CN1920009A - Producing method of repairable pigling pancreatic island cell - Google Patents
Producing method of repairable pigling pancreatic island cell Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
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Abstract
The invention relates the method for producing renovation pigling Langer-hans' insula cell. The method comprises the following steps: 1 choosing donator: choosing health pigling, at sterility opening abdomen, getting pancreatic, removing integument, vessel, connective tissue in Hank`s liquid at 4Deg.C; 2 separating and purifying Langer-hans' insula cell: separating Langer-hans' insula cell from external secretion tissue with finishing machine, centrifuging with Dextran non-sequence density gradient, getting Langer-hans' insula cell; 3 culturing Langer-hans' insula cell: carrying out CO2 culturing with non-serum microballoon carrier, using RPMI-1640 culture liquid, adding fibrin, packaging the Langer-hans' insula cell in the APA biological film to form microsphere, at 37Deg.C, culturing in the CO2 culture box, changing culture liquid every 48 hours, and getting the product.
Description
Technical field
The present invention relates to the production method of biomass cells technical field, particularly a kind of repairable pigling pancreatic island cell, but utilize this cell clinical application in diabetic subject's pancreatic islets transplantation.
Background technology
Diabetes are global diseases of harm humans health.Sickness rate is 3-5%; Mortality ratio is only second to cardiovascular diseases and tumour.Predict whole world diabetic subject in 2025 and will reach 300,000,000 people.Wherein type i diabetes patient (IDDM) will account for 5-10%; These crowds will rely on exogenous insulin throughout one's life.Long term injections Regular Insulin can make the local skin redness, itches, scleroma, infection, lipoatrophy or fibrosis hyperplasia.Every day, insulin injection was not only made troubles to the patient and misery, the more important thing is that the application of Regular Insulin only can be controlled hyperglycemia, can't prevent the generation of its chronic complicating diseases.Experimental results demonstrate for many years: the xenogenesis insulin cell is transplanted, and can not only reverse (IDDM) type i diabetes patient's hyperglycemia symptom, and can also prevent the generation and the development of chronic complicating diseases.But the graft application of islet cells is to the clinical several guardian technique problems of solution that need.The one, the source of islet cell donor.The 2nd, the separation of islet cells and purifying.The 3rd, islet cells cultivation, instructionization, induce.The 4th, the selection of transplantation site.These link gordian techniquies must be used modern biological new and high technology and solve, otherwise lack that part, all can cause the failure of islet cell transplantation treatment type i diabetes, cause the diabetes recurrence.
The present situation of islet cell transplantation treatment type i diabetes: nineteen nineties: the clinical application research of pancreatic islets transplantation has become the focus of transplanting boundary's research.According to the pertinent data report, existing three 70 many cases type i diabetes patients of country had accepted the pig pancreatic islets transplantation in 1989 to 2003.At the initial stage, both at home and abroad the investigator adopts people embryo pancreas islet to transplant, and the mechanism of type i diabetes morbidity to be the autoimmunization damage cause, though homotransplantation does not produce rejection, autoimmunity is kept away unavoidable to the destruction of islet cells and islet.Add effects limit such as people embryo donor shortage, ethics this work popularization and carry out.The Regular Insulin that it is found that pig is afterwards used more than ten years clinically, and security, validity fully proved, and is very similar to the insulin structure of pig from molecular biology and histologic analysis people, all is made up of A chain, B chain, 51 amino acid; The A chain all contains 21 amino acid, 30 amino acid of B chain.Different is that people B chain the 30th amino acids is a Threonine; The 30th L-Ala of pig B chain.Amino acid whose difference does not influence specific three-dimensional arrangement, so the biological function activity does not change.The pig pancreas islet causes the very big interest of investigator as donor like this.In order to obtain the purifying pancreas islet, when being separated, pancreas adopted collagenase digestion, and this method can obtain the purifying pancreas islet in the laboratory, contacts the side effect that the back generation can not be repaired with collagenase but ignored pancreas islet.Collagenase easily produces jelly in digestive process.This thing is difficult for wash-out, and easily sticking to influences islet cells absorption to nutritive substance in growth, renewal process on the microbial film, also affects the discharge of cell metabolism product simultaneously.Collagenase is a kind of stronger chemoattractant, is attached on the islet cells film scavenger cell is had strong chemotaxis in addition, does not so not only reduce repulsive interaction, and has strengthened immune rejection.The digestion of collagenase easily produces a large amount of thrombin, can increase the danger of transplanting the back intravascular coagulation, is unfavorable for after the pancreatic islets transplantation long-term survival in blood.Aspect cell cultures, the RPMI1640 nutrient solution is all adopted in each laboratory, and other adds calf serum or foetal calf serum.But the complicated component of serum easily causes bad culture effect.
Summary of the invention
The production method of a kind of repairable pigling pancreatic island cell that purpose of the present invention is developed at existing problem in the above-mentioned prior art just.
The objective of the invention is to realize by following scheme: the production method of repairable pigling pancreatic island cell of the present invention comprises following processing step:
(1), the selection of islet cell donor: donor is selected birth just, the healthy piglet of lactation not, and aseptic opening abdomen gets pancreas down, and removal coating, blood vessel, reticular tissue are also put in 4 ℃ of Hank ｀ s liquid;
(2), the separation of islet cells and purifying: make pancreas islet and external secretion separate tissue by mechanical mill.Centrifugal by Dextran (dextran-Shanghai reagent two factory's products) discontinuous density gradient again, obtain islet cells, sampling microscopy form, active purity, quantity, collection finish should be controlled at 4-6 hour from being separated to purifying;
(3), the cultivation of islet cells: use the microsphere supported CO that carries out of serum-free
2Cultivate, adopt the RPMI-1640 nutrient solution, and add fibronectin, in culturing process, islet cells is wrapped in the A-P-A microbial film forms microballoon, detect micro-capsule form, activity, counting in good time, contain CO for 37 ℃ at constant temperature
2Cultivate in the incubator, changed nutrient solution one time in 48 hours, regularly get nutrient solution and measure insulin content.The differentiation in micro-capsule of observed and recorded islet cells, growth, excreting insulin function are judged the termination incubation time, finally can obtain to have in a large number active and the bigger micro-capsule newborn piglet islet cells of purity.
In the present invention, described piglet is in the birth just 24 hours and be filial generation.
The separation of islet cells and the concrete steps of purifying are:
1. separate pancreas islet
A, employing VI6 type oscillatory type cell ball beveller make pancreas islet and other pancreas partly break away from (grinding 10min);
B, slurry is put into 4 ℃ of Hank ｀ s liquid, cross 500 μ m metal Stainless Steel nets;
C, not over the net organizing are again ground, and cross the net repeated multiple times, make slurry pass through 500 μ m metal Stainless Steel nets;
D, with the filtrate of collecting in 4 ℃ of environment, with centrifugal 5 minutes of 1500r/min, collecting precipitation thing R, abandoning supernatant;
2. purifying: centrifugal with the dextran discontinuous density gradient, operate as follows:
A, with 15ml, 27% Dextrna (dextran-Shanghai reagent two factory's products) and the abundant mixing of throw out, slowly add 4ml, 27% Dextran on this basis; 6ml, 23% Dextran; 4ml, 11% Dextran and the Hank ｀ s of 4ml;
B, with 4 ℃, the centrifugal 4min of 600r/min;
C, accelerate to the centrifugal 10min of 2500r/min then;
D, the two-layer islet cells of collection 11%-23%;
E, wash twice, abandoning supernatant, collecting cell precipitation S with RPMI-1640 liquid
F, with cell precipitation S and 10mlRPMI-1640 mixing, the sampling microscopy is observed pancreas islet form, activity, purity, quantity, surviving rate;
G, finish to be controlled at 4-6 hour from being separated to purifying.
Innovative point of the present invention is as follows:
＜one〉selection of donor
1. (not lactation) piglet that is no more than a day of birth just.Because of just leaving parent, do not contact with the external world.As long as the paternal maternal side of piglet is conscientiously checked, just can stop the generation of transmissible disease, the security after just the energy underwriter transplants.
2. utilize the characteristics of heterosis, hybrid vigor, the easy mutagenesis of cell easily shakes down.
3. porcine islet is mature on the whole at late embryogenesis.And exocrine pancreas, growth lags behind pancreas islet.Be easy to distinguish pancreas islet and external secretion tissue like this.Be convenient to the purifying of pancreas islet.
4. harsh one day piglet (not lactation) and tire pig relatively need not anyly be performed the operation; Not contact extraneous security big with young pig; Relatively pancreas islet is just ripe with becoming pig, and immunizing antigen is low; By cultivating instructionization, inducing the plasticity-that can bring into play hybridization, immature pancreas islet, low to aspect development and the cost that the mankind wish, be fit to industrialization production.
＜two〉separation and purification of islet cells.
Because pig pancreas islet differentiation, the late embryogenesis of reaching maturity, that is that all right when childbirth is ripe for the secretory tissue of pancreas.So should not separate with conventional collagenase digesting.Enzyme is to the side effect that can not repair of islet cells, be cause growth after the pancreatic islets transplantation, updating ability is low, active, function is poor, can not retain in the major cause of human body for a long time.Therefore adopt VI6 type oscillatory type cell ball shredder.(Johanna Otto company product) makes pancreas islet and external secretion separate tissue by mechanical mill.Centrifugal by Dextran (dextran-Shanghai reagent two factory's products) discontinuous density gradient again, obtain islet cells.
＜three〉use that serum-free is microsphere supported to be cultivated.
1. add Fibronectin (FN) in substratum, by volume every liter of substratum adds FN 5-30mg, and it has promoted the adherent adhesion growth of cell.Adhesion is the prerequisite that zooblast is finished growth.
2. in culturing process, islet cells is wrapped in the A-P-A microbial film forms microballoon, so not only protected islet cells to avoid the attack of rejection, also be the fusion of islet cells, the adherent condition of having created simultaneously.
A-P-A is the abbreviation of APA microcapsule technology.(reference: " foreign medical science surgery fascicle ", 2000 the 27th the 2nd phases of volume.P75-77)
Cellular segregation of the present invention, purifying, culture technique are compared with prior art, its advantage is: because the pancreatic islets transplantation thing is directly lost pancreas islet quantity in common purge process more, and often because dead easily with forced oscillation, high speed centrifugation, preparation time prolongation etc.Confirm that now cytoskeleton closely links to each other with extracellular matrix by striding film conglutnin molecule, forms a large amount of networks with 26S Proteasome Structure and Function.When cellular segregation or Transplanted cells, cytoskeleton, the extracellular matrix complex body breaks, conglutnin molecule transfer cell dead signal.Experiment shows that cell and its extracellular matrix dialysis will cause apoptosis by the numerator mediated dead signal of a kind of conglutnin.When the expert observes in the sepn process incomplete digestion in the laboratory, pancreas islet is embedded in the circle extracellular matrix, its apoptosis rate very low (＜10%) and pancreas islet after highly purified has high apoptosis rate (90%), and the former continues that good insulin secretion function is arranged.This shows isolating pancreatic islets transplantation object height degree non-activity and afunction, if added fibronectin in sepn process, can reduce the apoptosis of free pancreas islet, and the pancreas islet survival rate can reach more than 90%, and quantity is more, and volume is bigger.The islet cells of these bulks group is difficult for when implanting losing because of portal venous flow or pressure.
Technology wiring diagram of the present invention is:
Specific embodiments
The present invention further describes as follows below in conjunction with the concrete steps process:
One, the selection of xenogenesis pancreas islet:
1. the characteristic of newborn piglet pancreas islet:
(1) insulin molecule of people and pig all has A chain and B chain, all is made up of 51 amino acid.The A chain contains 21 amino acid, and the B chain contains 30 amino acid.And all there are two sulphur chains to connect between A, the B chain.Structure is very similar, and different have only the poor of an amino acid.Insulin human B chain the 30th amino acids is a Threonine.Pork insulin B chain the 30th amino acids is a L-Ala.(2) pork insulin is used for clinical treatment type i diabetes patient history of existing more than ten years, has fully proved security and validity after pork insulin is used for the people.(3) a little less than the neonatal pig pancreas islet immunogenicity, it is little to produce rejection.(4) porcine islet is containing in people's fresh serum substratum and can survive well and rise in value.The serum that shows the people is to the acellular toxic action of pig pancreas islet, the instructionization after helping transplanting, induces and grows.(5) just be born piggy particularly pancreatic cell also the differentiation finish.Cultivate the plasticity-of performance neonatal pig at different border environment.(6) neonatal pig contact fewly with extraneous, as long as maternal, paternal line does not have people, animal to suffer from transmissible disease altogether, neonatal pig does not just have transmissible disease, but the mother of newborn piglet, paternal line will have pedigree, case history, health check-up, epidemic prevention archives.After the approval of legal relevant department is arranged, can use.
2. biological safety measure:
(1) zooblast is cultivated from exsomatizing to and must be finished at short notice, but for human security, newborn piglet also will be done viral antibody inspection (hepatitis virus, immunodeficiency virus, cytomegalovirus) before getting pancreas, putting to death.(2) islet cells will be followed the tracks of detection, notice that abnormal conditions take place in culturing process.(3) before pancreatic islets transplantation to the mechanical property of micro-capsule, microbial film is to macromolecular controlled; The permeability of nutritive substance, the meta-bolites of islet cells own; The burst size of the form of cell, purity, quantity, Regular Insulin etc. all will conscientiously detect, and makes record.
Two, newborn piglet is dissected pre-treatment:
1. health check-up, number, weigh, whether source, sex, physical appearance, organ, four limbs unusual, body temperature, antiviral antibody inspection.
2. with tap water flush away body surface pollutent.
3. sterilize at body surface with alcohol.
4. the gauze with sterilization wraps up.
5. enter aseptic prosectorium by pass-through.
Three, newborn piglet is dissected and is got pancreas:
1. under sterile state, piglet is anaesthetized intraperitoneal injection (per kilogram of body weight consumption 17mg) with ketamine.
2. newborn piglet is fixed on (the dark * height of wide *) 1200 * 750 * 860mm on the autopsy table.
3. cut open the belly: at first check the piglet anesthesia level, whether body temperature, heartbeat, breathing is normal, carries out disinfection, cuts open the belly by the operation routine then.Along mesoduodenum, find out pancreas.＜1〉weighs and note down.＜2〉remove head of pancreas (upset) and collect body of pancreas and tail of pancreas (crevice), weigh, wash twice with 4 ℃ of Hank ｀ s liquid.
4. in aseptic pallet, remove the appreciable pancreas islet film of naked eyes, blood vessel, reticular tissue with operating scissors.
Four, separate pancreas islet:
1. adopt VI6 type oscillatory type cell ball beveller to make pancreas islet and other pancreas partly break away from (grinding 10min).
2. slurry is put into Hank ｀ s liquid, crossed 500 μ m metal Stainless Steel nets.
3. not over the net organizing again ground, and crosses the net repeated multiple times, makes slurry pass through 500 μ m metal Stainless Steel nets.
4. with 4 ℃ again of the filtrate of collecting, with centrifugal 5 minutes of 1500r/min, collecting precipitation thing R, abandoning supernatant.
Five, purifying:
Centrifugal with the dextran discontinuous density gradient, operate as follows:
1,, slowly adds 4ml, 27% Dextran on this basis with 15ml, 27% Dextrna and the abundant mixing of throw out R; 6ml, 23% Dextran; 4ml, 11% Dextran and the Hank ｀ s of 4ml;
2, with 4 ℃, the centrifugal 4min of 600r/min;
3, accelerate to the centrifugal 10min of 2500r/min then;
4, collect the two-layer islet cells of 11%-23%;
5, wash twice with RPMI-1640 liquid, abandoning supernatant, collecting cell precipitation S
6, with cell precipitation S and 10mlRPMI-1640 mixing, the sampling microscopy is observed pancreas islet form, activity, purity, quantity, surviving rate;
7, finish to be controlled at 4-6 hour from being separated to purifying.
Six, pancreas islet counting:
1. islet cells precipitation dithizone (DTZ) solution [the dithizone 10mg that gradient centrifugation is collected; The inferior maple 3ml of 99.5% dimethyl; 25% ammoniacal liquor, 50 μ l] dye, (islet cells takes on a red color or the raw meat redness under the mirror at the microscopically counting, shape is rounded, and the plentiful orange of endochylema is translucent, edge clear, other tissue and cell are not painted, and contrast is very distinct) islet cells diameter 50-300 μ m, differ in size, three countings of repeated sampling are averaged.
2. expect blue dyeing differentiation pancreas islet anyway with platform, viable cell is not painted, and dead cell is blue look.(mirror is observed down).
3. a patient once transplants and needs ten thousand pancreas islet of 36-40.(having 20-25 newborn piglet pancreas to provide approximately).
Seven, pancreas islet microcapsule technology:
Adopt the A-P-A microcapsule technology to make micro-capsule islet cells (reference: " (foreign medical science surgery fascicle ", 2000 the 27th volume the 2nd phase P75-77) with electrostatic high-pressure micro-capsule generator ten thousand islet cellss alive of 36-40.
Micro-capsule pancreas islet morphology is identified:
(1) light microscopy checking, the micro-capsule diameter should be 0.25-0.35mm
(2) in 37 ℃ of water bath with thermostatic control shakers, observed the micro-capsule breakage rate in 24 hours through vibrating with 150 times/min
(3) fluorescent dye: put 4 ℃ of storages with isotonic phosphate buffer liquid preparation pyridine orange (AO) [670 μ mol/L] and iodine third pyridine (PI) [750 μ mol/L] storage liquid, face with before be made into (AO) concentration be 0.67 μ mol/L and (PI) concentration be that the mixed solution of 57 μ mol/ dyes, viable cell is green and just looks at fluorescence, dead cell takes on a red color, between the difference clearly.
(4) formula of pancreas islet counting:
[N=quantitatively draws the microlitre number of suspension with micro sample adding appliance]
(5) pancreas islet reclaims formula behind the purifying:
Microscopy pancreas islet number * 100% before microscopy pancreas islet number/purifying behind the rate of recovery=purifying
(6) pancreas islet purity is estimated:
Recently estimate purity with pancreas islet number under the inverted microscope and external secretion histocyte quantity.
Eight, the micro-capsule islet cells is cultivated:
1. at 37 ℃ of constant temperature, CO
2Cultivate in the incubator, changed nutrient solution one time in 48 hours, regularly get nutrient solution and measure insulin content.The differentiation in micro-capsule of observation islet cells, growth, excreting insulin function are judged the termination incubation time.
2.RPMI-1640 nutrient solution adds fibronectin and carries out serum-free culture, the adherent conglomeration growth of inducing cell, differentiation.
Nine, cultured pancreas islet preferably will be transplanted the same day, and refrigeration cell growth, transplanting have certain influence.
Claims (3)
1, a kind of production method of repairable pigling pancreatic island cell is characterized in that: it comprises following processing step:
(1), the selection of islet cell donor: donor is selected birth just, the healthy piglet of lactation not, and aseptic opening abdomen gets pancreas down, and removal coating, blood vessel, reticular tissue are also put in 4 ℃ of Hank`s liquid;
(2), the separation of islet cells and purifying: make pancreas islet and external secretion separate tissue by mechanical mill.Centrifugal by the Dextran discontinuous density gradient again, obtain islet cells, sampling microscopy form, active purity, quantity, surviving rate were controlled at 4-6 hour from being separated to the purifying concluding time;
(3), the cultivation of islet cells: use that serum-free is microsphere supported to be cultivated, adopt the RPMI-1640 nutrient solution, do not contain bovine serum in the nutrient solution, and adding fibronectin (FN), add-on adds FN 5---30mg by every liter of culture volume, in culturing process, islet cells is wrapped in the A-P-A microbial film forms microballoon, detect micro-capsule form, activity, counting, at 37 ℃ of CO of constant temperature in good time
2Cultivate in the incubator, changed nutrient solution one time in 48 hours, regularly get nutrient solution and measure insulin content; The differentiation in micro-capsule of observed and recorded islet cells, growth, excreting insulin function are judged the termination incubation time, finally obtain the micro-capsule newborn piglet islet cells that activity is good, purity is high.
2, the production method of repairable pigling pancreatic island cell according to claim 1 is characterized in that: described piglet is in the birth just 24 hours and be filial generation.
3, the production method of repairable pigling pancreatic island cell according to claim 1 is characterized in that: the separation of islet cells and the concrete steps of purifying are:
1. separate pancreas islet
A, employing VI6 type oscillatory type cell ball beveller partly break away from pancreas islet and other pancreas, grind 10min;
B, slurry is put into 4 ℃ of Hank`s liquid, cross 500 μ m metal Stainless Steel nets;
C, not over the net organizing are again ground, and cross the net repeated multiple times, make slurry pass through 500 μ m metal Stainless Steel nets;
D, with the filtrate of collecting in 4 ℃ of environment, with centrifugal 5 minutes of 1500r/min, collecting precipitation thing R, abandoning supernatant;
2. purifying: centrifugal with the dextran discontinuous density gradient, operate as follows:
A, with 15ml, 27% Dextrna and the abundant mixing of throw out R, slowly add 4ml, 27% Dextran on this basis; 6ml, 23% Dextran; 4ml, 11% Dextran and the Hank`s of 4ml;
B, with 4 ℃ of centrifugal 4min of 600r/min;
C, accelerate to the centrifugal 10min of 2500r/min then;
D, the two-layer islet cells of collection 11%-23%;
E, wash twice, abandoning supernatant, collecting cell precipitation S with RPMI1640 liquid;
F, with cell precipitation S and 10mlRPMI1640 mixing, the sampling microscopy is observed pancreas islet form, activity, purity, quantity, surviving rate;
G, be controlled at 4-6 hour from being separated to the purifying concluding time.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008034387A1 (en) * | 2006-09-18 | 2008-03-27 | Zhengzhou Tongyuan Biotechnology Co., Ltd. | Method for preparing repairable piglet pancreatic island cells |
| CN104011546A (en) * | 2011-09-28 | 2014-08-27 | 胰岛科学股份有限公司 | Ex vivo maturation of islet cells |
| CN106963782A (en) * | 2017-03-28 | 2017-07-21 | 王斌 | A kind of preparation method and application of the compound microencapsulated transplantation body of fibronectin and functioning cell |
| CN110251731A (en) * | 2019-06-26 | 2019-09-20 | 山东百多安医疗器械有限公司 | A kind of used in tissue engineering islet cells micro-capsule and preparation method thereof |
| CN112063577B (en) * | 2020-08-14 | 2023-05-16 | 中国医科大学附属第一医院 | Combined culture medium for islet culture and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11253555A (en) * | 1998-03-13 | 1999-09-21 | Ltt Kenkyusho:Kk | Manufacture of barium alginate capsulated pancreatic islet, using specific gravity density gradient centrifugal method |
| US8288085B2 (en) * | 2004-12-22 | 2012-10-16 | Kyoto University | Method of separating pancreatic islet |
| CN100554407C (en) * | 2006-09-18 | 2009-10-28 | 郑州福恩生物工程技术有限公司 | The production method of repairable pigling pancreatic island cell |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008034387A1 (en) * | 2006-09-18 | 2008-03-27 | Zhengzhou Tongyuan Biotechnology Co., Ltd. | Method for preparing repairable piglet pancreatic island cells |
| CN104011546A (en) * | 2011-09-28 | 2014-08-27 | 胰岛科学股份有限公司 | Ex vivo maturation of islet cells |
| CN106963782A (en) * | 2017-03-28 | 2017-07-21 | 王斌 | A kind of preparation method and application of the compound microencapsulated transplantation body of fibronectin and functioning cell |
| CN110251731A (en) * | 2019-06-26 | 2019-09-20 | 山东百多安医疗器械有限公司 | A kind of used in tissue engineering islet cells micro-capsule and preparation method thereof |
| CN112063577B (en) * | 2020-08-14 | 2023-05-16 | 中国医科大学附属第一医院 | Combined culture medium for islet culture and preparation method thereof |
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| WO2008034387A1 (en) | 2008-03-27 |
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