CN104762257A - Method for preparing mesenchymal stem cell from umbilical cord - Google Patents
Method for preparing mesenchymal stem cell from umbilical cord Download PDFInfo
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- CN104762257A CN104762257A CN201510171887.5A CN201510171887A CN104762257A CN 104762257 A CN104762257 A CN 104762257A CN 201510171887 A CN201510171887 A CN 201510171887A CN 104762257 A CN104762257 A CN 104762257A
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Abstract
The invention relates to a method for preparing a mesenchymal stem cell from umbilical cord, and a mesenchymal stem cell prepared through the utilization of the method. The method comprises the following steps: (1) taking an umbilical cord sample; (2) removing a blood vessel and around tissues clung to the blood vessel; (3) preparing an umbilical cord tissue slice; (4) separating amnion substratum tissue; (5) treating the amnion substratum tissue; (6) cultivating in non-cell culture fluid; (7) adding cell culture fluid, and then continuously culturing; (8) trypsinizing; (9) filtering; and (10) centrifuging filtrate and removing supernatant, and repeatedly suspending to obtain the mesenchymal stem cell of the umbilical cord tissue. The mesenchymal stem cell prepared through the adoption of the method disclosed by the invention is high in purity, large in proliferation and differentiation potential, and extensive in application prospect in base research and clinical treatment.
Description
Technical field
The present invention relates to a kind of method preparing mescenchymal stem cell from umbilical cord, particularly relate to the amnion lower-hierarchy isolate mesenchymal stem cells from umbilical cord, belong to technical field of bioengineering.
Background technology
Stem cell is the cell that a class has self and multi-lineage potential.Different according to the precedence occurred in ontogenetic process, be divided into embryonic stem cell (ESC) and adult stem cell (ASC).Embryonic stem cell is the cell separated from the inner cell mass of blastaea, can unlimited self, can be divided into interior, in, the various biological cells and tissues of outer 3 germinal layers.But due to stem-cell research embryonic origin difficulty, itself there is the problems such as tumorigenicity, immunological rejection and ethics and limit it in clinical application.Along with the development of stem cells technology, oneself confirms to make it be programmed for the cell of embryonic stem cell sample again by the factor importing several key the terminally differentiated cells of adult now, this cell is called as inductive pluripotent stem cells, it can walk around ethics morals problem, but because need by virus vector quiding gene, there are the problems such as programming efficiency is low, Tumor formation, thus limit their application clinically.Adult stem cell is the general designation of the multipotential stem cell be present in fetus, children and adult's tissue, be found at Various Tissues organ now, as hemopoietic stem cell, neural stem cell, liver stem cells, cardiac stem cells and mescenchymal stem cell (MSC) etc.It is generally acknowledged, adult stem cell can only be divided into the cell type of its histoorgan, but research in recent years shows, the differentiation capability of these stem cells far exceedes the scope of traditional view limitation, as neural stem cell can be divided into blood cell, cord blood stem cell can be divided into neurocyte etc.
Mescenchymal stem cell derives from mesoblastic adult stem cell, is extensively present in whole body reticular tissue and organ interstitial, has good multiplication capacity and multi-lineage potential.Under different inductive conditions, mescenchymal stem cell not only can be divided into the mesoblastemas such as cartilage, bone, skeletal muscle, tendon, fat, simultaneously also can to ectodermic neurocyte and endoblastic liver oval sexual cell and various kinds of cell and tissue differentiation.Mescenchymal stem cell is easy to be separated and cultivate, be easy to importing and the expression of foreign gene, the cytokine profiles of secretion has the characteristic of immunity moderation, hematopoiesis support, and immunogenicity is weak, and genetic background is stablized and do not limited by the ethics problem run in embryo cleavage.Therefore, mescenchymal stem cell is the desirable seed cell source of organizational project, genetically engineered and cell therapy.
The clinical experimental study carried out at present confirms, has self, treatment and a rehabilitation that the mescenchymal stem cell of multi-lineage potential can be applied to various diseases.But, all research or treatment plan all must be based upon obtain enough mescenchymal stem cells basis on, isolate mesenchymal stem cells has very important researching value and economic interests as seed cell fast and effectively.In research and practical application, find that seed cell exists variety of problems, such as, although there is a large amount of mescenchymal stem cells in autologous bone marrow, but along with the age increases, mescenchymal stem cell number in autologous bone marrow can significantly reduce, and multiplication capacity also can significantly fail, simultaneously, from bone marrow collection mescenchymal stem cell complicated operation, unsuitable application.
Therefore, investigator starts other sources finding mesenchymal cell, and wherein, umbilical cord mesenchymal stem cells source is sufficient, virus contamination probability is low, immunogenicity is weak, cell is more original, also there is not society, ethics and legal dispute.People's umbilical cord is connected between parent and fetus, pregnancy duration provides nutrition for fetus, forms primarily of three parts: amnion lining epithelium, cord vessels and being positioned at is called as the mucus reticular tissue of magnificent Tong Shi glue (Wharton ' s Jelly) between the two.The main source of umbilical cord mesenchymal stem cells is magnificent Tong Shi glue and bleeding of the umbilicus.
The method be separated for umbilical cord mesenchymal stem cells at present mainly contains adherent method and enzyme digestion two kinds.Wherein adherent method is easy and simple to handle, low to requirement for experiment condition, easily grasps.The shortcoming of this method is, the cycle obtaining primary cell is long, and the buoyancy of liquid makes tissue block levitating, make it lose to grow the ability of cell, decrease cell quantity, in addition, be mixed with part endotheliocyte in product, need further to be screened by the collagenase treatment in later stage.Although adopt collagenase digestion can obtain cell at short notice, impurity is few, and costly, and condition is not easily grasped, may damaging cells if digestion time is long in normal temperature, causes cell not easily adherent and go down to posterity; If digestion time is short, liquid thickness, is difficult to by centrifugal acquisition q.s cell.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of umbilical cord mesenchymal stem cells preparation method not adopting digestive ferment to be different from again conventional adherent method is provided.The present inventor, by having carried out large quantity research and exploration to source for mesenchymal stem cells different in umbilical cord, experiment condition etc., finds can prepare high purity, highly active mescenchymal stem cell from the amnion lower-hierarchy of umbilical cord.The present inventor is also optimized experiment condition, does not first add cell culture fluid and cultivates for some time at 38.5 DEG C, and then add cell culture fluid cultivation, can significantly improve the rate of recovery of mescenchymal stem cell after finding to isolate amnion lower-hierarchy.Method of the present invention is simple to operate, and the mescenchymal stem cell purity obtained is high, has self and the multi-lineage potential of enhancing.
In order to realize object of the present invention, the invention provides a kind of method preparing mescenchymal stem cell from umbilical cord, comprising the steps: that (1) gets umbilical cord sample; (2) remove blood vessel and around it, be close to the tissue of blood vessel; (3) umbilical cord tissue sheet is prepared; (4) amnion lower-hierarchy is separated; (5) amnion lower-hierarchy is processed; (6) do not add cell culture fluid to cultivate; (7) add nutrient solution to continue to cultivate; (8) trysinization; (9) filter; (10) filtrate is centrifugal and remove supernatant, Eddy diffusion obtains umbilical cord tissue mescenchymal stem cell.
Preferably, method of the present invention comprises the steps: that (1) gets neonatal umbilical cord, repeatedly rinses well with the phosphate buffered saline buffer containing penicillin and Streptomycin sulphate, removes residual blood, obtains clean umbilical cord; (2) clean umbilical cord scalpel is longitudinally cut, remove vein blood vessel and arteries with tweezers and around it, be close to the tissue of blood vessel; (3) remaining tissue is shakeout, carefully reject gum part inside umbilical cord with tweezers inside umbilical cord upward, divest clean, until the tissue of remaining about 1mm thickness; (4) fixing umbilical cord tissue sheet Pictest skin side, to tear neat organized layer and amnion lower-hierarchy inside umbilical cord with tweezers, the umbilical cord epidermis that remaining thin layer is transparent and rete layer tissue; (5) amnion lower-hierarchy scissors is cut into about 1cm
2tissue, will be attached in culture dish near epidermis side and neatly arrange; (6) do not add cell culture fluid, be directly positioned over 5%CO
2, in 38.5 DEG C of incubators, leave standstill after one hour, be inverted two hours; (7) then add cell culture fluid 5mL, be placed in 5%CO
2, continue to cultivate in 37 DEG C of incubators, when the cell around tissue reaches 80% fusion, culture dish is taken out in incubator; (8) trysinization: first clean culture dish twice with phosphate buffered saline buffer, washing lotion is abandoned in suction, draw the phosphate buffered saline buffer containing 0.25% pancreatin and 0.1%EDTA, join in culture dish, digest 3 ~ 5 minutes, again cell culture fluid is joined in culture dish, repeatedly blow and beat, make umbilical cord tissue mescenchymal stem cell enter in Digestive system; (9) by Digestive system through 100 order sterilizing strainer filterings, remove umbilical cord tissue sheet, obtain containing the filtrate of umbilical cord tissue mescenchymal stem cell; (10) by filtrate under 900rpm centrifugal 5 minutes, remove supernatant, Eddy diffusion cell, obtain umbilical cord tissue mescenchymal stem cell.
Preferably, the phosphate buffered saline buffer in aforesaid method is composed as follows: 8g/L NaCl, 0.2g/LKCl, 1.15g/L Na
2hPO
4, 0.2g/L KH
2pO
4, pH 7.4.
Preferably, contain in aforesaid method in the phosphate buffered saline buffer of penicillin and Streptomycin sulphate, the concentration of penicillin is 100U/ml, and the concentration of Streptomycin sulphate is 0.1mg/ml.
Preferably, the cell culture fluid in aforesaid method is the D-MEM/F12 nutrient solution comprising 10-30% foetal calf serum and 5-15ng/mLEGF.Preferred, described cell culture fluid is the D-MEM/F12 nutrient solution comprising 20% foetal calf serum and 10ng/mL EGF.
The present invention also comprises the mescenchymal stem cell utilizing aforesaid method to prepare.
For the mescenchymal stem cell prepared according to the method for the invention, the standard of working out according to " international cell therapy association " (ISCT) carries out biological phenotype qualification, shows following technical characteristic:
1. the growth of plastic culture vessel fibroblast dimension sample is adhered under Standard culture conditions;
2. express CD29, CD44, CD90, CD105, do not express CD31, CD34, CD45, CD71;
3. externally neurocyte is induced to differentiate into.
The amnion lower-hierarchy of umbilical cord is the tissue being arranged in umbilical cord amnion lower floor position, the tissue (see Fig. 1) namely between rete layer and magnificent Tong Shi glue.Above-mentioned qualification result shows that the stem cell that the present invention is separated from the amnion lower-hierarchy of umbilical cord belongs to mescenchymal stem cell, and not containing hemopoietic stem cell.
The primordial stem cell prepared by the inventive method, because of its material therefor source and the performance of finished product cell, can solve the multinomial difficult problem perplexing medical circle in stem cell Application Areas at present.Method of the present invention does not adopt digestive ferment, avoid in mescenchymal stem cell sepn process, digestive ferment introduces the Pollution risk of animal derived protein and animal source pathogen to umbilical cord mesenchymal stem cells, more highly purified mescenchymal stem cell is obtained again than common paster method, eliminate ectoderm and the interference of endoblastic contaminating cell, improve cell purity.In addition, method of the present invention is through optimizing, improve the rate of recovery from amnion lower-hierarchy isolate mesenchymal stem cells, and the mescenchymal stem cell of preparation has higher propagation and differentiation potential, be conducive to it and play in later stage studies and clinical application and more importantly act on.
Accompanying drawing explanation
Fig. 1 is Cord structure schematic diagram.
A represents amnion, and B represents amnion lower-hierarchy, and C represents magnificent Tong Shi glue, and D represents perivascular cell, and E represents cord vessels.
Embodiment
In order to make those skilled in the art have understanding clearly to technical characteristic of the present invention, object and beneficial effect, below by embodiment, the present invention is further illustrated, but should not be construed as limitation of the present invention.
Embodiment 1: prepare mescenchymal stem cell from umbilical cord
Mescenchymal stem cell is prepared from umbilical cord according to following step:
(1) umbilical cord is chosen: select to comprise hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and virus of AIDS antibody mediated immunity 4 feminine genders, prepares to carry out caesarean healthy pregnant women.Intercept umbilical cord when cesarean section and be about 10cm, speed is placed in D-Hank balance liquid and delivers to cell culture chamber, processes in super clean bench to umbilical cord.
(2) umbilical cord pre-treatment: with phosphate buffered saline buffer (8g/L NaCl, 0.2g/L KCl, 1.15g/L Na containing penicillin and Streptomycin sulphate (concentration of penicillin is 100U/ml, and the concentration of Streptomycin sulphate is 0.1mg/ml)
2hPO
4, 0.2g/L KH
2pO
4, pH 7.4) repeatedly rinse umbilical cord tissue, remove residual blood, obtain clean umbilical cord.
(3) remove blood vessel: longitudinally cut by clean umbilical cord scalpel, remove vein blood vessel and arteries with tweezers and around it, be close to the tissue of blood vessel.
(4) prepare tissue: shakeout by remaining tissue, carefully reject gum part inside umbilical cord with tweezers inside umbilical cord upward, divest clean, until the tissue of remaining about 1mm thickness.
(5) be separated amnion lower-hierarchy: fixing umbilical cord tissue sheet Pictest skin side, tear neat organized layer and amnion lower-hierarchy, the umbilical cord epidermis that remaining thin layer is transparent and rete layer tissue inside umbilical cord with tweezers.
(6) organized processing: the amnion lower-hierarchy scissors of acquisition is cut into about 1cm
2tissue, will be attached in culture dish near epidermis side and neatly arrange.
(7) cultivate without nutrient solution: do not add cell culture fluid, be directly positioned over 5%CO
2, in 38.5 DEG C of incubators, leave standstill after one hour, be inverted two hours.
(8) add nutrient solution to cultivate: add cell culture fluid 5mL, be placed in 5%CO
2, continue cultivation about seven days in 37 DEG C of incubators, in culture dish, umbilical cord tissue starts have a large amount of cell to climb out of, and about fortnight, in time organizing peripheral cell to reach the degrees of fusion of 80%, is taken out by culture dish in incubator.
(9) trysinization: clean culture dish twice with phosphoric acid buffer, each consumption 10mL, washing lotion is abandoned in suction, draw the phosphoric acid buffer that 1mL contains 0.25% pancreatin and 0.1%EDTA, join in culture dish, digestion 3-5 minute, then D-MEM/F12 nutrient solution 5mL being contained 20% bovine serum and 10ng/mL EGF join in culture dish, repeatedly blow and beat, umbilical cord tissue mescenchymal stem cell is entered in Digestive system.
(10) filter: Digestive system, through 100 order sterilizing strainer filterings, removes umbilical cord tissue sheet, obtain the filtrate containing umbilical cord tissue mescenchymal stem cell.
(11) are centrifugal resuspended: by filtrate under 900rpm centrifugal 5 minutes, remove supernatant, use 3mLD-MEM/F12 nutrient solution Eddy diffusion uniform cell, obtain umbilical cord tissue mescenchymal stem cell.
Embodiment 2: the qualification of the mescenchymal stem cell of separation
Carry out identification and detection to the mescenchymal stem cell utilizing the method for embodiment 1 to prepare, identification and detection project comprises:
1. morphological observation: cell attachment becomes fiber-like to grow.
2. biological phenotype qualification: utilize flow cytomery cell phenotype.
1. the mescenchymal stem cell prepared by embodiment 1 respectively with the antibody incubation of fluorescently-labeled anti-CD29, CD31, CD34, CD44, CD45, CD71, CD90 and CD105, with the mouse IgG Isotype antibody of FITC and PE mark in contrast;
2. 4 DEG C of incubations 45 minutes, centrifugal collecting cell;
3. utilize phosphate buffered saline buffer to clean cell 3 times, then cell is resuspended in 300 μ l phosphate buffered saline buffers, carries out flow cytometry analysis, calculate the positive rate of cell surface marker thing.
Table 1 shows flow cytometry analysis result.
Table 1
As can be seen from Table 1, utilize mescenchymal stem cell prepared by method of the present invention, the positive rate of CD29, CD44, CD90 and CD105 is higher, is all greater than 95%; And the positive rate of CD31, CD34, CD45 and CD71 is very low, be all less than 1%.This illustrates that the purity of described mescenchymal stem cell is higher, does not have the pollution of hemopoietic stem cell.
3. safety index detects: carry out safety index detection to the mescenchymal stem cell that embodiment 1 obtains, comprise 1. microbial culture; 2. Viral diagnosis; 3. detection of mycoplasma; 4. the immunology detection of hepatitis B surface antigen, anti-HCV, syphilis antibody, virus of AIDS antibody is comprised.All batches of these four detections are feminine gender, illustrate that Product Safety prepared by method of the present invention is higher.
Embodiment 3: the contrast experiment of the umbilical cord mesenchymal stem cells differentiation potential of different sources
The object of the present embodiment is to identify under the condition that there is nerve cell inducer, and whether the potential that the umbilical cord mesenchymal stem cells of different sources is divided into neurocyte there are differences.
1. laboratory sample: the mescenchymal stem cell that (1) utilizes the method for embodiment 1 to prepare; (2) (concrete preparation method refers to the mescenchymal stem cell prepared from umbilical cord China Tong Shi glue, Yang Xiao is clear, " separation of people's umbilical cord China Tong Shi glue mesenchymal stem cells, cultivation, qualification and frozen, recovery ", " Nantong University's journal (medicine) ", 2010,30th volume, the 6th phase).
2. experimental technique:
(1) by the mescenchymal stem cell from embodiment 1 or umbilical cord China Tong Shi glue with 5 × 10
9the cell concn of individual/L is inoculated and is gone down to posterity in culture dish, carries out breeding, purifying cultivates;
(2) add containing Basic Fibroblast Growth Factor in the cell culture fluid of Secondary Culture, poly-lysine, the nerve cell inducer of dimethyl sulfoxide (DMSO) and oxidation inhibitor combination (beta-mercaptoethanol+all-trans-retinoic acid), cultivate and respectively get 6 holes after 10 days and carry out morphological observation, and add neuronspecific enolase antibody and microtubule-associated protein 2 (MAP-2) antibody, at the expression that fluorescence microscopy Microscopic observation mature neuron mark (neuronspecific enolase and MAP-2) is positive after incubation, statistics positive cell ratio (the results are shown in Table 2).
Table 2
As can be seen from Table 2, the differentiation potential of the mescenchymal stem cell utilizing the method for embodiment 1 to prepare will be significantly higher than the mescenchymal stem cell in magnificent Tong Shi glue source.The present invention has obvious advantage through the method preparing mescenchymal stem cell from umbilical cord amniotic membrane lower-hierarchy optimized relative to the method preparing mescenchymal stem cell from umbilical cord prior art.
Although present invention is described with reference to above-described embodiment, it being understood that the correction carry out it and change are included within the spirit and scope of the present invention.
Claims (7)
1. prepare a method for mescenchymal stem cell from umbilical cord, it is characterized in that, comprise the steps: that (1) gets umbilical cord sample; (2) remove blood vessel and around it, be close to the tissue of blood vessel; (3) umbilical cord tissue sheet is prepared; (4) amnion lower-hierarchy is separated; (5) amnion lower-hierarchy is processed; (6) cell free broth is cultivated; (7) add cell culture fluid to continue to cultivate; (8) trysinization; (9) filter; (10) filtrate is centrifugal and remove supernatant, Eddy diffusion obtains umbilical cord tissue mescenchymal stem cell.
2. the method for claim 1, is characterized in that, comprises the steps: that (1) gets neonatal umbilical cord, repeatedly rinses well with phosphate buffered saline buffer, removes residual blood, obtains clean umbilical cord; (2) clean umbilical cord scalpel is longitudinally cut, remove vein blood vessel and arteries with tweezers and around it, be close to the tissue of blood vessel; (3) remaining tissue is shakeout, carefully reject gum part inside umbilical cord with tweezers inside umbilical cord upward, divest clean, until the tissue of remaining about 1mm thickness; (4) in umbilical cord tissue sheet Pictest skin side, fixing umbilical cord tissue sheet Pictest skin side, to tear neat organized layer and amnion lower-hierarchy inside umbilical cord with tweezers, the umbilical cord epidermis that remaining thin layer is transparent and rete layer tissue; (5) amnion lower-hierarchy scissors is cut into about 1cm
2tissue, will be attached in culture dish near epidermis side and neatly arrange; (6) do not add nutrient solution, be directly positioned over 5%CO
2, in 38.5 DEG C of incubators, leave standstill after one hour, be inverted two hours; (7) then add cell culture fluid 5mL, be placed in 5%CO
2, continue to cultivate in 37 DEG C of incubators, when the cell around tissue reaches 80% fusion, culture dish is taken out in incubator; (8) trysinization: first clean culture dish twice with phosphate buffered saline buffer, washing lotion is abandoned in suction, draw the phosphate buffered saline buffer containing 0.25% pancreatin and 0.1%EDTA, join in culture dish, digestion 3-5 minute, again cell culture fluid is joined in culture dish, repeatedly blow and beat, make umbilical cord tissue mescenchymal stem cell enter in Digestive system; (9) by Digestive system through 100 order sterilizing strainer filterings, remove umbilical cord tissue sheet, obtain containing the filtrate of umbilical cord tissue mescenchymal stem cell; (10) by filtrate under 900rpm centrifugal 5 minutes, remove supernatant, Eddy diffusion cell, obtain umbilical cord tissue mescenchymal stem cell.
3. method as claimed in claim 2, is characterized in that, described phosphate buffered saline buffer composed as follows: the Na of the KCl of the NaCl of 8g/L, 0.2g/L, 1.15g/L
2hPO
4, the KH of 0.2g/L
2pO
4, pH is 7.4.
4. method as claimed in claim 2, is characterized in that, described containing in the phosphate buffered saline buffer of penicillin and Streptomycin sulphate, the concentration of penicillin is 100U/ml, and the concentration of Streptomycin sulphate is 0.1mg/ml.
5. method as claimed in claim 2, it is characterized in that, described cell culture fluid is the D-MEM/F12 nutrient solution of the EGF comprising 10-30% foetal calf serum and 5-15ng/mL.
6. method as claimed in claim 5, it is characterized in that, described cell culture fluid is the D-MEM/F12 nutrient solution of the EGF comprising 20% foetal calf serum and 10ng/mL.
7. the mescenchymal stem cell utilizing the method according to any one of claim 1 ~ 6 to prepare.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695401A (en) * | 2016-03-29 | 2016-06-22 | 南京大学医学院附属鼓楼医院 | Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells |
| CN109439623A (en) * | 2018-11-20 | 2019-03-08 | 潍坊市康华生物技术有限公司 | A method of preparing umbilical cord mesenchymal stem cells |
| CN109652362A (en) * | 2017-10-12 | 2019-04-19 | 北京弘润天源基因生物技术有限公司 | A kind of method that umbilical cord film saves and prepares stem cell |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
| CN112280733A (en) * | 2020-11-02 | 2021-01-29 | 上海坤爱生物科技股份有限公司 | Extraction method of amniotic stem cells |
| CN112430572A (en) * | 2020-12-09 | 2021-03-02 | 湖南源品细胞生物科技有限公司 | Processing method of umbilical cord mesenchymal stem cell collection |
| CN113498434A (en) * | 2020-01-22 | 2021-10-12 | 京东方科技集团股份有限公司 | Mesenchymal stem cell membrane and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695401A (en) * | 2016-03-29 | 2016-06-22 | 南京大学医学院附属鼓楼医院 | Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells |
| CN109652362A (en) * | 2017-10-12 | 2019-04-19 | 北京弘润天源基因生物技术有限公司 | A kind of method that umbilical cord film saves and prepares stem cell |
| CN109439623A (en) * | 2018-11-20 | 2019-03-08 | 潍坊市康华生物技术有限公司 | A method of preparing umbilical cord mesenchymal stem cells |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
| CN113498434A (en) * | 2020-01-22 | 2021-10-12 | 京东方科技集团股份有限公司 | Mesenchymal stem cell membrane and application thereof |
| CN112280733A (en) * | 2020-11-02 | 2021-01-29 | 上海坤爱生物科技股份有限公司 | Extraction method of amniotic stem cells |
| CN112280733B (en) * | 2020-11-02 | 2024-02-23 | 上海坤爱生物科技股份有限公司 | Amniotic stem cell extraction method |
| CN112430572A (en) * | 2020-12-09 | 2021-03-02 | 湖南源品细胞生物科技有限公司 | Processing method of umbilical cord mesenchymal stem cell collection |
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