CN109161518A - A method of being separately cultured primary hepatic cell - Google Patents
A method of being separately cultured primary hepatic cell Download PDFInfo
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- CN109161518A CN109161518A CN201811178787.5A CN201811178787A CN109161518A CN 109161518 A CN109161518 A CN 109161518A CN 201811178787 A CN201811178787 A CN 201811178787A CN 109161518 A CN109161518 A CN 109161518A
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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
- 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 belongs to technical field of cell culture, more particularly to a kind of method for being separately cultured primary hepatic cell, in vitro liver organization is stored in the tissue storage solution of pre-cooling, take out liver organization, and perfusate is inputted from the vena portae hepatica of liver organization, after perfusion, liver organization is put into another container, reinject collagenase solution, until there is turtleback crack in liver organization surface, then by liver organization stripping and slicing, and it is soaked in 4-8min in the collagenase solution, obtain the mature liver of digestion, the mature liver of digestion is cleaned with distilled water, it filters and collects hepatocyte suspension, hepatocyte suspension centrifugation, it collects cell precipitation and is resuspended with culture medium and cultivated, primary hepatic cell after being separated.The method provided by the invention for being separately cultured primary hepatic cell simplifies the agent prescription of traditional in-situ two-step perfusion method, simplifies preparation of reagents step, reduce costs, and the survival rate of separated cell, vigor and purity are improved.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a method of it is separately cultured primary hepatic cell.
Background technique
Liver is the vitals of human body, plays many important functions, have the homeostasis for keeping glucose, detoxification or
The effect of person's macromolecular synthesis, compromised liver function can make a significant impact health, and virus hepatitis and related liver disease can be tight
The health of the mankind is endangered again.Primary cell as a kind of external model, due to its can obtain simultaneously a large amount of characters it is uniform and
The cell of physiological status close in organism, has the advantages that prominent in terms of gene functional research.Liver cell is that liver is main
Parenchyma type, belong to well differentiated multi-functional, parenchymal viscera cell, in vitro in cultivating system it is existing support its
Value-added cultivating system also has the cultivating system for supporting its differentiation.But since liver cell is after in vitro, it will appear aging quickly
And the phenomenon that loss of function, eventually lead to cell death.Especially it is special will to lose it within several hours for primary hepatocyte
Property metabolic activity, brings puzzlement to the research work of liver cell.
It is widely used that in-situ two-step perfusion method in the prior art, is hepatocyte isolation methods classical in the world,
First from the no calcium of vein, oxygen-containing buffer early period, go out haemocyte and calcium ion, then change collagenase solution be perfused it is soft to organizing
Change.Although this method can the greater number of liver cell of sub-department, the formulatory agents such as used perfusate of operation link are complicated,
Prepare trouble.
Summary of the invention
A kind of method being separately cultured primary hepatic cell provided by the invention, simplifies traditional in-situ two-step perfusion method
Agent prescription, and then preparation of reagents step is simplified, it reduces costs, and the survival rate of separated cell and vigor are higher.
The object of the present invention is to provide a kind of methods for being separately cultured primary hepatic cell, comprising the following steps:
In vitro liver organization is stored in the tissue storage solution of pre-cooling by S1, spare；Described in 1L tissue storage solution according to
Following methods are prepared: HEPES (4- hydroxyethyl piperazineethanesulfonic acid, also known as N- (2- ethoxy) piperazine-N'-2- ethane sulfonic acid)
2.4g, 10000U penicillin, 100mg streptomysin, distilled water are settled to 1L, pH7.2-7.4；
S2 takes out liver organization, and inputs perfusate from the vena portae hepatica of liver organization, and perfusate described in 1L is according to following
Method is prepared: sodium chloride 8-9g, potassium chloride 0.2-0.4g, distilled water are settled to 1L；
S3 after perfusion, liver organization is put into another container, collagenase solution is reinjected, until liver organization
There is turtleback crack in surface, then by liver organization stripping and slicing, and is soaked in 4-8min in the collagenase solution, is digested
Mature liver；
S4 cleans the mature liver of digestion with distilled water, filters and collect hepatocyte suspension；
S5, hepatocyte suspension centrifugation are collected cell precipitation and are resuspended with culture medium and cultivated, the primary hepatic after being separated
Cell.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, in S2, the input time of the perfusate is
10min, perfusion flow velocity are 60mL/min, and perfusate temperature is 37 DEG C.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, collagenase solution described in 1L is matched in accordance with the following methods
System: II Collagenase Type 0.5g, HEPES 4.8g, disodium hydrogen phosphate 0.5g, sodium chloride 8.5g, potassium chloride 0.3g, calcium chloride 0.1g,
Distilled water is settled to 1L.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, in S3, the flow velocity of collagenase solution is 60mL/
Min, collagenase solution temperature are 50 DEG C, time 10min.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, in S3, after liver organization stripping and slicing with a thickness of 1-
3cm。
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, in S4, the apparatus structure for filtering use is as follows: packet
Include the 100 mesh gauzes, 150 mesh screens, 100 mesh gauzes, 200 mesh screens being sequentially overlapped from top to bottom.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, in S4, the apparatus structure for filtering use is as follows: from
Top to bottm successively includes 100 mesh gauzes, 150 mesh screens, 100 mesh gauzes, 200 mesh screens.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell, the sieve are stainless steel filter screen.
Preferably, the above-mentioned method for being separately cultured primary hepatic cell further includes buffer solution for cleaning step in S5, specifically such as
Under: hepatocyte suspension centrifugation, precipitating buffer solution for cleaning are centrifuged again, are collected cell precipitation and are resuspended with culture medium and cultivated, are obtained
Primary hepatic cell after to separation；Buffer described in 1L is prepared in accordance with the following methods: potassium chloride 0.2-0.4g, distilled water constant volume
To 1L.
Compared with prior art, the method provided by the invention for being separately cultured primary hepatic cell has below beneficial to effect
Fruit:
The method provided by the invention for being separately cultured primary hepatic cell simplifies the reagent of traditional in-situ two-step perfusion method
Formula, and then preparation of reagents step is simplified, it reduces costs, and the survival rate of separated cell, vigor and purity mention
It is high.It freezes experiment to show after freezing processing, liver cell still has higher adherent rate, can save, can be widely applied for a long time
In the research of the animals such as pig, the sheep of biological field and human body isolating hepatocytes, experiment basis has been established for liver cell research.
In addition, being settled to 1L, pH7.2-7.4 using HEPES 2.4g, 10000U penicillin, 100mg streptomysin, distilled water
The stock solution of formula stores in vitro liver organization, can be reserved for Activity of hepatocytes 12-36h, is the letter of the formulas such as subsequent perfusate
Change is laid a good foundation.We the liver organization stripping and slicing in turtleback crack will occur, it is only necessary to which impregnating 4-8min can be completed liver
Digestion, accelerate the digestion of collagenase solution.
Specific embodiment
The present invention is described in detail combined with specific embodiments below, but should not be construed as limitation of the invention.It is following
The test method of actual conditions is not specified in embodiment, operates usually according to normal condition, due to not being related to inventive point, thus it is not right
Its step is described in detail.
In following embodiments, liver organization used is in vitro pig liver tissue and sheep liver tissue, from local slaughterhouse
It is commercially available.The injection of perfusate and collagenase solution uses peristaltic pump.
Preferably, a kind of method being separately cultured primary hepatic cell provided by the invention, specifically includes following embodiment.
Embodiment 1
A method of being separately cultured primary hepatic cell, comprising the following steps:
In vitro liver organization is stored in the tissue storage solution of 4 DEG C of pre-coolings by S1, spare；Storage solution is organized described in 1L
It prepares in accordance with the following methods: HEPES (hydroxyethyl piperazine second thiosulfonic acid) 2.4g, 10000U penicillin, 100mg streptomysin, double steamings
Water is settled to 1L, pH7.2；
S2 takes out liver organization, and inputs perfusate from the vena portae hepatica of liver organization, and perfusate described in 1L is according to following
Method is prepared: sodium chloride 8g, potassium chloride 0.2g, distilled water are settled to 1L；The input time of the perfusate is 10min, perfusion
Flow velocity is 60mL/min, and perfusate temperature is 37 DEG C.
After perfusion, liver organization is put into another container (sterile petri dish) by S3, then injects glue to liver organization
Protoenzyme solution then by liver organization stripping and slicing, and is soaked in the clostridiopetidase A until turtleback crack occurs in liver organization surface
4min in solution obtains the mature liver of digestion；Collagenase solution described in 1L is prepared in accordance with the following methods: II Collagenase Type
0.5g, HEPES 4.8g, disodium hydrogen phosphate 0.5g, sodium chloride 8.5g, potassium chloride 0.3g, calcium chloride 0.1g, distilled water are settled to
1L.The flow velocity of collagenase solution is 60mL/min, and collagenase solution temperature is 50 DEG C, time 10min.After liver organization stripping and slicing
With a thickness of 1cm.
S4 cleans the mature liver of digestion with distilled water, filters and collects hepatocyte suspension (hepatocyte suspension is i.e.
Filter the liquid left after liver organization object)；
It is as follows to filter the apparatus structure used: including 100 mesh gauzes, 150 mesh screens, 100 being sequentially overlapped from top to bottom
Mesh gauze, 200 mesh screens.The sieve is stainless steel filter screen.
S5, hepatocyte suspension 500r/min are centrifuged 3min, collect cell precipitation and are resuspended with culture medium and cultivated, are separated
Primary hepatic cell afterwards.The formula of used medium in S5 are as follows: in every liter of DMEM culture medium add 100000U penicillin,
10mg streptomysin, 1g dimethyl sulfoxide, 1mg insulin, 1mmol niacinamide, 0.5mmol/L ascorbic acid, pH7.2.
Using the method for embodiment 1, porcine hepatocyte yield 2.1 × 108A/g liver organization, motility rate 99%, adherent rate
99%, purity 95%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 side B patent CN
After method secondary culture, for liver cell without aging death phenomenon, form is normal within 10 generations, and the speed of growth is normal.Referring to patent CN
After 102634480 B freeze and recover, the adherent rate of the liver cell of culture is up to 96% or more.Similar mode of operation measures sheep liver
Cell index of correlation is as follows: sheep liver cell yield 1.3 × 108A/g liver organization, motility rate 99%, adherent rate 99%, purity
95%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 B method secondary culture of patent CN
Afterwards, liver cell is without aging death phenomenon within 10 generations, and form is normal, and the speed of growth is normal.Referring to 102634480 B of patent CN
After freezing and recovering, the adherent rate of the liver cell of culture is up to 96% or more.
Embodiment 2
A method of being separately cultured primary hepatic cell, comprising the following steps:
In vitro liver organization is stored in the tissue storage solution of 4 DEG C of pre-coolings by S1, spare；Storage solution is organized described in 1L
Prepare in accordance with the following methods: HEPES 2.4g, 10000U penicillin, 100mg streptomysin, distilled water are settled to 1L, pH7.4；
S2 takes out liver organization, and inputs perfusate from the vena portae hepatica of liver organization, and perfusate described in 1L is according to following
Method is prepared: sodium chloride 9g, potassium chloride 0.4g, distilled water are settled to 1L；The input time of the perfusate is 10min, perfusion
Flow velocity is 60mL/min, and perfusate temperature is 37 DEG C.
After perfusion, liver organization is put into another container (sterile petri dish) by S3, then injects glue to liver organization
Protoenzyme solution then by liver organization stripping and slicing, and is soaked in the clostridiopetidase A until turtleback crack occurs in liver organization surface
4min in solution obtains the mature liver of digestion；Collagenase solution described in 1L is prepared in accordance with the following methods: II Collagenase Type
0.5g, HEPES 4.8g, disodium hydrogen phosphate 0.5g, sodium chloride 8.5g, potassium chloride 0.3g, calcium chloride 0.1g, distilled water are settled to
1L.The flow velocity of collagenase solution is 60mL/min, and collagenase solution temperature is 50 DEG C, time 10min.After liver organization stripping and slicing
With a thickness of 3cm.
S4 cleans the mature liver of digestion with distilled water, filters and collects hepatocyte suspension (hepatocyte suspension is i.e.
Filter the liquid left after liver organization object)；
It is as follows to filter the apparatus structure used: including 100 mesh gauzes, 150 mesh screens, 100 being sequentially overlapped from top to bottom
Mesh gauze, 200 mesh screens.The sieve is stainless steel filter screen.
S5, hepatocyte suspension 500r/min are centrifuged 3min, and precipitating buffer solution for cleaning, 500r/min is centrifuged 3min again,
It collects cell precipitation and is resuspended with culture medium and cultivated, the primary hepatic cell after being separated.The formula of used medium in S5
Are as follows: 100000U penicillin, 10mg streptomysin, 1g dimethyl sulfoxide, 1mg insulin, 1mmol are added in every liter of DMEM culture medium
Niacinamide, 0.5mmol/L ascorbic acid, pH7.2.Buffer described in 1L is prepared in accordance with the following methods: potassium chloride 0.2g, double steamings
Water is settled to 1L.
Using the method for embodiment 2, porcine hepatocyte yield 2.2 × 108A/g liver organization, motility rate 99%, adherent rate
99%, purity 96%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 side B patent CN
After method secondary culture, for liver cell without aging death phenomenon, form is normal within 10 generations, and the speed of growth is normal.Referring to patent CN
After 102634480 B freeze and recover, the adherent rate of the liver cell of culture is up to 96% or more.Similar mode of operation measures sheep liver
Cell index of correlation is as follows: sheep liver cell yield 3.4 × 108A/g liver organization, motility rate 99%, adherent rate 99%, purity
95%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 B method secondary culture of patent CN
Afterwards, liver cell is without aging death phenomenon within 10 generations, and form is normal, and the speed of growth is normal.Referring to 102634480 B of patent CN
After freezing and recovering, the adherent rate of the liver cell of culture is up to 96% or more.
Embodiment 3
A method of being separately cultured primary hepatic cell, comprising the following steps:
In vitro liver organization is stored in the tissue storage solution of 4 DEG C of pre-coolings by S1, spare；Storage solution is organized described in 1L
Prepare in accordance with the following methods: HEPES 2.4g, 10000U penicillin, 100mg streptomysin, distilled water are settled to 1L, pH7.2；
S2 takes out liver organization, and inputs perfusate from the vena portae hepatica of liver organization, and perfusate described in 1L is according to following
Method is prepared: sodium chloride 8.5g, potassium chloride 0.3g, distilled water are settled to 1L；The input time of the perfusate is 10min, is filled
Flow liquid flow velocity is 60mL/min, and perfusate temperature is 37 DEG C.
After perfusion, liver organization is put into another container (sterile petri dish) by S3, then injects glue to liver organization
Protoenzyme solution then by liver organization stripping and slicing, and is soaked in the clostridiopetidase A until turtleback crack occurs in liver organization surface
8min in solution obtains the mature liver of digestion；Collagenase solution described in 1L is prepared in accordance with the following methods: II Collagenase Type
0.5g, HEPES 4.8g, disodium hydrogen phosphate 0.5g, sodium chloride 8.5g, potassium chloride 0.3g, calcium chloride 0.1g, distilled water are settled to
1L.The flow velocity of collagenase solution is 60mL/min, and collagenase solution temperature is 50 DEG C, time 10min.After liver organization stripping and slicing
With a thickness of 2cm.
S4 cleans the mature liver of digestion with distilled water, filters and collects hepatocyte suspension (hepatocyte suspension is i.e.
Filter the liquid left after liver organization object)；
It is as follows to filter the apparatus structure used: including 100 mesh gauzes, 150 mesh screens, 100 being sequentially overlapped from top to bottom
Mesh gauze, 200 mesh screens.The sieve is stainless steel filter screen.
S5, hepatocyte suspension 500r/min are centrifuged 3min, and precipitating buffer solution for cleaning, 500r/min is centrifuged 3min again,
It collects cell precipitation and is resuspended with culture medium and cultivated, the primary hepatic cell after being separated.The formula of used medium in S5
Are as follows: 100000U penicillin, 10mg streptomysin, 1g dimethyl sulfoxide, 1mg insulin, 1mmol are added in every liter of DMEM culture medium
Niacinamide, 0.5mmol/L ascorbic acid, pH7.2.Buffer described in 1L is prepared in accordance with the following methods: potassium chloride 0.4g, double steamings
Water is settled to 1L.
Using the method for embodiment 3, porcine hepatocyte yield 2.6 × 108A/g liver organization, motility rate 99%, adherent rate
99%, purity 95%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 side B patent CN
After method secondary culture, for liver cell without aging death phenomenon, form is normal within 10 generations, and the speed of growth is normal.Referring to patent CN
After 102634480 B freeze and recover, the adherent rate of the liver cell of culture is up to 96% or more.Similar mode of operation measures sheep liver
Cell index of correlation is as follows: sheep liver cell yield 2.1 × 108A/g liver organization, motility rate 99%, adherent rate 99%, purity
94%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 B method secondary culture of patent CN
Afterwards, liver cell is without aging death phenomenon within 10 generations, and form is normal, and the speed of growth is normal.Referring to 102634480 B of patent CN
After freezing and recovering, the adherent rate of the liver cell of culture is up to 96% or more.
Comparative example 1
By filtering use apparatus structure replace with such as flowering structure: including be sequentially overlapped from top to bottom 100 mesh gauzes,
100 mesh gauzes, 100 mesh screens, 200 mesh screens.The sieve is stainless steel filter screen.Remaining operation while example 1.
Using the method for comparative example 1, porcine hepatocyte yield 8.6 × 107A/g liver organization, motility rate 99%, adherent rate
98%, purity 94%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 side B patent CN
After method secondary culture, for liver cell without aging death phenomenon, form is normal within 10 generations, and the speed of growth is normal.Referring to patent CN
After 102634480 B freeze and recover, the adherent rate of the liver cell of culture is up to 95% or more.Similar mode of operation measures sheep liver
Cell index of correlation is as follows: sheep liver cell yield 8.8 × 107A/g liver organization, motility rate 99%, adherent rate 98%, purity
94%；It can be passed on when primary hepatic cell confluency degree reaches 80-90%, according to 102634480 B method secondary culture of patent CN
Afterwards, liver cell is without aging death phenomenon within 10 generations, and form is normal, and the speed of growth is normal.Referring to 102634480 B of patent CN
After freezing and recovering, the adherent rate of the liver cell of culture is up to 95% or more.
Comparative example 2
Embodiment 1 and 4 the method for embodiment are separately cultured primary hepatocyte in 102634480 B of referenced patent CN.Pig
Liver cell yield 6.5 × 107A/g liver organization, motility rate 99%, adherent rate 98%, purity 93%；When primary hepatic cell converges
The right 80-90% that reaches can be passed on, and after 102634480 B method secondary culture of patent CN, liver cell is without declining within 10 generations
The old phenomena of mortality, form is normal, and the speed of growth is normal.After freezing and recover referring to 102634480 B of patent CN, the liver of culture
The adherent rate of cell is up to 95% or more.Similar mode of operation measures sheep liver cell index of correlation, as follows: sheep liver cell yield
6.2×107A/g liver organization, motility rate 99%, adherent rate 98%, purity 93%；When primary hepatic cell confluency degree reaches 80-
90% can pass on, after 102634480 B method secondary culture of patent CN, within 10 generations liver cell without aging death phenomenon,
Form is normal, and the speed of growth is normal.After freezing and recover referring to 102634480 B of patent CN, the adherent rate of the liver cell of culture
Up to 95% or more.
Comparative example 3
Following document the methods separate primary hepatocyte in referenced patent CN 102634480B.
P.O.Seglen.Pregaration of rat liver cells:II Effects of ions and
chelators on tissue dispersion.Experimental Cell Research,1973,76(1),25-30.
Porcine hepatocyte yield 7.8 × 106A/g liver organization, motility rate 85%, adherent rate 83%, purity 80%；Work as liver
Primary cell convergence degree, which reaches 80-90%, to pass on, after 102634480 B method secondary culture of patent CN, liver when 10 generation
The cell ageing death rate is up to 56%.After freezing and recover referring to 102634480 B of patent CN, the adherent rate of the liver cell of culture
Up to 64% or more.Similar mode of operation measures sheep liver cell index of correlation, as follows: sheep liver cell yield 7.7 × 106A/g liver
Dirty tissue, motility rate 86%, adherent rate 84%, purity 80%；It can pass on, press when primary hepatic cell confluency degree reaches 80-90%
After 102634480 B method secondary culture of patent CN, liver cell aging death rate is up to 59% when 10 generation.Referring to patent CN
After 102634480 B freeze and recover, the adherent rate of the liver cell of culture is up to 71% or more.
It should be noted that involved in the present invention when numberical range, it is thus understood that two endpoints of each numberical range with
And any one numerical value can be selected between two endpoints, since the step method of use is identical as embodiment, go to live in the household of one's in-laws on getting married in order to prevent
It states, the present invention describes preferred embodiment., but technology people in the art although preferred embodiments of the present invention have been described
Member is once know basic creative concept, then additional changes and modifications can be made to these embodiments.So appended right
It is required that being intended to be construed to includes preferred embodiment and all change and modification for falling into the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (8)
1. a kind of method for being separately cultured primary hepatic cell, which comprises the following steps:
In vitro liver organization is stored in the tissue storage solution of pre-cooling by S1, spare；Tissue storage solution is according to following described in 1L
Method is prepared: HEPES 2.4g, 10000U penicillin, 100mg streptomysin, distilled water are settled to 1L, pH7.2-7.4；
S2 takes out liver organization, and inputs perfusate from the vena portae hepatica of liver organization, and perfusate described in 1L is in accordance with the following methods
Prepare: sodium chloride 8-9g, potassium chloride 0.2-0.4g, distilled water are settled to 1L；
S3 after perfusion, liver organization is put into another container, collagenase solution is reinjected, until liver organization surface
There is turtleback crack, then by liver organization stripping and slicing, and be soaked in 4-8min in the collagenase solution, it is mature to obtain digestion
Liver；
S4 cleans the mature liver of digestion with distilled water, filters and collect hepatocyte suspension；
S5, hepatocyte suspension centrifugation are collected cell precipitation and are resuspended with culture medium and cultivated, and the primary hepatic after being separated is thin
Born of the same parents.
2. the method according to claim 1 for being separately cultured primary hepatic cell, which is characterized in that in S2, the perfusion
The input time of liquid is 10min, and perfusion flow velocity is 60mL/min, and perfusate temperature is 37 DEG C.
3. the method according to claim 1 for being separately cultured primary hepatic cell, which is characterized in that clostridiopetidase A described in 1L is molten
Liquid is prepared in accordance with the following methods: II Collagenase Type 0.5g, HEPES 4.8g, disodium hydrogen phosphate 0.5g, sodium chloride 8.5g, potassium chloride
0.3g, calcium chloride 0.1g, distilled water are settled to 1L.
4. the method according to claim 3 for being separately cultured primary hepatic cell, which is characterized in that in S3, clostridiopetidase A is molten
The flow velocity of liquid is 60mL/min, and collagenase solution temperature is 50 DEG C, time 10min.
5. the method according to claim 3 for being separately cultured primary hepatic cell, which is characterized in that in S3, liver organization
After stripping and slicing with a thickness of 1-3cm.
6. the method according to claim 1 for being separately cultured primary hepatic cell, which is characterized in that in S4, filtering is used
Apparatus structure it is as follows: including the 100 mesh gauzes, 150 mesh screens, 100 mesh gauzes, 200 mesh screens being sequentially overlapped from top to bottom.
7. the method according to claim 6 for being separately cultured primary hepatic cell, which is characterized in that the sieve is stainless
Steel filter screen.
8. the method according to claim 1-7 for being separately cultured primary hepatic cell, which is characterized in that in S5 also
Specific as follows including buffer solution for cleaning step: hepatocyte suspension centrifugation, precipitating buffer solution for cleaning are centrifuged again, collect cell
It precipitates and is resuspended with culture medium and cultivated, the primary hepatic cell after being separated；Buffer described in 1L is prepared in accordance with the following methods:
Potassium chloride 0.2-0.4g, distilled water are settled to 1L.
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| CN111647549A (en) * | 2020-07-28 | 2020-09-11 | 内蒙古自治区农牧业科学院 | Method for separating single cells in animals and plants |
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