CN104928176A - Primary hepatocyte separating system and method with tissue fixing and multi-layer filtering device - Google Patents
Primary hepatocyte separating system and method with tissue fixing and multi-layer filtering device Download PDFInfo
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- CN104928176A CN104928176A CN201510418641.3A CN201510418641A CN104928176A CN 104928176 A CN104928176 A CN 104928176A CN 201510418641 A CN201510418641 A CN 201510418641A CN 104928176 A CN104928176 A CN 104928176A
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a primary hepatocyte separating system and method with a tissue fixing and multi-layer filtering device. The primary hepatocyte separating system comprises a hepatic tissue fixing device, a multi-layer filtering device, a magnetic force stirrer, a liquid storage container and peristaltic pumps. A first peristaltic pump, the liquid storage container and a second peristaltic pump are connected in sequence through a pipeline between the hepatic tissue fixing device and the multi-layer filtering device. The hepatic tissue fixing device is of a hollow cuboid structure, the size of the hollow cuboid shape can be adjusted, and accordingly hepatic tissue with different sizes can be well fixed. The multi-layer filtering device is divided into an end cover part, a middle part and a bottom part which are all in a hollow cylinder shape and are connected through threads. Screens with different hole diameters are arranged in the parts, anti-blocking magnetic beads are arranged, by layer-by-layer filtering with cell perfusate, primary hepatocyte with high purity can be obtained, and repeated using of perfusate can be achieved. The invention provides a whole set of device system for separating primary hepatocyte from hepatic tissue, easiness and quickness are achieved, and primary hepatocyte high in purity is obtained economically.
Description
Technical field
The present invention relates to field of biomedicine technology, specifically a kind of primary hepatocyte separation system and method containing organizing fixing and multilayer filtering device.
Background technology
Collagenase perfusion method is primary hepatocyte separation method main at present.This method carries out collagenase perfusion by existing vascular system to bulk hepatic tissue, or fritter hepatic tissue is carried out to the method acquisition primary hepatocytes such as multi-point lancet puncture collagenase perfusion.The hepatocyte suspension be separated tentatively screens out larger tissue block, manadesma etc. by filter screen, then adopts the method for density gradient centrifugation to obtain purer liver cell.Current China viral hepatitis, pathogenesis of cirrhosis rate still occupy high-order, and Metabolic liver disease sickness rate rises year by year, and hepatic diseases is still the principal disease posed a health risk, and research and the demand for the treatment of for primary hepatocyte of hepatic diseases increase day by day.Particularly bioartificial liver's R&D work and clinical application possible in the future, need the primary hepatocyte that a large amount of purity is higher.
And current domestic employing collagenase perfusion obtains the working method of primary hepatocyte, lack the perfusion equipment of whole set of system, have the following disadvantages in operation: 1, hepatic tissue is without proprietary stationary installation, artificial hand fix fritter hepatic tissue carry out the puncture of multidigit point time, have and the risk of pricking wound occur.Particularly be engaged in scientific research, during the Hematogenic infectious disease patient liver organization primary hepatocytes such as isolated viral hepatitis, if there is pricking wound occupational exposure, operator can be being caused to infect corresponding pathogenic agent; 2, hepatic tissue is without proprietary stationary installation, and in collagenase perfusion process, the situation of hepatic tissue displacement, the disengaging of perfusion shower nozzle, liquid splash easily occurs, and these liquid splashes, to positions such as eye conjunctivas, mucous membrane occupational exposure can occur; 3, lack multilayer filtration system, need complicated density gradient centrifugation could obtain the higher primary hepatocyte of purity; And block for preventing, the perfusate containing collagenase can not recycle.
Summary of the invention
The object of the present invention is to provide a kind of primary hepatocyte separation system and method containing organizing fixing and multilayer filtering device, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
Containing the primary hepatocyte separation system organizing fixing and multilayer filtering device, be made up of hepatic tissue stationary installation, multilayer filtering device, magnetic stirring apparatus, the first peristaltic pump, liquid storage container and the second peristaltic pump, described hepatic tissue stationary installation is arranged in multilayer filtering device, connects the first peristaltic pump, liquid storage container and the second peristaltic pump successively between hepatic tissue stationary installation and multilayer filtering device by pipeline;
The hollow square body structure that described hepatic tissue stationary installation is made up of multiple retaining plate and portable plate, described retaining plate and portable plate all have sieve aperture, it is all stiff that described retaining plate comprises the first retaining plate, the second retaining plate and the 3rd retaining plate that are closely connected, and the first retaining plate, the second retaining plate and the 3rd retaining plate form cubical three fixed faces jointly; Described portable plate comprises the first portable plate and the second portable plate, first portable plate is slided on the second pulley track by the first pulley, second portable plate is slided on the first pulley track by the second pulley, first portable plate is provided with the equal intersection seam in multiple interval, one end of intersection seam is closed and is connected with the first portable plate main body, the other end of intersection seam opens and holds the second portable plate and passes, and the first portable plate and the second portable plate form cubical two active face; Second retaining plate and the second portable plate all offer the fixed orifices between sieve aperture, be equipped with fixed plate in fixed orifices, multiple fixed plate penetrates after in fixed orifices and forms a cubical face; Described first pulley and the second pulley are equipped with thumbwheel switch;
Described multi-layer filtrating equipment is made up of the end cap portions of hollow cylindrical, pars intermedia and bottom three part, cubical hollow space is provided with in described end cap portions, and hollow space and hepatic tissue stationary installation are coincide, the interior lower end of end cap portions is provided with the first screen cloth, the lower end of end cap portions is also provided with end cap portions screw thread, the top of described pars intermedia is provided with the threaded upper ends be connected with end cap portions screw thread, and end cap portions screw thread and threaded upper ends form the first threaded connection place, the inside of described 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, described second screen cloth is placed with the first magnetic bead, the lower end of described pars intermedia is provided with lower end screw thread, the top of described bottom is provided with the bottom thread be connected with lower end screw thread, lower end screw thread and bottom thread form the second threaded connection place, the inside of described bottom is provided with the 3rd screen cloth, the space that second screen cloth and the 3rd screen cloth are formed is cell enrichment cavity, described 3rd screen cloth is placed with the second magnetic bead, the space, bottom being positioned at the 3rd screen cloth is the temporary storage chamber body of perfusate, the sidewall of described bottom is provided with liquid exit.
As the further preferred version of the present invention: the aperture of described first screen cloth, the second screen cloth and the 3rd screen cloth reduces successively.
As the present invention's further preferred version: described retaining plate, portable plate and fixed plate are made up of metallic substance, stainless steel or polycarbonate; The shell of described multi-layer filtrating equipment is made up of transparent polycarbonate material.
Contain the separation method of the primary hepatocyte separation system organizing fixing and multilayer filtering device, concrete steps are as follows:
(1) hepatic tissue is fixed
First carry out sterilising treatment to each parts of reactor, each step of isolating hepatocytes is all carried out in aseptic super clean bench laboratory, is placed on by hepatic tissue in space that three retaining plates surround; Second portable plate is installed, slides the second portable plate until compress hepatic tissue, then locking first pulley along the first pulley track; First portable plate being installed, making the second portable plate at correct position through intersecting seam, slide the first portable plate until compression hepatic tissue, then locking second pulley along the second pulley track; Be inserted through fixed plate in fixed orifices on the second portable plate and the second retaining plate, thus formed six faces cubes stationary installation for be fixed for cellular segregation hepatic tissue;
(2) assembling of system
End cap portions, pars intermedia and bottom is good by corresponding helical assembly, and place the first magnetic bead and the second magnetic bead, to form multilayer filtering device; The hepatic tissue stationary installation being fixed with hepatic tissue is placed in hollow space, and liquid line connects peristaltic pump, magnetic stirring apparatus and liquid storage container;
(3) hepatic tissue collagenase perfusate perfusion
Perfusion shower nozzle inserts in hepatic tissue vascular by sieve aperture, or thrust in hepatic tissue by puncture needle multi-point, open the second peristaltic pump, by collagenase perfusate perfusion in liquid storage container in hepatic tissue, liver cell under the effect of collagenase in hepatic tissue is separated gradually, and with perfusate toward dirty, when multilayer filtering device liquid level is more than the first mesh screen height, open magnetic stirring apparatus adjusting rotary speed, make the first magnetic bead and the second magnetic bead slow circumvolve, and open the first peristaltic pump immediately, with corresponding first peristaltic pump corresponding speed the perfusate in temporary for perfusate storage chamber body discharged by liquid exit and be back in liquid storage container, in perfusing course, can fixed plate be removed, and help liver cell fully to dissociate by blade cuts,
(4) primary hepatocyte is collected
The second peristaltic pump is stopped at the end of perfusion, first peristaltic pump works on, perfusate in suction multilayer filtering device, when perfusate liquid level stops suction lower than during the second threaded connection place height, and stop magnetic stirring apparatus work, turn on by screw thread and be separated pars intermedia and bottom, with pipettor transfer primary hepatocyte suspension in cell therefor, with further centrifugal acquisition primary hepatocyte.
Compared with prior art, the invention has the beneficial effects as follows:
1, the invention provides the apparatus system being separated primary hepatocyte from hepatic tissue of a whole set of structure improvement, the primary hepatocyte that easy, quick, economic acquisition purity is higher;
2, can hepatic tissue be fixed, avoid free-hand fixing hepatic tissue, during multi-point lancet puncture, pricking wound occur;
3, fixing hepatic tissue, avoids hepatic tissue to be subjected to displacement, causes perfusate to splash;
4, there is multilayer filtration system, the collagenase perfusate after filtration, perfusion can be repeated in hepatic tissue, the consumption of collagenase can be reduced, cost-saving.
Accompanying drawing explanation
Fig. 1 is enforcement schematic diagram of the present invention.
Fig. 2 is the structural representation of hepatic tissue stationary installation in the present invention.
Fig. 3 is the cross sectional representation of the first retaining plate in hepatic tissue stationary installation, the 3rd retaining plate;
Fig. 4 is the cross sectional representation of the 3rd retaining plate, the second portable plate in hepatic tissue stationary installation;
Fig. 5 is the cross-sectional structure schematic diagram of fixed plate in hepatic tissue stationary installation;
Fig. 6 is the cross sectional representation of the first portable plate in hepatic tissue stationary installation;
Fig. 7 is the Split type structure schematic diagram of multilayer filtering device in the present invention.
Fig. 8 is the one-piece construction schematic diagram of multilayer filtering device in the present invention.
In figure: 1-first retaining plate; 2-fixed orifices; 3-second pulley track; 4-first pulley; 5-second retaining plate; 6-hepatic tissue stationary installation; 7-the 3rd retaining plate; 8-second portable plate; 9-first portable plate; 10-intersects seam; 11-first pulley track; 12-second pulley; 13-sieve aperture; 14-fixed plate; 15-hollow space; 16-end cap portions; 17-first screen cloth; 18-end cap portions screw thread; 19-threaded upper ends; 20-pars intermedia; 21-second screen cloth; 22-lower end screw thread; 23-first magnetic bead; 24-bottom thread; Bottom 25-; 26-the 3rd screen cloth; 27-liquid exit; The temporary storage chamber body of 28-perfusate; 29-second magnetic bead; 30-first threaded connection place; 31-cell mass receiving cavity; 32-second threaded connection place; 33-cell enrichment cavity; 34-magnetic stirring apparatus; 35-magnetic stirring apparatus speed governing button; 36-multilayer filtering device; 37-hepatic tissue; 38-first peristaltic pump; 39-liquid storage container; 40-second peristaltic pump.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Refer to Fig. 1 ~ 8, in the embodiment of the present invention, containing the primary hepatocyte separation system organizing fixing and multilayer filtering device, be made up of hepatic tissue stationary installation, multilayer filtering device, magnetic stirring apparatus, the first peristaltic pump, liquid storage container and the second peristaltic pump, it is characterized in that, described hepatic tissue stationary installation is arranged in multilayer filtering device, connects the first peristaltic pump, liquid storage container and the second peristaltic pump successively between hepatic tissue stationary installation and multilayer filtering device by pipeline.
The hollow square body structure that hepatic tissue stationary installation 6 is made up of multiple retaining plate and portable plate, retaining plate, portable plate and fixed plate are made up of metallic substance, stainless steel or polycarbonate; Retaining plate and portable plate all have sieve aperture 13, described retaining plate comprise the first retaining plate 1, second retaining plate 5 of being closely connected and the 3rd retaining plate 7 all stiff, the first retaining plate 1, second retaining plate 5 and the 3rd retaining plate 7 form cubical three fixed faces jointly; Portable plate comprises the first portable plate 9 and the second portable plate 8, first portable plate 9 is slided on the second pulley track 3 by the first pulley 4, second portable plate 8 is slided on the first pulley track 11 by the second pulley 12, first portable plate 9 is provided with the equal intersection seam 10 in multiple interval, one end of intersection seam 10 is closed and is connected with the first portable plate 9 main body, the other end of intersection seam 10 opens and holds the second portable plate 8 and passes, and the first portable plate 9 and the second portable plate 8 form cubical two active face; Second retaining plate 5 and the second portable plate 8 all offer the fixed orifices 2 between sieve aperture, be equipped with fixed plate 14 in fixed orifices 2, multiple fixed plate penetrates after in fixed orifices 2 and forms a cubical face.
First pulley 4 and the second pulley 12 are equipped with thumbwheel switch, after moving to required position, locking thumbwheel switch, portable plate is fixed on retaining plate, by the collocation of retaining plate and portable plate, and according to hepatic tissue size, realize the expansion of cubic space and reduce, better to fix hepatic tissue.
Hepatic tissue stationary installation 6 is the quantity adjustable of the cubes configuration of hollow adjustable size, retaining plate wherein and portable plate.For this stationary installation retaining plate and the adjustment of portable plate quantity, the hepatic tissue stationary installation forming size adjustable hollow cube configuration is all considered as the protection domain of patent of the present invention.
Multi-layer filtrating equipment 36 is made up of the end cap portions 16 of hollow cylindrical, pars intermedia 20 and bottom 25 3 part, and the shell of multi-layer filtrating equipment is made up of transparent polycarbonate material, can observe the height of liquid storage tank and inner chamber liquid level easily; This device total height is 10cm, and except end cap portions 16, the diameter of interior hollow-cylindrical is 8cm, and cubic capacity is about 500ml.
Cubical hollow space 15 is provided with in end cap portions 16, and hollow space 15 coincide with hepatic tissue stationary installation 6, the interior lower end of end cap portions 16 is provided with the first screen cloth 17, first screen cloth 17 is 20 orders, its aperture 0.8mm, relatively large hepatic tissue chip and manadesma etc. can be filtered, and allow little liver cell agglomerate to pass through; The lower end of end cap portions 16 is also provided with end cap portions screw thread 18, and the top of pars intermedia 20 is provided with the threaded upper ends 19 be connected with end cap portions screw thread 18, and end cap portions screw thread 18 and threaded upper ends 19 form the first threaded connection place 30; It is 300 orders that the inside of pars intermedia 20 is provided with the second screen cloth 21, second screen cloth 21, and 50 μm, its aperture, allows unicellular size and less material to pass through; The space that first screen cloth 17 and the second screen cloth 21 are formed is cell mass receiving cavity 31.
Second screen cloth 21 is placed with the first magnetic bead 23, when magnetic stirring apparatus 34 works, first magnetic bead 23 rotarily drives the motion of liquid vortex sample, blocks and promotes the abundant effect that collagenase is rolled into a ball liver cell, make it become single primary hepatocyte to prevent the second screen cloth 21; The lower end of pars intermedia 20 is provided with lower end screw thread 22, and the top of bottom 25 is provided with the bottom thread 24 be connected with lower end screw thread 22, and lower end screw thread 22 and bottom thread 24 form the second threaded connection place 32.
To be provided with the 3rd screen cloth the 26, three screen cloth 26 be 1250 orders in the inside of bottom 25,10 μm, its aperture, unicellular not by; The space that second screen cloth 21 and the 3rd screen cloth 26 are formed is cell enrichment cavity the 33, three screen cloth 26 is placed with the second magnetic bead 29, second magnetic bead 29 to rotarily drive liquid vortex sample and move, and blocks to prevent the second screen cloth 26; The space, bottom 25 being positioned at the 3rd screen cloth 26 is the temporary storage chamber body 28 of perfusate, and the sidewall of bottom is provided with liquid exit 27.
Contain the separation method of the primary hepatocyte separation system organizing fixing and multilayer filtering device, concrete steps are as follows:
(1) hepatic tissue is fixed
First carry out sterilising treatment to each parts of reactor, each step of isolating hepatocytes is all carried out in aseptic super clean bench or hundred grades of laboratories, is placed on by hepatic tissue in space that three retaining plates surround; Second portable plate 8 is installed, slides the second portable plate 8 until compress hepatic tissue, then locking first pulley 4 along the first pulley track 11; First portable plate 9 being installed, making the second portable plate 8 at correct position through intersecting seam 10, slide the first portable plate 9 until compression hepatic tissue, then locking second pulley 12 along the second pulley track 2; Be inserted through fixed plate in fixed orifices 2 on the second portable plate 8 and the second retaining plate 5, thus formed six faces cubes stationary installation for be fixed for cellular segregation hepatic tissue;
(2) assembling of system
By good by corresponding helical assembly to end cap portions 16, pars intermedia 20 and bottom 25, and place the first magnetic bead 23 and the second magnetic bead 29, to form multilayer filtering device 36; The hepatic tissue stationary installation 6 being fixed with hepatic tissue is placed in hollow space 15, and liquid line connects peristaltic pump, magnetic stirring apparatus 34 and liquid storage container 39;
(3) hepatic tissue collagenase perfusate perfusion
Perfusion shower nozzle inserts in hepatic tissue vascular by sieve aperture 13, or thrust in hepatic tissue by puncture needle multi-point, open the second peristaltic pump 40, by collagenase perfusate perfusion in liquid storage container 39 in hepatic tissue, liver cell under the effect of collagenase in hepatic tissue is separated gradually, and with perfusate toward dirty, when multilayer filtering device 36 liquid level is more than the first mesh screen 17 height, open magnetic stirring apparatus 34 adjusting rotary speed, make the first magnetic bead 23 and the second magnetic bead 29 slow circumvolve, and open the first peristaltic pump 38 immediately, with corresponding first peristaltic pump 38 corresponding speed the perfusate in temporary for perfusate storage chamber body 28 discharged by liquid exit 27 and be back in liquid storage container 39, in perfusing course, fixed plate 14 can be removed, and help liver cell fully to dissociate by blade cuts,
(4) primary hepatocyte is collected
The second peristaltic pump 40 is stopped at the end of perfusion, first peristaltic pump 38 works on, perfusate in suction multilayer filtering device 36, when perfusate liquid level stops suction lower than during the second threaded connection place 32 height, and stop magnetic stirring apparatus 34 to work, turn on by screw thread and be separated pars intermedia 20 and bottom 25, with pipettor transfer primary hepatocyte suspension in cell therefor, with further centrifugal acquisition primary hepatocyte.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.Any Reference numeral in claim should be considered as the claim involved by limiting.
Claims (4)
1. one kind contains the primary hepatocyte separation system organizing fixing and multilayer filtering device, be made up of hepatic tissue stationary installation (6), multilayer filtering device (36), magnetic stirring apparatus (34), the first peristaltic pump (38), liquid storage container (39) and the second peristaltic pump (40), it is characterized in that, described hepatic tissue stationary installation (6) is arranged in multilayer filtering device (36), connects the first peristaltic pump (38), liquid storage container (39) and the second peristaltic pump (40) successively between hepatic tissue stationary installation (6) and multilayer filtering device (36) by pipeline;
The hollow square body structure that described hepatic tissue stationary installation (6) is made up of multiple retaining plate and portable plate, described retaining plate and portable plate all have sieve aperture (13), it is all stiff that described retaining plate comprises the first retaining plate (1), the second retaining plate (5) and the 3rd retaining plate (7) that are closely connected, and the first retaining plate (1), the second retaining plate (5) and the 3rd retaining plate (7) form cubical three fixed faces jointly, described portable plate comprises the first portable plate (9) and the second portable plate (8), first portable plate (9) is above slided at the second pulley track (3) by the first pulley (4), second portable plate (8) is above slided at the first pulley track (11) by the second pulley (12), first portable plate (9) is provided with equal intersection seam (10) in multiple interval, one end of intersection seam (10) is closed and is connected with the first portable plate (9) main body, the other end of intersection seam (10) opens and holds the second portable plate (8) and passes, first portable plate (9) and the second portable plate (8) form cubical two active face, second retaining plate (5) and the second portable plate (8) all offer the fixed orifices (2) be positioned between sieve aperture (13), is equipped with fixed plate (14) in fixed orifices (2), multiple fixed plate penetrates after in fixed orifices (2) and forms a cubical face, described first pulley (4) and the second pulley (12) are equipped with thumbwheel switch,
Described multi-layer filtrating equipment (36) is by the end cap portions (16) of hollow cylindrical, pars intermedia (20) and bottom (25) three part composition, cubical hollow space (15) is provided with in described end cap portions (16), and hollow space (15) coincide with hepatic tissue stationary installation (6), the interior lower end of end cap portions (16) is provided with the first screen cloth (17), the lower end of end cap portions (16) is also provided with end cap portions screw thread (18), the top of described pars intermedia (20) is provided with the threaded upper ends (19) be connected with end cap portions screw thread (18), end cap portions screw thread (18) and threaded upper ends (19) form the first threaded connection place (30), the inside of described pars intermedia (20) is provided with the second screen cloth (21), the space that first screen cloth (17) and the second screen cloth (21) are formed is cell mass receiving cavity (31), described second screen cloth (21) is placed with the first magnetic bead (23), the lower end of described pars intermedia (20) is provided with lower end screw thread (22), the top of described bottom (25) is provided with the bottom thread (24) be connected with lower end screw thread (22), lower end screw thread (22) and bottom thread (24) form the second threaded connection place (32), the inside of described bottom (25) is provided with the 3rd screen cloth (26), the space that second screen cloth (21) and the 3rd screen cloth (26) are formed is cell enrichment cavity (33), described 3rd screen cloth (26) is placed with the second magnetic bead (29), bottom (25) space being positioned at the 3rd screen cloth (26) is the temporary storage chamber body (28) of perfusate, the sidewall of described bottom is provided with liquid exit (27).
2. the primary hepatocyte separation system of and multilayer filtering device fixing containing tissue according to claim 1, is characterized in that, the aperture of described first screen cloth (17), the second screen cloth (21) and the 3rd screen cloth (26) reduces successively.
3. the primary hepatocyte separation system containing organizing fixing and multilayer filtering device according to claim 1, it is characterized in that, described retaining plate, portable plate and fixed plate are made up of metallic substance, stainless steel or polycarbonate material; The shell of described multi-layer filtrating equipment (36) is made up of transparent polycarbonate material.
4. the separation method containing the primary hepatocyte separation system organizing fixing and multilayer filtering device as claimed in claim 1, it is characterized in that, concrete steps are as follows:
(1) hepatic tissue is fixed
First carry out sterilising treatment to each parts of reactor, each step of isolating hepatocytes is all carried out in aseptic super clean bench or hundred grades of laboratories, is placed on by hepatic tissue in space that three retaining plates surround; Second portable plate (8) is installed, slides the second portable plate (8) until compress hepatic tissue, then locking first pulley (4) along the first pulley track (11); First portable plate (9) being installed, making the second portable plate (8) at correct position through intersecting seam (10), slide the first portable plate (9) until compression hepatic tissue, then locking second pulley (12) along the second pulley track (2); Be inserted through fixed plate (14) in fixed orifices (2) on the second portable plate (8) and the second retaining plate (5), thus formed six faces cubes stationary installation for be fixed for cellular segregation hepatic tissue;
(2) assembling of system
By end cap portions (16), pars intermedia (20) and bottom (25) good by corresponding helical assembly, and place the first magnetic bead (23) and the second magnetic bead (29), to form multilayer filtering device (36); The hepatic tissue stationary installation being fixed with hepatic tissue is placed in hollow space (15), and liquid line connects peristaltic pump, magnetic stirring apparatus (34) and liquid storage container (39);
(3) hepatic tissue collagenase perfusate perfusion
Perfusion shower nozzle inserts in hepatic tissue vascular by sieve aperture (13), or thrust in hepatic tissue by puncture needle multi-point, open the second peristaltic pump (40), by collagenase perfusate perfusion in liquid storage container (39) in hepatic tissue, liver cell under the effect of collagenase in hepatic tissue is separated gradually, and with perfusate toward dirty, when multilayer filtering device (36) liquid level is more than the first mesh screen (17) height, open magnetic stirring apparatus (34) adjusting rotary speed, make the first magnetic bead (23) and the second magnetic bead (29) slow circumvolve, and open the first peristaltic pump (38) immediately, with corresponding first peristaltic pump (38) corresponding speed, the perfusate in temporary for perfusate storage chamber body (28) is discharged by liquid exit (27) and is back in liquid storage container (39), in perfusing course, fixed plate (14) can be removed, and help liver cell fully to dissociate by blade cuts,
(4) primary hepatocyte is collected
The second peristaltic pump (40) is stopped at the end of perfusion, first peristaltic pump (38) works on, perfusate in suction multilayer filtering device (36), when perfusate liquid level stops suction lower than during the second threaded connection place (32) height, and stop magnetic stirring apparatus (34) to work, turn on by screw thread and be separated pars intermedia (20) and bottom (25), with pipettor transfer primary hepatocyte suspension in cell therefor, with further centrifugal acquisition primary hepatocyte.
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| 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 |
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| 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 |
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| CN104928176A true CN104928176A (en) | 2015-09-23 |
| CN104928176B CN104928176B (en) | 2017-03-08 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733072A (en) * | 2020-06-08 | 2020-10-02 | 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 and method and application |
| CN112410179A (en) * | 2020-12-09 | 2021-02-26 | 桂林医学院 | Simple in-vitro liver perfusion device for digesting and separating liver cells |
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| EP0909811A2 (en) * | 1997-10-16 | 1999-04-21 | B. BRAUN CAREX S.p.A. | Bioreactor for culturing animal and/or human cells, particularly hepatocytes |
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| 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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| EP0909811A2 (en) * | 1997-10-16 | 1999-04-21 | B. BRAUN CAREX S.p.A. | Bioreactor for culturing animal and/or human cells, particularly hepatocytes |
| US20020012982A1 (en) * | 2000-07-13 | 2002-01-31 | Invitrogen Corporation | Methods and compositions for rapid protein and peptide extraction and isolation using a lysis matrix |
| 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 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733072A (en) * | 2020-06-08 | 2020-10-02 | 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 and method and application |
| 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 |
| CN112410179A (en) * | 2020-12-09 | 2021-02-26 | 桂林医学院 | Simple in-vitro liver perfusion device for digesting and separating liver cells |
| 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 |
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| CN104928176B (en) | 2017-03-08 |
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