CN104928176B - Primary hepatocyte piece-rate system and method containing the fixing and multilayer filtering device of tissue - Google Patents
Primary hepatocyte piece-rate system and method containing the fixing and multilayer filtering device of tissue Download PDFInfo
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- CN104928176B CN104928176B CN201510418641.3A CN201510418641A CN104928176B CN 104928176 B CN104928176 B CN 104928176B CN 201510418641 A CN201510418641 A CN 201510418641A CN 104928176 B CN104928176 B CN 104928176B
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- 238000001914 filtration Methods 0.000 title claims abstract description 42
- 210000003494 hepatocyte Anatomy 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 11
- 230000002440 hepatic effect Effects 0.000 claims abstract description 76
- 239000004744 fabric Substances 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 32
- 239000011324 bead Substances 0.000 claims abstract description 22
- 210000004027 cell Anatomy 0.000 claims abstract description 15
- 238000003760 magnetic stirring Methods 0.000 claims abstract description 14
- 230000010412 perfusion Effects 0.000 claims description 20
- 239000011796 hollow space material Substances 0.000 claims description 9
- 210000005229 liver cell Anatomy 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 7
- 238000003780 insertion Methods 0.000 claims description 6
- 230000037431 insertion Effects 0.000 claims description 6
- 210000004185 liver Anatomy 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 239000004425 Makrolon Substances 0.000 claims description 4
- 229920000515 polycarbonate Polymers 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 230000002792 vascular Effects 0.000 claims description 4
- 239000007769 metal material Substances 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 230000006835 compression Effects 0.000 claims description 2
- 238000007906 compression Methods 0.000 claims description 2
- 230000000149 penetrating effect Effects 0.000 claims description 2
- 239000011148 porous material Substances 0.000 abstract 1
- 238000004064 recycling Methods 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 64
- 238000010586 diagram Methods 0.000 description 4
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 206010019799 Hepatitis viral Diseases 0.000 description 2
- 206010073310 Occupational exposures Diseases 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
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- 238000006073 displacement reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000000887 face Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 231100000675 occupational exposure Toxicity 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000002324 hematogenic effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of primary hepatocyte piece-rate system and method containing the fixing and multilayer filtering device of tissue, it is made up of hepatic tissue fixing device, multilayer filtering device, magnetic stirring apparatus, liquid storage container and peristaltic pump, the first peristaltic pump, liquid storage container and the second peristaltic pump are sequentially connected by pipeline between hepatic tissue fixing device and multilayer filtering device, hepatic tissue fixing device is hollow cube configuration, its hollow cube size can adjust, preferably to fix different size of hepatic tissue；Multi-layer filtrating equipment is divided into end cap portions, pars intermedia, bottom, it is hollow cylindrical, mutually it is connected by screw thread, there is the screen cloth of different pore size in each portion, and it is equipped with anti-clogging magnetic bead, by the higher primary hepatocyte of purity to the successively filtration containing cell perfusate, can be obtained, and can achieve the recycling of perfusate.The present invention provides a whole set of structurally consummate apparatus system for separating primary hepatocyte from hepatic tissue, the higher primary hepatocyte of easy, quick, economic acquisition purity.
Description
Technical field
The present invention relates to field of biomedicine technology, specifically a kind of primary liver containing the fixing and multilayer filtering device of tissue
Cell separation system and method.
Background technology
Clostridiopetidase A perfusion method is primary hepatocyte separation method main at present.The method is by existing vascular system to bulk
Hepatic tissue carries out clostridiopetidase A perfusion, or carries out the acquisition of the methods such as multi-point lancet puncture clostridiopetidase A perfusion to fritter hepatic tissue
Primary hepatocyte.Detached hepatocyte suspension tentatively screens out larger tissue block, manadesma etc. by filter screen, then adopts density level bands
The method of degree centrifugation obtains purer liver cell.Current China virus hepatitis, pathogenesis of cirrhosis rate still occupy high-order, metabolic
The hepatopathy incidence of disease rises year by year, and liver diseases are still the principal disease for posing a health risk, and the research of liver diseases and treatment are for original
Increasingly increase for hepatocellular demand.Particularly bioartificial liver's R＆D work and in the future possible clinical practice, need big
The higher primary hepatocyte of the purity of amount.
And the domestic at present method of operating using clostridiopetidase A perfusion acquisition primary hepatocyte, lack the perfusion of whole set of system
Equipment, is had the following disadvantages in operation：1st, hepatic tissue manually fixes fritter liver with hand nothing proprietary fixing device
Tissue carry out multidigit point puncture when, have occur pricking wound risk.Particularly scientific research, isolated viral hepatitis are being engaged in
Deng Hematogenic infectious disease patient's liver organization primary hepatocyte when, if occur pricking wound occupational exposure, may result in operator infect
Corresponding pathogen；2nd, hepatic tissue during collagenase perfusion, is susceptible to hepatic tissue displacement, fills nothing proprietary fixing device
Note shower nozzle departs from, the situation of liquid splash, and these liquid splashes can occur mucous membrane occupational exposure to positions such as eye conjunctivas；3rd, lack
Weary multilayer filtration system, needs complicated density gradient centrifugation obtain the higher primary hepatocyte of purity；And for preventing
Obstruction, the perfusate containing clostridiopetidase A can not be recycled.
Content of the invention
It is an object of the invention to provide a kind of primary hepatocyte piece-rate system containing the fixing and multilayer filtering device of tissue
And method, to solve the problems, such as to propose in above-mentioned background technology.
For achieving the above object, the present invention provides following technical scheme：
Primary hepatocyte piece-rate system containing the fixing and multilayer filtering device of tissue, by hepatic tissue fixing device, many metafiltration
Device, magnetic stirring apparatus, the first peristaltic pump, liquid storage container and the second peristaltic pump composition is crossed, the hepatic tissue fixing device is arranged
In multilayer filtering device, between hepatic tissue fixing device and multilayer filtering device by pipeline be sequentially connected the first peristaltic pump,
Liquid storage container and the second peristaltic pump；
The hollow square body structure that the hepatic tissue fixing device is made up of multiple fixed plates and portable plate, the fixation
Sieve aperture is all had on plate and portable plate, and the fixed plate includes the first fixed plate of tight linking, the second fixed plate and the 3rd admittedly
Fixed board is all stiff, and the first fixed plate, the second fixed plate and the 3rd fixed plate collectively form cubical three stationary planes；Institute
Stating portable plate includes the first portable plate and the second portable plate, and the first portable plate is sliding on second pulley track by first pulley
Dynamic, the second portable plate is slided on first pulley track by second pulley, and it is equal that the first portable plate is provided with multiple intervals
Intersect seam, the one end for intersecting seam is closed and is connected with the first portable plate main body, the other end for intersecting seam opens and accommodate the second work
Dynamic plate is passed through, and the first portable plate and the second portable plate constitute cubical two active face；Second fixed plate and the second portable plate
On all offer fixing hole between sieve aperture, be equipped with stator in fixing hole, after multiple stators are penetrated in fixing hole
Form a cubical face；Thumbwheel switch is equipped with the first pulley and second pulley；
The multilayer filtering device is made up of the end cap portions of hollow cylindrical, pars intermedia and three part of bottom, the end cap
Cubical hollow space being provided with portion, and hollow space being coincide with hepatic tissue fixing device, the interior lower end of end cap portions is provided with
First screen cloth, the lower end of end cap portions be additionally provided with end cap portions screw thread, and the top of the pars intermedia is provided with and is connected with end cap portions screw thread
Threaded upper ends, end cap portions screw thread form the first threaded connection place with threaded upper ends；The inside of the pars intermedia is provided with the second screen cloth,
The space that first screen cloth and the second screen cloth are formed is cell mass receiving cavity, is placed with the first magnetic bead, institute on second screen cloth
The lower end for stating pars intermedia is provided with lower end screw thread, and the top of the bottom is provided with the bottom thread being connected with lower end screw thread, lower end spiral shell
Line and bottom thread form the second threaded connection place, and the inside of the bottom is provided with the 3rd screen cloth, the second screen cloth and the 3rd screen cloth
The space of formation is cell enrichment cavity, is placed with the second magnetic bead on the 3rd screen cloth, empty in the bottom of the 3rd screen cloth
Between be the temporary storage chamber body of perfusate, the side wall of the bottom is provided with liquid outlet.
As the further preferred version of the present invention：The aperture of first screen cloth, the second screen cloth and the 3rd screen cloth is successively
Reduce.
As the present invention further preferred version：The fixed plate, portable plate and stator are by metal material, stainless
Steel or Merlon are made；The shell of the multilayer filtering device is made up of transparent makrolon material.
The separation method of the primary hepatocyte piece-rate system containing the fixing and multilayer filtering device of tissue, comprises the following steps that：
(1) hepatic tissue is fixed
Sterilization treatment is first carried out to each part of reactor, and each step of isolating hepatocytes is all in aseptic super-clean bench laboratory
In carry out, hepatic tissue is placed in the space that three fixed plates are surrounded；Second portable plate is installed, along first pulley track sliding
The second portable plate is moved until hepatic tissue is compressed, then locking first pulley；First portable plate is installed so that the second portable plate is being closed
Right position is put through seam is intersected, and slides the first portable plate until compressing hepatic tissue, then locking second is slided along second pulley track
Wheel；In fixing hole on the second portable plate and the second fixed plate, insertion insertion stator, is consequently formed the cube in six faces
Fixing device is used for being fixed for the hepatic tissue of cell separation；
(2) assembling of system
End cap portions, pars intermedia and bottom is good by corresponding helical assembly, and the first magnetic bead and the second magnetic bead is placed, with
Form multilayer filtering device；The hepatic tissue fixing device for being fixed with hepatic tissue is placed in hollow space, and liquid line connects
Connect peristaltic pump, magnetic stirring apparatus and liquid storage container；
(3) hepatic tissue clostridiopetidase A perfusate perfusion
Perfusion shower nozzle is inserted in hepatic tissue vascular by sieve aperture, or is pierced in hepatic tissue by puncture needle multi-point, is opened
Second peristaltic pump, by clostridiopetidase A perfusate perfusion in liquid storage container to hepatic tissue, in the presence of clostridiopetidase A in hepatic tissue
Liver cell is gradually disengaged, and with perfusate toward dirty, when multilayer filtering device liquid level is more than the first mesh screen height, opens magnetic force
Agitator adjusts rotating speed, so that the first magnetic bead and the second magnetic bead is slowly rotated, and opens the first peristaltic pump immediately, compacted with corresponding first
Perfusate in temporary for perfusate storage chamber body is discharged by liquid outlet and is back in liquid storage container by the corresponding speed of dynamic pump；Fill
During stream, stator is can remove, and help liver cell is cut by blade and fully dissociate；
(4) primary hepatocyte is collected
Stop the second peristaltic pump at the end of perfusion, the first peristaltic pump works on, aspirate the perfusion in multilayer filtering device
Liquid, stops suction, and stops magnetic stirring apparatus work, twist by screw thread when perfusate liquid level is less than the second threaded connection place height
Open and pars intermedia and bottom is separated, with pipettor transfer primary hepatocyte suspension to cell therefor, to be centrifuged acquisition further
Primary hepatocyte.
Compared with prior art, the invention has the beneficial effects as follows：
1st, the present invention provides a whole set of structurally consummate apparatus system for separating primary hepatocyte from hepatic tissue, easy,
Fast, the higher primary hepatocyte of economic acquisition purity；
2nd, hepatic tissue can be fixed, it is to avoid free-hand fixing hepatic tissue, during multi-point lancet puncture, pricking wound occurs；
3rd, fixing hepatic tissue, it is to avoid hepatic tissue is subjected to displacement, causes perfusate to splash；
4th, there is multilayer filtration system, the clostridiopetidase A perfusate after filtration, perfusion is repeated in hepatic tissue, can reduce collagen
The consumption of enzyme, cost-effective.
Description of the drawings
Fig. 1 is the enforcement schematic diagram of the present invention.
Fig. 2 is the structural representation of hepatic tissue fixing device in the present invention.
Fig. 3 is the first fixed plate, the cross-sectional view of the 3rd fixed plate in hepatic tissue fixing device；
Fig. 4 is the 3rd fixed plate, the cross-sectional view of the second portable plate in hepatic tissue fixing device；
Fig. 5 is the cross-sectional structure schematic diagram of stator in hepatic tissue fixing device；
Fig. 6 is the cross-sectional view of the first portable plate in hepatic tissue fixing device；
Fig. 7 is the Split type structure schematic diagram of multilayer filtering device in the present invention.
Fig. 8 is the overall structure diagram of multilayer filtering device in the present invention.
In figure：The first fixed plate of 1-；2- fixing hole；3- second pulley track；4- first pulley；The second fixed plate of 5-；6-
Hepatic tissue fixing device；The 3rd fixed plate of 7-；The second portable plate of 8-；The first portable plate of 9-；10- intersects seam；11- first pulley rail
Road；12- second pulley；13- sieve aperture；14- stator；15- hollow space；16- end cap portions；The first screen cloth of 17-；18- end cap portions
Screw thread；19- threaded upper ends；20- pars intermedia；The second screen cloth of 21-；22- lower end screw thread；The first magnetic bead of 23-；24- bottom thread；
25- bottom；The 3rd screen cloth of 26-；27- liquid outlet；The temporary storage chamber body of 28- perfusate；The second magnetic bead of 29-；30- first is threaded
Place；31- cell mass receiving cavity；The second threaded connection place of 32-；33- cell enrichment cavity；34- magnetic stirring apparatus；35- magnetic force
Agitator speed governing button；36- multilayer filtering device；37- hepatic tissue；The first peristaltic pump of 38-；39- liquid storage container；40- second is compacted
Dynamic pump.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
Fig. 1～8 are referred to, in the embodiment of the present invention, the primary hepatocyte containing the fixing and multilayer filtering device of tissue is separated
System, by hepatic tissue fixing device, multilayer filtering device, magnetic stirring apparatus, the first peristaltic pump, liquid storage container and the second peristaltic pump
Composition, it is characterised in that the hepatic tissue fixing device is arranged in multilayer filtering device, hepatic tissue fixing device and many metafiltration
Cross between device and the first peristaltic pump, liquid storage container and the second peristaltic pump are sequentially connected by pipeline.
The hollow square body structure that hepatic tissue fixing device 6 is made up of multiple fixed plates and portable plate, fixed plate, activity
Plate and stator are made up of metal material, stainless steel or Merlon；Sieve aperture 13 is all had in fixed plate and portable plate, described solid
Fixed board includes that the first fixed plate 1, the second fixed plate 5 and the 3rd fixed plate 7 of tight linking is all stiff, the first fixed plate 1,
Second fixed plate 5 and the 3rd fixed plate 7 collectively form cubical three stationary planes；Portable plate includes the first portable plate 9 and
Two portable plates 8, the first portable plate 9 are slided on second pulley track 3 by first pulley 4, and the second portable plate 8 is slided by second
Wheel 12 is slided on first pulley track 11, and the first portable plate 9 is provided with the equal intersection seam 10 in multiple intervals, intersects seam 10
One end is closed and is connected with 9 main body of the first portable plate, intersects the other end opening of seam 10 and the second portable plate 8 of receiving is passed through, the
One portable plate 9 and the second portable plate 8 constitute cubical two active face；All open up on second fixed plate 5 and the second portable plate 8
The fixing hole 2 having between sieve aperture, is equipped with stator 14 in fixing hole 2, multiple stators are formed after penetrating in fixing hole 2
A cubical face.
Thumbwheel switch is equipped with first pulley 4 and second pulley 12, after required position is moved to, locking pulley
Switch, portable plate is fixed in fixed plate, by the collocation of fixed plate and portable plate, and according to hepatic tissue size, is realized vertical
The expansion in cube space and reduce, preferably to fix hepatic tissue.
Hepatic tissue fixing device 6 is the cube configuration of hollow adjustable size, the number of fixed plate therein and portable plate
Amount is adjustable.This fixing device fixed plate and portable plate quantity are adjusted, is formed size and can adjust hollow cube configuration
Hepatic tissue fixing device is accordingly to be regarded as the protection domain of patent of the present invention.
Multilayer filtering device 36 is made up of the end cap portions 16 of hollow cylindrical, pars intermedia 20 and 25 3 part of bottom, multilayer
The shell of filtering device is made up of transparent makrolon material, in that context it may be convenient to observe the height of liquid storage tank and inner chamber liquid level；
The device total height is 10cm, and in addition to end cap portions 16, a diameter of 8cm of interior hollow-cylindrical, total measurement (volume) are about 500ml.
Cubical hollow space 15 is provided with end cap portions 16, and hollow space 15 is coincide with hepatic tissue fixing device 6, end
The interior lower end of cap 16 is provided with the first screen cloth 17, and the first screen cloth 17 is 20 mesh, and its aperture 0.8mm can filter relatively large liver
Tissue debris and manadesma etc., and allow little liver cell agglomerate to pass through；The lower end of end cap portions 16 is additionally provided with end cap portions screw thread 18, in
Between the top in portion 20 be provided with the threaded upper ends 19 being connected with end cap portions screw thread 18, end cap portions screw thread 18 and threaded upper ends 19 form the
One threaded connection place 30；The inside of pars intermedia 20 is provided with the second screen cloth 21, and the second screen cloth 21 is 300 mesh, 50 μm of its aperture, it is allowed to
Unicellular size and less material pass through；The space that first screen cloth 17 and the second screen cloth 21 are formed is cell mass receiving cavity
31.
The first magnetic bead 23 is placed with second screen cloth 21, when magnetic stirring apparatus 34 works, 23 rotating band hydrodynamic of the first magnetic bead
Body vortex sample moves, to prevent the second screen cloth 21 from blocking and promoting the abundant effect that clostridiopetidase A is rolled into a ball to liver cell so as to be changed into
Single primary hepatocyte；The lower end of pars intermedia 20 is provided with lower end screw thread 22, and the top of bottom 25 is provided with and is connected with lower end screw thread 22
Bottom thread 24, lower end screw thread 22 and bottom thread 24 form the second threaded connection place 32.
The inside of bottom 25 is provided with the 3rd screen cloth 26, and the 3rd screen cloth 26 is 1250 mesh, 10 μm of its aperture, unicellular can not lead to
Cross；The space that second screen cloth 21 and the 3rd screen cloth 26 are formed is cell enrichment cavity 33, is placed with the second magnetic on the 3rd screen cloth 26
Pearl 29, the second magnetic bead 29 rotarily drive the motion of liquid vortex sample, to prevent the second screen cloth 26 from blocking；Bottom positioned at the 3rd screen cloth 26
In portion 25, space is the temporary storage chamber body 28 of perfusate, and the side wall of bottom is provided with liquid outlet 27.
The separation method of the primary hepatocyte piece-rate system containing the fixing and multilayer filtering device of tissue, comprises the following steps that：
(1) hepatic tissue is fixed
First sterilization treatment is carried out to each part of reactor, each step of isolating hepatocytes is all in aseptic super-clean bench or hundred
Level is carried out in laboratory, and hepatic tissue is placed in the space that three fixed plates are surrounded；Second portable plate 8 is installed, along first
Pulley track 11 slides the second portable plate 8 up to hepatic tissue is compressed, then locking first pulley 4；First portable plate 9 is installed, is made
The second portable plate 8 is obtained in correct position through seam 10 is intersected, the first portable plate 9 is slided up to compression along second pulley track 3
Hepatic tissue, then locking second pulley 12；In fixing hole 2 on the second portable plate 8 and the second fixed plate 5, insertion insertion is fixed
Piece, is consequently formed the cube fixing device in six faces for being fixed for the hepatic tissue of cell separation；
(2) assembling of system
End cap portions 16, pars intermedia 20 and bottom 25 is good by corresponding helical assembly, and place the first magnetic bead 23 and the
Two magnetic beads 29, to form multilayer filtering device 36；The hepatic tissue fixing device 6 for being fixed with hepatic tissue is placed on hollow space 15
Interior, and liquid line connects peristaltic pump, magnetic stirring apparatus 34 and liquid storage container 39；
(3) hepatic tissue clostridiopetidase A perfusate perfusion
Perfusion shower nozzle is inserted in hepatic tissue vascular by sieve aperture 13, or is pierced in hepatic tissue by puncture needle multi-point, is beaten
The second peristaltic pump 40 is opened, by clostridiopetidase A perfusate perfusion in liquid storage container 39 to hepatic tissue, liver group in the presence of clostridiopetidase A
Liver cell in knitting is gradually disengaged, and with perfusate toward dirty, when 36 liquid level of multilayer filtering device is more than 17 height of the first mesh screen
When, open magnetic stirring apparatus 34 and rotating speed is adjusted, so that the first magnetic bead 23 and the second magnetic bead 29 is slowly rotated, and it is compacted to open first immediately
Perfusate in temporary for perfusate storage chamber body 28 is passed through liquid outlet 27 with the corresponding speed of corresponding first peristaltic pump 38 by dynamic pump 38
Discharge and be back in liquid storage container 39；In perfusing course, stator 14 is can remove, and help liver cell is cut by blade and fill
Decompose from；
(4) primary hepatocyte is collected
Stop the second peristaltic pump 40 at the end of perfusion, the first peristaltic pump 38 works on, in suction multilayer filtering device 36
Perfusate, stop suction when perfusate liquid level is less than the second 32 height of threaded connection place, and stop 34 work of magnetic stirring apparatus
Make, turn on by screw thread and pars intermedia 20 and bottom 25 is separated, with pipettor transfer primary hepatocyte suspension to cell therefor, with
Centrifugation obtains primary hepatocyte further.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of spirit or essential attributes without departing substantially from the present invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Any reference in claim should not be considered as and limit involved claim.
Claims (2)
1. a kind of primary hepatocyte piece-rate system containing the fixing and multilayer filtering device of tissue, by hepatic tissue fixing device (6), many
Layer filtering device (36), magnetic stirring apparatus (34), the first peristaltic pump (38), liquid storage container (39) and the second peristaltic pump (40) group
Become, it is characterised in that hepatic tissue fixing device (6) are arranged in multilayer filtering device (36), hepatic tissue fixing device (6)
The first peristaltic pump (38), liquid storage container (39) and the second peristaltic pump are sequentially connected by pipeline and multilayer filtering device (36) between
(40)；
The hollow square body structure that hepatic tissue fixing device (6) are made up of multiple fixed plates and portable plate, the fixation
Sieve aperture (13) is all had on plate and portable plate, and the fixed plate includes first fixed plate (1) of tight linking, the second fixed plate
(5) and the 3rd fixed plate (7) is all stiff, the common structure of the first fixed plate (1), the second fixed plate (5) and the 3rd fixed plate (7)
Become cubical three stationary planes；The portable plate includes the first portable plate (9) and the second portable plate (8), the first portable plate (9)
By first pulley (4) in the upper slip of second pulley track (3), the second portable plate (8) is slided first by second pulley (12)
Wheel track (11) is upper to slide, and the first portable plate (9) is provided with equal intersection seam (10) in multiple intervals, intersects one end of seam (10)
Close and be connected with the first portable plate (9) main body, intersect the other end opening of seam (10) and the second portable plate of receiving (8) is passed through,
First portable plate (9) and the second portable plate (8) constitute cubical two active face；Second fixed plate (5) and the second portable plate
(8) fixing hole (2) between sieve aperture (13) is all offered on, is equipped with stator (14), multiple fixations in fixing hole (2)
Piece forms a cubical face after penetrating in fixing hole (2)；Cunning is equipped with first pulley (4) and second pulley (12)
Wheel switch；
Multilayer filtering device (36) are by the end cap portions (16) of hollow cylindrical, pars intermedia (20) and (25) three part group of bottom
Become, in end cap portions (16), be provided with cubical hollow space (15), and hollow space (15) and hepatic tissue fixing device (6)
It coincide, the interior lower end of end cap portions (16) is provided with the first screen cloth (17), and the lower end of end cap portions (16) is additionally provided with end cap portions screw thread
(18), top described pars intermedia (20) is provided with the threaded upper ends (19) being connected with end cap portions screw thread (18), end cap portions screw thread
(18) the first threaded connection place (30) is formed with threaded upper ends (19)；The inside of pars intermedia (20) is provided with the second screen cloth
(21) space that, the first screen cloth (17) is formed with the second screen cloth (21) is cell mass receiving cavity (31), second screen cloth
(21) the first magnetic bead (23) is placed with, and the lower end of pars intermedia (20) is provided with lower end screw thread (22), bottom (25)
Top is provided with the bottom thread (24) being connected with lower end screw thread (22), and lower end screw thread (22) and bottom thread (24) form the second spiral shell
Line junction (32), the inside of bottom (25) are provided with the 3rd screen cloth (26), the second screen cloth (21) and the 3rd screen cloth (26) shape
The space for becoming is cell enrichment cavity (33), is placed with the second magnetic bead (29), positioned at the 3rd screen cloth on the 3rd screen cloth (26)
(26) the interior space in bottom (25) is that perfusate is temporary storage chamber body (28), and the side wall of the bottom is provided with liquid outlet (27)；
The aperture of the first screen cloth (17), the second screen cloth (21) and the 3rd screen cloth (26) is sequentially reduced；
The fixed plate, portable plate and stator (14) are made up of metal material, stainless steel or makrolon material；The multilayer
The shell of filtering device (36) is made up of transparent makrolon material.
2. a kind of separation of the primary hepatocyte piece-rate system containing the fixing and multilayer filtering device of tissue as claimed in claim 1
Method, it is characterised in that comprise the following steps that：
(1) hepatic tissue is fixed
Sterilization treatment is first carried out to each part of reactor, and each step of isolating hepatocytes is all in aseptic super-clean bench or hundred grades of realities
Testing in room is carried out, and hepatic tissue is placed in the space that three fixed plates are surrounded；Second portable plate (8) is installed, is slided along first
Wheel track (11) slides the second portable plate (8) up to hepatic tissue is compressed, then locking first pulley (4)；First portable plate is installed
(9) so that the second portable plate (8) is in correct position through seam (10) is intersected, movable along second pulley track (3) slip first
Plate (9) is until compression hepatic tissue, then locking second pulley (12)；Consolidating on the second portable plate (8) and the second fixed plate (5)
Determine insertion insertion stator (14) in hole (2), the cube fixing device for being consequently formed six faces is divided for being fixed for cell
From hepatic tissue；
(2) assembling of system
Will be good by corresponding helical assembly to end cap portions (16), pars intermedia (20) and bottom (25), and place the first magnetic bead (23)
With the second magnetic bead (29), to form multilayer filtering device (36)；During the hepatic tissue fixing device for being fixed with hepatic tissue is placed on
In absolutely empty (15), and liquid line connects peristaltic pump, magnetic stirring apparatus (34) and liquid storage container (39)；
(3) hepatic tissue clostridiopetidase A perfusate perfusion
Perfusion shower nozzle is inserted in hepatic tissue vascular by sieve aperture (13), or is pierced in hepatic tissue by puncture needle multi-point, is opened
Second peristaltic pump (40), by clostridiopetidase A perfusate perfusion in liquid storage container (39) to hepatic tissue, liver in the presence of clostridiopetidase A
Liver cell in tissue is gradually disengaged, and with perfusate toward dirty, when multilayer filtering device (36) liquid level is more than the first mesh screen
(17) during height, open magnetic stirring apparatus (34) and rotating speed is adjusted, so that the first magnetic bead (23) and the second magnetic bead (29) is slowly rotated, and
First peristaltic pump (38) is opened immediately, with corresponding first peristaltic pump (38) corresponding speed by temporary for perfusate storage chamber body (28)
Perfusate is discharged by liquid outlet (27) and is back in liquid storage container (39)；In perfusing course, stator (14) is removed, and
Cut by blade and help liver cell fully to dissociate；
(4) primary hepatocyte is collected
Stop the second peristaltic pump (40) at the end of perfusion, the first peristaltic pump (38) works on, suction multilayer filtering device (36)
In perfusate, stop suction when perfusate liquid level is less than the second threaded connection place (32) height, and stop magnetic stirring apparatus
(34) work, turn on by screw thread and pars intermedia (20) and bottom (25) is separated, primary hepatocyte suspension is shifted to corresponding with pipettor
Container in, to be centrifuged acquisition primary hepatocyte further.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510418641.3A CN104928176B (en) | 2015-07-16 | 2015-07-16 | Primary hepatocyte piece-rate system and method containing the fixing and multilayer filtering device of tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510418641.3A CN104928176B (en) | 2015-07-16 | 2015-07-16 | Primary hepatocyte piece-rate system and method containing the fixing and multilayer filtering device of tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104928176A CN104928176A (en) | 2015-09-23 |
| CN104928176B true CN104928176B (en) | 2017-03-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510418641.3A Expired - Fee Related CN104928176B (en) | 2015-07-16 | 2015-07-16 | Primary hepatocyte piece-rate system and method containing the fixing and multilayer filtering device of tissue |
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| Country | Link |
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| CN (1) | CN104928176B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111733072B (en) * | 2020-06-08 | 2023-06-30 | 中国科学院宁波工业技术研究院慈溪生物医学工程研究所 | Circulating tumor cell screening and separating device, method and application |
| CN112410179B (en) * | 2020-12-09 | 2023-09-29 | 桂林医学院 | Simple in-vitro liver perfusion device for digestion and separation of liver cells |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0909811A2 (en) * | 1997-10-16 | 1999-04-21 | B. BRAUN CAREX S.p.A. | Bioreactor for culturing animal and/or human cells, particularly hepatocytes |
| CN101732771A (en) * | 2009-12-17 | 2010-06-16 | 中日友好医院 | Cell reactor and artificial liver support system comprising same |
| WO2013030761A1 (en) * | 2011-08-29 | 2013-03-07 | Stempeutics Research Private Limited | A system for isolating stromal vascular fraction (svf) cells from the adipose tissue and a method thereof |
| CN104312903A (en) * | 2014-10-29 | 2015-01-28 | 贵阳医学院 | Experimental apparatus for in-vitro circulating perfusion and digestion of liver tissues |
| CN204918596U (en) * | 2015-07-16 | 2015-12-30 | 中南大学湘雅医院 | Former liver cell piece -rate system that takes place of containing device is filtered to fixed multilayer that reaches of hepatic tissue |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002006456A1 (en) * | 2000-07-13 | 2002-01-24 | Invitrogen Corporation | Methods and compositions for rapid protein and peptide extraction and isolation using a lysis matrix |
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2015
- 2015-07-16 CN CN201510418641.3A patent/CN104928176B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0909811A2 (en) * | 1997-10-16 | 1999-04-21 | B. BRAUN CAREX S.p.A. | Bioreactor for culturing animal and/or human cells, particularly hepatocytes |
| CN101732771A (en) * | 2009-12-17 | 2010-06-16 | 中日友好医院 | Cell reactor and artificial liver support system comprising same |
| WO2013030761A1 (en) * | 2011-08-29 | 2013-03-07 | Stempeutics Research Private Limited | A system for isolating stromal vascular fraction (svf) cells from the adipose tissue and a method thereof |
| CN104312903A (en) * | 2014-10-29 | 2015-01-28 | 贵阳医学院 | Experimental apparatus for in-vitro circulating perfusion and digestion of liver tissues |
| CN204918596U (en) * | 2015-07-16 | 2015-12-30 | 中南大学湘雅医院 | Former liver cell piece -rate system that takes place of containing device is filtered to fixed multilayer that reaches of hepatic tissue |
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| CN104928176A (en) | 2015-09-23 |
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