CN102304492A - Primary culture method for liver cells of crucian carp - Google Patents
Primary culture method for liver cells of crucian carp Download PDFInfo
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Abstract
The invention provides a new primary culture method for liver cells of crucian carp, which mainly comprises the following steps: a, separating liver tissues from crucian carp; b, digesting cut liver tissues by using a stepwise digestion process and obtaining inner cell mass; and c, purifying and culturing obtained inner cell mass. In the method disclosed by the invention, the raw material is obtained from common crucian carp aquatic experimental animals, in-vitro tissue blocks are soaked in chlorhexidine gluconate (chlorhexidine) to be disinfected for reducing pollution. The tissues are digested by a stepwise digestion process, blood cell lysing solution is used to reduce the blood cell content in cells, and the liver cells are purified by percoll gradient centrifugation. The liver cells are purified by combined material preparation, the stepwise digestion process, blood cell lysing solution and percoll. The method can culture adequate liver cells, the survival rate of the liver cells is over 90 percent, and the method meets requirements of primary culture.
Description
Technical field
The present invention relates to the former breeding method of being commissioned to train of a kind of new crucian carp liver cell, belong to cytobiology and fields of biotechnology.
Background technology
Liver is the main detoxifcation organ of body, is the target organ of many poisonous substance damages, also is simultaneously the metabolic vitals of chemicals in the body.Environment complicated and changeable in the body is to causing that at body research chemicals hepar damnification brings certain degree of difficulty; The primary cultured cell isolated time is short; Great changes will take place as yet for biological character; To a certain extent can the reactant internal state; Utilize primary cultured cell Physiology and biochemistry and hereditary property stability, the toxicity and liver cell the replying and the mechanism of detoxifying of research chemicals to poisonous substance.
Fish are the vertebratess that live in the water body, are to be used for detecting the good biological indication body of water pollutant.The former foster liver cell of being commissioned to train keeps preferably and has kept hepatocellular integrity and metabolic activity, reactant intracellular metabolite situation truly.Simultaneously, liver cell is that the external source chemicals transform and metabolic important place, also is the main action target spot that allogenic material causes chemistry and biological damage.Therefore external liver cell is ideal models of research in vitro toxicity aspect research chemicals bio-transformation and the toxic action mechanism many advantages being arranged.The fish cultured cells has the incomparable advantage of live body fish as experimental subjects: (1) experiment material cost is low, and the maintenance of cell does not need a large amount of water, inflation and cultivation equipments of changing; (2) good reproducibility, experiment condition can accurately be controlled, and pair cell is furtherd investigate, and no matter aspect theory research, still aspect practical application, far reaching significance is arranged all.Crucian carp is the important food fish of China, occupies critical role in China aquatic products market, with crucian as experimental subjects, no matter with scientific research powerful pushing effect is arranged all on the market economy.
Liver parenchyma is main the composition by a large amount of liver cells, and it is few to contain a matter, and liver cell has multiple function, and metabolism is vigorous, and is high in external required condition." Chinese comparative medicine journal; 2006 14 3 phases of volume; In Xiphophorus helleri hepatic tissue blocking cultural method; That sterilization is adopted to tissue block is 70% alcohol immersion 30s; Experiment mentions that soak time should be strict controlled in the 30S; The long time can influence tissue activity, reduces cell attachment ability and survival rate.With respect to ethanol, Tubulicid is more suitable for being used for the sterilization of tissue block.
Though some separation are arranged and identify hepatocellular method at present, yet isolating liver cell purity is lower, active relatively low.This experiment improves, uses for reference existing cell culture processes, and desire provides good experimental model for research environment pollutes to hepatocellular murder by poisoning of crucian carp and intoxicating mechanism through setting up the liver cell culture method of higher degree.We find in modelling, from the separate tissue to the cell, obtain, and there are 3 key factors the centre: 1. sterile culture; 2. digestive process; 3. the adherent degree of cell growth, these three factors can determine the growth conditions of cell, affect carrying out of follow-up test.
Summary of the invention
The objective of the invention is in order in the former breeding method of being commissioned to train of crucian carp liver cell, to obtain purity and active relative higher liver cell.
On the basis that existing cell culture processes is improved, used for reference, the invention provides the former breeding method of being commissioned to train of a kind of crucian carp liver cell, it comprises following processing step:
A, from the crucian carp of health, separate hepatic tissue
At first cut off its gill portion arcus haemalis, sacrificed by exsanguination carries out disinfection with alcohol wipe crucian carp body surface, in Bechtop, dissects crucian carp, and takes out hepatic tissue, and isolating hepatic tissue is soaked in 10min in the glucose Tubulicid;
B, employing substep digestion method digest the acquisition cell mass to the hepatic tissue that shreds
With soaking tissue D-hank ' the s rinsing 2 times of glucose Tubulicid, shred into 2-3mm
3Tissue block; Adopt substep digestion method digestion tissue in order to improve the digestion effect:
Pour the tissue that shreds into centrifuge tube, insert in 28 ℃ the water-bath; Trypsinase and the EDTA digestion that at first adds tissue mass 4-5 times volume, during do not stop to shake up, after 15min digestion finishes, the centrifugal supernatant of abandoning.D-hank ' the s that adds 3ml with the bottom settlings mixing after the centrifugal supernatant of abandoning, residual trypsinase is removed with EDTA, stop digesting;
The type enzyme and the CaCl that in gained deposition tissue, add 3-4 times of volume
2Insert 28 ℃ of water-baths, during do not stop to shake up, every supernatant liquor of 3/4 of behind 10min, taking out moves into another centrifuge tube; In beaker, add new type enzyme and CaCl
2Continue the digestion tissue block;
After 30min digestion finishes, the supernatant liquor cell suspension of collecting is passed through 200 order bore diameter stainless steel filter screens, filter tissue block; The centrifugal supernatant of abandoning, the serum free medium that adds 3ml with the bottom settlings mixing after the centrifugal supernatant of abandoning, with residual type enzyme and CaCl
2Remove, stop digestion;
C. the cell mass that obtains is carried out purifying and cultivation
1. use erythrocyte cracked liquid to remove hemocyte: in centrifuge tube, to add 10ml D-hank ' s; Sedimentation cell group is dispersed into the individual cells suspension with pipettor piping and druming, adds the erythrocyte cracked liquid of 1ml simultaneously, blow and beat mixing gently; Turn upside down 6-8 time, leave standstill 5min; Centrifugally abandon red part, stay pipe end white cell mass; Add D-hank ' s with white cell mass mixing, piping and druming is also centrifugal, and the D-hank ' s that is dissolved in 3ml is made into hepatocyte suspension;
2. add Percoll reagent to isolating liver cell purifying: get hepatocyte suspension and be suspended on the Percoll parting liquid II of 2.5 times of volumes the centrifugal supernatant of abandoning; It is resuspended with the D-hank ' s of 5ml to get sedimentation cell group, rifle head piping and druming mixing; 1500rpm, 5min is centrifugal, abandons supernatant, obtains purifying liver cell later;
Wherein, described D-hank ' s solution is: with 8g NaCl, 0.4g KCl, 0.13g Na
2HPO
412H
20,0.06g KH
2PO4,0.35g NaHCO
3Constant volume is in the 1L pure water, and autoclave sterilization treats that temperature descends the back in 4 ℃ of preservations;
Wherein saidly be formulated as with Percoll parting liquid II: with Percoll reagent stoste and 10 * D-hank ' s proportional arrangement parting liquid I with 9:1, the parting liquid I that usefulness is configured to and 1 * D-hank ' s are configured to required parting liquid II by the volume ratio of 2:3 earlier;
Wherein the concentration of the glucose Tubulicid described in the step a is preferably 0.1%.
Wherein the trypsinase concentration described in the step b is preferably 0.25%; The working concentration of EDTA is preferably 0.02%; The volume ratio of trypsinase and EDTA is preferably 1:1.
Wherein the concentration of the type enzyme described in the step b is preferably 0.1%, CaCl
2Concentration 3mmol/L preferably.
Wherein the culture temperature of the described cultural method described in the step c is 28 ℃.
Wherein the substratum pH value of the cultural method described in the step c is 7-8.
Wherein centrifugal speed is 1000rmp in the said digestive process described in the step b, 5min.
Wherein centrifugal rotational speed is 1500rpm in the erythrocyte cracked liquid centrifugal process described in the step c, 5min.
Wherein the serum among the step c described in the step c is preferably used new-born calf serum; Described use substratum is: add earlier Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of 20% new-born calf serum, 100U/ml penicillin, 100U/ml with the DMEM/F12 substratum, change new-born calf serum, the 100U/ml penicillin of DMEM/F12 substratum adding 10%, Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of 100U/ml after 8 hours into.
Wherein, choose in a step when organizing and select near near the hepatic tissue the courage, the hepatic tissue at this place is bigger, and easy and pancreas separates, and avoids obscuring of other cells.Concrete dissection step is: opening with tweezers and see green courage, is large stretch of hepatic tissue around courage.With near the tissue the close courage of another tweezers tweezer.The hepatic tissue that exsomatizes is immersed in the glucose Tubulicid.
Wherein digestive ferment is preferably the digestion of tissue and the processing step of purifying among the step b:
(1) the glucose liquor hibitane is outwelled.Add D-hank ' s rinsing 2 times.
(2) with eye scissors the gained tissue is cut into about 2-3mm
3Tissue block.Digest in 28 ℃ the water-bath.
(3) trypsinase that adds 4-5 times of volume of tissue mass digests with EDTA, during do not stop to shake up.
(4) after 15min digestion finishes, 1000rpm, the centrifugal supernatant of abandoning of 5min.
(5) D-hank ' s that adds 3ml with the bottom settlings mixing after 1000rpm, 5min is centrifugal, and residual trypsinase and EDTA are removed, and stops digestion.
(6) the type enzyme and the CaCl of 3-4 times of volume of adding in the deposition tissue of gained
2Insert 28 ℃ of water-baths, during do not stop to shake up.
(7) every another centrifuge tube of supernatant liquor immigration of 3/4 that behind 10min, takes out.
(8) in beaker, add new type enzyme and CaCl
2Continue the digestion tissue block.
(9) after 30min digestion finishes the supernatant liquor cell suspension of collecting is passed through 200 order bore diameter stainless steel filter screens, filter tissue block.
(10) 1000rpm, the centrifugal supernatant of abandoning of 5min adds behind the serum free medium mixing of 3ml centrifugally, removes residual type enzyme and CaCl
2, stop digestion.
(11) in the centrifuge tube of collecting cell, add the D-hank ' s of 10ml, and the erythrocyte cracked liquid mixing of 1ml, turn upside down and leave standstill 5min after 6-8 time.
(12) 1500rpm, 5min is centrifugal to abandon red part, stays pipe end white cell mass; Add D-hank ' s solution with white cell mass mixing after 1500rpm, 5min is centrifugal, removes residual erythrocyte cracked liquid.
(13) if hemocyte separation for the first time is not thorough, can repeating step (11) (12).
(14) become the parting liquid I with Percoll stoste and 10 * D-hank ' s with the proportional arrangement of 9:1.
(15) be made into the parting liquid II with the parting liquid I that is configured to and 1 * D-hank ' s by the volume ratio of 2:3.
(16) erythrocyte cracked liquid is handled the D-hank ' s that the white cell mass of managing the end in the back is dissolved in 3ml, add 2.5 times of parting liquid II that volume is 7.5ml.
(17) 4500rpm, the centrifugal supernatant liquor of abandoning of 5min.It is resuspended with the D-hank ' s of 5ml to get sedimentation cell group, rifle head piping and druming mixing.1500rpm, 5min is centrifugal.
(18) substratum (Streptomycin sulphate of 20% new-born calf serum, 100U/ml penicillin, 100U/ml and the DMEM/F12 substratum of 20ug/ml hepatocyte growth promoting factors) resuspending, counting cells density makes its density reach 1 * 10
6Individual/ml.
(19) liver cell is cultivated in porous culture plate or culturing bottle, used culture medium prescription is: Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of first new-born calf serum with DMEM/F12 substratum adding 20%, 100U/ml penicillin, 100U/ml.CO in 28 ℃
2Cultivate in the incubator.
(20) mistake changed the low culture medium culturing of serum content into after 8 hours.Said substratum is: Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of the new-born calf serum of DMEM/F12 substratum adding 10%, 100U/ml penicillin, 100U/ml.
(21) wherein, the pH of above-mentioned parting liquid I is 7.6, and osmotic pressure is 335mOsm.
(21) wherein, need frequent mixing between the period of digestion, behind the 10min 3/4 supernatant liquor is moved into another centrifuge tube.In beaker, add new Digestive system and continue the digestion tissue block.Digest 30min altogether.
Crucian carp liver cell cultured method of former generation of the present invention adopts the laboratory animal crucian carp, and cost is low, and the source is wide.Cell cultures operation and environmental requirement are high, in order to reduce the outside pollution that brings as far as possible, guarantee aseptic technique, use the glucose Tubulicid to soak the hepatic tissue that exsomatizes in the present invention, have obtained good result, do not find between effective date that cell is contaminated.Certainly; The glucose Tubulicid is not the determinative of sterile culture; But can reduce the possibility of bacterial contamination in operation; The survival of toxicity pair cell and the growth that add it does not have adverse influence; Be suitable for the sterilization to tissue, we also find in the experiment, and hemocyte quantity is less than unsoaked hepatic tissue behind the tissue block collecting cell that Tubulicid soaked; Cause the reason of this phenomenon it be unclear that, but effect is sure.Not only pollution can be reduced so increase the operation of this part, a part of hemocyte can also be eliminated.
Adding 0.02%EDTA in 0.25% trypsinase can chelating Ca
2+And Mg
2+, can help improving tryptic digestion effect.Because add EDTA, so will use D-hank ' s to stop digestion.Behind centrifugal removal trypsinase and the EDTA, add type enzyme and CaCl
2, Ca
2+Can improve the digestion ability of type enzyme effectively, and stop digestion with serum free medium.
In the cell suspension that digested, add erythrocyte cracked liquid and can effectively reduce the hemocyte content in the isolated cell group.Use the erythrocyte cracked liquid purifying cells obviously to reduce with the untapped hemocyte quantity of comparing.
Percoll cellular segregation liquid is as purification media; Be a kind of novel density gradient separation liquid; Silica gel particle by the policapram pyrrolidone constitutes; Granular size differs; Through the discontinuous density gradient that can form after centrifugal; The cellular segregation of different densities is opened, thereby reached the cell purification effect.Though use Percoll reagent can reduce hepatocellular quantity, because the cultivation of impurity cell or hemocyte and cell debris pair cell has very big influence, therefore with Percoll reagent liver cell being carried out purifying is a necessary step.Use Percoll purifying crucian carp liver cell, be significantly higher than non-purifying group on cell survival rate and the purity, the purifying group has clear superiority on hepatocyte function and adherent rate, and along with the increase of time, this advantage is obvious all the more.
The present invention has contrasted the influence that new-born calf serum and foetal calf serum pair cell are cultivated simultaneously, and the result finds, cultivate 24h after, new-born calf serum group liver cell is adherent many than the foetal calf serum group, so select new-born calf serum as the substratum serum composition when cultivating.
In sum, the inventive method raw material comes from common experimental hydrocole animal crucian carp, soaks the in vitro tissue piece with the glucose Tubulicid, and the tissue block that exsomatizes is carried out disinfection, and reduces and pollutes.Utilize the substep digestion method that tissue is digested, use erythrocyte cracked liquid to reduce the content of hemocyte in the cell, and use the Percoll gradient centrifugation, the purifying liver cell.Unite draw materials, substep digestion method, erythrocyte cracked liquid and three kinds of mode purifying of Percoll liver cell, the liver cell abundant amount that the present invention obtains, and survival rate reaches more than 90% meets former generation culture requirement.
Description of drawings
The firm isolated cells of Fig. 1 (magnification 10 * 20).
Fig. 2 cultivates the cell (magnification 10 * 40) of 12h.
Fig. 3 cultivates the cell (magnification 10 * 10) of 48h.
Embodiment
The former breeding method of being commissioned to train of embodiment 1 crucian carp liver cell
1 main raw prescription and source
1) serum free medium: the DMEM/F12 substratum adds Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of 100U/ml penicillin, 100U/ml.
2) culture medium prescription is: the Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors that in DMEM/F12 substratum (the Shanghai rising sun flies biological company limited), add new-born calf serum, 100U/ml penicillin, 100U/ml.
3) digestive ferment: 0.25% the trypsin Shanghai rising sun flies biological company limited), 0.02% EDTA and 0.1% type enzyme (the Shanghai rising sun flies biological company limited), 3mmol/LCaCl
2
4) erythrocyte cracked liquid buys in Shanghai that the rising sun flies biological company limited.
5) Percoll parting liquid: become the parting liquid I with the proportional arrangement of 9:1 with Percoll stoste (the Shanghai rising sun flies biological company limited) and 10 * D-hank ' s; The parting liquid I that usefulness is configured to and 1 * D-hank ' s are made into the parting liquid II by the volume ratio of 2:3.
6) D-hank ' s solution: with 8g NaCl, 0.4g KCl, 0.13g Na
2HPO
412H
20,0.06g KH
2PO4,0.35g NaHCO
3Constant volume is in the 1L pure water, and autoclave sterilization treats that temperature descends the back in 4 ℃ of preservations.
7) 0.1% type enzyme: accurately take by weighing type enzyme powder 0.1g and be dissolved among the D-hank ' s of 100mL, 4 ℃ of mixings that spend the night, regulating the pH value is about 7.4.With the filtration sterilization of injection filter, bottle packing (centrifuge tube) is frozen in-20 ℃.
8) laboratory animal: 3 of healthy crucian carps, body weight 300g.Experimental implementation is abideed by national Animal Protection Law and is carried out.
2 operation stepss
1) takes suitable anatomy mode, from the crucian carp of health, isolate hepatic tissue.
The test fish was supported 3 days temporarily, got healthy crucian carp, at first cut off its gill portion arcus haemalis; Bloodletting 5min with the sterilization of alcohol wipe fish body surface, dissects crucian carp in Bechtop; And get hepatic tissue, in order to reduce the pollution of bacterium, soak in vitro tissue 10min with glucose Tubulicid (chlorhexidine).Use D-hank ' s rinsing 2 times to there not being color.
2) with eye scissors tissue is cut into 2-3mm
3Fragment, place beaker.
3) in order to improve the method digestion tissue that the digestion effect adopts substep digestion.Pour the tissue that shreds into centrifuge tube, insert in 28 ℃ the water-bath.Trypsinase and the EDTA digestion that at first adds tissue mass 4-5 times volume, during do not stop to shake up, after 15min digestion finishes.
4) the centrifugal supernatant that goes, the D-hank ' s that adds 3ml is with organizing 1000rpm behind the mixing, and 5min is centrifugal, removes residual trypsinase and EDTA, stops digesting.
5) in the deposition tissue of gained, add the type enzyme and the CaCl of 3-4 times of volume
2Insert 28 ℃ of water-baths, during do not stop to shake up.
6) every another centrifuge tube of supernatant liquor immigration of 3/4 that behind 10min, takes out.In beaker, add new type enzyme and CaCl
2Continue the digestion tissue block.
7) after 30min digestion finishes the supernatant liquor cell suspension of collecting is passed through 200 order bore diameter stainless steel filter screens, filter tissue block.
8) the centrifugal supernatant of abandoning, the serum free medium that adds 3ml with bottom settlings cell mass mixing after 1000rpm, 5min is centrifugal, removes residual type enzyme and CaCl
2, stop digestion.
9) abandon supernatant, in the gained sedimentation cell, add D-hank ' the s re-suspended cell of 10ml.The erythrocyte cracked liquid that adds 1ml then behind the mixing, turns upside down 6-8 time, leaves standstill 5min.1500rpm, 5min is centrifugal, abandons cleer and peaceful red cell part, stays the cell mass of white.
10), add the Percoll parting liquid II of 7.5ml simultaneously with D-hank ' the s re-suspended cell group of 3ml.4500rpm, 5min is centrifugal.Abandon supernatant liquor, get cell mass.
11) it is resuspended with the D-hank ' s of 5ml to get cell mass, rifle head piping and druming mixing.1500rpm, 5min is centrifugal.Obtain purifying cell later.
12) substratum (Streptomycin sulphate of 20% new-born calf serum, 100U/ml penicillin, 100U/ml and the DMEM/F12 substratum of 20ug/ml hepatocyte growth promoting factors) resuspending, counting cells density makes its density reach 1 * 10
6Individual/ml.
13) adding contains the Streptomycin sulphate of 20% new-born calf serum, 100U/ml penicillin, 100U/ml and the DMEM/F12 substratum of 20ug/ml hepatocyte growth promoting factors, and 28 ℃ place CO
2In the incubator.Change the Streptomycin sulphate that contains 10% new-born calf serum, 100U/ml penicillin, 100U/ml and the DMEM/F12 substratum of 20ug/ml hepatocyte growth promoting factors after 8 hours into.
Use method provided by the present invention, it is high to have obtained purity, quantity foot, the cell that growth conditions is good.More than the method triplicate, the result is approximate, and repetition rate is high.
Test case
1, viable count: carry out viable count with platform phenol indigo plant.
The blue counting of platform phenol: cell is from tissue digestion; Through repeatedly washing and centrifugal; Certain damage can take place; Separation through cellular segregation liquid can be with the same smudge cells of intact cell; Hemocyte is separated; The cell of purifying dyes through trypan blue, and the viable cell number reaches more than 90%, satisfies former generation culture requirement.
2, morphology is identified: observation post gets the formalness characteristic of cell under the OLYMPUS inverted microscope.
Morphological observation: the down visible rounded or similar round (Fig. 1) of firm isolating liver cell of light microscopic, cell is single dispersion state, and boundary is clear, and kernel is high-visible, and cell is bright.Behind the cultivation 12h (Fig. 2), cellular form is still rounded, arranges closely.Begin to occur adherent, stretching, extension phenomenon.Behind the cultivation 48h (Fig. 3), cell closely links to each other, and forms island and connects.Show that the cell growing state is good.
Claims (9)
1. former breeding method of being commissioned to train of crucian carp liver cell, it comprises following processing step:
A. from the crucian carp of health, separate hepatic tissue
At first cut off crucian carp gill portion arcus haemalis, sacrificed by exsanguination carries out disinfection with alcohol wipe crucian carp body surface, in Bechtop, dissects crucian carp, and takes out hepatic tissue, and isolating hepatic tissue is soaked in 10min in the glucose Tubulicid;
B. adopt the substep digestion method that the hepatic tissue that shreds is digested the acquisition cell mass
With soaking tissue D-hank ' the s rinsing 2 times of glucose Tubulicid, shred into 2-3mm
3Tissue block; Adopt substep digestion method digestion tissue in order to improve the digestion effect:
Pour the tissue that shreds into centrifuge tube, insert in 28 ℃ the water-bath; Trypsinase and the EDTA digestion that at first adds tissue mass 4-5 times volume, during do not stop to shake up, after 15min digestion finishes, the centrifugal supernatant of abandoning; D-hank ' the s that adds 3ml with the bottom settlings mixing after the centrifugal supernatant of abandoning, residual trypsinase is removed with EDTA, stop digesting;
The type enzyme and the CaCl that in gained deposition tissue, add 3-4 times of volume
2Insert 28 ℃ of water-baths, during do not stop to shake up, every supernatant liquor of 3/4 of behind 10min, taking out moves into another centrifuge tube; In beaker, add new type enzyme and CaCl
2Continue the digestion tissue block;
After 30min digestion finishes, the supernatant liquor cell suspension of collecting is passed through 200 order bore diameter stainless steel filter screens, filter tissue block; The centrifugal supernatant of abandoning, the serum free medium that adds 3ml with the bottom settlings mixing after the centrifugal supernatant of abandoning, with residual type enzyme and CaCl
2Remove, stop digestion;
C. the cell mass that obtains is carried out purifying and cultivation
1. use erythrocyte cracked liquid to remove hemocyte: in centrifuge tube, to add 10ml D-hank ' s; Sedimentation cell group is dispersed into the individual cells suspension with pipettor piping and druming, adds the erythrocyte cracked liquid of 1ml simultaneously, blow and beat mixing gently; Turn upside down 6-8 time, leave standstill 5min; Centrifugally abandon red part, stay pipe end white cell mass; Add D-hank ' s with white cell mass mixing, piping and druming is also centrifugal, and the D-hank ' s that is dissolved in 3ml is made into hepatocyte suspension;
2. add Percoll reagent to isolating liver cell purifying: get hepatocyte suspension and be suspended on the Percoll parting liquid II of 2.5 times of volumes the centrifugal supernatant of abandoning; It is resuspended with the D-hank ' s of 5ml to get sedimentation cell group, rifle head piping and druming mixing; 1500rpm, 5min is centrifugal, abandons supernatant, obtains purifying liver cell later;
Wherein, described D-hank ' s solution is: with 8g NaCl, 0.4g KCl, 0.13g Na
2HPO
412H
20,0.06g KH
2PO4,0.35g NaHCO
3Constant volume is in the 1L pure water, and autoclave sterilization treats that temperature descends the back in 4 ℃ of preservations;
Wherein saidly be formulated as with Percoll parting liquid II: with Percoll reagent stoste and 10 * D-hank ' s proportional arrangement parting liquid I with 9:1, the parting liquid I that usefulness is configured to and 1 * D-hank ' s are configured to required parting liquid II by the volume ratio of 2:3 earlier;
2. cultural method according to claim 1, the concentration of glucose Tubulicid are 0.1%.
3. cultural method according to claim 1 is characterized in that: trypsinase concentration is 0.25%, and the working concentration of EDTA is 0.02%, and the volume ratio of trypsinase and EDTA is 1:1.
4. cultural method according to claim 1 is characterized in that: the concentration of collagenase is 0.1%, CaCl
2Concentration be 3mmol/L.
5. cultural method according to claim 1 is characterized in that: described culture temperature is 28 ℃.
6. cultural method according to claim 1 is characterized in that: described substratum PH is 7-8.
7. cultural method according to claim 1 is characterized in that, centrifugal speed is 1000rmp in the said digestive process of b step, 5min.
8. cultural method according to claim 1 is characterized in that, adds among the described step c that centrifugal rotational speed is 1500rpm in the erythrocyte cracked liquid centrifugal process, 5min.
9. cultural method according to claim 1 is characterized in that the serum among the described step c is selected new-born calf serum for use; Described use substratum is: add earlier Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of 20% new-born calf serum, 100U/ml penicillin, 100U/ml with the DMEM/F12 substratum, change new-born calf serum, the 100U/ml penicillin of DMEM/F12 substratum adding 10%, Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of 100U/ml after 8 hours into.
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| CN111575236A (en) * | 2020-03-19 | 2020-08-25 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Preparation method of human liver cancer tissue and liver tissue active single cell suspension |
| CN113957035A (en) * | 2021-08-02 | 2022-01-21 | 中国农业科学院饲料研究所 | Method for separating and culturing duck embryo primary hepatocytes |
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| CN104293731A (en) * | 2014-10-13 | 2015-01-21 | 中国水产科学研究院淡水渔业研究中心 | Separation culture method of primary hepatocyte of jian carp |
| CN105754932A (en) * | 2016-04-15 | 2016-07-13 | 上海大学 | Method for extracting and cultivating primary cyprinidae hepatocytes |
| CN105754932B (en) * | 2016-04-15 | 2021-04-09 | 上海大学 | Method for extracting and culturing primary hepatocytes of carp |
| CN108300688A (en) * | 2018-02-07 | 2018-07-20 | 上海赛立维生物科技有限公司 | Primary hepatocyte detaches and cultural method |
| CN109161515A (en) * | 2018-08-06 | 2019-01-08 | 南京农业大学 | The isolated culture method of megalobrama amblycephala primary hepatocyte |
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| CN109971700A (en) * | 2019-03-12 | 2019-07-05 | 南京师范大学 | A kind of primary gill cell culture processes of fugu obscurus |
| CN109971700B (en) * | 2019-03-12 | 2021-09-28 | 南京师范大学 | Culture method of primary gill cells of takifugu obscurus |
| CN110684718B (en) * | 2019-10-30 | 2021-05-11 | 南京师范大学 | Method for efficiently separating and culturing primary hepatocytes of fugu obscurus |
| CN110684718A (en) * | 2019-10-30 | 2020-01-14 | 南京师范大学 | Method for efficiently separating and culturing primary hepatocytes of fugu obscurus |
| CN111575236A (en) * | 2020-03-19 | 2020-08-25 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Preparation method of human liver cancer tissue and liver tissue active single cell suspension |
| CN111575236B (en) * | 2020-03-19 | 2024-04-26 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Preparation method of active single cell suspension of human liver cancer tissue and liver tissue |
| CN113957035A (en) * | 2021-08-02 | 2022-01-21 | 中国农业科学院饲料研究所 | Method for separating and culturing duck embryo primary hepatocytes |
| CN113957035B (en) * | 2021-08-02 | 2023-08-04 | 中国农业科学院饲料研究所 | Duck embryo primary liver cell separation culture method |
| CN115109745A (en) * | 2022-07-19 | 2022-09-27 | 南京师范大学 | Method for efficiently separating and primarily culturing pelteobagrus fulvidraco hepatocytes |
| CN115109745B (en) * | 2022-07-19 | 2024-05-31 | 南京师范大学 | Efficient separation and primary culture method for liver cells of pelteobagrus fulvidraco |
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