CN102634480B - Method for isolating and culturing liver primary cells - Google Patents
Method for isolating and culturing liver primary cells Download PDFInfo
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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, as a kind of external model, has its outstanding advantage aspect gene functional research: can obtain a large amount of proterties than the sample of homogeneous simultaneously; With situation in organism, substantially be consistent, can be in the situation that approach the expression of physiological status research gene; Get rid of many complicated factors and disturb, as interference neural, hormone; Primary hepatocyte has experiment in vitro circulation ratio preferably, substantially maintained the metabolic function of liver, particularly well retain the level with cytochrome P 450 enzymes consistent in body (cytochrome P450), substantially retained the original metabolic function of liver and cytodifferentiation state.
Liver cell (Hepatocytes) is the topmost parenchyma type of liver, under physiological situation, accounts for 60%, belongs to well differentiated multi-functional, parenchymal viscera cell.Liver cell can highly break up, and can enter the cell cycle very soon again and breeds, and in culture system, according to the difference of research purpose, the culture system of existing its propagation of support, also have the culture system of supporting its differentiation in vitro.After liver cell is in vitro, occur very soon that in unfavorable environment liver cell is old and feeble and lose function, the pH value is not suitable for, without the hormone support, causes necrocytosis, and the activation of gene causes apoptosis etc.Primary hepatocyte, under general culture condition, is just lost its gap in several hours and is connected, and loses its tissue specificity Metabolic activity in several days.Liver cell separation method commonly used in prior art is perfusion method, and traditional perfusion method operation link is many, and the time is long, and liver cell easily damages, and vigor is low, efficiency is low, and purity is not high.Therefore, the requirement of studying to meet cytobiology, tissue culture technique etc. in the urgent need to the pig liver primary cell that the preparation vitro culture flushes, function is good.
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 defect existed in above-mentioned prior art.
The method of separation and Culture liver primary cell provided by the invention comprises following steps:
(1) input perfusate from the hepatic vein of liver; Described perfusate, its 1L formula 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) input collagenase perfusion solution, after hepatic tissue is turtleback and splits, stop perfusion;
(3) will digest ripe liver is soaked in collagenase solution;
(4) add scavenging solution and stop digestion, filter, collect hepatocyte suspension;
(5) centrifugal, abandon supernatant, with the resuspended cultivation of perfect medium.
Described step 1) the in vitro liver that liver is 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 liver temperature drop to 10 ℃, keep the liver within 1-4 ℃ of in vitro 3h.After below liver temperature drop to 10 ℃, liver can be placed in to ice chest or other and can keep the container of 1-4 ℃ to preserve liver being no more than 3h.
The inventive method adopts peristaltic pump to carry out perfusion, with bubble in the emptying peripump tubing of aseptic PBS.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 formula 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 Collagenase Type 0.5g, distilled water is settled to 1L.
Wherein said step (3) soak time is 5-10min.
Wherein said step (4) scavenging solution formula 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.
Wherein said step (4) filtration is that the liver cell suspension is sieved by two-layer gauze and two confluent monolayer cells, and gauze and cell sieve all infiltrate with scavenging solution, and described cell sieves upper strata 100Mu， lower floor 200 orders.
The formula of separation and Culture liver primary cell of the present invention several solns used is in Table 1.
Table 1 perfusate, collagenase perfusate, scavenging solution formula table
Annotate: with hydrochloric acid, pH is adjusted to 7.2-7.4, ddH
2o is settled to 1000ml, filtration sterilization.
Wherein, described step (5) is centrifugal is that liver cell filtrate is added respectively in centrifuge tube, 400rpm, and 3min, abandon supernatant, recentrifuge 500rpm, 3min, abandon supernatant.In an embodiment of the present invention, centrifuge tube specification used is 50ml.
In wherein said step (5), its formula of perfect medium is: the DMEM substratum interpolation final concentration containing 20% foetal calf serum (FBS) is 200-600U/ml penicillin, 20-60 μ g/ml Streptomycin sulphate, the 1%-2% dimethyl sulfoxide (DMSO), 1-10mg/L Regular Insulin, the 1-10mmol/L nicotinamide, 0.1-1mmol/L xitix, with the NaHCO of 1M
3regulate pH to 7.2-7.4.
The resuspended culture condition of wherein said step (5) is for making 5 * 10
5the cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.
DMSO can allow liver cell stablize and copy, 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 extend the liver cell lifetime, keeps for a long time hepatocyte activity and function.Substratum in the present invention adopts above-mentioned additive and adds the FBS of high level to substitute expensive somatomedin (as 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 taking-up 0.5ml of 0.05ml cell suspension dilution and add the 1.5mlEP pipe, then add 0.5ml0.4% trypan blue dye liquor, dyeing 2min.Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.By the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and tally.Microscopic observation after standing 2min, 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 and dye navy blue dead cell counting, calculating motility rate, result shows, Cell viability is greater than 95%.The large lattice total cellular score of count plate four, counting according to the following formula: the large lattice cell count of cell count/ml=4 * 10
4* extension rate/4.Make 5 * 10 after cell counting
5the cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.Change liquid after 4h and remove dead cell and attached cell not, after 20h, cell state is good, obtains the pig liver primary cell that can be used for subsequent experimental research.
Separation and Culture liver primary cell Microscopic observation liver cell of the present invention is rounded, and shape is full, bright, and karyon is clear, and the nonparenchymal cell pollution rate is low, and cell debris and other nonparenchymal cells pollute few.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 the liver cell that more traditional perfusion method obtains is every all to be 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 twice replacing perfusate, save the gradient centrifugation process except heteroproteose cell, adopt while separating and remove thoroughly hemocyte with perfusate, thoroughly remove by filter the slower reticular tissue of digestion during the digestion liver cell, the low speed centrifugal fibrocyte that is reduced in short-term obtain the liver primary cell that purity is higher, and the inventive method is simple to operate, and reduce and pollute probability.Perfusate provided by the invention and scavenging solution formula are simple, and the preparation starting material easily obtain, and do not use DNase in scavenging solution, but adopt increasing scavenging solution consumption to wash intercellular adhesion open, cost-saving; Without antithrombotics such as using heparin sodium, process; Without using the coated culture dish of collagen or other matrix, use the ordinary cells culture dish 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 the researchs such as Transplanted cells and drug screening provide high-quality liver cell.
The accompanying drawing explanation
Fig. 1 is fresh separated Pup pig hepatocyte under 4 * mirror.
Fig. 2 is fresh separated Pup pig hepatocyte under 10 * mirror.
Fig. 3 is fresh separated Pup pig hepatocyte under 20 * mirror.
Fig. 4 cultivates the 24h Pup pig hepatocyte under 4 * mirror.
Fig. 5 cultivates the 24h Pup pig hepatocyte under 10 * mirror.
Fig. 6 cultivates the 24h Pup pig hepatocyte under 20 * mirror.
Fig. 7 cultivates the 12h Pup pig hepatocyte under 4 * mirror.
Fig. 8 cultivates the 12h Pup pig hepatocyte under 10 * mirror.
Fig. 9 cultivates the 12h Pup pig hepatocyte under 20 * mirror.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, in embodiment, chemical reagent, except special instruction, is commercially available.
The separation of embodiment 1 primary hepatocyte
The present embodiment liver used is the fresh pig liver gathered from slaughterhouse, after in vitro, use immediately the syringe THPV that 4 ℃ of UW liquid of 3 times of precoolings to liver volume (purchased from U.S. Dupont Critical Care company) are poured into to liver with 80ml/min speed, make below the rapid uniform decrease in temperature to 10 of liver ℃, then liver is put into and is transported to laboratory within ice chest (keeping 2 ℃ of left and right) 3 hours and carries out subsequent operations.
Get the liver of 100g left and right, starting peristaltic pump first take 37 ℃ of perfusates of constant temperature from hepatic vein (the 1L formula is NaCl 9.397g, KCl 0.235g, HEPES 2.383g, distilled water is settled to 1L, pH value 7.3) the open perfusion 12min of flow velocity 60ml/min, then with identical speed perfusion 0.05%IV Collagenase Type (purchased from sigma company) solution, its 1L formula of this collagenase solution is NaCl 9.397g, KCl 0.235g, HEPES 2.383g, Na
2hPO
40.504g, CaCl
20.1g, IV Collagenase Type 0.5g, distilled water is settled to 1L, about 3min, the hepatic tissue under coating is the turtleback rear end perfusion that splits.Take off rapidly that digestion is ripe to be to liver surface the liver that turtleback splits and to be placed in another aseptic plate, be soaked in 0.05%IV Collagenase Type solution.Cut towards periphery several lower livers by the liver center, passivity is torn Glisson's capsule, allows liver continue the about 7min of digestion in 100ml 0.05%IV Collagenase Type.
Treat that liver digests " mud shape ", add the 500ml scavenging solution, the formula 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, allow the liver cell suspension digested pass through gauze and the moistening cell sieve (upper strata 100Mu， lower floor 200 orders) of two-layer scavenging solution that two-layer scavenging solution infiltrated.Again the suspension after filtering is proceeded to respectively in some 50ml centrifuge tubes, 400rpm, 3min, abandon supernatant, the scavenging solution that to add with the precipitation volume ratio be 8: 1, recentrifuge 500rpm, 3min, abandon supernatant, obtains the liver primary cell separated.
The separation of embodiment 2 primary hepatocytes
The present embodiment liver used is the fresh animal liver gathered from slaughterhouse, after in vitro, use immediately the syringe THPV that 4 ℃ of UW liquid of 4 times of precoolings to liver volume (purchased from U.S. Dupont Critical Care company) are poured into to liver with 100ml/min speed, make below the rapid uniform decrease in temperature to 10 of liver ℃, then liver is put into and is transported to laboratory within ice chest (keeping 1 ℃ of left and right) 3 hours and carries out subsequent operations.
Get the liver of 100g left and right, starting peristaltic pump first take 37 ℃ of perfusates of constant temperature from hepatic vein (the 1L formula is NaCl 9g, KCl 0.2g, HEPES 4.8g, distilled water is settled to 1L, pH value 7.2) the open perfusion 15min of flow velocity 55ml/min, then with identical speed perfusion 0.05%II Collagenase Type (purchased from sigma company) solution, its 1L formula of this collagenase solution is NaCl9g, KCl 0.2g, HEPES 4.8g, Na
2hPO
40.6g, CaCl
20.14g, II Collagenase Type 0.05g, distilled water is settled to 1L, about 3min, the hepatic tissue under coating is the turtleback rear end perfusion that splits.Take off rapidly that digestion is ripe to be to liver surface the liver that turtleback splits and to be placed in another aseptic plate, be soaked in 0.05%II Collagenase Type solution.Cut towards periphery several lower livers by the liver center, passivity is torn Glisson's capsule, allows liver continue the about 10min of digestion in 100ml 0.05%II Collagenase Type.
Treat that liver digests " mud shape ", add the 600ml scavenging solution, the formula 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, allow the liver cell suspension digested pass through gauze and the moistening cell sieve (upper strata 100Mu， lower floor 200 orders) of two-layer scavenging solution that two-layer scavenging solution infiltrated.Again the suspension after filtering is proceeded to respectively in some 50ml centrifuge tubes, 400rpm, 3min, abandon supernatant, the scavenging solution that to add with the precipitation volume ratio be 7: 1, recentrifuge 500rpm, 3min, abandon supernatant, obtains the liver primary cell separated.
The separation of embodiment 3 primary hepatocytes
The present embodiment experiment sucking pig is purebred Large White, from suitable new grand kind of pig farm, Beijing.Be prepared in advance 37 ℃ of water-baths and peristaltic pump (ALC-B6 type constant flow pump, Shanghai Alcott bio tech ltd), with aseptic PBS emptying pipe bubble.Gather by the following method the liver of sucking pig, working method is: after 5 age 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.After the sucking pig of Baoding, open abdomen for T-shaped mouthful, expose and separate hepatic vein and postcava, threading is standby.Cut an osculum on portal vein, insert the gavage pin No. 16, bulldog clamp is fixed.The ligation hepatic artery, stomach left and right vein, splenic vein, mesenteric vein.Cut off the liver ligament, other blood vessels and mesentery and by hepatic metastasis in aseptic plate.
Cut off immediately postcava after starting peristaltic pump.(the 1L formula is NaCl 8g first to take 37 ℃ of perfusates of constant temperature, KCl 0.4g, HEPES2.4g, 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 Collagenase Type solution purchased from sigma company, (the 1L formula is collagenase solution: NaCl 8g, KCl 0.4g, HEPES 2.4g, Na
2hPO
40.6g, CaCl
20.1g IV Collagenase Type 0.5g, distilled water is settled to 1L), about 3min, the hepatic tissue under coating is the turtleback rear end perfusion that splits.Take off rapidly that digestion is ripe to be to liver surface the liver that turtleback splits and to be placed in another aseptic plate, be soaked in 0.05%IV Collagenase Type solution.Cut towards periphery several lower livers by the liver center, passivity is torn Glisson's capsule, allows liver continue the about 5-10min of digestion in 100ml 0.05%IV Collagenase Type.
Treat that liver digests " mud shape ", (the 1L formula 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, allow the liver cell suspension digested pass through gauze and the moistening cell sieve (upper strata 100Mu， lower floor 200 orders) of two-layer scavenging solution that two-layer scavenging solution infiltrated.Again the suspension after filtering is proceeded to respectively in some 50ml centrifuge tubes, 400rpm, 3min, abandon supernatant, the scavenging solution that to add with the precipitation volume ratio be 5: 1, recentrifuge 500rpm, 3min, abandon supernatant, and precipitation is the liver primary cell.
The cultivation of embodiment 4 primary hepatocytes
Resuspended with perfect medium.The DMEM substratum that in the present invention, perfect medium is 20%FBS has added 600U/ml penicillin, 20 μ g/ml Streptomycin sulphates, and 2% dimethyl sulfoxide (DMSO), 10mg/L Regular Insulin, the 10mmol/L nicotinamide, the 1mmol/L xitix, separately use the NaHCO of 1M
3regulate pH to 7.2.
Separate with the resuspended embodiment 1,2,3 of 10ml perfect medium the liver primary cell obtained respectively, get 100 times of taking-up 0.5ml of 0.05ml cell suspension dilution and add the 1.5mlEP pipe, then add 0.5ml 0.4% trypan blue dye liquor, dyeing 2min.Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.By the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and tally.Microscopic observation after standing 2min, 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 and dye navy blue dead cell counting, calculating motility rate, result shows, Cell viability all is greater than 95%.The large lattice total cellular score of count plate four, counting according to the following formula: the large lattice cell count of cell count/ml=4 * 10
4* extension rate/4.After cell counting, according to substratum, with the precipitation volume ratio, be to make 5 * 10 at 250: 1
5the cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.Change liquid after 4h and remove dead cell and attached cell not, after 20h, cell state is good, 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/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 |
Cultivate latter 0 hour, 24 hours 4 times of mirrors, under 10 times of mirrors and 20 times of mirrors, cell growth state is shown in Fig. 1-6.
The Microscopic observation liver cell is rounded, and shape is full, bright, and karyon is clear, and the nonparenchymal cell pollution rate is low, and cell debris and other nonparenchymal cells pollute few.The indices 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 the 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.The DMEM substratum that in the present invention, perfect medium is 20%FBS has added 200U/ml penicillin, 60 μ g/ml Streptomycin sulphates, and 1% dimethyl sulfoxide (DMSO), 1mg/L Regular Insulin, the 1mmol/L nicotinamide, the 0.1mmol/L xitix, separately use the NaHCO of 1M
3regulate pH to 7.4.
Separate with the resuspended embodiment 1,2,3 of 10ml perfect medium the liver primary cell obtained respectively, get 100 times of taking-up 0.5ml of 0.05ml cell suspension dilution and add the 1.5mlEP pipe, then add 0.5ml 0.4% trypan blue dye liquor, dyeing 2min.Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.By the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and tally.Microscopic observation after standing 2min, 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 and dye navy blue dead cell counting, calculating motility rate, result shows, Cell viability all is greater than 95%.The large lattice total cellular score of count plate four, counting according to the following formula: the large lattice cell count of cell count/ml=4 * 10
4* extension rate/4.After cell counting, according to substratum, with the precipitation volume ratio, be to make 5 * 10 at 300: 1
5the cell suspension of/ml is cultivated in 37 ℃, 5% CO2gas incubator.Change liquid after 4h and remove dead cell and attached cell not, after 20h, cell state is good, 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/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 rear 12 hours 4 times of mirrors, under 10 times of mirrors and 20 times of mirrors, cell growth state is shown in Fig. 7-9.
The Microscopic observation liver cell is rounded, and shape is full, bright, and karyon is clear, and the nonparenchymal cell pollution rate is low, and cell debris and other nonparenchymal cells pollute few.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 the 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 can go down to posterity.Take the 10cm Tissue Culture Dish as example, siphon away substratum, clean 3 times repeatedly with 1ml PBS (believing because of China purchased from Beijing button), add 2ml 0.05% pancreatin, 37 ℃ of digestion 1min, add 2ml perfect medium (formula is with embodiment 4) to stop digestion, with pipettor, blows and beats gently, make the cell detachment on the culture dish wall, what form cell suspension proceeds to centrifuge tube, 800rpm, centrifugal 5min.Remove supernatant, add the resuspended rear 10cm Tissue Culture Dish that fills 9ml preheating perfect medium toward each of 2ml perfect medium to add 1ml.After 4h, Microscopic observation passage cell adherent rate reaches more than 99.5%, and after 60-72h, passage cell degree of converging reaches 80-90% and can go down to posterity again.To the observation in 10 generations of going down to posterity, liver cell is without the aging death phenomenon, and form is normal, and the speed of growth and state are all normal.
The test of the frozen and resuscitation effect of embodiment 7
Frozen: plan frozen front 12h and change fresh perfect medium into, cell grows to degree of converging 80% can be frozen.Two resist+1mg/L Regular Insulin+the 10%DMSO of preparation frozen storing liquid: 50%DMEM+40%FBS+, 4 ℃ of pre-cold standbies.The rear frozen storing liquid with precooling of cell dissociation collection re-suspended cell to concentration slowly is about 5 * 10
6/ ml, put into after cryopreservation tube and be placed on Virahol cell cryopreservation box (purchased from U.S. Nalgene company) and be placed in-80 ℃ of refrigerators, is transferred to the liquid nitrogen container persistence after 16-18h.
Recovery: preheating water bath to 40 ℃.Take out the cryopreservation tube of preserving the liver primary cell from liquid nitrogen container, be placed in rapidly warm water and constantly stir, make it to melt within 1 minute.Open cryopreservation tube in ultra-clean box, cell suspension is slowly splashed in the centrifuge tube that fills the 5ml perfect medium.Centrifugal 5 minutes of 800rpm, abandon supernatant.Every pipe precipitation adds the 10ml perfect medium, and piping and druming evenly, is transferred to cell in the 10cm culture dish, and 37 ℃ of cultivations are observed adherent rate more than 95% after 4h, 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 every experiment of field of biology.
Claims (1)
1. the method for a separation and Culture liver primary cell comprises following steps:
1) input perfusate from the hepatic vein of in vitro liver, described perfusate, its 1L formula is: NaCl 8-9.4g, KCl 0.2-0.4g, HEPES 2.4-4.8g, distilled water is settled to 1L, pH value 7.2-7.4;
Wherein in step 1), described in vitro liver is: fresh in vitro liver THPV is with the 0-4 more than 3 times ℃ of precooling UW liquid of 80-100ml/min speed injection liver volume, after below liver temperature drop to 10 ℃, liver is placed in to ice chest and keeps 1-4 ℃, the shelf time is no more than 3h;
Wherein perfusion described in step 1) is with the open perfusion 10-15min of 37 ℃ of perfusates of constant temperature, flow velocity 50-60ml/min;
2) input collagenase perfusate, after hepatic tissue is turtleback and splits, stop perfusion; Described step 2) collagenase perfusate perfusion flow velocity 50-60ml/min;
Step 2 wherein) the collagenase perfusate in, its 1L formula 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 Collagenase Type 0.5g, distilled water is settled to 1L;
3) will digest ripe liver is soaked in the collagenase perfusate; Described step 3) soak time is 5-10min;
4) add scavenging solution and stop digestion, filter, collect hepatocyte suspension; Wherein said step 4) scavenging solution formula 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;
Described step 4) filtration is that the liver cell suspension is sieved by two-layer gauze and two confluent monolayer cells, and gauze and cell sieve all infiltrate with scavenging solution, and described cell sieves upper strata 100Mu， lower floor 200 orders;
5) centrifugal, abandon supernatant, with the resuspended cultivation of perfect medium;
Wherein, described step 5) is centrifugal is that liver cell filtrate is added in centrifuge tube, 400rpm, and 3min, abandon supernatant, by the volume ratio 5-8:1 with precipitation, adds scavenging solution, recentrifuge 500rpm, 3min, abandon supernatant;
Wherein, in described step 5), its formula of perfect medium is: the DMEM substratum interpolation final concentration containing 20%FBS is 200-600U/ml penicillin, 20-60 μ g/ml Streptomycin sulphate, the 1%-2% dimethyl sulfoxide (DMSO), 1-10mg/L Regular Insulin, the 1-10mmol/L nicotinamide, the 0.1-1mmol/L xitix, with the NaHCO of 1M
3regulate pH to 7.2-7.4.
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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 |
| 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 |
| CN106834210B (en) * | 2017-02-21 | 2021-01-26 | 广州柏赛柯生物技术有限公司 | Method for separating and preparing primary hepatic cells |
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| CN109055305B (en) * | 2018-09-17 | 2021-03-26 | 河南科技大学 | Method for separating and extracting 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 |
| 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 |
| 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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