US 2928614 A
Description (OCR text may contain errors)
March l5, 1960 c. F. EMANur-:L ETAI- 2,928,614
HYDRAULIC ATISSUE HoMoGENIzER Filed April 16, 195e BY I ATTORNEY coniined liquid suspension of tissue matter.
HYDRAULIC rrssUE noMooENrzEn Carl F. Emanuel and Israel L. Chaikoi, Berkeley, Calif.; said Chaikott assigner to said Emanuel Application April 16, 195s, serial No. 729,024
` 7 claims. (01.241-39) This invention relates to an explosive pulverization device. More particularly it relates to a device based on pressure release principles and adapted to homogenize animal and plant tissues with greater ease and reproducibility than heretofore possible.
In particular, the instant invention relates to an apparatus for controlled release of the cellular components from various tissues rapidly and at low temperatures.
The investigator who wishes to disperse tissues in a liquid medium often has one of three objectives in mind. He may desire to completely comminute the tissues and cells preliminary to some isolation procedure. .Alternatively he may wish to control the degree of tissue breakdown so that some particular cell inclusions remain intact. Finally he may desire to produce homogenates very rapidly and in a reproducible fashion for purposes of metabolic investigations.
Ideally the samedevice should be capable of producing only whole cells, or alternatively, the intact products of whole cells (i.e., nuclei, mitochondria, and microsomes), or alternatively the dispersed contents of nuclei plus the other inclusions. Moreover the desired results should be accomplished rapidly, at low temperatures, and should yield metabolically active products.
The prime objects of this invention is to provide a` device for uniformly comminuting tissue materials.
A further object of this invention is to provide a device which can prepare uniform dispersions of tissue fragments in liquid media.
Further objects and the advantages of the instant invention will be apparent from the description which follows.
Advantageously, the homogenizer of the instant invention provides for application of a high pressure upon a VA piston which applies the pressure forces the suspension through a small annular orifice into a region of low pressure. The sudden pressure drop realized as the suspension passes through the orice causes communition of the tissue to a degree predetermined by the orice size.
Referring now to the drawing wherein there is illustrated in cross section and semi-diagrammatically a preferred embodiment of the apparatus comprising the instant invention, the device as a whole is shown at 10.
Device 10, hereinafter called the homogenizer, cornprises an elongated cylindrical vessel or container 12, its chamber i4 being adapted to hold a roughly ground-up tissue suspended in a buier liquid, shown at 15. As illustrated in the drawing, chamber 14 has an open top land a tapered bottom outlet 16. Tapered outlet 16 terminates with a vertical downspout 18 of slightly decreasing diameter, top to bottom. The drawing exaggerates the taper on downspout 13 for ease of viewing. Taperedv downspout i3 terminates in a nozzle 20. Desirably vessel 12 may be provided with a cooling jacket 24 to chill the contents. For ease of construction and because of `the high pressures sustained by homogenizer during opera-I ltio`ns,"/ess`el 12 is preferably made of a strong corrosionice resistant material such as stainless steel. Forming vesselV 12 out of metal allows certain advantages; accurate machining of chamber 14 is facilitated.- Also cooling jacket 24 may then be a metallic sleeve 26 shrunk-lit around a recessed portion 28 in ther outer wall of vessel 12. The inlet and outlet taps 30 provided in cooling jacket 24 can be built into the sleeve.
The base of homogenizer 10 Vis a cup-shaped member 32 suitably constructed to be secured underneath chamber 14, e.g., by a threaded connection 34. Animportant aspect of the interconnection between base 32 and vessel 12 lies in the need for accurate alignment. Desirably, iiat annular opposing surfaces 36, 38 are provided on vessel 12 and base 372, respectively, to give a large area for actual contact so that stresses incurred during operation are transmitted through at Vsurfacesl't, 38 rather I than through the threaded connection 34. If desired, in-
terrupted threads may be used for connection 34 so that the operator can more easily thread vessel 12 and base 32 into contact at opposing surfaces 36, 38.
Ordinarily a plastic, glass or metallic retainer cup '40 will be Vloosely positioned inside of base member` 32. However, for the occasionswhen contact of the homogenizate with air should be minimized, an annular groove '42 is provided on the bottom of vessel 12. A collapsible receiver such as a rubber balloon may be hung from groove 42 to receive the homogenizate issuing through nozzle 20, Base 32 is provided with a vent passage 44. A tap 46 can be built into vent passage 44 to allow connection with a rubber or plastic hose.
Loosely disposed in the upper portion of chamber 14 is a piston 4S. One or more gaskets like O-ring Sti should be provided in recessedy portions of piston 48 to insure a uniform contact between the piston 48 and the wall of chamber 14. The Vgasket 50 barely touches the wall of chamber 14 when piston 48 is at rest. Application of a moving force to piston'4 48 causes the G-ring gasket 50 to expand, completely sealingthe annular space between pistonV and chamber wall against leakage from chamber 1.4 and consequent loss of chamber pressure.
Removably attached to piston 48 and depending from the interior thereof is a center rod V52. This center rod S2 is secured to the body vof piston 48, suitably through at an oifset pin 54. One or more such pins may be ernployed. Alternatively, other suitable modes of interconnection may be employed, eg., an Allen head screw. In any event center rod 52is detachable by release of securing element 54' so that the rod may readily be slipped out the bottom of piston 48. In removing and replacing center rod 52, due care should be taken to avoid damaging the gasket 56 (one or more'O-ring's) employed to maintain center rod S2 accurately positioned relative to the body of piston 48. Gasket 56 serves also to prevent leakage from chamber 14 to the hollow interior region of piston 48 where center rod 52 is disposed; gasket 56 thereby prevents loss of pressure from chamber 1,4 and permits atmospheric pressure to exist at the .top extremity of center rod 52 during operation of the homogenizer. Atmospheric pressure always exists at the discharge end of nozzle 20, and consequently at the botinch) throughout the portion of its length which passes downwardly through nozzle 20. As shown in the drawing, a relatively loose tit of the center rod 52 in piston 48 is actually desirable since-downward movement of piston 48 then allows or permits a very slight lateral movement of the rod. This slight lateral movement amounts to aV pumping 'action which facilitatesA smooth builtdirectly Ainto the body of vessel 12.
- laboratory press.
gassen 3 and eeient ejection fof Vhomogenate through the annular orifice 58 spaced between center rod A52 and Ynozzle 20.
rl`he presence of an accurately sized annular orice 58 is critical inthe device ofthe instant invention.
centerrod and nozzle must be accurately dimensioned. `For best results it is `imperative Vthat `the center rod be machined to -a roundness and constant diameter within Vi0.0001 inch.
VVon these elementsby operation ofthe homogenizer.
suitably, center rod '52 can be fabricated from stainless fsteel. 'T he nozzlematerial,however, should be dissimilar fromthe centerrodin order to prevent seizing at minimum orifice widths. Thus nozzle 20 can comprise a "finely'ground carbide (e.g., tungsten, borium, chromium,
etc.) linsert 60 mounted in athreadedhousing `62 which 1n turn Vis threaded into a central recess 64 in the'bottom- Vmost portion of vessel 12. 4A compressible seat is providedby O-ring 66 in order to'prevent leakage around A,thernozzle HA carbide ,insert 60 scapable of being ground with diamond `dust and vpolished toan inside diameter accurate within ).0001f inch, giving nozzle 20 the desired uniform and predetermined inside diameter.
The specific orifice size depends on the diameters of i rod152 and of nozzle 20. The actual width ofthe j annular orice at room temperature should bepr'e-sele'cted To` insure uniformity and a constantf orifice width, the
i piston is pushed ,so'that' center rod 52 closes 4the nozzle atalevel between 6 and 130 microns.(0.00025 to 0.005
pinch) bygappropriately forming nozzle 20 and center rod 52. .To attain a desirable flexibility voioperation, orifice size may be changed by substitutionof different 4 nozzle inserts 60 and, or, alternatively different center orifice widths of 6 microns,'130 microns or each. of several intermediate `widths can be employed as desired. `VThe drawing furtherA illustrates certain optional features which have been found useful. At thelower end of chamberd14 a valved` outlet passage '7,0 may be provided. Outletpassage 70 vterminates at a larger threaded opening 72 so that a vacuum release valve 74 can be A suitable port (not shown) provided in the threaded opening 72 is brought into Vorout ,of alignment vwith passage 76 Vin the valve 74 by` rotating or turning the valve body 78. A knurled knob 801is providedto facilitate rotation of valve body78fin'to the open or closed position.
At thetop of piston 48 a threaded recess 82 is provided-so that the piston may ,be securedfto some suitable mechanism for forcing the piston down.A The drawing illustrates Vthe connection to upper platen 84 o'f a Carver Ansimilar threaded recess 85 is provided on the underside of base member 32.
The operation of the homogenizer is as follows. A
tissue suspension `can be introduced into the cylinder'v in three ways:
(l) The` base 32 is separated from'tlie vessel cylinderv 12, andpiston48 ispulled back so as to withdraw center.
rod 52 from theoritice 58. The mixture is then 'poured through the'nozzle 20 directly into Vthe inverted chamber 14; thel piston is `then pushed up (with the cylinder still inverted) until the center rod obstruets the nozzle opening. A'
' (2) The piston 'fro'm the cylinder, the opening at the'bottom vof the nozzle `20 is closedwith a stopper, and the tissue mixopening.
(3) The tissue mixture containing fragments no larger than -2to 3 can also be'introduced through the vacuum-release valve 74. In this case, homogenizer 10 is Vcompletely assembled and threaded into the Carver press with the head of Vpiston 48 resting at the botto'ni of chamber 14. The vacuum-release valve 74 is opened by oneturn, counter clockwise of its knurled knob 80. A rlfygon or rubber tube is then attached to the vacuum release valve,t-t he openend of thetube .being immersed in the tissuesuspension. The suction created by pull ing the homogenizer downward draws the tissue mixture into the bottom of'chamber 14. Av\/'acuumreleased valve 7-4 isthen closed.
Regardless of how chamber 14 is filled, the assembled apparatus containing the tissue mixture is placed in a device adapted to force piston 48 down, c g., in la Caiver press withits platens-apart. `The lower platen is raised slightly `until :the homogenizer piston alrnost` tonches the upperfplaten. The proper size bolt isman ually screwed into recess 182 of piston 48 to insure that homogenizer 10 is centered in the press.` The piston; is now-rapidly -pushed into `the cylinder. This application of .force to piston '48 places the ,suspension in chamber 14- under a jhigh pressure, forcing the suspension out"-o`f chamber 14through `annular orifice 58. A sudden re lease of pressureoccurs at theinstant of passage through orifice 58 to cup `41) .effecting an explosive pulverization orV comminution ofthe .tissuesf One excursion o'f piston 48 (2 to 3 minutes)fissu'icient to comminute the tissue to anextent Adetermined by thesize of the ,annular orifice,
Y anddisperse Athe -fragments `inthe liquid buffer. The
piston can now be readily withdrawn frornchamber 14 by opening vacuum-release valve 74.
" For most purposes, the homogenate is collected in the retainer cup normally disposed inside b ase 32. Some foaming occurs upon homogenization whichrnay result A in a slight loss when the volumeof the recovered homogenate is high (e.g., exceeds'50 ml. with a chamber 14 `ol 1.5 inch'diameter). :Foamiloss maybe avoided and air contact avoided by collecting the/homogenate ina collapsed rubber bag or balloonhu'ng from groove 42. In this waythe comminution products can be completely recovered. Ordinarily comminuted Yconnective tissue quickt 1y floats tothe top of the homogenate.
To homogenize large volumes of a tissue or cell suspension, retainer -cup 40 is removed and a delivery tube is attached to vent tap 46. The homogenizer is filled by method 3, described above, and homogenization of the tissue is carried fout. The homogenizate Adrains out f through vent passage 44. to the deivery tube. In this fashion, filling and homogenizationcan be done repeatedly without dismantling-the alsiparatus.V
In a specific homogenizer ll() built `with Va chamber l1/2in diameter the bestresultsare -achieved with at least.20 ml. of suspension. In fact, for thissize homogenizer little or not comminution will result with less than 10 m1. At high dilution of tissue in buffer, e.g., 1 gm./ v20 ml., the greatest efficiency results. Satisfactory come minution, or homogenization, will result with as much as `2.5 ml. of buffer per giri; of tissue. Still higheritissue concentrations interfere with properoperation.
Y eral it should'belborne inmindthat operation of the A'48 with'its center rod 52 is separated 'Y ture is poured into the open top end'of the cylinder.'V
TheV piston is then replaced and pushed past the O-ring device depends upon the velocity of the tissue suspension passing through annular orice 58. Accordingly the tissue suspension'should'be reasonably fluid.
'I'he homogenizer of the instant invention is particularly adapted for biochemical and biological laboratory medium, aridby circulating brine in cooling jacket 24,`
vfected at near freezing pointitemperatures. YFor smaller quantities oftissueor 4where thermal stability is not too hornogenization of large `quantities of tissue can be eft critical, one or more of these aforementioned cooling expedients can be omitted.
Whereas the invention has been described in connection with a particular embodiment, it will be understood that this is only for the purpose of explanation and that the invention is to be limited only by the spirit and scope of the appended claims.
What is claimed is:
1. A pressure release comminuting device comprising: a container adapted to hold a suspension of solid particulate matter suspended in a liquid medium, said container having a bottom outlet terminating in a nozzle; a closetting piston disposed in said container above the bottom outlet thereof; an elongated rod extending from said pistion through said bottom outlet and nozzle opening; the dimensions of the portion of said rod extending through said nozzle being uniform, and the dimensions of said nozzle opening being uniform, whereby a small predetermined annular orice is Provided between the ro-d and the nozzle through which the suspension is forced when said piston is advanced toward the bottom of said container. Y
2. A homogenizer for pressure release of cellular cornponents which comprises: a container adapted to hold a suspension of solid particulate matter suspended in a liquid medium, said container having a bottom outlet terminating in a nozzle; a close-fitting piston disposed in said container above the bottom outlet thereof; an elongated rod removably attached to said piston and extending from said piston through said bottom outlet and nozzle opening; the dimensions of the portion of said rod extending through said nozzle being uniform, and the dimensions of said nozzle being uniform, whereby a small predetermined annular orifice is provided between the rod and the nozzle through which the suspension is forced when said piston is advanced toward the bottom of said container.
3. The apparatus of claim 2 wherein a cup-shaped base member is provided, said base member and said container being attachable into an integral unit and the homogenate discharged through said orifice being collected inthe cup portion of said base member.
4. The apparatus of claim 2 wherein a cooling jacket surrounds the container.
5. The apparatus of claim 1 wherein the nozzle is a separate insert made of tungsten carbide ground to an internal diameter accuracy of 10.0001 inch, andY wherein the external diameter of the center rod is dimensioned to an accuracy of $00001 inch.
6. The apparatus of claim 1 wherein a set of nterchangeable rods are provided each rod being of different uniform dimensions whereby an annular orifice ranging from between about 6 to about 130 microns at room temperature may be preselected. Y
7. The apparatus of claim 1 wherein a valved Side opening is provided in said container adjacent said bottom outlet, said valved side opening being adapted to release the vacuum generated in said container by movement of said piston away from the bottom of said container.
References Cited in the ile of this patent UNITED STATES PATENTS