US 3708111 A
Description (OCR text may contain errors)
United States Patent n91 Sheeler et al. I
v 1 1 23,708,111 [451 Jan.2, 1973 [5 APPARATUS AND METHOD FOR GRADIENT ZONAL CENTRIFUGATION Inventors; Phillip Sheeler, 19034 Killock Way, Northridge, Calif. 91324; John R. Wells, 12035 Herbert Street, Los Angeles, Calif. 90066 Filed: Sept. 14, 1970 Appl. No.: 71,882
Related US. Application Data Continuation-impart of Ser. No. 886,720, Dec. 19, 1969, abandoned. 1
US. Cl ..,....233/32, 233/46, B04b/1/00 References Cited UNITED STATES PATENTS Anderson Cohn Field of Search ....233/27, 28, 32, l R, 45,213 R,
3,125,5l6 3/l964 Kaldewey ..233/39 Primary Examiner-Jordan Franklin Assistant Examiner-George H. Krizmanich Attorney-4. Jordan Kunik 57 ABSTRACT cylindrical chamber divided into a plurality of sectorshaped compartments by vertical septa radiating from a central core. Either the annular floor or ceiling of said chamber is formed with sloping converging walls intermediate the spaced outer and inner walls of said chamber to converge either to an annular V-shaped groove or to a plurality of funnel-shaped recesses terminating, respectively, in an annular apex or a plurality of apices at which a plurality of spaced apart gradient removal ports are located, and toward each of which said converging sloping walls cause the gradient zones to be constricted and concentrated for improved definition and separation for subsequent fraction collection and analysis.
28 Claims, 9 Drawing Figures APPARATUS AND METHOD FOR GRADIENT ZONAL CENTRIFUGATION CROSS-REFERENCE TO RELATED APPLICATION The present application is a continuation-in-part of a co-pending United States Patent application entitled APPARATUS AND METHOD FOR GRADIENT ZONAL CENTRIFUGATION, Ser. No. 886,720, filed Dec. 19, 1969, by the same inventors, Phillip Sheeler and John R. Wells, now abandoned.
BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION This invention relates to zonal centrifuge rotors and, more particularly, to an improved reorienting gradient zonal rotor for separation of biological cell components and the like. The apparatus is utilized to provide quantitative separations of tissue cells or cell particles, or the like, in density gradients for the purpose of collecting discrete fractions thereof for subsequent analysis in research and clinical studies.
Zonal centrifuge rotors are used for quantitative density gradient centrifugation for fractionating various types of biological materials ranging from cellular constituents of tissues at the large end of the spectrum to molecular components of those tissues representing the small end of the spectrum. The rotor can be used to separate cells of different sizes from one another as, for example, red from white blood cells, different types or sizes of cellspresent in a culture of microorganisms such as bacteria, algae, protazoa, and the like. When cells of a tissue to be'studied are mechanically broken, the rotor may be used to separate and isolate the various constituent particles of those cells such as cell nuclei, mitochondria, chloroplast, membranes, and ribosomes. Also, various cellular molecules may be isolated by centrifugation, such as nucleic acids, polysaccharides, and certain proteins.
2. Description of the Prior Art The prior art relating to the subject of the present invention is described in a recent treatise entitled, The Development of Zonal Centrifuges and Ancillary Systems for Tissue Fractionation and Analysis, edited by Dr. Norman G. Anderson, National Cancer Institute Monograph 21, June 1966, U. S. Department of Health, Education and Welfare, Public Health Service, National Cancer Institute, Bethesda, Maryland, Superintendent of Documents, U. S. Government Printing Office, Washington, D. C.
Zonal centrifuges are described therein as comprising a rotor having a circular chamber with rotating-seal means for introducing and removing materials from said chamber while the rotor is spinning. Rotating-seal zonal rotors suffer disadvantages such as (a) crossing over at the seal when gradient solutions of high viscosity are pumped into the rotor or when samples are in troduced under high pressure; (b) inadvertently long loading and unloading times due to the requirement of gravity feed; (c) continued sedimentation of particles during the unloading period; and (d) since the rotor is loaded and unloaded while spinning at 3,000 to 5,000 rpm and the seal assembly must be manually removed and later inserted during this rotation, considerable dexterity is demanded of the operator. Furthermore, such rotating-seal zonal rotors are expensive, require special centrifuges or modification of older models,
and demand extensive maintenance and cleaning between runs.
Where prior art reorienting gradient zonal rotors have a funnel-shaped sloping floor with a single outlet located at the axial center thereof, such rotors are usually mounted upon a fly wheel which, in turn, is mounted on the centrifuge rotor shaft. This entails time-consuming operational difficulties involving extra manipulations that may reduce the possibility of obtaining optimum or desired results.
, SUMMARY OF THE INVENTION The zonal centrifuge rotor of the present invention is loaded when the rotor is at rest. A manifold distributor is utilized to load density gradients simultaneously into each of a plurality of sector compartments at a point of entry at the apex of the annular V-shaped groove forming the floor of one embodiment of the rotor chamber, said annular groove apex being concentric with and being displaced radially from the axis of said rotor.
' Thereafter, the rotor is accelerated during which time the density gradient undergoes reorientation in which the vertically arranged isodense layers gradually become concentrically arranged about the axis of rotation.
The sample may be introduced by initially placing it in the bottom of the V-shaped groove after which it becomes displaced upwardly upon introduction of the density gradient, with both the gradient and the sample undergoing reorientation during acceleration. Alternatively, after preliminary acceleration of the rotor, the sample may be spread onto the reoriented density gradient through an opening in the top cover of the rotor by using a syringe, or, the like.
Thereafter, therotor is accelerated to the higher speed desired for particle separation during which said particles become arranged as a series of concentric zones about the axis of rotation. After separation is achieved, the rotor is decelerated during which time both the density gradient and the separated particle zones undergo reorientation. With the rotor once again at rest, the separated particles are arranged as a series of disc-shaped layers in the now vertical density gradient. Finally, the density gradient and the separated particles are pumped from the rotor beginning with the dense end of the gradient at the bottom of the V-shaped groove and the material is collected as a series of fractions.
A unique feature of the present invention is the provision of a circular V-shaped groove forming the floor of the rotor chamber in which a removable set of radially arranged partitions or septa are located. This V-shaped groove is one of the important features which distinguishes the configuration of the new rotor from the flat-floor rotating-seal rotors or other reorienting gradient rotors. The septa are integrally formed upon a cylindricalcore which surrounds the interior circular wall of the rotor chamber. The core has a plurality of channels leading downwardly and obliquely outwardly into the septa where they terminate at the V-shaped vertex of each of the septa which correspond in angular dimension to the V-shaped floor of the rotor. The materials are introduced into and withdrawnfrom the respective multiple sectors of the rotor chamber through said channels and through a removable distributor having multiple channels which are connectable to respective channels in the septa core.
The zonal rotor of the present invention, however, not only offers all of the advantages of the prior art rotating-seal zonal rotors, but is considerably simpler in design and construction, does not require a rotatingseal and can be operated with ease by any laboratory technician. Also, the rotor of the present invention has an axial recess adaptable for connection to the tapered drive shaft of a centrifuge such as disclosed in U.S. Pat. No. 2,827,229, Mar. 18, 1958, as embodied in Model RC-2 produced by Ivan Sorvall, Inc. The reorientinggradient zonal rotor described herein employs an annular V-shaped groove as its floor, the annular apex of said groove being displaced radially from the axis of said rotor, and this is in contrast to the prior art rotating-seal zonal rotors which have a solid, flat bottom. The presence of this annular V-shaped floor also distinguishes the rotor described herein from that of the prior art which has a funnel-shaped floor whose apex exit port is at the axis of rotation. Such prior art funnelshaped rotors must be mounted on a fly wheel or the like which, in turn, is connected to the drive shaft of the centrifuge.
One alternative embodiment of the invention comprises the location of the annular V-shaped groove formed by two converging annular slopes in the roof of the rotor chamber instead of the floor, and wherein the removal of the gradient zones is effected through the top of the rotor. A still further embodiment of the invention comprises the formation of a plurality of funnel-shaped recesses in circular array either in the floor or ceiling of the rotor chamber whereby the removal of the gradient zones is effected through the apices of said recesses and wherein the advantageous constriction and concentration of said zones is effected by the walls of the recesses sloping towards said apices from all directions instead of from inward and outward radial directions in the case of the V-shaped annular grooves.
The rotor of the present invention is loaded and unloaded while the rotor is at rest by. means of a manifold distributor which may be connected to a peristaltic pump or the like. The use of a peristaltic pump with a rotating-seal zonal rotor may result in crossing over. Since the gradient in the rotor of the present invention is eluted with the rotor at rest, not further particle sedimentation occurs as obtains in rotating-seal zonal rotors where the particles continue to sediment as the rotor continues to rotate during the entire unloading procedure. Thus, the reorienting gradient rotor of the BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a vertical section view, partly in elevation and partly broken away, and some parts being omitted;
FIG. 2 is a top view taken on line 2--2 of FIG. 1;
FIG. 3 is an enlarged detail view, in section, of the manner in which the distributor channels may be connected to the septa channels;
FIG. 4 is an exploded view, partially in section and partially in elevation, of the apparatus shown in FIG. 1, some parts being shown schematically and some parts being omitted;
FIG. 5 is a vertical central section view, partly broken away, some parts being omitted, illustrating another embodiment of the invention;
FIG. 6 is a view of the top of the rotor shown in FIG. 5 with the cover removed, and other parts being omitted;
FIG. 7 is a greatly enlarged fragmentary view of the upper central portion of the apparatus shown in FIG. 5;
FIG. 8 is an exploded diagrammatical view, in vertical central section, illustrating another embodiment of the invention; and
FIG. 9 is a view taken on line 9-9 of FIG. 8.
DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring now to the drawings in detail, there is shown the upper fragmentary portion of a cone-shaped hub 21 mounted upon the upper end of a centrifuge shaft, not shown, connected to a suitable rotating power source such as an electric motor or the like.
Mounted on hub 21 is a centrifuge rotor, generally designated 22, made of aluminum or the like, having an axial core portion 23 encircling a bottom conicallyshaped axial recess or aperture 24 which fits intimately with hub 21. The upper narrowed end of aperture 24 terminates in an outwardly extending annular shoulder 26 which forms a boundary between aperture 24 and an upwardly extending axial aperture 27. The upper end of hub 21 terminates in a threaded stub 28 which extends upwardly into aperture 27 and is threadably engaged by nut 29 bearing downwardly upon washer 31, resting on shoulder 26, to secure rotor 22 to hub 21.
The upper outer portion of rotor 22 is annularly threaded at 32 to accommodate the interior threaded portion 33 of the downwardly extending rim 34 of circular cover plate 36 made of aluminum or the like whereby said cover is removably secured upon said rotor. The top of cover plate 36 may be provided with a plurality of recesses 37 to accommodate a tool for tightening said cover plate upon and for removing it from said rotor. Cover plate 36 has a central aperture 38, the purpose of which will be described hereinafter. The top surface of the outer portion of rotor 22 has an annular recess 39 'which accommodates O-ring 40 made of a suitable resilient material such as rubber, nylon, or the like, to form a liquid-tight seal between cover plate 36 and the adjacent portion of said rotor.
Rotor 22 has an annular centrifugation chamber, generally designated 41 (FIG. 4) which is formed by vertical circular outer wall 42 and a spaced apart vertical circular inner wall 43, both of said walls being concentrically located in respect of the axial center of said rotor. The lower end of outer wall 42 is intersected by the upper end of inwardly and downwardly sloping annular groove wall 44, while the lower end of inner wall 43 is intersected by the upper end of outwardly and downwardly sloping annular groove wall 46. Groove walls 44 and 46 converge to form a V-shaped annular groove, generally designated 47, which is disposed concentrically around the axis of rotor 22. At least a portion of chamber 41 concentrically surrounds recess 24.
In some embodiments, the convergence of sloping groove walls 44 and 46 may form a sharp vertex. In the embodiment shown in the drawings herein, the bottom of groove 47 is in the form of a narrow annular floor 48 whose radius is substantially equidistant from the respective radii of walls 42 and 43. In other embodiments, the upper edges of the V-shaped groove need not intersect respective walls 42 and 43 directly but may be joined by intermediate short annular slopes or curves. Furthermore, it is contemplated that walls 44 and 46 of the V-shaped groove may be curved in any suitable manner provided they perform the function of constricting the radial area of the materials being removed therefrom as will be described hereinafter.
Removably positioned in annular chamber 41 are radially arrayed partitions or septa 51 that divide said chamber into'a plurality of substantially equal sector chambers. The inner vertical edges of septa 51 are integrally formed upon a tubular core 52, the inner cylindrical surface 53 thereof having upper and lower inwardly extending annular bosses 54 and 56 which form a smooth, sliding fit over the circular surface 43 of core portion 23 of rotor 22. On the upper portion of vertical edge 58 of each septum is an integrally formed, outwardly extending boss 59 which'serves in conjunction with bosses 54 and 56 to align said septa within chamber 41. The removal of septa 51 from, and their insertion into, chamber 41 is accomplished by raising and lowering cylindrical core 52 relative to core portion 23 of rotor .22. Although six septa 51 are disclosed in the embodiment shown in the drawings herein, it is understood that any suitable number such as 4, 8, 12, or the like, can be provided as may be necessary or desired.
The bottom of each septum 51 is bounded by a pair of converging edges 61 and 62, the inwardly angled edge 61 matching the angle of groove wall 44, and the outwardly angled edge 62 matching the angle of groove wall 46, to form a close fit between said respective edges and groove walls as shown in FIG. 1. Septa edges 61 and 62 converge to narrow horizontal annular edge 63 which is substantially parallel to annular floor 48 of groove 47 and is spaced a short distance apart from said floor to allow liquid communication between adjacent sectors which would ensure that isodense layers occupy identical-positions in each sector due to hydrostatic pressure, and to facilitate introducing materials into and removing the same from the sector chambers by way of septa channels described hereinafter.
In some embodiments, the septa 51 may comprise separate plates which are inserted into chamber 41 in suitable radial array whereby the separate sector chambers may be filled with materials to be centrifuged and drained thereof after configuration by suitable means, the profiles of said septa conforming in size and shape substantially equal to the cross-sectional area of said chamber and to the particular configuration of walls 44 and 46 of the V-shaped groove.
In the preferred embodiment illustrated and described herein, however, septa 51 are formed into a unitary component with cylindrical core 52 whereby the filling and draining of the sector chambers within the centrifuging chamber 41 may be accomplished by a vertical channel 66 for each septa within said core, said vertical channel communicating with an outwardly and downwardly inclining channel 67' whose lower outlet port 68 is located in edge 63.
Removably mounted on top of the axial core portion 23 of rotor 22 is a circular distributor block 71 which is located within axial aperture 38 of cover plate 35. Distributor block 71 may be removably secured in position upon rotor 22 by means of a plurality of spaced apart threaded bolts 72. Distributor 71 has an integrally formed, upwardly extending axial boss 73 which has a vertical axial channel 74 whose lower end terminates in a plurality of radially spaced apart downwardly and outwardly extending channels 76 which are equal in number to the number of septa 51. Each channel 76 communicates with a corresponding vertical channel 66 in a respective septum 51.
One typical arrangement for connecting each channel 76 with its respective channel 66 is shown in fragmentary enlarged detail view in FIG. 3, where core 52 in the region of channel 66 is provided with a recess 77 in which is secured an O-ring 78. The upper end of an elongated tubular adapter 79 extends into block 71 by means of a press fit to join channel 76 while the lower end of said adapter extends by a loose fit into core 52 to join channel 66 thereby forming a unitary passage between channel 76 and channel 66. A liquid-tight juncture is provided by an integrally formed annular flange 81 on tube 79 bearing against O-ring 78 when block 71 is urged downwardly and securely upon the core portion 23 of rotor 22. Other suitable means may be provided for ensuring a leak-tight juncture between channels 76 in block 71 and corresponding channels 66 in core 52.
When distributor 71 is bolted in position, it secures core 52 and septa 51 firmly in position. Port 74 at the top of distributor 71 is connected by means of flexible tubing to the pump when the rotor is being filled and being emptied of the density gradient and distributed particles, but the tube is disconnected during centrifugation.
OPERATION In operation, channel 74 of axial boss 73 is connected to a suitable source of continuous liquid density gradient which comprises a stream of fluid whose density progressively changes from someinitial minimum value to some final maximum value. These gradients are routinely prepared in laboratories engaged in density gradient centrifugation and are prepared by commercially available apparatus known as a gradient engine. The density gradient may be transmitted by way of suitable flexible tubing 83 to port 74 by means of a peristaltic pump 84 of the like, connected to a source 85 of said gradient. Before centrifugation is begun and while the rotor is stationary, the density gradient enters by way of channels 76 in distributor 71 in a continuous liquid stream and passes through channels 66 and 67 of core 52 and septa 51, respectively, and emerges from the terminal ports 68 at the bottom of said septa into the groove vertex whereby each of the sector chambers are filled simultaneously. The liquid is then displaced upwardly by the denser fluid flowing immediately beparatus herein, the sample whose particles are to be separated may be introduced first by way of channels 66 and 67 into the bottom of V-shaped groove 47 after which the density gradient is introduced thereby lifting the sample toward the top of chamber 41. Thereafter, the flexible tubing to the pump is removed from distributor 71 and the rotor is rotated to produce reorientation of both the gradient and the sample simultaneously. Following centrifugation and the deceleration of the rotor, the flexible tubing from the pump is connected to the distributor 71 and by reverse pumping the separated layers of sample particles are sequentially removed from chamber 41 by way of ports 68 and channels 67 and 66, and transmitted to a suitable fraction collector 86.
Alternatively, the isodense gradient is introduced first into the sector compartments of the rotor by the means described hereinbefore after which distributor 71 is removed and the gradient is preliminarily reoriented by rotation of the rotor into a plurality of vertical cylindrical isodense layers, as described in the National Cancer Monograph 21 cited hereinbefore. Thereafter, the sample is introduced by spraying or the like on to the reoriented gradient, after which centrifugation takes place during which time the sample initially forms a vertical cylindrical zone at the inner circular wall 43 of the rotor, after which the particles of said sample migrate or sediment through the isodense layers and become arranged as a series of concentric zones about the axis of rotation. After separation is achieved, the rotor is decelerated during which time both the density gradient and the separated particle zones undergo reorientation. With the rotor once again at rest, the separated particles of the sample are arranged as a series of spaced apart horizontal discshaped layers in the now vertical density gradient. The flexible tubing from the pump is then attached to port 74 of distributor 71 and the density gradient, along with the separated sample particles, is pumped from the rotor chamber 41 beginning with the dense end of the gradient atthe bottom of V-shaped groove 47 and is collected as a series of fractions.
It is understood that when density gradients and samples are introduced into annular chamber 41 for centrifugation, the top surface of these materials will be a suitable distance below the top of rotor 22 so that when centrifugation takes place and the mass of liquids rises to impinge upon the bottom surface of cover plate 35 to form a vertical cylindrical column of liquid within chamber 41, the diameter of the inner surface of said rotating cylinder of liquid, at least at the upper portion thereof, will be somewhat greater than the diameter of aperture 38 so that no spillage will take place through said aperture.
The events occurring within the reorienting gradient rotor take place as follows: As'the rotor is initially accelerated, the density gradient and the sample begin to undergo a reorientation within chamber 41. Each isodense layer bows inwardly to form a paraboloid of revolution about the axis of rotation. With continued acceleration, a centrifugal force is attained which is sufficient to cause the isodense layers to achieve verticality. The sample zone also has undergone reorientation so that initially it has formed a single cylindrical inner zone around the axis of rotation. The sample particles subsequently sediment radially away from the axis of rotation under the g force and their rate of sedimentation will be determined by their respective sizes and densities. Accordingly, the sample particles form several separate spaced apart concentric vertical cylindrical columns throughout the density gradient. After the desired separation of particles has been achieved, the rotor is slowly decelerated during which time the density gradient which now contains the separated particles again reorients in' chamber 41 resulting in a horizontal array whereby the separate zones of separated particles are present in the gradient as a plurality of spaced apart horizontal disc-shaped rings in vertical array within the density gradient.
While the sloping walls 44 and 46 of annular V- shaped groove 47 and may be symmetrical as to their respective angular arrays from a horizontal plane, and in such a configuration may provide satisfactory operating conditions for the apparatus described herein, a preferred embodiment is shown in the drawings herein wherein sloping wall 44 is arrayed approximately 30 and sloping wall 46 is arrayed approximately 45 from the horizontal plane. With such an asymmetric array, the bottom of the vertical annular zone formed by the sample during the early stage of centrifugation will be located substantially at the bottom of wall 43 which is at a higher level than the bottom of outer wall 42. Thus, during subsequent centrifugation, the initial narrow cylindrical sample zone around core 23 of the rotor migrates from inner wall 43 toward outer wall 42 through rotor chamber 41. Since sedimentation of the sample particles takes place during centrifugation in a direction perpendicular to the axis of the initial cylindrical sample zone, those migrating particles that ultimately reach the outer annular wall 42 do not enter into the portion of the density gradient that occupies groove 47 defined by walls 44 and 46. By providing that the bottom end of wall 42 is lower than the bottom end of wall 43, the latter being the approximate locus of the bottom end of the initial cylindrical sample zone, the asymmetric configuration of the V-shaped groove ensures that uniform sedimentation of the complete sample zone will take place by virtue of the unimpeded migration of the total vertical dimension of said sample zone through chamber 41.
The vertical length of the sample zone can be readily determined by empirical means with the foreknowledge of the dimensions of V-shaped groove 47, whereby a sufficient amount of density gradient material is caused to fill said groove so that the lower end of the sample zone extends to a level above or slightly above the vertex between the outer vertical wall 42 of the rotor chamber and the upper end of the outer sloping wall 44 of the V-shaped groove.
Another advantage of the V-shaped groove of the centrifugation chamber of the apparatus herein lies in the greatly improved efficiency in recovering the discrete horizontal sample layers separately from the rotor. This is accomplished by pumping the density gradient out of rotor chamber 41 from the bottom of the annular V-shaped groove by means described hereinbefore, beginning with that portion occupying the bottom of the V-shaped groove (i.e. the flow being opposite to that which obtains when the density gradient is loaded into the rotor). As pumping continues, the fluid contents of the rotor slowly descend toward and through the annular V-shaped groove. The discrete layers of separated particles are sequentially channeled by the converging walls of the V-shaped groove so that they are drawn in the proper sequence through channels 67 and 66 of the septa-core component of the apparatus and collected in separate test tubes after passing from the rotor proper.
Before unloading chamber 41 of the rotor, the sample particle zones which are to be collected are arranged as a series of horizontal layers suspended in the gradient and separated by some vertical distance. As the gradient is being pumped out of the rotor, all of the sample layers slowly descend toward the rotor floor. Before the V-shaped groove is reached, the surface area of each sample zone remains constant and the distances between sample zones also remain constant. As the fluid descends into the region of the V-shaped groove, however, the surface area of each sample zone becomes reduced, and since the fluid volume of each zone must remain constant, it follows that each zone becomes deeper in vertical dimension. By the same token, the vertical distance between sample zone layers also increases and this phenomenon ensures against mixing that might otherwise occur between sample zone layers. The surface area of each zone layer reaches a minimum value when the vertex of the V- shaped groove is reached and at this time each sample zone is drawn into the exit ports 68 and through channels 67 and 66 for removal from the rotor. Since all of the particles in one sample zone layer located at the vertex of the V-shaped groove are closer to an exit port than particles in other zone layers not yet near the vertex, the possibility of mixing between zone layers is minimized.
Converging walls of the V-shaped groove ensure that each zone layer will be focused into the vertex of the groove and pumped in the proper sequence from the rotor. Thus, the annular V-shaped groove acts in a manner similar to a funnel; it guarantees that fluid flow from the rotor will be orderly and that particles suspended in the separate layers in the density gradient will leave the rotor in exactly the same order as they were arranged vertically before unloading was initiated.
If no groove existed in the floor of the rotor, each sample zone would maintain the same maximum surface area throughout its descent and would be spread over the entire annular flat rotor floor. Under such conditions, the removal of a sample particle zone layer at the rotor floor would be most inefficient, due in large part to the fact that particles in other zone layers located higher in the gradient would be physically as close to exit port 68 as particles lodged in the margins of the sample zone already resting on the flat chamber floor. Accordingly, mixing between zone layers would occur and, consequently, the resolution achieved during centrifugation would be lost during the collecting operation. It should also be noted that in the absence of a V-shaped groove, the distance between sample zone layers would remain the same during their descent to the flat floor of the rotor, whereas the presence of a V- shaped groove produces an increase in the distance between the sample zone layers as they descend to the bottom of the chamber and thereby further reduces the chances for mixing to occur.
The V-shaped groove plays a similarly vital role during the loading of the rotor although its effectiveness is not immediately obvious. The liquid density gradient which is to be loaded into the rotor may be thought of as a sequence of horizontal zones which differ slightly in their densities. As fluid emerges from the ports at the bottom of the core-septa section, it slowly fills the V- shaped groove. The surface area of each zone slowly increases as the fluid level is elevated in the groove and finally attains a constant value once above the groove. In other words, a more dense portion of the gradient is always layered below a lighter portion and there is no mixing as would occur if the groove were not present. If no groove were present, fluid emerging at the base of the core would have to spread instantaneously over the entire floor surface area. Thus, the V-shaped groove provides for a smooth and orderly increase in the surface area of each zone entering the rotor.
In the design of the V-shaped groove, it is desirable to keep the volume of the density gradient occupying said groove to a minimum. This could be accomplished by providing very shallow angles for the groove walls, but this would result in less efficient funneling during the unloading operation since a very shallow angle would be almost as deficient as no groove at all. As a corollary, steep angles in the V-shaped groove would result in efficient loading and unloading but would severely restrict the usable volume of rotor chamber 41. Any angles between 10 and from the horizontal plane could be used, but practical angles would be in the range of 20 60. In one satisfactory embodiment where the V-shaped groove was symmetrical, the groove walls 44 and 46 were arrayed at approximately 47 from the horizontal. In other embodiments where the asymmetrical V- shaped groove is desired, respective angles of 30 for wall 44, and 45 for wall 46 from the horizontal plane have been found useful. Other suitable asymmetric arrays for groove walls 44 and 46 may be arranged to accomplish successful results with the apparatus herein.
It is to be understood that the provision of the annular V-shaped groove 47 herein is also useful without employing the structural features of core 52 and its channels 66 and channels 67 in septa 51. It is possible to utilize separate septa 51 as partitioning elements and to load and unload the density gradient relative to the various sector chambers by means of one or more tubular elements that are inserted into chamber 41 with the lower ends of said tubes being located at annular floor 48 whereby the introduction and removal of the gradient can be achieved in a manner that takes advantage of the V-shaped groove as described hereinbefore for fractionally collecting each of the separate ring-disc particle zones without contamination or interference from other particle zones.
In another embodiment of the invention, the annular V-shaped groove which has heretofore been illustrated and described, in connection with FIGS. 1 4, as being located in the floor of the rotor chamber, may instead be located and formed in cover plate 91 as shown diagrammatically in FIG. 5.
1n this embodiment, rotor 92 has an annular chamber 93 having a flat annular floor 94. Surrounding the central hub 96 of rotor 92 is a tubular core 97, the inner cylindrical surface 98 of which is slightly spaced apart from said core portion by means of inwardly extending annular bosses 99 and 101 on the upper and lower ends thereof which form a smooth, sliding fit over the circular surface 102 of hub 96. Formed integrally with tubular core 97 and extending radially outwardly therefrom in chamber 93 is a plurality of partitions or septa 103 which divide saidchamber into a plurality of substantially equal sector chambers. The bottom horizontal edges 104 of septa 103 rest on floor 94 of rotor chamber 93. Formed intermediate the inner and outer edges of each septum 103 at the bottom thereof is an upwardly extending recess 105 which permit hydrostatic equilibration between adjacent compartments of the rotor.
The upper end of each septum 103 terminates in an asymmetric apex comprising outer sloping edge 106 and inner sloping edge 107. Said septa apices extend upwardly into the top of rotor 92.
Mounted on the hub 96 of rotor 92 is a distributor block 108, secured thereon by means of circularly spaced apart bolts 109. lntegrally formed upon and extending axially upwardly from block 108 is a tubular conduit stem 111 having a vertical axial channel 112, the lower end of which terminates in a plurality of radially spaced apart downwardly and outwardly extending channels 113 which are equal in number to the number of septa 103. Each channel 113 communicates directly with a corresponding vertical channel 114 in core 97, the bottom open end of channel '1 14 terminating at the bottom edge of said core slightly spaced apart above floor 94 of annular chamber 93. Suitable connecting means, as described hereinbefore, for example, may be provided between channels 113 and respective channels 114 to serve as leakproof connections therebetween. Cover plate 91 has a central aperture for freely accommodating block 108.
Cover plate 91 has an annular V-shaped groove formed in its bottom surface which comprises an outwardly sloping wall 116 and an inwardly sloping wall 117 whose angles of inclination conform with the angles of corresponding edges 106 and 107 of septa 103 and with which they make a close fit. The apex of each septum 103 terminates in a narrow, flat, horizontal top edge 118 while sloping walls 116 and 117 of cover plate 91 converge into an annular apex located above top edges 118 of septa 103.
Located in cover plate 91 in circularly spaced apart arrayis a plurality of outlet ports 119, each of which is located directly opposite a corresponding top edge 118 of a respective septum 103. Mounted onthe top central portion of cover plate 91 by means of bolts 121 is a circular distributor block 122 whose diameter is somewhat greater than that of the annular apex in the bottom surface of cover plate 91. Each outlet port 119 has a corresponding vertical channel 123 in cover plate 91 which, in turn, is aligned with and communicates with a corresponding vertical channel 124 in block 122. Here, also, suitable means are provided to form leak-tight seals between channels 123 and respective channels 124.
Channels 124 intersect with corresponding horizontal channels 126 in block 122 which extend inwardly and communicate with an annular channel 127 formed between the central portion of block 122 and conduit stem 111 extending axially therethrough. As shown in FIG. 7, annular channel 127 terminates in and intersects with channel 128 in a laterally extending tube 129. Both stern 111 and tube 129 are connectable to suitable apparatus for charging the rotor with materials to be treated therein and for removing materials therefrom.
Since the vertical channels 112 and 127 are concentrically oriented, with channel 127 leading to a peripheral port 128, the leak-tight integrity of the latter is ensured by the provision of a static O-ring seal 131 below channel 126, and O-ring seal 132 above and spaced apart from channels 127 and port 128.
Since chamber 93 of rotor 92 is loaded and unloaded under pressure, said chamber must be completely sealed off. This is accomplished by the provision of an O-ring seal .133 near the lower peripheral end of axial core 96 which provides a seal between the tubular septa core 97 and said axial core 96. Another O-ring seal 134 is provided to seal the junction between cover plate 91 and tubular septa core 97.
In operating the embodiment of the apparatus shown in FIGS. 5, 6, and 7, and with the rotor 92 at rest, the sector-shaped compartments in rotor chamber 93 are filled with a dense cushion solution introduced through inlet channel 112 in stem 111 and passed through channels 113 and 114 into said chamber. The density gradient is then loaded, with the dense end first, through peripheral channel 128, vertical channel 127, horizontal channels 126, and vertical channels 124, 123, under pressure, thereby displacing the initial dense cushion solution out through the core channels 114, 113, and 112. Eventually, the entire rotor chamber is filled with a density gradient whose lighter region is at the vertex of the inverted V-shaped groove formed in the interior surface of cover plate 91, and whose densest region is represented by a residual volume of the cushion solution at the rotor floor.
The sample to be fractionated is then introduced through the cover plate channels by way of the apex of the annular inverted V-shaped groove, thereby displacing additional cushion solution out of channels 114, 113 and 111. Connections to channels 111 and 112 are removed. Rotor 92 is then accelerated to reorient the density gradient and the sample, and thereafter centrifugation is carried out as described hereinbefore. After the desired separation has been achieved, rotor 92 is then slowly decelerated to rest. Thereafter, a dense solution is pumped through the core channels 112, 113 and 114, to the bottom of rotor chamber 93 to cause displacement of the gradient and of the separated sample zones sequentially up and out through the cover plate channels 124, 126, 127 and 128. The fractions are then collected, utilizing a fraction collector.
One advantage of providing the inverted annular V- shaped recess at the top of the rotor chamber lies in the ability to introduce the sample last at the top of the gradient with the rotor at rest. A further advantage lies in the ability to displace the contents of the rotor under controlled pressure which ensures an even flow through all of the inlet and outlet channels. This is in contrast to the configuration in FIGS. 1 4 where the contents of the rotor chamber were removed by aspiration or by other negative pressure means.
It will be noted that slopes 116 and 117 forming the annular V-shaped groove in cover plate 91 in FIG. 5 are asymmetric in a manner comparable to the asymmetry of slopes 44 and 46 forming the annular V- shaped groove in rotor 22 of FIG. 1, whereby the same functional results are obtained in both embodiments.
A still further embodiment of the invention is shown schematically in rotor 140 in FIGS. 8 and 9 wherein the annular chamber floor of rotor chamber 141 has a plurality of generally funnel-shaped recesses 142 in circular array around the axis of said rotor chamber. Along the radial line between contiguous recesses 142 as shown at the left side of rotor 140 (FIG. 8) the floor at the bottom of the rotor chamber 141 is V-shaped in the form of low outer slope 143 and low inner slope 144. The funnel-shaped recesses 142 may be machined into the floor of the rotor chamber with a symmetrical sloping conical surface 146, but since slopes 143 and 144 are arrayed at somewhat different angles, the top perimeters of recesses 142 as shown in FIG. 9 turn out to be asymmetrical instead of circular in shape if the annular floor of chamber 141 were otherwise flat.
The conical wall 146 of each recess 142 converges into a downwardly extending apex 147. Located in chamber 141 would be a tubular core and radially extending, integrally formed septa similar to core 52 and septa 51,- as shown in FIGS. 1 and 2, with the lower portions of said septa descending into respective funnel shaped recesses 142 and performing the same function as described hereinbefore in connection with FIGS. 1, 2 and 4. As in the previous embodiments, rotor 1411 would be enclosed by a cover plate 148.
, Whereas in the embodiment in FIGS. 1 and 2 the draining of the materials from the rotor chamber after reorientation caused said materials to flow toward the apex of the annular V-shaped groove generally from walls arrayed on opposite sides of the apex of said groove, the embodiment in FIGS. 8 and 9 permits the materials to flow toward the apex of each funnel from all directions substantially evenly, whereby each separated zone of particles is more efiiciently constricted at the point of removal at the apex 147 of each funnel recess and the corresponding adjacent exit port of the respective septa. Thus, the converging walls in a funnel-shaped recess may serve to improve the definition and complete collection ofeach particle zone over that which is possible in the two-sided annular V- shaped groove of FIGS. 1 and 2. v
The apices 147 of each funnel recess 142 are equidistantly aligned in circular array from the axial center of rotor 140. It will also be noted that the upper edge of each funnel-shaped recess 142 is preferably contiguous to the upper edge of an adjacent funnelshaped recess 142 whereby the downward flow of the rotor contents into each of said funnels is controlled equivalently in orderto catch particles moving inwardly from the outer wall 150 of chamber 141.
Furthermore, there is provided an annular shelf 149 adjacent the bottom end of the inner wall 151 which would act to limit the sample after loading to eliminate wall effects on the centrifugal, ascending surface of each funnel. This shelf would be sufficiently wide so that the sample would not move into the funnels during the initial reorientation.
since the mouths of the respective exhaust ports 67 of septa 51 are located in respective apices of the funnel recess 142 and since said apices are in a circular array equidistant from the axial center of the rotor 140, it is manifest that the introduction and removal of materials into and from the rotor chamber 141 of rotor of FIG. 8 takes place substantially in the same manner as the comparable introduction and removal of materials in annular chamber 42 of rotor 22 of FIGS. 1 and 2. Hence, the two embodiments, namely FIGS. 1 and 2 on the one hand, and FIGS. 8 and 9 on the other hand, are substantially equivalent in structure and function except for the exercise of choice where necessary or desired. In collecting particle zones in the funnel-shaped recesses of FIGS. 8 and 9, the materials are caused to flow toward the outlet port from all radial directions around that focal point, namely, apex 147, in funnel recess 142, rather than from principally opposite or radially inward and outward directions from slopes 44 and 46 in the annular V-shaped groove in the embodiment of FIGS. 1 and 2. In the latter case, this also includes some flow of materials from generally opposite lateral directions along the arcuate path of the annular apex 48. Where it is desired or necessary to concentrate the specimen zones to a higher degree of resolution, it may be useful to utilize the funnel-shaped recess as embodied in FIGS. 8 and 9, although a satisfactorily high resolution of concentration is achieved in'the annular V-shaped groove in the embodiment of F168. 1 and 2.
The V-shaped array of slopes 143 and 144 of the floor of rotor chamber 141 between the respective adjacent funnel recesses 142 are provided in order to cause a suitable flow of materials toward said funnel recesses in the areas therebetween so that a continuous flow of materials is caused to flow into said funnels which might otherwise remain on some portions of said floor if it were otherwise flat. Thus, the V-shaped configuration of slopes 143 and 144 in FIG. 8 is comparable to the V-shaped array of slopes 44 and 46 in FIG. 1.
It is to be understood that the configuration of the funnel-shaped recesses 142 and V-shaped array of slopes 143 and 144 in FIGS. 8 and 9, may be transposed and incorporated, instead, in the cover plate of the rotor in a manner comparable to that shown in FIG. 5, in which case the floor of the rotor chamber would be flat as shown in FIG. 5.
In all of the embodiments illustrated and described herein, the removal of the gradient occurs at a plurality of spaced apart locations within the rotor chamber intermediate the inner and outer walls thereof. The points of removal are each located at an apex of converging'floor or ceiling slopes that are inclined either into an annular V-shaped groove or into a plurality of funnel-shaped recesses where, in both cases, said inclined walls produce a desirable constriction and concentration of the gradient so that superior separation between gradient zones is achieved. The converging sloping walls may be incorporated either in the floor or ceiling of the rotor chamber, as may be desired or required.
Although the present invention has been described with reference to particular embodiments, methods, and examples, it will be apparent to those skilled in the art that variations and modifications can be substituted therefor without departing from the principles and true spirit of the invention. The Abstract given above is for the convenience of technical searchers and is not to be used for interpreting the scope of the invention or claims.
1. A gradient zonal centrifuge rotor comprising an annular chamber in said rotor, said chamber being bounded by coaxial spaced apart annular inner and outer vertical walls, a floor at the bottom of said chamber, said floor comprising a V-shaped annular groove, the inner and outer respective annular upper edges of said V-shaped groove intersecting the respective bottom edges of said inner and outer walls, a plurality of removable spaced apart radially arrayed separate septa in said chamber dividing said chamber into a plurality of chamber sectors, the respective vertical edges of each of said septa extending substantially to the respective inner and outer walls, each of said septa having a profile conforming substantially in size and shape to the cross-sectional dimensions of the combined chamber and V-shaped groove, distributor means secured to said rotor and means interconnecting the distributor means with said groove for introduction and removal of material into and from said chamber.
2. A centrifuge rotor according to claim 1 wherein said means interconnecting the distributor means with said groove comprises a channel in each of said septa for introducing materials into and removing materials from said chamber in the area of the apex of said groove.
3. A centrifuge rotor according to claim 2 wherein said a distributor means is located axially on top of said rotor through which said materials are transmitted to and collected from said septa channels.
4. A centrifuge rotor according to claim 1 wherein the lower portions of said septa are V-shaped and wherein the apex of the V-shaped portion of each septum is spaced apart a short distance from the apex of the V-shaped groove.
5. A centrifuge rotor according to claim 4 wherein the apex of the V-shaped groove is in the form of a nar row flat annular floor and wherein the bottom edge of each septum has a flat edge.
6. A gradient zonal centrifuge rotor comprising an annular chamber in said rotor, said chamber being bounded by coaxial spaced apart annular inner and outer vertical walls, a floor at the bottom of said chamber, said floor comprising a V-shaped annular groove, the inner a nd outer respective annular upper edges of said V-shaped groove intersecting the respective bottom edges of said inner and outer walls, a cylindrical core removably mounted within said chamber adjacent said inner wall, a plurality of spaced apart radially arrayed separate septa integrally formed on said core, said septa dividing said chamber into a plurality of sector chambers, each of said septa having a profile conforming substantially in size and shape to the cross-sectional dimensions of the combined chamber and V-shaped groove, distributor means secured to said rotor and means interconnecting the distributor means with said groove for introduction and removal of material into and from said chamber.
7. A rotor according to claim 6 wherein said means interconnecting the distributor means with said groove comprises a plurality of vertical channels in said core equal in number to said septa, each of said channels extending through said core and into a corresponding septum and terminating in an open port at the bottom of the V-shaped portion of said septum.
8. A rotor according to claim 7 wherein the bottom of the V-shaped portion of each septum is slightly displaced above the bottom of said V-shaped groove, whereby liquid may flow between adjacent sector chambers.
9. A rotor according to claim 7 wherein said distributor means is removably mounted axially on the top of said rotor and includes a plurality of radially extending channels in said distributor equal in number to said channels in said core, each of said distributor channels being detachably connectable to a corresponding channel in said core for introducing materials into and removing said materials from said sector chambers in the area of the apex of said V-shaped groove.
10. A rotor according to claim 1 wherein the upper ends of said distributor channels converge into a single channel at the top of said distributor.
11. A gradient zonal centrifuge apparatus comprising a rotor, an annular chamber in said rotor, said chamber being bounded by coaxially spaced apart inner and outer circular walls and opposite end walls, a plurality of spaced apart port means arrayed in a circle in said rotor intermediate said inner and outer side walls for removing effluents from said chamber, one of said end walls of the interior of said chamber being formed in sloping surfaces converging towards each of said removing ports and distributor means connected to said port means for introduction and removal of materials into and from said chamber.
i2. A rotor according to claim 11 and further comprising a plurality of removable spaced apart, radially arrayed septa in said chamber dividing said chamber into a plurality of chamber sectors, each of said septa having a profile conforming substantially to the crosssectional shape of said chamber and of said converging sloping surfaces where said removing port means are located.
13. Apparatus according to claim 11 wherein said sloping surfaces comprise an outer annular sloping surface and an inner annular sloping surface, said annular surfaces converging to form an annular V-shaped apex in said chamber, said apex being located coaxially relative to the axis of said rotor and intermediate the respective radii of said inner and outer walls.
14. Apparatus according to claim 13 wherein said port means are located in circular array in said annular apex and wherein gradient zones in said chamber, when removed therefrom, are constricted and concentrated in both inward and outward radial directions by said respective annular surfaces relative to said annular apex.
15. Apparatus according to claim 11 wherein said sloping surfaces comprise a plurality of circularly arrayed funnel-shaped recesses.
16. Apparatus according to claim wherein said port means includes a port in the apex of each funnelshaped recess, said ports being arrayed in a circle relative to the axis of said rotor and located radiallyintermediate the respective radii of said inner and outer circular walls and wherein gradient zones in said chamber, when being removed therefrom through said ports, are constricted and concentrated in all angled directions relative to the apex of each funnel-shaped recess.
17. Apparatus according to claim 15 wherein an outer edge of each funnel-shaped recess is contiguous to the outer edge of an adjacent funnel-shaped recess.
18. Apparatus according to claim 15 and further comprising a narrow annular shelf located adjacent the inner wall of said chamber, the inner edge of the upper end of said funnel-shaped recess being spaced apart from said inner wall by the width of said shelf.
19. A gradient zonal centrifuge rotor comprising an annular chamber in said rotor, said chamber being bounded by coaxially spaced apart annular inner and outer walls, a floor at the bottom of said chamber, a removable cover on said rotor enclosing said chamber, the inner surface of said cover comprising an inverted V-shaped annular groove, the inner and outer respective annular lower edges of said groove intersecting the respective upper edges of said inner and outer walls, distributor means secured to said rotor and means interconnecting the distributor means with said groove for introduction and removal of material into and from said chamber.
20. A rotor according to claim 19 wherein the annular apex of said groove is disposed concentrically around the axis of said rotor and located intermediate said inner and outer walls.
21. A rotor according to claim 19 wherein said groove comprises an outwardly and downwardly sloping annular outer wall, and an inwardly and downwardly sloping annular inner wall, said sloping walls converging upwardly toward each other to form the apex of said V-shaped annular groove, the angle of said outer groove wall from the horizontal being smaller than the angle of said inner groove wall from the horizontal.
22. A rotor according to claim 19 wherein said meansinterconnecting said distributor means with said groove comprises a plurality of spaced apart exit ports arranged in circular array in said cover, said ports each communicatingwith said chamber in the region of said annular apex.
23. A rotor according to claim 19 and further comprising a plurality of removably spaced apart radially arrayed septa in said chamber dividing said chamber into a plurality of chamber sector, each of said septa having a profile conforming substantially to a cross-sectional shape of said chamber and said V-shaped groove.
24. A rotor according to claim 23 wherein said means interconnecting said distributor means with said groove comprises a channel in each of said septa for intum channel.
26. A rotor according to claim 19 and further comprising a plurality of spaced apart exit ports in said cover communicating with the apex of said V-shaped annular groove, a collector mounted on said cover, and a plurality of channels in said collector, each of said channels communicating with a corresponding port in said cover for removing materials from said chamber.
27. A gradient zonal centrifuge rotor comprising an annular chamber in said rotor, said chamber being bounded by coaxial spaced apart annular inner and outer side walls and opposite end walls, said chamber being bounded at a selected one of said end walls by a V-shaped annular groove forming a portion of said chamber, the inner and outer respective annular edges of said groove intersecting adjacent respective edges of said inner and outer side walls, a plurality of spaced apart radially arrayed septa in said chamber dividing said chamber into a plurality of chamber sectors, the respective vertical edges of each of said septa extending substantially to the respective inner and outer side walls, each of said septa having a profile conforming substantially in size and shape to the cross-sectional dimensions of the combined chamber and V-shaped groove, distributor means secured to said rotor and means interconnecting the distributor means with said groove for introduction and removal of material into and from said chamber.
2%. The method of separating particles of biological cells or the like in the annular chamber of a centrifuge rotor which comprises introducing a quantity of said cells in suspension into said chamber, introducing a density gradient into said chamber, spinning said rotor to form vertical cylindrical columns of isodense layers of density gradient therein and causing said cells to migrate through said isodense layers by centrifugation until said particles have formed separate vertical cylindrical zones in accordance with their respective characteristics, causing said isodense layers and said particle zones to become reoriented into disc-shaped rings in spaced apart horizontal array within said chamber during deceleration of centrifugation, and removing and collecting said particle zones separately when said rotor is at rest by causing each particle zone to flow through an annular V-shaped space forming part of said chamber whereby the surface area of each zone-becomes reduced and the distance between successive sample zones is increased thereby reducing the possibility of interference of contamination between particle zones, said removal taking place substantially at the apex of said V-shaped space.
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