35-day observation period. In addition, Bruni and Chang, Biomat. Art. Cells Art. Org.. 17:403 (1989),demollslldled the use of micro~illca~sulated hepatocytes to lower bilirubin levels in hyperbilirubinemia.
Microrns~rslll~trd hepatocytes were injected into the peritoneal cavity of Grunn rats.
Bilirubin dropped from 14mg/lOOml to 6 mg/lOOml, and remained depressed after 90 days.
10 Again, the composite microreactors of the invention can be used as described above to treat these hepatic ~ ç~es Parkinson's Disease Parkinson's disease is a neuronal system rlice~e~ involving a degeneration of the nigrostriatal dop~minergic system. Experimental work in both rodents and nonhllm~n 15 primates has shown that transplantation of fetal tissue cont~ining substantial nigra (dopaminergic) neurons from ventral mesencephalon to dopamine-depleted striatum reinstates near-normal dopamine internalvation and reduces motor abnormalities. In addition, implantation of adrenal chromaffin cells has been shown to reverse chemically-in~lllrer1 Parkinson's disease in rodents.
Widner et al., Transplant. Proc.. 23 :793 (1991), reported evidence of fetal nigral allograft survival and function up to 10 months after transplantation and immuno~u~ e;,~ion (cyclosporine. azathioprine, and prednisone) in a human Parkinson's patient. Beginning from the second month after the transplantation, they observed a progressive decrease in limb rigidity~ increased movement speed in a number of arm. hand. and foot movements~ and 25 prolonged "on" periods (>80% increase) after a single dose of L-dopa.
Thus~ transplantation of fetal neural tissue, or cells genetically engineered to produce dopamine and nerve growth factors or other n~;u-ot opic factors, should have a great potential as a new therapeutic approach in patients with neurological disorders. However, in the case of transplanted xenogeneic donor tissue, rejection would pose a serious problem, even by the 30 combined approach of using an immllnnprivileged site and by employing immlln~ u~ es~ive drugs. Therefore the composite microreactors of the invention permit a novel approach to this problem, i.e.. the delivery of dopamine for the treatment of Parkinson's disease using encapsulated donor tissue harvested from ~nim~l~ or genetically engineered cells.
Al7heimer's l~isease An estimated 2.5 to 3.0 million Americans are afflicted with Alzheimer's (1i~ç~eThe disease is char~ct.?ri7t-cl by a progressive loss of cognitive function associated with degeneration of basal ro~ cholinergic neurons. Studies in ~nim~l~ in~lic~t~ that Nerve CA 0221~039 1997-09-09 WO 96/28029 PCI~/US96/03135 Growth Factor (NGF), e.g., brain-derived n~ul~Llvphic factor (BDNF) and n~urol,o~ i.l-3 (NT-3), available from Regeneron and Amgen, respectively, and other n~u,ollopic factors normally act to support the viability and function of these neuron cells, and that continuous infusion of NGF into the ventricles can prevent injury-in~ucecl degeneration of cholinergic neurons as described in Williams et al., P.N.A.S.~ USA. 83:9231 (1986). This tre~tment correlates with improved cognitive function in rodents with memory i~ ;""ent as described in Fisher et al., Neurobiol. A~in~ 10:89 (1989).
These studies suggest that composite microreactors cn~ ;--i l Ig grafts of recombinant or natural NGF-secreting tissue such as astroglial cells or developing skin, can be used to 10 treat patients suffering from Alzheimer's ~ e~e.
Genç Therapy Gene therapy is an approach to treating a broad range of fli.ce~es by deliveringtherapeutic genes directly into the human body. Diseases that can potentially be cured by gene therapy include ~ e~çs associated with the aging population such as cancer, heart 15 ~ e~ce Alzheimer's ~ e~e, high blood pressure, atherosclerosis and arthritis; viral infectious diseases such as acquired immllnç deficiency syndrome (AIDS) and herpes; and inherited diseases such as diabetes, hemophilia, cystic fibrosis, and muscular dy~ hy.
In one particular example, a favored approach for human gene therapy involves the transplantation of genetically-altered cells into patients, e.g., as described Rosenberg, et al., 20 New En~. J. Med., ~:570-578 (1988). This approach requires the surgical removal of cells from each patient to isolate target cells from nolllalg~l cells. Genes are introduced into these cells via viral vectors or other means, followed by transplantation of the genetically-altered cells back into the patient. Although this approach is useful for purposes such as enzyme replacement therapy (for example, for transplantation into a patient of cells that secrete a 25 horrnone that ~ e~ced cells can no longer secrete), transplantation strategies are less likely to be suitable for treating diseases such as cystic fibrosis or cancer, where the diseased cells themselves must be corrected. Other problems commonly encountered with this approach include technical problems, including inefficient tr~n.~duction of stem cells, low ~ es~ion of the transgene, and growth of cells in tissue culture which may select for cells that are 30 predisposed to cancer.
The composite microreactors of the invention are well suited to avoid these problems, because they allow the use of standard human cell lines of, e.g., fibroblast cells, epithelial cells such as HeLa cells, and hepatoma cells such as HepG2, as the implanted cells, rather than requiring the surgical removal of cells from the patient. These cell lines are genetically 35 altered as required bv standard techniques and are ene~rslll:~tt-d and implanted into the patient. These cell lines are much easier to obtain. culture. and work with than individual p~tient~' cells. Moreover, since the composite microreactors prevent the patient's immlme CA 0221~039 1997-09-09 system from recognizing and ~tt~rking the impl~nt~l cells, any human cell lines can be used, making the technique of gene therapy more universally applicable.
Hypo~alhy, uidism Acute and chronic ~y~ JLullls of lly~op~dLhyloidism result from ullLl~dled 5 hypocalcemia, and are shared by both hereditary and acquired hypopaldLhyroidism. The hereditary form typically occurs as an isolated entity without other endocrine or clerm~tologic m~nifeet~tions or, more typically, in association w-vith other abnc rm~lities such as defective development of the thymus or failure of other endocrine organs such as the thyroid or ovary.
Acquired hy~Jup~rdLllyloidism is usually the result of inadv~l LellL surgical removal of all the 10 p~Lhyloid glands, and is a problem in patients undergoing operations secondary to parathyroid adenoma or hyperplasia. Hy~opoldLhyloidism has been treated in hypocalcemic rats by the ~lminietration of microencapsulated parathyroid cells that served as a bioartificial parathyroid. Parathyroid cells can also be encapsulated in the composite microreactors of the invention for use in ~llminictration to animal and human patients.
1 5 Osteoporosis The term osteoporosis covers diseases of diverse etiology that cause a reduction in the mass of bone per unit volume. These rli~ee-e can be treated by the ~flminictration of composite microreactors collL~ g cells that secrete insulin-like growth factor (IGF-l), estrogen in postmenopausal woman to reduce the negative calcium balance and decrease 20 urinary hydroxyproline, androgens in the treatment of osteoporotic men with gonadal deficiency, or calcitonin for use in established osteoporosis.
Reproductive Disorders There are numerous disorders of the ovary and female reproductive tract that can be treated with progestrogens, estrogens. and other hormones. These include progestrogen, e.g., 25 proge~,LeLulle, therapy to inhibit ~iluiLal y gonadotropins (precocious puberty in girls), and for prophylaxis to prevent hyperplasia in PCOD. Estrogen therapy is used in the treatment of gonadal failure, control of fertility. and in the management of dysfunctional uterine bleeding.
Androgens, gonadotropins. and other hormones are used to treat disorders of the testis, e.g., androgen therapy in hypogonadal men, or gonadotropins to establish or restore fertility in 30 patients with gonadotropin deficiency. Accordingly, these rlici~cc~c can be treated with composite microreactors c~,.~ lE the ~ u~l;ate hormone-producing cells.
Huntington's Disease Huntington's disease is characterized by a combination of choreoathetotic movements and progressive ~l~?ml?nti~ usually beginning in midadult life. Distinctive for the disease is 35 atrophy of the caudate nucleus and. to a lesser extent. other structures of the basal ganglia (putamen and globus pallidus). Rodent cells that secrete n~;:ul~llopic factors have been - impl~ntt-fl into the brains of baboons that have a condition similar to Huntington's disease and CA 0221~039 1997-09-09 WO 96/28029 PCr/US96/03135 reversed some of the damaged nerve networks that, in Huntington's patients, lead to pro~les:jivt; loss of eontrol over the body. Similarly, Huntington's disease in human p~tientc ean be treated by the ~rlminictration of eomposite mieroreaetors that eontain human or recombinant cells that seerete the ~ lu~liate nt;~ue,~ L,hie faetors.
Sp;n~1 Cord Tr~uries The majority of spinal eord injuries result from damage to the surrounding vertebral eolumn, from fraeture, disloeation, or both. Tre~tment of sueh injuries involves the ~rlminictr~tion of nerve growth faetors sueh as eiliary n~:u-oL-op,ie faetor (CNTF), insulin-like growth faetor (IGF- l ), and n~u ol.oL,ie faetors, to enhanee the repair of the eentral and peripheral nervous system. Thus, eomposite mieroreaetors eo~ g eells that seerete sueh faetors, either naturally or through genetie engineering, ean be used to treat spinal eord injuries.
Mood (orAffeetive) Disorders Mood disorders are a group of mental disorders sueh as sehi_ophrenia eharaeterized l 5 by extreme exaggerations and disturbanees of mood and affeet assoeiated with physiologie (vegetative), eognitive. and psyehomotor dysfunetions. Many mood disorders are assoeiated with medieal ~1ice~ces that ean be treated with eomposite mieroreaetors eo.l~ i,-p the a~,p"oL,.iate eells sueh as hypothyroidism, Parkinson's t1ice~ce ~17heimer's disease, and m~lign~ncies as diseussed herein. In addition, it has been shown that the n~ e~ cmitt~r 5-hydroxyindol aeetie aeid (5-HIAA), a serotonin metabolite, is redueed in the cerebral spinal fluid of depressed patients. Defieits in other neurotransmitters sueh as dopamine and gamma-aminobutyric acid (GABA) have also been identified in patients with major depression.
Therefore, composite mieroreactors cont:~ining cells that secrete these neurotransmitter are useful to treat these defieiencies.
Motor Neuron Diseases Degenerative motor neuron diseases include ALS (see above), heritable motor neuron diseases (spinal muscular atrophy (SMA), and those associated w ith other degenerative disorders such as olivopontocerebellar atrophies and peroneal muscular atrophy. These 11iccz~cçc can be treated by ~-lminictration of composite microreaetors eol~ i1-g cells that secrete neurotropic faetors like brain-derived n~urolloL,hic factor (BDNF), and n~ulollo,l?hin-3 (NT-3).
Acquiredlmmunodefieiency Syndrome (AIDS) AIDS is caused by an underlying defect in cell-mediated i ~ y due to the human immunodeficiency virus (HIV), and causes persistent constitutional symptoms and/or ~lice~cçC sueh as seeondary infeetions, neoplasms~ and neurologie (iice~ce Patients can be treated to ameliorate symptoms by immlm~logic ~ngment~tion with composite microreactors that eontain cells genetieally engineered to seerete, e.g.~ recombinant human IL-2 (to decrease CA 0221~039 1997-09-09 suppressor cell activity resulting in an increased T cell adjuvant activity), or recombinant human INF-r (macrophage ~llgment~ti~ n). AIDS-related tumors such as Kaposi's s~.;ollla ean be treated with encapsulated eells that seerete human illL~lrelull-a, interleukin-2 and tumor neerosis factor (TNF).
S .An~yotrophic Lateral Sclerosis (T.ou Gehri~'sDisP~c~o.) ALS is the most frequently encountered form of pro~,les~ive motor neuron clicç~ee, and is eharacterized by progressive loss of motor neurons, both in the eerebral cortex and in the anterior homs of the spinal cord, together with their homologs in motor nuclei of the br~in~t~ m ALS can be treated with composite microreactors that contain cells that seerete nerve growth fac~ors such as myotrophin, insulin-like growth factor (IGF-1), ciliary neurotropic factor (CNTF), brain-derived neurotrophic factor (BDNF) and n~uloL,u~hin-3 (NT-3). Animal studies with these factors (IGF-l is available from Cephalon, CNTF from Regeneron, and ~T-3 from Amgen), have demonstrated that they can stem the degenerative effects caused by nerve damage or disease.
1 5 Cancer In most cases. cancer oriEinz~tçs from a single stem cell which proliferates to form a clone of malignant cells. Growth is not properly regulated by the normal biochemical and physical influences in the environment. There is also a lack of normal, coordinated cell differentiation. Cancer cells develop the capacity for discontinuous growth and dissemination to other parts of the body.
Various cancers can be treated according to the invention by the ~riminictration of composite microreactors cont~inin~ cells that secrete hltelr~lull-a (IFN-a) (for solid tumors7 hairy cell leukemia. Kaposi's sarcoma, osteosarcoma. and various lymphomas), reeombinant interleukin-2 (IL-') (for melanoma, renal earcinoma. and Kaposi's sarcoma); tumor necrosis factor (w/IL-2 for Kaposi's sarcoma); recombinant human IFN-a and recombinant human colony stimulating factor-granulocyte macrophage (CSF-gm) (for Kaposi's sarcoma);
recombinant human INF-y (for macrophage ~llgm.ont~tion), CSF (for aggressive ehemotherapy, bone marrow transplantation, priming of leukemic cells to enhance sensitivity to ehemotherapy and to support dose int~n.cification): ciliary neurotropic factor (CNTF) and insulin-like gro~th factor (IGF-1) (for peripheral neuropathies caused by chemotherapy);
adrenal gland cells (for pain relief when injected into the lower spine to secrete natural painkillers) and progestrogen-producing cells (for palliation in endometrial and breast carcinoma).
Duchenne'~ Muscular Dvstrophy D~l~hPnne's ~ly~lophy is an X-linked recessive disorder characterized by progressive wP~knPcc of girdle muscles, inability to walk after age 12, kyphoscoliosis (curvature of the spine). and respiratory failure after the fourth decade. This disease can be treated by -CA 0221~039 1997-09-09 mini~tration of composite microreactors c~ p myoblast cells and growth factors.
Myoblasts have been injected into young boys with Dll- henne's mn~clll~r dy~llu~hy to d~ whether the cells can supply a structural protein that is mi~in~ Researchers have observed muscle strength improvement in several of the boys.
F,pilepsy The epilepsies are a group of disorders characterized by chronic, recurrent, ~uoxy~ al changes in neurologic function caused by abnormalities in the electrical activity of the brain. In some forms of focal epilepsy, inhibitory interneurons appear to be pler~ lLially lost. Tre~tment with neurotropic factors and other neuropeptides such as has been found effective. Therefore, the composite microreactors of the invention c~ i. .g cells secreting these factors can be used to treat epilepsy.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intt?n~ l to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications within the scope of the invention will be a~>pa~ to those skilled in the art to which the invention pertains.
What is claimed is:

Claims (25)

1. A composite microreactor which includes:
(a) an internal particle which includes:
(i) a source of a therapeutic substance;
(ii) an internal particle matrix which contacts the source;
(b) a super matrix, e.g., a gel super matrix, in which the internal particle is embedded, the composite microreactor preferably providing a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from coming into contact with the source and wherein a component of the composite microreactor, e.g., the internal particle, the super matrix, or both, is geometrically stabilized.

2. The composite microreactor of claim 1, wherein the source of a therapeutic substance is a living cell.

3. The composite microreactor of claim 1, wherein the source of a therapeutic substance is an islet.

4. The composite microreactor of claim 1, wherein the internal particle matrix is a gel.

5. The composite microreactor of claim 1, further comprising an internal particle coating which comprises a polyaminoacid.

6. The composite microreactor of claim 5, wherein the internal particle coating is polylysine having a molecular weight of less than 15 kDa.

7. The composite microreactor of claim 1, wherein the super matrix comprises a gel.

8. The composite microreactor of claim 1, wherein the super matrix comprises alginate.

9. The composite microreactor of claim 1, wherein the diameter of the internal particle is between 50 and 700 microns and the diameter of the composite microreactor is between 300 and 1500 microns.

10. The composite microreactor of claim 1, wherein the composite microreactor includes between 2 and 100 internal particles.

11. The composite microreactor of claim 1, wherein the internal particle matrix is geometrically stabilized by allowing it to age for between 2 hours and 14 days.

12. The composite microreactor of claim 5, wherein the internal particle coating has a lower molecular weight exclusion number than does the super matrix.

13. A a composite microreactor which includes:
(a) an internal particle which includes:
(i) a source of a therapeutic substance;
(ii) an internal particle matrix which contacts the first source; and (iii) an internal particle coating of a polyamino acid enclosing the internal particle matrix.
(b) a super matrix in which the internal particle is embedded;
the composite microreactor preferably providing a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from coming into contact with the source.

14. A composite microreactor which includes:
(a) an internal particle which includes:
(i) a source of a therapeutic substance;
(ii) an internal particle matrix which contacts the source; and (iii) an internal particle coating enclosing the internal particle matrix;
(b) a gel super matrix in which the internal particle is embedded;
the composite microreactor preferably providing a molecular weight cutoff that prevents molecules larger than about 400,000 daltons from coming into contact with the sources.

15. A double composite microreactor which includes:
(1) an internal particle which includes:
(a) a source of a therapeutic substance;
(b) an internal particle matrix which contacts the source; and (2) a particle which includes:
(a) the internal particle or particles of (1) (b) a particle matrix in which the internal particle is embedded: and (3) a super matrix in which the particle of (2) is embedded.;

16. A double composite microreactor which includes:
(1) an internal particle which includes:
(a) an islet;
(b) an internal particle matrix of alginate in which the islet is embedded;

(c) an internal particle coating of polylysine enclosing the first internal particle matrix;
(2) a geometrically stabilized particle which includes:
(a) the internal particle of (1);
(b) a particle matrix of alginate in which the internal particles are embedded; and (c) a particle coating of polylysine enclosing the particle; and (3) a gel super matrix of agarose in which the particle of (2) is embedded.

17. A method of improving the performance of a microcapsule which comprises (a) forming a first component of a microcapsule;
(b) treating the first component so as to geometrically stabilize the first component;
and (c) combining the geometrically stabilized first component with a second component of a microcapsule.

18. A method of improving the performance of a composite microreactor which comprises:
(a) forming a first component of a composite microreactor;
(b) combining the first component with a second component of a composite microcapsule; and performing one or both of the following steps (c) and (d):
(c) prior to combining the first and the second component, geometrically stabilizing the first component; and/or (d) prior to combining the first and the second component with a third component, geometrically stabilizing the second component.

19. A method of making a microcapsule having reduced volume comprising:
(a) forming a component of a microcapsule; and (b) applying a volume reducing coating to the component, thereby producing a microcapsule including a component and a coating and having a volume less than the volume of the uncoated component.

20. A method of making a composite microreactor having reduced volume comprising:
(a) forming a first component of a composite microreactor;
(b) combining the first component with a second component of a composite microreactor; and performing one or both of the following steps (c) and (d):
(c) prior to combining the first component with the second component, applying avolume reducing coating to the first component; and/or (d) applying a volume reducing coating to the second component, thereby producing a composite microreactor having a reduced volume.

21. A microcapsule comprising:
a source of a therapeutic substance, PEO of at least 1-8 million Da in molecular weight, and a matrix, the source and PEO embedded in the matrix.

22. A microcapsule, which includes:
a source of a therapeutic substance, and a matrix which is not a liquid.

23. A method of implanting a living donor cell into a recipient comprising:
providing a microcapsule, e.g., a composite microreactor, of the invention whichcontains the living donor cell, and implanting the microcapsule into the recipient animal.

24. The method of claim 23, further comprising testing the recipient for antibodies to the living cell. .

25. The method of claim 23, wherein no adjunctive immunosuppression is administered to the recipient.