CN102304492B - Primary culture method for liver cells of crucian carp - Google Patents
Primary culture method for liver cells of crucian carp Download PDFInfo
- Publication number
- CN102304492B CN102304492B CN 201110248769 CN201110248769A CN102304492B CN 102304492 B CN102304492 B CN 102304492B CN 201110248769 CN201110248769 CN 201110248769 CN 201110248769 A CN201110248769 A CN 201110248769A CN 102304492 B CN102304492 B CN 102304492B
- Authority
- CN
- China
- Prior art keywords
- digestion
- hank
- cell
- tissue
- add
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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 a kind of new crucian carp Primary Hepatocyte Culture method, 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, is also simultaneously the vitals of chemicals metabolism in body.Environment complicated and changeable in body is to causing that at body specializes in chemistry thing hepar damnification brings certain difficulty, the primary cultured cell isolated time is short, not yet great changes will take place 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 of specializes in chemistry thing and liver cell are replied and the mechanism of detoxifying poisonous substance.
Fish are the vertebratess that live in water body, are to detect the good biological indication body of water pollutant.Primary Culture hepatocyte keeps preferably and has kept hepatocellular integrity and metabolic activity, reactant intracellular metabolite situation truly.Simultaneously, liver cell is the important place of the conversion of external source chemicals and metabolism, is also the Main Function target spot that allogenic material causes chemistry and biological damage.Therefore hepatocyte is having many advantages aspect the bio-transformation of specializes in chemistry thing and toxic effect mechanism, is the ideal model of research in vitro toxicity.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 cell is furtherd investigate, and no matter aspect theory research, or aspect practical application, far reaching significance is arranged.Crucian carp is the important food fish of China, and fish market occupies critical role in China, with crucian as experimental subjects, no matter on the market economy and scientific research powerful pushing effect is arranged.
Liver parenchyma is main the composition by a large amount of liver cells, contains a matter few, and liver cell has several functions, and metabolism is vigorous, and is high in external required condition." Chinese comparative medicine magazine ", 14 3 phases of volume in 2006, 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 30S, the long time can affect tissue activity, reduces cell attachment ability and survival rate.With respect to ethanol, Tubulicid is more suitable for the sterilization for tissue block.
Although some separation are arranged and identify hepatocellular method at present, yet the liver cell purity of separating is lower, active relatively low.This experiment improves, uses for reference existing cell culture processes, and wish provides good experimental model for research environment pollutes to the hepatocellular murder by poisoning of crucian carp and intoxicating mechanism by setting up the liver cell culture method of higher degree.We find in model is set up, and obtain from the separate tissue to the cell, there are 3 key factors the centre: 1. sterile culture; 2. digestive process; 3. the adherent degree of Growth of Cells, 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 to obtain purity and active relatively high liver cell in crucian carp Primary Hepatocyte Culture method.
On the basis that existing cell culture processes is improved, used for reference, the invention provides a kind of crucian carp Primary Hepatocyte Culture method, it comprises following processing step:
A, separate hepatic tissue from the crucian carp of health
At first cut off its gill section arcus haemalis, sacrificed by exsanguination carries out disinfection with alcohol wipe crucian carp body surface, dissects crucian carp in Bechtop, and takes out hepatic tissue, and the hepatic tissue that separates 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 digestion effect:
Pour the tissue that shreds into centrifuge tube, insert in the water-bath of 28 ℃; At first add the trypsinase of tissue mass 4-5 times volume and EDTA digestion, during do not stop to shake up, after 15min digestion is complete, 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 and EDTA are removed, stop digestion;
The typeⅡ Collagen enzyme and the CaCl that add 3-4 times of volume in the gained precipitate
2Insert 28 ℃ of water-baths, during do not stop to shake up, take out 3/4 supernatant liquor and move into another centrifuge tube after 10min; Add new typeⅡ Collagen enzyme and CaCl in centrifuge tube
2Continue the digestion tissue block;
After 30min digestion is complete, 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 typeⅡ Collagen 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: to add 10ml D-hank ' s in centrifuge tube, sedimentation cell is rolled into a ball with pipettor piping and druming be dispersed into the individual cells suspension, add simultaneously the erythrocyte cracked liquid of 1ml, blow and beat gently mixing, turn upside down 6-8 time, standing 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 the liver cell purifying of Percoll reagent to separating: get hepatocyte suspension and be suspended on the Percoll parting liquid II of 2.5 times of volumes, the centrifugal supernatant of abandoning; D-hank ' the s that gets the sedimentation cell use 5ml of group is resuspended, 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 descends until temperature rear in 4 ℃ of preservations;
Wherein said Percoll parting liquid II is formulated as: first use Percoll reagent stoste and 10 * D-hank ' s with the proportional arrangement parting liquid I of 9:1, the parting liquid I that use is configured to and 1 * D-hank ' s are configured to required parting liquid II by the volume ratio of 2:3;
Liver cell after purifying is suspended with substratum again, and wherein said substratum is: the Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors that add 20% new-born calf serum, 100U/ml penicillin, 100U/ml in the DMEM/F12 substratum.
Wherein the concentration of the glucose Tubulicid described in step a is preferably 0.1%.
Wherein the trypsinase concentration described in 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 typeⅡ Collagen enzyme described in step b is preferably 0.1%, CaCl
2Concentration 3mmol/L preferably.
Wherein the culture temperature of the described cultural method described in step c is 28 ℃.
Wherein the substratum pH value of the cultural method described in step c is 7-8.
Wherein in the described digestive process described in step b, centrifugal speed is 1000rmp, 5min.
Wherein in the erythrocyte cracked liquid centrifugal process described in step c, centrifugal rotational speed is 1500rpm, 5min.
Wherein the substratum described in the step c described in step c is: first add 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 Streptomycin sulphate and 20ug/ml hepatocyte growth promoting factors that the DMEM/F12 substratum adds 10% new-born calf serum, 100U/ml penicillin, 100U/ml after 8 hours into.
Wherein, choose in a step when organizing and select near near the hepatic tissue courage, the hepatic tissue at this place is larger, 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 in step b, digestive ferment is preferably the digestion of tissue and the processing step of purifying:
(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 the water-bath of 28 ℃.
(3) add the trypsinase of tissue mass 4-5 times volume and EDTA digestion, during do not stop to shake up.
(4) 15min digestion complete after, 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) add typeⅡ Collagen enzyme and the CaCl of 3-4 times of volume in the precipitate of gained
2Insert 28 ℃ of water-baths, during do not stop to shake up.
(7) supernatant liquor of taking-up 3/4 moves into another centrifuge tube after 10min.
(8) add new typeⅡ Collagen enzyme and CaCl in beaker
2Continue the digestion tissue block.
(9) 30min digestion complete after with the supernatant liquor cell suspension collected by 200 order bore diameter stainless steel filter screens, filter tissue block.
(10) 1000rpm, the centrifugal supernatant of abandoning of 5min adds after the serum free medium mixing of 3ml centrifugally, removes residual typeⅡ Collagen enzyme and CaCl
2, stop digestion.
(11) add the D-hank ' s of 10ml in the centrifuge tube of collecting cell, and the erythrocyte cracked liquid mixing of 1ml, rear standing 5min turns upside down 6-8 time.
(12) 1500rpm, 5min is centrifugal abandons 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 processed after the white cell mass at the pipe end be dissolved in the D-hank ' s of 3ml, the parting liquid II that to add 2.5 times of volumes be 7.5ml.
(17) 4500rpm, the centrifugal supernatant liquor of abandoning of 5min.D-hank ' the s that gets the sedimentation cell use 5ml of group is resuspended, 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) suspends again, and counting cells density makes its density reach 1 * 10
6Individual/ml.
(19) liver cell is cultivated in porous culture plate or culturing bottle, culture medium prescription used is: the Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors that first add 20% new-born calf serum, 100U/ml penicillin, 100U/ml with the DMEM/F12 substratum.CO in 28 ℃
2Cultivate in incubator.
(20) mistake after 8 hours, changes the low culture medium culturing of serum content into.Described substratum is: the DMEM/F12 substratum adds Streptomycin sulphate and the 20ug/ml hepatocyte growth promoting factors of 10% new-born calf serum, 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, after 10min, 3/4 supernatant liquor is moved into another centrifuge tube.Add new Digestive system and continue the digestion tissue block in beaker.Altogether digest 30min.
The method of crucian carp Primary Hepatocyte Culture 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 as far as possible the outside pollution that brings, guarantee aseptic technique, use in the present invention the glucose Tubulicid to soak the hepatic tissue that exsomatizes, and 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 in operation the possibility of bacterial contamination, the toxicity that adds it does not have adverse influence to cell survival and growth, be suitable for the sterilization to tissue, in experiment, we also find, after the tissue block collecting cell that Tubulicid soaked, hemocyte quantity is less than unsoaked hepatic tissue, 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.
Add the 0.02%EDTA can chelating Ca in 0.25% trypsinase
2+And Mg
2+, can be conducive to improve tryptic digestion effect.Because add EDTA, so will use D-hank ' s to stop digestion.After centrifugal removal trypsinase and EDTA, add typeⅡ Collagen enzyme and CaCl
2, Ca
2+Can effectively improve the digestion ability of typeⅡ Collagen enzyme, and stop digestion with serum free medium.
Add erythrocyte cracked liquid can effectively reduce Content of Hemocytes in isolated cell group in the cell suspension that digested.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, a kind of novel density gradient separation liquid, silica gel particle by the policapram pyrrolidone consists of, granular size differs, by the discontinuous density gradient that can form after centrifugal, the cellular segregation of different densities is opened, thereby reached the cell purification effect.Although use Percoll reagent can reduce hepatocellular quantity, because the cultivation to cell of impurity cell or hemocyte and cell debris has a great impact, therefore with Percoll reagent, liver cell carried out the step that purifying is necessity.Use the Percoll purification of Carassius auratus hepatocytes, be significantly higher than non-purifying group on cell survival rate and 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 new-born calf serum and foetal calf serum simultaneously on the impact of cell cultures, found that, after cultivating 24h, 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 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 liver cells of Percoll, the liver cell abundant amount that the present invention obtains, and survival rate reaches more than 90%, meets the requirement of former culture.
Description of drawings
The cell (magnification 10 * 20) that Fig. 1 has just separated.
Fig. 2 cultivates the cell (magnification 10 * 40) of 12h.
Fig. 3 cultivates the cell (magnification 10 * 10) of 48h.
Embodiment
Embodiment 1 crucian carp Primary Hepatocyte Culture method
1 main raw formula 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 add new-born calf serum, 100U/ml penicillin, 100U/ml in DMEM/F12 substratum (the Shanghai rising sun flies biological company limited).
3) digestive ferment: 0.25% trypsin Shanghai rising sun flies biological company limited), 0.02% EDTA and 0.1% typeⅡ Collagen enzyme (the Shanghai rising sun flies biological company limited), 3mmol/LCaCl
2
4) erythrocyte cracked liquid is bought in the Shanghai rising sun and is flown 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; 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.
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 descends until temperature rear in 4 ℃ of preservations.
7) 0.1% typeⅡ Collagen enzyme: accurately take typeⅡ Collagen enzyme powder 0.1g and be dissolved in the D-hank ' s of 100mL, 4 ℃ of mixings that spend the night, regulating the pH value is 7.4 left and right.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 carried out in accordance with national Animal Protection Law.
2 operation stepss
1) take suitable anatomy mode, isolate hepatic tissue from the crucian carp of health.
The test fish was supported 3 days temporarily, got healthy crucian carp, at first cut off its gill section 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 without color.
2) with eye scissors, tissue is cut into 2-3mm
3Fragment, be placed in beaker.
3) adopt the method digestion tissue of substep digestion in order to improve digestion effect.Pour the tissue that shreds into centrifuge tube, insert in the water-bath of 28 ℃.At first add the trypsinase of tissue mass 4-5 times volume and EDTA digestion, during do not stop to shake up, after 15min digestion is complete.
4) the centrifugal supernatant that goes, the D-hank ' s that adds 3ml is with organizing 1000rpm after mixing, and 5min is centrifugal, removes residual trypsinase and EDTA, stops digesting.
5) add typeⅡ Collagen enzyme and the CaCl of 3-4 times of volume in the precipitate of gained
2Insert 28 ℃ of water-baths, during do not stop to shake up.
6) supernatant liquor of taking-up 3/4 moves into another centrifuge tube after 10min.Add new typeⅡ Collagen enzyme and CaCl in centrifuge tube
2Continue the digestion tissue block.
7) 30min digestion complete after with the supernatant liquor cell suspension collected by 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 typeⅡ Collagen enzyme and CaCl
2, stop digestion.
9) abandon supernatant, add D-hank ' the s re-suspended cell of 10ml in the gained sedimentation cell.Then the erythrocyte cracked liquid that adds 1ml after mixing, turns upside down 6-8 time, standing 5min.1500rpm, 5min is centrifugal, abandons cleer and peaceful red cell part, stays the cell mass of white.
10) with D-hank ' the s re-suspended cell group of 3ml, add simultaneously the Percoll parting liquid II of 7.5ml.4500rpm, 5min is centrifugal.Abandon supernatant liquor, get cell mass.
11) get cell mass and use the D-hank ' s of 5ml resuspended, 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) suspends again, and counting cells density makes its density reach 1 * 10
6Individual/ml.
13) add the Streptomycin sulphate that contains 20% new-born calf serum, 100U/ml penicillin, 100U/ml and the DMEM/F12 substratum of 20ug/ml hepatocyte growth promoting factors, 28 ℃ are placed in CO
2In 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, obtained purity high, quantity foot, the cell that growth conditions is good.More than the method triplicate, 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 occur, separation by cellular segregation liquid can be with the same smudge cells of intact cell, hemocyte is separated, the cell of purifying is through Trypan Blue, and the viable cell number reaches more than 90%, satisfies the requirement of former culture.
2, Morphological Identification: observation post gets the formalness feature of cell under the OLYMPUS inverted microscope.
Morphological observation: the rounded or similar round (Fig. 1) of the visible liver cell that has just separated under light microscopic, cell is single dispersion state, and boundary is clear, and kernel is high-visible, and cell is bright.After cultivation 12h (Fig. 2), cellular form is still rounded, arranges closely.Begin to occur adherent, stretching, extension phenomenon.After cultivation 48h (Fig. 3), cell closely is connected, and forms island and connects.Show that Growth of Cells is all right.
Claims (8)
1. crucian carp Primary Hepatocyte Culture method, it comprises following processing step:
A, separate hepatic tissue from the crucian carp of health
At first cut off crucian carp gill section arcus haemalis, sacrificed by exsanguination carries out disinfection with alcohol wipe crucian carp body surface, dissects crucian carp in Bechtop, and takes out hepatic tissue, and the hepatic tissue that separates 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 digestion effect:
Pour the tissue that shreds into centrifuge tube, insert in the water-bath of 28 ℃; At first add the trypsinase of tissue mass 4-5 times volume and EDTA digestion, during do not stop to shake up, after 15min digestion is complete, 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 and EDTA are removed, stop digestion;
The typeⅡ Collagen enzyme and the CaCl that add 3-4 times of volume in the gained precipitate
2Insert 28 ℃ of water-baths, during do not stop to shake up, take out 3/4 supernatant liquor and move into another centrifuge tube after 10min; Add new typeⅡ Collagen enzyme and CaCl in centrifuge tube
2Continue the digestion tissue block;
After 30min digestion is complete, 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 typeⅡ Collagen 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: to add 10ml D-hank ' s in centrifuge tube, sedimentation cell is rolled into a ball with pipettor piping and druming be dispersed into the individual cells suspension, add simultaneously the erythrocyte cracked liquid of 1ml, blow and beat gently mixing, turn upside down 6-8 time, standing 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 the liver cell purifying of Percoll reagent to separating: get hepatocyte suspension and be suspended on the Percoll parting liquid II of 2.5 times of volumes, the centrifugal supernatant of abandoning; D-hank ' the s that gets the sedimentation cell use 5ml of group is resuspended, 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 descends until temperature rear in 4 ℃ of preservations;
Wherein said Percoll parting liquid II is formulated as: first use Percoll reagent stoste and 10 * D-hank ' s with the proportional arrangement parting liquid I of 9:1, the parting liquid I that use is configured to and 1 * D-hank ' s are configured to required parting liquid II by the volume ratio of 2:3;
2. cultural method according to claim 1, the concentration of glucose Tubulicid is 0.1%.
3. cultural method according to claim 1, it is characterized in that: trypsinase concentration is that the working concentration of 0.25%, EDTA is 0.02%, and the volume ratio of trypsinase and EDTA is 1:1.
4. cultural method according to claim 1, it is characterized in that: the concentration of collagenase is 0.1%, CaCl
2Concentration be 3mmol/L.
5. cultural method according to claim 1, it is characterized in that: described substratum PH is 7-8.
6. cultural method according to claim 1, is characterized in that, in the described digestive process of b step, centrifugal speed is 1000rmp, 5min.
7. cultural method according to claim 1, is characterized in that, adds in described step c that in the erythrocyte cracked liquid centrifugal process, centrifugal rotational speed is 1500rpm, 5min.
8. cultural method according to claim 1, it is characterized in that, described substratum is: first add 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 Streptomycin sulphate and 20ug/ml hepatocyte growth promoting factors that the DMEM/F12 substratum adds 10% new-born calf serum, 100U/ml penicillin, 100U/ml after 8 hours into.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110248769 CN102304492B (en) | 2011-08-27 | 2011-08-27 | Primary culture method for liver cells of crucian carp |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110248769 CN102304492B (en) | 2011-08-27 | 2011-08-27 | Primary culture method for liver cells of crucian carp |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102304492A CN102304492A (en) | 2012-01-04 |
| CN102304492B true CN102304492B (en) | 2013-06-26 |
Family
ID=45378410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110248769 Expired - Fee Related CN102304492B (en) | 2011-08-27 | 2011-08-27 | Primary culture method for liver cells of crucian carp |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102304492B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105754932B (en) * | 2016-04-15 | 2021-04-09 | 上海大学 | Method for extracting and culturing primary hepatocytes of carp |
| CN108300688B (en) * | 2018-02-07 | 2021-02-02 | 上海赛立维生物科技有限公司 | Primary hepatocyte separation and culture method |
| CN109161515B (en) * | 2018-08-06 | 2021-12-03 | 南京农业大学 | Isolation culture method of primary hepatocytes of megalobrama amblycephala |
| 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 |
| 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 |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587392A (en) * | 2004-09-02 | 2005-03-02 | 浙江大学 | External tissue cultivating method for live cell |
| CN101538552A (en) * | 2009-04-24 | 2009-09-23 | 扬州大学 | Method for separating and cultivating rat hepatocytes |
| CN102041242A (en) * | 2010-05-12 | 2011-05-04 | 北京汇智泰康医药技术有限公司 | Method for separating and culturing primary hepatocytes |
-
2011
- 2011-08-27 CN CN 201110248769 patent/CN102304492B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587392A (en) * | 2004-09-02 | 2005-03-02 | 浙江大学 | External tissue cultivating method for live cell |
| CN101538552A (en) * | 2009-04-24 | 2009-09-23 | 扬州大学 | Method for separating and cultivating rat hepatocytes |
| CN102041242A (en) * | 2010-05-12 | 2011-05-04 | 北京汇智泰康医药技术有限公司 | Method for separating and culturing primary hepatocytes |
Non-Patent Citations (8)
| Title |
|---|
| A METHOD OF OBTAINING A SUSPENSION OF INTACT PARENCHYMAL CELLS FROM ADULT RAT LIVER;JENNIFER J. GALLAI-HATCHARD等;《Journal of Cell Science》;19710131;第8卷(第1期);73-86页 * |
| Comparison of Cytologic and Biochemical Properties Between Liver Ceils Isolated from Adult Rats by Trypsin Perfusion and Those Isolated by Collagenase Perfusion;M. Miyazaki等;《Research in Experimental Medicine》;19840731;第184卷(第3期);191-204页 * |
| JENNIFER J. GALLAI-HATCHARD等.A METHOD OF OBTAINING A SUSPENSION OF INTACT PARENCHYMAL CELLS FROM ADULT RAT LIVER.《Journal of Cell Science》.1971,第8卷(第1期),73-86页. |
| M. Miyazaki等.Comparison of Cytologic and Biochemical Properties Between Liver Ceils Isolated from Adult Rats by Trypsin Perfusion and Those Isolated by Collagenase Perfusion.《Research in Experimental Medicine》.1984,第184卷(第3期),191-204页. |
| 壬基酚对鲫鱼原代肝细胞增殖和抗氧化功能的影响;张毅 等;《应用生态学报》;20090228;第20卷(第2期);352-357页 * |
| 张毅 等.壬基酚对鲫鱼原代肝细胞增殖和抗氧化功能的影响.《应用生态学报》.2009,第20卷(第2期),352-357页. |
| 张高峰 等.采用Percoll法分离、纯化鲫肝细胞.《中国水产科学》.2007,第14卷(第2期),208-214页. |
| 采用Percoll法分离、纯化鲫肝细胞;张高峰 等;《中国水产科学》;20070331;第14卷(第2期);208-214页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102304492A (en) | 2012-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102304492B (en) | Primary culture method for liver cells of crucian carp | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN107475190B (en) | Method for clinical-level efficient preparation and cryopreservation of fat SVF cells and application thereof | |
| CN102191218B (en) | Complete medium and human amnion-derived mesenchymal stem cell culture method | |
| CN101984049B (en) | Method for separating mesenchymal stem cells from dispose tissues | |
| CN112458040B (en) | Isolation culture method of rumen epithelial cells of adult yaks | |
| CN103667187A (en) | Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank | |
| CN101705206B (en) | Novel highly efficient method for preparing mice pancreatic single cell suspension | |
| CN105754932A (en) | Method for extracting and cultivating primary cyprinidae hepatocytes | |
| CN102492623A (en) | Method for preparing and culturing culture medium for supravital culture of seawater scuticociliatida ciliates | |
| CN107129962B (en) | A kind of primary culture method of Hirudo japonica salivary gland cell | |
| CN104293731A (en) | Separation culture method of primary hepatocyte of jian carp | |
| CN106916779A (en) | A kind of sea mollusk primary cell separation and cultivate reagent box and application thereof | |
| CN110527661B (en) | Carassius xyphoides larva cell line and construction method and application thereof | |
| CN104818238A (en) | Isolated culture method for chicken enterocyte | |
| CN101955909A (en) | Novel primary culture method of pancreatic duct epithelial cells of rat | |
| CN101451121B (en) | Construction method of Epinephelus fuscoguttatus heart cell line | |
| CN106497863A (en) | A kind of separation of cornea of rats endothelial cell, purifying and cultural method | |
| CN102120983B (en) | Separation and culture method of epidermal stem cells of Beijing ducks | |
| CN108774630A (en) | A kind of primary culture method of Microhyla ornata Skeletal Muscle Cell | |
| CN114107181A (en) | Sturgeon embryonic cell line, culture medium and preparation method thereof | |
| CN106906177A (en) | A kind of naked mole interstitial glands is isolated and purified and cultural method | |
| CN102258004B (en) | Method for preserving/culturing oocytes in vitro to inhibit oocytes from germinal vesicle breakdown | |
| CN105132287A (en) | Method for culturing myxobolus in vitro |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| ASS | Succession or assignment of patent right |
Owner name: SHANGHAI VOCATIONAL AND TECHNICAL COLLEGE OF AGRIC Effective date: 20120210 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20120210 Address after: 201306 Shanghai city Pudong New Area Lingang New City Shanghai City Ring Road No. 999 Applicant after: Shanghai Maritime University Co-applicant after: SHANGHAI VOCATIONAL TECHNICAL COLLEGE OF AGRICULTURE & FORESTRY Address before: 201306 Shanghai city Pudong New Area Lingang New City Shanghai City Ring Road No. 999 Applicant before: Shanghai Maritime University |
|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130626 Termination date: 20140827 |
|
| EXPY | Termination of patent right or utility model |