CN102634480A - Method for isolating and culturing liver primary cells - Google Patents
Method for isolating and culturing liver primary cells Download PDFInfo
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- CN102634480A CN102634480A CN201210083696XA CN201210083696A CN102634480A CN 102634480 A CN102634480 A CN 102634480A CN 201210083696X A CN201210083696X A CN 201210083696XA CN 201210083696 A CN201210083696 A CN 201210083696A CN 102634480 A CN102634480 A CN 102634480A
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Abstract
The invention provides a method for isolating and culturing liver primary cells. The method comprises the following steps: (1) inputting perfusate from hepatic portal vein of a liver; (2) inputting collagenase perfusate; (3) soaking the maturely digested liver in the collagenase perfusate; (4) adding a cleaning solution to stop digestion, filtering, and collecting hepatic cell suspension; and (5) centrifuging, discarding supernatant, resuspending with complete medium, and culturing. The operation of the method provided by the invention is simple, is low in cost and is stable and efficient, and the hepatic cells are high in yield, high in vitality and easy for adherence. The method can be generalized in laboratories, and is a conventional method for scientific research.
Description
Technical field
The present invention relates to cell field, particularly, relate to a kind of method of separation and Culture liver primary cell.
Background technology
Primary cell has its outstanding advantage: can obtain the sample of a large amount of proterties than homogeneous simultaneously as a kind of external model aspect gene functional research; Basically be consistent with situation in the organism, can under situation, study expression of gene near physiological status; Get rid of many complicated factors and disturb, like interference neural, hormone; Primary hepatocyte has good in-vitro experiment circulation ratio; Basically kept the metabolic function of liver; Particularly well kept with body in the level of consistent cytochrome P 450 enzymes (cytochrome P450), keep original metabolic function of liver and cytodifferentiation state basically.
Liver cell (Hepatocytes) is the topmost parenchyma type of liver, accounts for 60% under the physiological situation, belongs to well differentiated multi-functional, parenchymal viscera cell.Liver cell can highly break up, and can get into the cell cycle very soon again and breeds, and in the vitro culture system, according to the difference of research purpose, the culture system of existing its propagation of support also has the culture system of supporting its differentiation.After liver cell is exsomatized, in unfavorable environment, occur the old and feeble and mistake function of liver cell very soon, the pH value is not suitable for, does not have the hormone support and causes necrocytosis, and the activation of gene causes apoptosis etc.Primary hepatocyte is just lost its slit in several hours and is connected its tissue specificity Metabolic activity of forfeiture in several days under general culture condition.Liver cell separation method commonly used in the prior art is a perfusion method, and traditional perfusion method operation link is many, and the time is long, and liver cell damages easily, and vigor is low, efficient is low, and purity is not high.Therefore, press for the preparation pig liver primary cell that vitro culture flushes, function is good to satisfy the requirement of researchs such as cytobiology, tissue culture technique.
Summary of the invention
The object of the present invention is to provide a kind of method of separation and Culture liver primary cell, to overcome the defective that exists in the above-mentioned prior art.
The method of separation and Culture liver primary cell provided by the invention comprises following steps:
(1) imports perfusate from the hepatic vein of liver; Described perfusate, its 1L prescription is: NaCl 8-9.4g, KCl 0.2-0.4, HEPES 2.4-4.8g, distilled water is settled to 1L, pH value 7.2-7.4;
(2) after input collagenase perfusion solution, hepatic tissue are turtleback and split, stop perfusion;
(3) will digest sophisticated liver is soaked in the collagenase solution;
(4) add scavenging solution and stop digestion, filter, collect hepatocyte suspension;
(5) centrifugal, abandon supernatant, with the resuspended cultivation of perfect medium.
The liver of said step 1) is the stripped liver of fresh collection, or fresh in vitro liver THPV is with 0-4 ℃ of precooling UW liquid of 80-100ml/min speed injection liver volume more than 3 times, below the liver temperature drop to 10 ℃, keeps the liver within the 1-4 ℃ of stripped 3h.After below the liver temperature drop to 10 ℃, can place ice chest or other can keep 1-4 ℃ container to preserve liver in liver and be no more than 3h.
The inventive method adopts peristaltic pump to carry out perfusion, with bubble in the aseptic PBS emptying peripump tubing.Wherein said step (1) is with the open perfusion 10-15min of 37 ℃ of perfusates of constant temperature, flow velocity 50-60ml/min.
Wherein said step (2) collagenase perfusion solution perfusion flow velocity 50-60ml/min.
The collagenase perfusate of wherein said step (2), its 1L prescription is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na
2HPO
40.5-0.6g, CaCl
20.10-0.15g, II type or IV Collagen Type VI enzyme 0.5g, distilled water is settled to 1L.
Wherein said step (3) soak time is 5-10min.
Wherein said step (4) scavenging solution prescription is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na
2HPO
40.5-0.6g, CaCl
20.10-0.15g distilled water is settled to 1L.
It is that the liver cell suspension is sieved through two-layer gauze and two confluent monolayer cells that wherein said step (4) is filtered, and gauze and cell sieve all soak into scavenging solution, said cell sieve upper strata 100 orders, lower floor's 200 orders.
The prescription of the used several solns of separation and Culture liver primary cell of the present invention is seen table 1.
Table 1 perfusate, collagenase perfusate, scavenging solution formula table
Annotate: transfer to 7.2-7.4 to pH with hydrochloric acid, ddH
2O is settled to 1000ml, filtration sterilization.
Wherein, said step (5) is centrifugal to be that liver cell filtrating is added respectively in the centrifuge tube, 400rpm, and 3min abandons supernatant, recentrifuge 500rpm, 3min abandons supernatant.In an embodiment of the present invention, used centrifuge tube specification is 50ml.
Its prescription of perfect medium is in the wherein said step (5): the DMEM substratum interpolation final concentration that contains 20% foetal calf serum (FBS) is a 200-600U/ml penicillium mould; 20-60 μ g/ml Streptomycin sulphate; The 1%-2% DMSO 99.8MIN., 1-10mg/L Regular Insulin, 1-10mmol/L nicotinamide; 0.1-1mmol/L xitix is with the NaHCO of 1M
3Regulate pH to 7.2-7.4.
The resuspended culture condition of wherein said step (5) is for processing 5 * 10
5The cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.
DMSO can let liver cell stablize and duplicate, and keeps its specific function, suppresses the nonparenchymal cell hypertrophy; Regular Insulin can promote cell proliferation and differentiation; Nicotinamide impels the differentiation of liver cell quick clone hyperplasia; Xitix can prolong the liver cell lifetime, keeps hepatocyte activity and function for a long time.Substratum among the present invention adopts above-mentioned additive and the FBS that adds high level to substitute expensive growth factor (like EGF, HGF etc.), can keep hepatocellular vigor and function equally, has also reduced cost simultaneously.
With the cell in the resuspended centrifuge tube of 10ml perfect medium, get 100 times of 0.05ml cell suspension dilutions and take out 0.5ml adding 1.5mlEP pipe, add 0.5ml0.4% trypan blue dye liquor again, dyeing 2min.Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.With the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and the tally.Mirror is observed down after leaving standstill 2min, and visible viable cell is full circle, and light transmission is good, and karyon is clear.Get any several visual field to the viable cell refusing to dye with dye navy blue dead cell counting, calculate motility rate, the result shows that the cell motility rate is greater than 95%.Count plate four big lattice TCSs are counted according to following formula: the big lattice cell count of cell count/ml=4 * 10
4* extension rate/4.Process 5 * 10 after the cell counting
5The cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.Change liquid behind the 4h and remove dead cell and attached cell not, cell state is good behind the 20h, obtains can be used for the pig liver primary cell of subsequent experimental research.
The observation liver cell is rounded down for separation and Culture liver primary cell mirror of the present invention, and shape is full, and is bright, and karyon is clear, and the nonparenchymal cell pollution rate is low, and cell debris pollutes few with other nonparenchymal cells.The yield of method separation and Culture liver primary cell of the present invention is 6.5 * 10
7, the liver cell motility rate is 95%-99%, and the cultivation adherent rate is 95%-99%, and purity is 90%-95%, and liver cell each item that more traditional perfusion method obtains all is significantly improved.
The method of separation and Culture liver primary cell provided by the invention is compared with four traditional step perfusion methods; Save the operation of changing perfusate for twice, save the gradient centrifugation process and remove heteroproteose cell, adopt when separating and remove hemocyte completely with perfusate; Thoroughly remove by filter the slower reticular tissue of digestion during the digestion liver cell; The centrifugal in short-term fibrocyte that is reduced to of low speed promptly obtains the higher liver primary cell of purity, and the inventive method is simple to operate, and reduces and pollute probability.Perfusate provided by the invention and scavenging solution prescription are simple, and the preparation starting material are prone to obtain, and do not use DNase in the scavenging solution, but adopt increasing scavenging solution consumption to wash intercellular adhesion open, practice thrift cost; Need not to use antithrombotics processing such as heparin sodium; Need not to use collagen or other matrix to encapsulate petridish, use the ordinary cells petridish can realize high adherent rate.Operation is simple for the inventive method, and cost is low, and good stability can be widely used in the research of the extensive isolating hepatocytes of biological field, for researchs such as Transplanted cells and drug screening provide high-quality liver cell.
Description of drawings
Fig. 1 is a fresh separated sucking pig liver cell under 4 * mirror.
Fig. 2 is a fresh separated sucking pig liver cell under 10 * mirror.
Fig. 3 is a fresh separated sucking pig liver cell under 20 * mirror.
Fig. 4 cultivates 24h sucking pig liver cell under 4 * mirror.
Fig. 5 cultivates 24h sucking pig liver cell under 10 * mirror.
Fig. 6 cultivates 24h sucking pig liver cell under 20 * mirror.
Fig. 7 cultivates 12h sucking pig liver cell under 4 * mirror.
Fig. 8 cultivates 12h sucking pig liver cell under 10 * mirror.
Fig. 9 cultivates 12h sucking pig liver cell under 20 * mirror.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, the conventional means that used technique means is well known to those skilled in the art among the embodiment, chemical reagent is commercially available except that specifying among the embodiment.
The separation of embodiment 1 primary hepatocyte
The used liver of present embodiment is the fresh pig liver of gathering from the slaughterhouse; Use the syringe THPV to pour into liver to 3 times of 4 ℃ of UW liquid of precooling (available from U.S. Dupont Critical Care company) with 80ml/min speed immediately after exsomatizing to liver volume; Make below the rapid uniform decrease in temperature to 10 of liver ℃, again liver is put into and be transported to the laboratory within ice chest (keeping about 2 ℃) 3 hours and carry out subsequent operations.
Get the liver about 100g, (the 1L prescription is: NaCl 9.397g, KCl 0.235g with 37 ℃ of perfusates of constant temperature earlier from hepatic vein to start peristaltic pump; HEPES 2.383g, distilled water is settled to 1L, pH value 7.3) the open perfusion 12min of flow velocity 60ml/min; With identical speed perfusion 0.05%IV Collagen Type VI enzyme (available from sigma company) solution, its 1L prescription of this collagenase solution is NaCl 9.397g, KCl 0.235g then; HEPES 2.383g, Na
2HPO
40.504g, CaCl
20.1g, IV Collagen Type VI enzyme 0.5g, distilled water is settled to 1L, and the hepatic tissue under the about 3min, coating is the turtleback back of splitting and finishes perfusion.Take off rapidly that digestion is ripe to be the liver that turtleback splits to liver surface and to place another aseptic plate, be soaked in the 0.05%IV Collagen Type VI enzyme solution.Cut several livers down towards periphery by the liver center, passivity is torn Glisson's capsule, lets liver in 100ml 0.05%IV Collagen Type VI enzyme, continue the about 7min of digestion.
Treat that liver digests " mud shape ", add the 500ml scavenging solution, the prescription of scavenging solution of the present invention is NaCl 9.397g, KCl 0.235g, HEPES 2.383g, Na
2HPO
40.504g, CaCl
20.1g distilled water is settled to 1L.Stop digestion, good liver cell suspension passes through gauze and the moistening cell sieve (upper strata 100 orders, lower floor's 200 orders) of two-layer scavenging solution that two-layer scavenging solution soaked into to let digestion.Change the suspension after filtering respectively in some 50ml centrifuge tubes again, 400rpm, 3min abandons supernatant, and adding with the precipitation volume ratio is 8: 1 scavenging solution, recentrifuge 500rpm, 3min abandons supernatant, obtains isolating liver primary cell.
The separation of embodiment 2 primary hepatocytes
The used liver of present embodiment is the fresh animal liver of gathering from the slaughterhouse; Use the syringe THPV to pour into liver to 4 times of 4 ℃ of UW liquid of precooling (available from U.S. Dupont Critical Care company) with 100ml/min speed immediately after exsomatizing to liver volume; Make below the rapid uniform decrease in temperature to 10 of liver ℃, again liver is put into and be transported to the laboratory within ice chest (keeping about 1 ℃) 3 hours and carry out subsequent operations.
Get the liver about 100g, (the 1L prescription is: NaCl 9g, KCl 0.2g with 37 ℃ of perfusates of constant temperature earlier from hepatic vein to start peristaltic pump; HEPES 4.8g, distilled water is settled to 1L, pH value 7.2) the open perfusion 15min of flow velocity 55ml/min; With identical speed perfusion 0.05%II Collagen Type VI enzyme (available from sigma company) solution, its 1L prescription of this collagenase solution is NaCl9g, KCl 0.2g then; HEPES 4.8g, Na
2HPO
40.6g, CaCl
20.14g, II Collagen Type VI enzyme 0.05g, distilled water is settled to 1L, and the hepatic tissue under the about 3min, coating is the turtleback back of splitting and finishes perfusion.Take off rapidly that digestion is ripe to be the liver that turtleback splits to liver surface and to place another aseptic plate, be soaked in the 0.05%II Collagen Type VI enzyme solution.Cut several livers down towards periphery by the liver center, passivity is torn Glisson's capsule, lets liver in 100ml 0.05%II Collagen Type VI enzyme, continue the about 10min of digestion.
Treat that liver digests " mud shape ", add the 600ml scavenging solution, the prescription of scavenging solution of the present invention is NaCl 9g, KCl 0.2g, HEPES 4.8g, Na
2HPO
40.6g, CaCl
20.14g distilled water is settled to 1L.Stop digestion, good liver cell suspension passes through gauze and the moistening cell sieve (upper strata 100 orders, lower floor's 200 orders) of two-layer scavenging solution that two-layer scavenging solution soaked into to let digestion.Change the suspension after filtering respectively in some 50ml centrifuge tubes again, 400rpm, 3min abandons supernatant, and adding with the precipitation volume ratio is 7: 1 scavenging solution, recentrifuge 500rpm, 3min abandons supernatant, obtains isolating liver primary cell.
The separation of embodiment 3 primary hepatocytes
Present embodiment experiment sucking pig is purebred Large White, plants a pig farm from Beijing along new swelling.Be prepared in advance 37 ℃ of water-baths and peristaltic pump (ALC-B6 type constant flow pump, Shanghai Alcott bio tech ltd) are with aseptic PBS emptying pipe bubble.Gather the liver of sucking pig through following method, working method is: behind the 5 ages in days experiment sucking pig fasting 12h, adopt 3% vetanarcol (sterile saline preparation) intraperitoneal injection of anesthesia (1.6ml/kg), clean suckling pig skin with warm water, and soak 75% alcohol 5min.Behind the sucking pig of Baoding, T type mouth is opened abdomen, exposes and separation hepatic vein and postcava, and threading is subsequent use.On portal vein, cut an osculum, insert No. 16 and irritate the stomach pin, bulldog clamp is fixed.The ligation hepatic artery, stomach left and right sides vein, splenic vein, mesenteric vein.Cut off the liver ligament, other blood vessels and mesentery also are transferred to liver in the aseptic plate.
Cut off postcava immediately after starting peristaltic pump.(the 1L prescription is: NaCl 8g, KCl 0.4g, HEPES2.4g with 37 ℃ of perfusates of constant temperature earlier; Distilled water is settled to 1L, pH value 7.4) the open perfusion 10min of flow velocity 50ml/min, then with identical speed perfusion 0.05%IV Collagen Type VI enzyme solution available from sigma company; (the 1L prescription is collagenase solution: NaCl 8g; KCl 0.4g, HEPES 2.4g, Na
2HPO
40.6g, CaCl
20.1g IV Collagen Type VI enzyme 0.5g, distilled water is settled to 1L), the hepatic tissue under the about 3min, coating is the turtleback back of splitting and finishes perfusion.Take off rapidly that digestion is ripe to be the liver that turtleback splits to liver surface and to place another aseptic plate, be soaked in the 0.05%IV Collagen Type VI enzyme solution.Cut several livers down towards periphery by the liver center, passivity is torn Glisson's capsule, lets liver in 100ml 0.05%IV Collagen Type VI enzyme, continue the about 5-10min of digestion.
Treat that liver digests " mud shape ", (the 1L prescription is: NaCl 8g, KCl 0.4g, HEPES 2.4g, Na to add the 400ml scavenging solution
2HPO
40.6g, CaCl
20.1g distilled water is settled to 1L), stop digestion, good liver cell suspension passes through gauze and the moistening cell sieve (upper strata 100 orders, lower floor's 200 orders) of two-layer scavenging solution that two-layer scavenging solution soaked into to let digestion.Change the suspension after filtering respectively in some 50ml centrifuge tubes again, 400rpm, 3min abandons supernatant, and adding with the precipitation volume ratio is 5: 1 scavenging solution, recentrifuge 500rpm, 3min abandons supernatant, and deposition is the liver primary cell.
The cultivation of embodiment 4 primary hepatocytes
Resuspended with perfect medium.Perfect medium is that the DMEM substratum of 20%FBS has added 600U/ml penicillium mould among the present invention, 20 μ g/ml Streptomycin sulphates, and 2% DMSO 99.8MIN., 10mg/L Regular Insulin, the 10mmol/L nicotinamide, the 1mmol/L xitix is in addition with the NaHCO of 1M
3Regulate pH to 7.2.
Separate the liver primary cell that obtains with the resuspended embodiment 1,2,3 of 10ml perfect medium respectively, get 100 times of 0.05ml cell suspension dilutions and take out 0.5ml adding 1.5mlEP pipe, add 0.5ml 0.4% trypan blue dye liquor again, dyeing 2min.Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.With the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and the tally.Mirror is observed down after leaving standstill 2min, and visible viable cell is full circle, and light transmission is good, and karyon is clear.Get any several visual field to the viable cell refusing to dye with dye navy blue dead cell counting, calculate motility rate, the result shows that the cell motility rate is all greater than 95%.Count plate four big lattice TCSs are counted according to following formula: the big lattice cell count of cell count/ml=4 * 10
4* extension rate/4.Be to process 5 * 10 at 250: 1 according to substratum and precipitation volume ratio after the cell counting
5The cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.Change liquid behind the 4h and remove dead cell and attached cell not, cell state is good behind the 20h, and dikaryocyte is island and connects, and can be used for experiment.
The present invention compares as shown in table 2 below with the liver cell that traditional perfusion method (seeing accompanying reference 1-3) obtains:
Table 2
| Perfusion method of the present invention | The tradition perfusion method [1-3] | |
| Yield (individual/the gram hepatic tissue) | 6.5×10 7 | 1.25×10 7 |
| Motility rate (%) | 99 | 80-90 |
| Adherent rate (%) | 98 | 80-90 |
| Purity (%) | 93 | 80-90 |
Cultivated back 0 hour, 24 hours 4 times of mirrors, cell growth state is seen Fig. 1-6 under 10 times of mirrors and the 20 times of mirrors.
The observation liver cell is rounded down for mirror, and shape is full, and is bright, and karyon is clear, and the nonparenchymal cell pollution rate is low, and cell debris pollutes few with other nonparenchymal cells.Each item index of the liver primary cell of separation and Culture all is significantly higher than traditional perfusion method.Operation is simple for the inventive method, and cost is low, and good stability can be widely used in the research of the extensive isolating hepatocytes of biological field, for researchs such as Transplanted cells and drug screening provide high-quality liver cell.(reference: [1] P.O.Seglen.Preparation of rat liver cells:I.Effect of Ca
2+On enzymatic dispersion of isolated, perfused liver [J] Experimental Cell Research, 1972,74 (2), 450-454.
[2]P.O.Seglen.Preparation?of?rat?liver?cells：II.Effects?of?ions?and?chelators?on?tissue?dispersion.Experimental?Cell?Research，1973，76(1)，25-30.
[3]P.O.Seglen.Preparation?of?rat?liver?cells：III.Enzymatic?requirements?for?tissue?dispersion.Experimental?Cell?Research，1973，82(2)，391-398.)
The cultivation of embodiment 5 primary hepatocytes
Resuspended with perfect medium.Perfect medium is that the DMEM substratum of 20%FBS has added 200U/ml penicillium mould among the present invention, 60 μ g/ml Streptomycin sulphates, and 1% DMSO 99.8MIN., 1mg/L Regular Insulin, the 1mmol/L nicotinamide, the 0.1mmol/L xitix is in addition with the NaHCO of 1M
3Regulate pH to 7.4.
Separate the liver primary cell that obtains with the resuspended embodiment 1,2,3 of 10ml perfect medium respectively, get 100 times of 0.05ml cell suspension dilutions and take out 0.5ml adding 1.5mlEP pipe, add 0.5ml 0.4% trypan blue dye liquor again, dyeing 2min.Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.With the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and the tally.Mirror is observed down after leaving standstill 2min, and visible viable cell is full circle, and light transmission is good, and karyon is clear.Get any several visual field to the viable cell refusing to dye with dye navy blue dead cell counting, calculate motility rate, the result shows that the cell motility rate is all greater than 95%.Count plate four big lattice TCSs are counted according to following formula: the big lattice cell count of cell count/ml=4 * 10
4* extension rate/4.Be to process 5 * 10 at 300: 1 according to substratum and precipitation volume ratio after the cell counting
5The cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.Change liquid behind the 4h and remove dead cell and attached cell not, cell state is good behind the 20h, and dikaryocyte is island and connects, and can be used for experiment.
The present invention compares as shown in table 3 below with the liver cell that traditional perfusion method obtains:
Table 3
| Perfusion method of the present invention | The tradition perfusion method [1-3] | |
| Yield (individual/the gram hepatic tissue) | 6.5×10 7 | 1.25×10 7 |
| Motility rate (%) | 99 | 80-90 |
| Adherent rate (%) | 95 | 80-90 |
| Purity (%) | 95 | 80-90 |
Cultivate back 12 hours 4 times of mirrors, cell growth state is seen Fig. 7-9 under 10 times of mirrors and the 20 times of mirrors.
The observation liver cell is rounded down for mirror, and shape is full, and is bright, and karyon is clear, and the nonparenchymal cell pollution rate is low, and cell debris pollutes few with other nonparenchymal cells.Hepatocellular yield, motility rate, adherent rate and purity all are significantly higher than traditional perfusion method.Operation is simple for the inventive method, and cost is low, and good stability can be widely used in the research of the extensive isolating hepatocytes of biological field, for researchs such as Transplanted cells and drug screening provide high-quality liver cell.
Embodiment 6 effect test that goes down to posterity
When reaching 80-90%, liver primary cell degree of converging to go down to posterity.With the 10cm Tissue Culture Dish is example, siphons away substratum, cleans with 1ml PBS (believing because of China available from Beijing button); 3 times repeatedly, add 2ml 0.05% pancreatin, 37 ℃ of digestion 1min; Add 2ml perfect medium (prescription is with embodiment 4) and stop digestion, blow and beat gently, make the cell detachment on the petridish wall with pipettor; What form cell suspension changes centrifuge tube, 800rpm, centrifugal 5min over to.Remove supernatant, add the resuspended back of 2ml perfect medium and add 1ml toward the 10cm Tissue Culture Dish that each fills 9ml preheating perfect medium.Behind the 4h, mirror is observed down the passage cell adherent rate and is reached more than 99.5%, and passage cell degree of converging reaches 80-90% and can go down to posterity again behind the 60-72h.To the observation in 10 generations of going down to posterity, liver cell does not have the aging death phenomenon, and form is normal, and the speed of growth and state are all normal.
Embodiment 7 frozen and resuscitation effect tests
Frozen: plan frozen preceding 12h and change fresh perfect medium into, cell is long can be frozen to degree of converging 80%.Preparation frozen storing liquid: 50%DMEM+40%FBS+ is two to be resisted+1mg/L Regular Insulin+10%DMSO, and 4 ℃ of precoolings are subsequent use.Cell dissociation collect the back with the frozen storing liquid of precooling slowly re-suspended cell to concentration be about 5 * 10
6/ ml puts into and is placed on Virahol cell cryopreservation box (available from U.S. Nalgene company) behind the frozen pipe and places-80 ℃ of refrigerators, is transferred to liquid nitrogen container behind the 16-18h and forever preserves.
Recovery: preheating water bath to 40 ℃.From liquid nitrogen container, take out the frozen pipe of preserving the liver primary cell, place warm water and constantly stirring rapidly, make it within 1 minute, to melt.Open frozen pipe in the ultra-clean box, cell suspension is slowly splashed in the centrifuge tube that fills the 5ml perfect medium.Centrifugal 5 minutes of 800rpm abandons supernatant.Every pipe deposition adds the 10ml perfect medium, and piping and druming is even, and to the 10cm petridish, 37 ℃ of cultivations are observed adherent rate more than 95% behind the 4h with cell transfer, and the liver cell shape is full, and growth conditions is good.
The liver primary cell of experimental result explanation separation and Culture of the present invention is after frozen, recovery, and its liver cell shape is full, and growth conditions is good, and good stability can continue to be applied in the experiments of field of biology.
Claims (10)
1. the method for a separation and Culture liver primary cell comprises following steps:
1) from the hepatic vein input perfusate of the liver that exsomatizes, described perfusate, its 1L are filled a prescription and are: NaCl 8-9.4g, and KCl 0.2-0.4, HEPES 2.4-4.8g, distilled water is settled to 1L, pH value 7.2-7.4;
2) after input collagenase perfusate, hepatic tissue are turtleback and split, stop perfusion;
3) will digest sophisticated liver is soaked in the collagenase perfusate;
4) add scavenging solution and stop digestion, filter, collect hepatocyte suspension;
5) centrifugal, abandon supernatant, with the resuspended cultivation of perfect medium.
2. the method for claim 1 is characterized in that, the liver of said step 1) is the stripped liver of fresh collection; Or fresh in vitro liver THPV is with 0-4 ℃ of precooling UW liquid of 80-100ml/min speed injection liver volume more than 3 times, below the liver temperature drop to 10 ℃, keeps the liver within the 1-4 ℃ of stripped 3h.
3. the method for claim 1 is characterized in that, said step 1) is with the open perfusion 10-15min of 37 ℃ of perfusates of constant temperature, flow velocity 50-60ml/min.
4. the method for claim 1 is characterized in that, said step 2) collagenase perfusate perfusion flow velocity 50-60ml/min.
5. method as claimed in claim 4 is characterized in that, said step 2) the collagenase perfusate, its 1L prescription is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na
2HPO
40.5-0.6g, CaCl
20.10-0.15g, II type or IV Collagen Type VI enzyme 0.5g, distilled water is settled to 1L.
6. the method for claim 1 is characterized in that, said step 3) soak time is 5-10min.
7. the method for claim 1 is characterized in that, said step 4) scavenging solution prescription is NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, Na
2HPO
40.5-0.6g, CaCl
20.10-0.15g distilled water is settled to 1L.
8. the method for claim 1 is characterized in that, it is that the liver cell suspension is sieved through two-layer gauze and two confluent monolayer cells that said step 4) is filtered, and gauze and cell sieve all soak into scavenging solution, said cell sieve upper strata 100 orders, lower floor's 200 orders.
9. the method for claim 1 is characterized in that, said step 5) is centrifugal to be that liver cell filtrating is added in the centrifuge tube 400rpm respectively; 3min abandons supernatant, presses and sedimentary volume ratio 5-8: 1 adds the described scavenging solution of claim 7; Recentrifuge 500rpm, 3min abandons supernatant.
10. the method for claim 1 is characterized in that, its prescription of perfect medium is in the said step 5): the DMEM substratum interpolation final concentration that contains 20%FBS is a 200-600U/ml penicillium mould; 20-60 μ g/ml Streptomycin sulphate; The 1%-2% DMSO 99.8MIN., 1-10mg/L Regular Insulin, 1-10mmol/L nicotinamide; 0.1-1mmol/L xitix is with the NaHCO of 1M
3Regulate pH to 7.2-7.4.
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| CN104894056A (en) * | 2015-06-19 | 2015-09-09 | 中国长江三峡集团公司中华鲟研究所 | Method for constructing spleen tissue cell line of acipenser dabryanus |
| CN104946528A (en) * | 2015-07-16 | 2015-09-30 | 中南大学湘雅医院 | Primary hepatic tissue cell separation system and method |
| CN105010307A (en) * | 2015-07-08 | 2015-11-04 | 中国检验检疫科学研究院 | Cryopreservation solution and cryopreservation resuscitation method for liver primary cells |
| CN105420181A (en) * | 2015-12-09 | 2016-03-23 | 南方医科大学南方医院 | Method for separating in-vitro mesenchymal cells |
| CN106834210A (en) * | 2017-02-21 | 2017-06-13 | 广州柏赛柯生物技术有限公司 | A kind of liver primary cell method for separating and preparing |
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| CN110331128A (en) * | 2019-07-16 | 2019-10-15 | 新疆医科大学第一附属医院 | The separation method of liver cell and hepatic stellate cells in a kind of Alveolar echincoccus animal |
| CN110724661A (en) * | 2019-07-17 | 2020-01-24 | 湖南农业大学 | Separation method of mouse primary hepatocytes, mouse primary hepatocytes prepared by separation method and application of mouse primary hepatocytes |
| CN110724663A (en) * | 2019-10-08 | 2020-01-24 | 王克强 | Isolated culture method of liver stem cells |
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| CN104894056B (en) * | 2015-06-19 | 2017-11-24 | 中国长江三峡集团公司中华鲟研究所 | A kind of construction method of acipenser dabryanus spleen tissue cell line |
| CN104894056A (en) * | 2015-06-19 | 2015-09-09 | 中国长江三峡集团公司中华鲟研究所 | Method for constructing spleen tissue cell line of acipenser dabryanus |
| CN105010307A (en) * | 2015-07-08 | 2015-11-04 | 中国检验检疫科学研究院 | Cryopreservation solution and cryopreservation resuscitation method for liver primary cells |
| CN104946528A (en) * | 2015-07-16 | 2015-09-30 | 中南大学湘雅医院 | Primary hepatic tissue cell separation system and method |
| CN105420181A (en) * | 2015-12-09 | 2016-03-23 | 南方医科大学南方医院 | Method for separating in-vitro mesenchymal cells |
| CN108070551A (en) * | 2016-11-15 | 2018-05-25 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of human primary hepatocyte |
| CN106834210A (en) * | 2017-02-21 | 2017-06-13 | 广州柏赛柯生物技术有限公司 | A kind of liver primary cell method for separating and preparing |
| CN109207429A (en) * | 2018-05-04 | 2019-01-15 | 窦科峰 | α -1,3- galactosyl transferase gene knock-out pig hepatic cell line of immortalization and its preparation method and application |
| CN109055305B (en) * | 2018-09-17 | 2021-03-26 | 河南科技大学 | Method for separating and extracting milk cow liver stem cells |
| CN109055305A (en) * | 2018-09-17 | 2018-12-21 | 河南科技大学 | A kind of separating and extracting process of milk cow liver stem cells |
| CN109161518A (en) * | 2018-10-10 | 2019-01-08 | 郑州大学第附属医院 | A method of being separately cultured primary hepatic cell |
| CN110331128A (en) * | 2019-07-16 | 2019-10-15 | 新疆医科大学第一附属医院 | The separation method of liver cell and hepatic stellate cells in a kind of Alveolar echincoccus animal |
| CN110724661A (en) * | 2019-07-17 | 2020-01-24 | 湖南农业大学 | Separation method of mouse primary hepatocytes, mouse primary hepatocytes prepared by separation method and application of mouse primary hepatocytes |
| CN110724663A (en) * | 2019-10-08 | 2020-01-24 | 王克强 | Isolated culture method of liver stem cells |
| 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 |
| CN111647549B (en) * | 2020-07-28 | 2023-08-11 | 内蒙古自治区农牧业科学院 | Method for separating single cells in animals and plants |
| CN114752549A (en) * | 2022-05-11 | 2022-07-15 | 中国农业科学院兰州畜牧与兽药研究所 | Sheep primary hepatocyte isolation culture method |
| CN114752549B (en) * | 2022-05-11 | 2022-11-25 | 中国农业科学院兰州畜牧与兽药研究所 | Sheep primary hepatocyte isolation culture method |
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