CN112608892B - Method for serum-free separation and subculturing of umbilical cord mesenchymal stem cells by using platelet lysate - Google Patents

Method for serum-free separation and subculturing of umbilical cord mesenchymal stem cells by using platelet lysate Download PDF

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CN112608892B
CN112608892B CN202011567586.1A CN202011567586A CN112608892B CN 112608892 B CN112608892 B CN 112608892B CN 202011567586 A CN202011567586 A CN 202011567586A CN 112608892 B CN112608892 B CN 112608892B
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mesenchymal stem
stem cells
cells
umbilical cord
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CN112608892A (en
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肖海蓉
李新峰
王正
刘冰
汤乐
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Shenzhen Boya Perception Pharmaceutical Co ltd
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Abstract

The present invention relates to a method of platelet lysate for serum-free separation and subculturing umbilical cord mesenchymal stem cells. In one aspect, the invention relates to a method for subculturing umbilical cord mesenchymal stem cells, comprising the steps of: inoculating primary P0 generation mesenchymal stem cells into a culture bottle, supplementing a passage complete culture medium, culturing in a CO2 incubator until the cell fusion degree reaches 70-80%, discarding the old culture medium, washing the cells with D-hanks liquid, adding a pancreatin solution of a weighting group to digest the cells so as to enable the cells to fall off, adding the D-hanks liquid for dilution, combining all cell suspensions into a centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells; the subculture method from the generation P0 to the generation P1 is continued to obtain umbilical cord mesenchymal stem cells of the subsequent generation, for example, to the generation P10. And also relates to a subculture complete medium for umbilical cord mesenchymal stem cells. The method and the culture medium used in the method have excellent technical effects as described in the specification.

Description

Method for serum-free separation and subculturing of umbilical cord mesenchymal stem cells by using platelet lysate
Technical Field
The invention belongs to the technical field of biology, and relates to a method for separating and passaging umbilical cord mesenchymal stem cells from umbilical cords. The invention also relates to a culture medium and a related test solution used in the culture of the umbilical cord mesenchymal stem cells, and also relates to the application of the serum-free culture medium in the separation and passage of umbilical cord mesenchymal stem cells. The method of the invention can be used for separating, culturing and subculturing the umbilical cord mesenchymal stem cells, and can show the excellent technical effects of the invention. In particular, the invention relates to methods for isolating and subculturing mesenchymal stem cells from umbilical cord using serum-free medium. In particular, the serum-free medium used in the isolated culture and subculture of the umbilical cord mesenchymal stem cells of the present invention contains platelet lysate.
Background
Mesenchymal stem cells (mesenchymal stem cell, MSC), such as human mesenchymal stem cells, were originally isolated from bone marrow, were derived from a class of tissue stem cells of mesoderm that have multipotent differentiation potential and self-renewal capacity, and were capable of differentiating into various adult cells such as osteoblasts, chondrocytes, adipocytes, endothelial cells, neural cells, myocytes, hepatocytes, etc., under specific conditions in vivo and in vitro (Caplan AI. Mesenchyal stem cells J ortho Res.1991,9:641-650.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999;284:143-147). Recent studies have shown that mesenchymal stem cells have immunoregulatory and hematopoietic support effects and are easy for exogenous gene expression. Therefore, the mesenchymal stem cells not only tissue engineer seed cells in bone, cartilage and myocardial construction and carrier cells important in gene therapy, but also have wide application prospects in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft versus host reaction functions. Mesenchymal stem cells have the characteristic of in vitro adherent growth, and by utilizing the characteristic, people have successfully isolated and cultured the mesenchymal stem cells from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.
Stem cells are ancestors of human cells, and all cells in our body are derived from stem cells. When cells in the body die or the injury degenerates, stem cells grow and transform into cells that can replace them. As seed cells, the seed cells are mainly used for treating various refractory diseases of tissue cell and organ injury which cannot be naturally repaired by organisms clinically; as immunoregulatory cells, for the treatment of immune rejection and autoimmune diseases. Human mesenchymal stem cells are important members of stem cell families, originate from early-stage mesoderm, belong to multipotent stem cells, and MSCs are originally found in bone marrow, and are increasingly attracting attention due to their multipotent differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation, self replication and the like. Initial clinical studies were carried out by Lazarus et al in 1995 who collected autologous MSCs from patients with hematological neoplasms in remission, amplified in vitro for 4-7 weeks, and then injected intravenously into patients, and patients were divided into 3 groups, each given different doses of MSCs, with no adverse side effects observed after injection, suggesting that MSCs are safe and reliable for transplantation therapy. Clinical reports of autologous MSCs are gradually increased, and disease types relate to hematopoietic reconstitution after radiotherapy and chemotherapy, graft Versus Host Disease (GVHD), heart system diseases and the like, and all the reports prove that the clinical intravenous infusion is safe and reliable.
The isolated culture and subculture process of mesenchymal stem cells are key steps related to the safety of stem cells used as therapeutic drugs. The composition of the culture process, in particular of the culture medium, is a major influencing parameter. In the methods described in the prior literature, when mesenchymal stem cells are isolated and subcultured, the culture medium used requires addition of serum, such as fetal bovine serum, and in particular, 10% fetal bovine serum is usually required, and for example, MSC complete medium (DMEM-F12 medium containing 10% fetal bovine serum) is usually used for isolating and culturing the mesenchymal stem cells for subculturing.
However, on the one hand, the cost of fetal bovine serum is quite high, which is disadvantageous for the culture of mesenchymal stem cells; on the other hand, fetal bovine serum, an exogenous substance of animal origin, is present in stem cells and presents a potential risk to the safety of the clinical use of the cells. Thus, serum-free isolation and subculturing of mesenchymal stem cells is of interest.
Umbilical cord mesenchymal stem cells (Umbilical cord Mesenchymal Stem Cells, UCMSCs) are multifunctional stem cells existing in neonatal umbilical cord tissues, can differentiate into various tissue cells, and have wide clinical application prospects. The application of the inactivated umbilical cord serum culture system can successfully amplify the human umbilical cord mesenchymal stem cells, the cultured cells have the basic characteristics of the mesenchymal stem cells, a theoretical basis is provided for establishing a mesenchymal stem cell library and clinical application, and the yield of the method is quite limited. Umbilical cord Mesenchymal Stem Cells (MSCs) have high differentiation potential and can differentiate towards multiple directions. It has wide clinical application prospect in the aspects of tissue engineering such as bones, cartilage, muscles, tendons, ligaments, nerves, livers, endothelium and cardiac muscles. MSCs are reported to be separated from human umbilical cords, the cell content and proliferation capacity of the MSCs are superior to those of bone marrow MSCs, the immunogenicity of the MSCs is lower than that of the bone marrow MSCs, and the MSCs have the advantages of convenience in material acquisition, no ethical disputes and the like, so the MSCs are more and more concerned by researchers. The main uses of umbilical cord mesenchymal stem cells include: can have stronger immunoregulation effect, can be used for treating autoimmune diseases such as lupus erythematosus, scleroderma and the like, reduces the immune rejection reaction after cell or organ transplantation, and improves the success rate of cell or organ transplantation; compared with single hematopoietic stem cell transplantation, mesenchymal stem cells and hematopoietic stem cells co-transplantation can remarkably improve the treatment effects of diseases such as leukemia, refractory anemia and the like; can repair damaged or diseased tissue and organ, and can be used for treating bone and muscle degeneration diseases, cardiovascular and cerebrovascular diseases, liver diseases, brain and spinal nerve injury, senile dementia, etc. A typical separation culture method of umbilical mesenchymal stem cells comprises taking fresh healthy umbilical cord, washing with PBS, removing blood vessel with scissors forceps, removing Fahrenheit gum tissue, cutting the obtained tissue, adding alpha-MEM culture solution, placing in a 37 deg.C 5% CO2 incubator, culturing, wherein the culture solution contains 10% FBS,100U/ml penicillin, and 100U/ml streptomycin. After 5-7 days of umbilical cord tissue culture, part of cells can climb out from the periphery of the tissue block, the form of the umbilical cord tissue culture is tiny, after one week, the cells start to proliferate rapidly to form cell colonies with different sizes, and after the umbilical cord tissue culture is full, the umbilical cord tissue culture is digested with 0.25% trypsin for passage.
The prior art discloses a plurality of culture methods related to isolated culture of umbilical cord mesenchymal stem cells. For example, CN102965338A (application number 2012105103791) discloses a method for extracting and culturing human umbilical cord mesenchymal stem cells. Umbilical cord tissue is cut up, digested by type I collagenase and transferred into a culture flask containing a culture medium for continuous culture. The culture medium is low-sugar DMEM basal medium and is added with fetal calf serum, fibroblast growth factor, epithelial cell growth factor, cell transcription factor and cholesterol. The extraction and culture method can be used for long-term culture of human umbilical cord mesenchymal stem cells and maintain the activity of the stem cells. The invention solves the problems of over-fast cell aging and differentiation in the current human umbilical cord mesenchymal stem cell culture, and can obtain the human umbilical cord mesenchymal stem cells with stem cell characteristics for a long time.
CN103421739a (application number 2013101962521) provides a simple, efficient method for isolating umbilical cord mesenchymal stem cells, comprising: a step of isolating umbilical cord mesenchymal stem cells using a cannula and culturing umbilical cord mesenchymal stem cells using a trypLETM digestion and serum-free medium. It is believed that the invention provides faster access to umbilical cord mesenchymal stem cells entirely from the mother, with less damage to the cells; experiments prove that the method can protect mesenchymal stem cells to the greatest extent, has higher activity and higher activity, and can more effectively perform adipogenesis and osteogenesis induced differentiation.
CN104232573a (application No. 2014104597914) discloses a growth medium for culturing stem cells, which contains a basal medium and an additive containing an antibody against human vascular endothelial growth factor, an antibody against human epidermal growth factor receptor 1, an antibody against human epidermal growth factor receptor 2 and guanosine-5-trisodium triphosphate. The invention also provides application of the growth medium in culturing umbilical cord mesenchymal stem cells. The invention also provides a method for culturing umbilical cord mesenchymal stem cells, which comprises the following steps: umbilical cord mesenchymal stem cells were inoculated into the growth medium as described above for culture. By the technical scheme, the invention is believed to greatly improve the expansion capacity of umbilical cord mesenchymal stem cells in vitro.
CN105112365A (application number: 2015105051216) provides a serum-free culture medium of human umbilical cord mesenchymal stem cells, belongs to the technical field of stem cells, comprises a DMEM basic culture medium and further comprises the following components: recombinant human insulin, human serum albumin, transferrin, fibronectin, vitamin C, biotin, stem cell growth factors and stem cell factors. The serum-free culture medium of the human umbilical cord mesenchymal stem cells provided by the invention is not required to be coated in a culture bottle, is simple and convenient to operate, has high cell proliferation rate, maintains good stem cell naive morphology and stem cell characteristics, has good cell induction differentiation potential, and reduces the cost.
CN105112360a (application number: 2015104231387) provides a method for bulk culturing umbilical cord mesenchymal stem cells, comprising: obtaining umbilical cord Wangton glue; carrying out P0-generation culture on umbilical cord Wangton glue until the cell fusion degree is 40% -50%, and then transferring P1-generation culture until the cell fusion degree is 85% -95%; inoculating and culturing the P1 generation cells in a plate culture medium, and separating suspended cells which are not adhered by the plate culture medium; the suspension cells are cultured in a medium containing leukemia inhibitory factor and fibroblast-like growth factor for the P2 generation. It is believed that the method for tissue adherence culture of the invention directly cultures the P0 cells with low early pleiotropic changes to the P1 generation, and then uses a tissue culture plate for adherence screening to keep the uniformity of cell differentiation; and then, leukemia inhibitory factor and fibroblast-like growth factor are used for increasing the adherence performance of cells in P2 generation culture and inhibiting differentiation, so that umbilical cord mesenchymal stem cells which have lower differentiation degree and tend to be consistent and have stronger adherence capability are obtained and are favorable for collection.
CN105602893a (2015109956392) discloses a serum-free method for culturing umbilical cord mesenchymal stem cells, the steps of the invention include: (1) Preparing a coating liquid of fibronectin, recombinant human epidermal growth factor and recombinant human fibroblast growth factor, and coating a plastic culture bottle; (2) Taking human umbilical cord tissue Wharton's jelly, shearing, and adding the human umbilical cord tissue Wharton's jelly into the step (1); (3) adding the serum-free culture medium into the solution obtained in the step (2); (4) Culturing in a 5% carbon dioxide incubator at 37 ℃ for 10-12 days to grow mesenchymal stem cells; (5) phosphate buffer washing and Triple enzyme digestion; (6) adding phosphate buffer to terminate digestion; (7) collecting mesenchymal stem cell supernatant; (8) centrifuging; (9) collecting the precipitate to obtain the required mesenchymal stem cells. The method of the invention is believed to enable the mesenchymal cells to be passaged for 20 generations, maintain the characteristics of the mesenchymal stem cells, treat diseases and have application prospects.
CN106399237a (application number 2016110843258) discloses a primary isolation method of umbilical cord mesenchymal stem cells. The separation method comprises the steps of firstly obtaining the Wharton's jelly from an umbilical cord, then mixing the Wharton's jelly with a DMEM/F12 culture medium containing 20% FBS, carrying out crisscross grinding on the mixture in a mortar for 30-50 rounds, suspending the mixture for 15s every 10 rounds, and gathering the Wharton's jelly again; the ground Wharton's jelly is resuspended in a medium and filtered, and the filtrate is cultured in the medium until mesenchymal stem cells are obtained. The invention is believed to replace the scissors shearing operation in the tissue block adherence culture method in a specific grinding mode and frequency, achieves the aim of rapidly treating umbilical cord tissue, remarkably shortens the appearance time of climbing out cells, has no influence on the proliferation capacity of passage cells, and improves the primary separation efficiency of umbilical cord mesenchymal stem cells as a whole.
CN107653226a (application number: 2017111313893) relates to a method for isolated culture of human umbilical cord mesenchymal stem cells, comprising the following steps: taking an umbilical cord of a fetus of term caesarean section in vitro, and placing the umbilical cord into a sterile tissue preservation solution for preservation; washing with physiological saline for several times to remove residual blood stain; removing two umbilical veins and two umbilical arteries of the tissue block by using toothed forceps, and completely cutting the Huatong glue by using sterile scissors; transferring the Huatong glue sheared in the step into a culture medium, oscillating and centrifuging; transferring the centrifuged pellet fraction to a cell culture flask; after 5 th to 6 th days of culture, part of cells can climb out from the periphery of the tissue small block, the culture medium is replaced every 3 days, and the culture is continued; the cell fusion degree reaches more than 80% around day 14, and the cells grow in a vortex manner; and then, the mesenchymal stem cells can be obtained every 3 days. The invention is believed to be easier than conventional tissue mass methods and to have higher stem cell purity and yield.
Although the prior art discloses some methods of culturing umbilical cord mesenchymal stem cells such as those described above, these methods often require the use of media containing relatively concentrated fetal bovine serum.
It is still desirable in the art to provide a new method for isolated culture or even subculture of mesenchymal stem cells, and in particular to provide a new method for isolated culture or even subculture of mesenchymal stem cells without fetal bovine serum.
Disclosure of Invention
The present invention aims to provide a novel method for preparing umbilical cord mesenchymal stem cells, in particular to provide a method suitable for separating umbilical cord mesenchymal stem cells from umbilical cord and a method for passaging the obtained stem cells, and such a method is expected to exhibit characteristics such as high cell yield and/or high cell viability and/or other excellent properties. The present invention has unexpectedly found that technical effects according to one or more aspects of the invention can be obtained using the method of the invention. The present invention has been completed based on this finding.
To this end, a first aspect of the present invention provides a method of isolated culturing primary umbilical cord mesenchymal stem cells, comprising the steps of:
(1) Treating umbilical cord samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections by using surgical scissors, the small sections are placed in a plate, repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting off tissues, peeling off the epidermis, removing arteries and veins to obtain the tissue of the Whatman's jelly, cutting the Whatman's jelly into small blocks, and weighing;
(4) Inoculating the tissue into a culture flask according to a specified tissue amount, adding a primary complete culture medium, fully and uniformly mixing to ensure that the tissue blocks are evenly paved on the bottom of the flask, and culturing in a CO2 incubator;
(5) Culturing until the 3 rd full culture medium is supplemented, continuously culturing until the 5 th full culture medium is supplemented, continuously culturing until the 7 th full culture medium is replaced until the cell fusion degree reaches more than 80%, removing old culture medium, cleaning cells with D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution to dilute, centrifuging, and re-suspending cell sediment with the primary full culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
The method according to the first aspect of the present invention, wherein in step (3), the Whatman's jelly is cut into 0.25cm pieces 2 Small blocks of size.
The method according to the first aspect of the present invention, wherein in the step (4), the step of inoculating the culture flask with the prescribed tissue amount means that 1.5g of the Whatman tissue mass obtained in the step (3) is inoculated into a T225 culture flask.
The method according to the first aspect of the present invention, wherein in the step (4), 15ml of the primary complete medium is added per flask, and the mixture is thoroughly mixed, so that the tissue mass is uniformly spread on the bottom of the flask, and cultured in a CO2 incubator.
The method according to the first aspect of the invention, wherein in step (4), CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
The method according to the first aspect of the present invention, wherein in step (5), the culture is continued until the 3 rd d is supplemented with 10ml of the primary complete medium.
The method according to the first aspect of the present invention, wherein in step (5), the culture is continued until 5d is supplemented with 10ml of the primary complete medium.
The method according to the first aspect of the present invention, wherein in step (5), 5ml of the recombinant pancreatin solution is added per bottle to digest the cells for 2min.
The method according to the first aspect of the present invention, wherein in step (5), 25ml of D-hanks liquid is added per bottle for dilution.
The method according to the first aspect of the invention, wherein in step (5), centrifugation is performed at 100xg for 10min.
The method according to the first aspect of the invention, wherein the formula of the D-Hanks liquid is composed as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000ml. For example, the preparation method is as follows: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The method according to the first aspect of the present invention, wherein the primary complete medium is formulated with DMEM-F12 medium as a substrate and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.
The method according to the first aspect of the invention, wherein the primary complete medium is replaced with a primary supplemental medium. The method according to the first aspect of the invention, wherein the primary supplemental medium is formulated with DMEM-F12 medium as a substrate and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose.
The method according to the first aspect of the invention, wherein the DMEM-F12 medium formulation consists of: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The method according to the first aspect of the invention, further comprising detecting the primary umbilical cord mesenchymal stem cells obtained by the isolated culture. Such as detecting cell morphology and/or immunophenotype identification. In one embodiment, the immunophenotyping is the detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary umbilical cord mesenchymal stem cells obtained by the invention are positive in CD73, CD90 and CD105 (more than 98 percent), and are negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (less than 2 percent).
Further, the second aspect of the present invention provides a method for isolating and subculturing umbilical cord mesenchymal stem cells, comprising (a) isolating and culturing primary umbilical cord mesenchymal stem cells and (b) subculturing umbilical cord mesenchymal stem cells, wherein
(a) The stage of isolated culture of primary umbilical cord mesenchymal stem cells comprises the following steps:
(a1) Treating umbilical cord samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(a2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections by using surgical scissors, the small sections are placed in a plate, repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(a3) Cutting off tissues, peeling off the epidermis, removing arteries and veins to obtain the tissue of the Whatman's jelly, cutting the Whatman's jelly into small blocks, and weighing;
(a4) Inoculating the tissue into a culture flask according to a specified tissue amount, adding a primary complete culture medium, fully and uniformly mixing to ensure that the tissue blocks are evenly paved on the bottom of the flask, and culturing in a CO2 incubator;
(a5) Culturing until the 3 rd full culture medium is supplemented, continuously culturing until the 5 th full culture medium is supplemented, continuously culturing until the 7 th full culture medium is replaced until the cell fusion degree reaches more than 80%, removing old culture medium, cleaning cells with D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution for dilution, centrifuging, and re-suspending cell sediment with the primary full culture medium to obtain primary umbilical cord mesenchymal stem cells (namely P0 generation);
(b) The stage of subculturing umbilical cord mesenchymal stem cells comprises the following steps:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
The method according to the second aspect of the present invention, wherein in the step (a 3), the Whatman's jelly is cut into 0.25cm 2 Small blocks of size.
The method according to the second aspect of the present invention, wherein the step (a 4) of inoculating the culture flask with a prescribed tissue amount means that 1.5g of the mass of the Whatman tissue obtained in the step (3) is inoculated into a T225 culture flask.
The method according to the second aspect of the present invention, wherein in the step (a 4), 15ml of the primary complete medium is added per flask, and the mixture is thoroughly mixed so that the tissue mass is uniformly spread on the bottom of the flask, and cultured in a CO2 incubator.
The method according to the second aspect of the invention, wherein in step (a 4), CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
The method according to the second aspect of the present invention, wherein in step (a 5), the culture is continued until the 3 rd d is supplemented with 10ml of the primary complete medium.
The method according to the second aspect of the present invention, wherein in step (a 5), the culture is continued until the 5 th d is supplemented with 10ml of the primary complete medium.
The method according to the second aspect of the present invention, wherein in step (a 5), 5ml of the recombinant pancreatin solution is added per bottle to digest the cells for 2min.
The method according to the second aspect of the present invention, wherein in step (a 5), 25ml of D-hanks liquid is added per bottle for dilution.
The method according to the second aspect of the present invention, wherein in step (a 5), centrifugation is performed at 100xg for 10min.
The method according to the second aspect of the present invention, wherein the formulation of the D-Hanks liquid is as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000ml. For example, the preparation method is as follows: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The method according to the second aspect of the present invention, wherein the primary complete medium is formulated with DMEM-F12 medium as a substrate and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.
The method according to the second aspect of the invention, wherein the primary complete medium is replaced with a primary supplemental medium. The method according to the second aspect of the invention, wherein the primary supplemental medium is formulated with DMEM-F12 medium as a substrate and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose.
The method according to the second aspect of the present invention, wherein the DMEM-F12 medium formulation is composed as follows: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The method according to the second aspect of the present invention, further comprising detecting the primary umbilical cord mesenchymal stem cells obtained by the isolated culture. Such as detecting cell morphology and/or immunophenotype identification. In one embodiment, the immunophenotyping is the detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary umbilical cord mesenchymal stem cells obtained by the invention are positive in CD73, CD90 and CD105 (more than 98 percent), and are negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (less than 2 percent).
In the context of the present invention, reference is made to a passaging complete medium formulated on DMEM-F12 medium as a substrate and comprising: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
Further, a third aspect of the present invention provides a medium for isolated culture of mesenchymal stem cells, which may also be referred to as a primary complete medium, prepared on a DMEM-F12 medium as a substrate and comprising: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.
The culture medium according to the third aspect of the invention is prepared by taking DMEM-F12 culture medium as a matrix and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose. The primary complete medium comprising thioglycerol and fructose may also be referred to as primary supplemental medium.
The culture medium according to the third aspect of the invention, wherein the mesenchymal stem cell isolated culture is performed according to a method comprising the steps of:
(1) Treating umbilical cord samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections by using surgical scissors, the small sections are placed in a plate, repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting off tissues, peeling off the epidermis, removing arteries and veins to obtain the tissue of the Whatman's jelly, cutting the Whatman's jelly into small blocks, and weighing;
(4) Inoculating the tissue into a culture flask according to a specified tissue amount, adding a primary complete culture medium, fully and uniformly mixing to ensure that the tissue blocks are evenly paved on the bottom of the flask, and culturing in a CO2 incubator;
(5) Culturing until the 3 rd full culture medium is supplemented, continuously culturing until the 5 th full culture medium is supplemented, continuously culturing until the 7 th full culture medium is replaced until the cell fusion degree reaches more than 80%, removing old culture medium, cleaning cells with D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution to dilute, centrifuging, and re-suspending cell sediment with the primary full culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
The culture medium according to the third aspect of the invention, wherein in the step (3) of the mesenchymal stem cells isolated culture, the Whatman's jelly is cut into 0.25cm 2 Small blocks of size.
According to the culture medium of the third aspect of the invention, in the step (4) of separating and culturing the mesenchymal stem cells, the step of inoculating the mesenchymal stem cells into a culture flask according to a specified tissue amount refers to the step of weighing 1.5g of the Whatman tissue block obtained in the step (3) and inoculating the Whatman tissue block into a T225 culture flask.
In the culture medium according to the third aspect of the invention, in the step (4) of isolated culture of the mesenchymal stem cells, 15ml of primary complete culture medium is added to each bottle, and the mixture is fully and uniformly mixed, so that the tissue blocks are uniformly paved on the bottle bottom, and the tissue blocks are cultured in a CO2 incubator.
The culture medium according to the third aspect of the invention, wherein in the step (4) of the mesenchymal stem cells are isolated and cultured, CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
The culture medium according to the third aspect of the invention, wherein in the step (5) of the isolated culture of mesenchymal stem cells, the culture is continued until the 3 rd d is supplemented with 10ml of the primary complete culture medium.
The culture medium according to the third aspect of the invention, wherein in the step (5) of the isolated culture of mesenchymal stem cells, the culture is continued until 5d is supplemented with 10ml of the primary complete culture medium.
The culture medium according to the third aspect of the invention, wherein in the step (5) of the isolated culture of mesenchymal stem cells, 5ml of the recombinant pancreatin solution is added per bottle to digest the cells for 2min.
The culture medium according to the third aspect of the invention, wherein in the step (5) of the isolated culture of mesenchymal stem cells, 25ml of D-hanks solution is added per bottle for dilution.
The culture medium according to the third aspect of the invention, wherein in the step (5) of the mesenchymal stem cells are isolated and cultured, the culture medium is centrifuged at 100Xg for 10min.
The culture medium according to the third aspect of the invention, wherein the mesenchymal stem cell isolated culture is the D-Hanks fluid having the following formulation: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000ml. For example, the preparation method is as follows: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The culture medium according to the third aspect of the invention, wherein the DMEM-F12 medium formula consists of: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The culture medium according to the third aspect of the invention, wherein the step of isolated culture of mesenchymal stem cells further comprises detecting primary umbilical cord mesenchymal stem cells obtained by the isolated culture. Such as detecting cell morphology and/or immunophenotype identification. In one embodiment, the immunophenotyping is the detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary umbilical cord mesenchymal stem cells obtained by the invention are positive in CD73, CD90 and CD105 (more than 98 percent), and are negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (less than 2 percent).
Furthermore, the fourth aspect of the present invention provides a primary umbilical cord mesenchymal stem cell, wherein all of CD73, CD90 and CD105 are positive, all of CD19, CD11b, CD31, CD45, HLADR and CD34 are negative, and the primary umbilical cord mesenchymal stem cell is isolated and cultured according to the following steps:
(1) Treating umbilical cord samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections by using surgical scissors, the small sections are placed in a plate, repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting off tissues, peeling off the epidermis, removing arteries and veins to obtain the tissue of the Whatman's jelly, cutting the Whatman's jelly into small blocks, and weighing;
(4) Inoculating the tissue into a culture flask according to a specified tissue amount, adding a primary complete culture medium, fully and uniformly mixing to ensure that the tissue blocks are evenly paved on the bottom of the flask, and culturing in a CO2 incubator;
(5) Culturing until the 3 rd full culture medium is supplemented, continuously culturing until the 5 th full culture medium is supplemented, continuously culturing until the 7 th full culture medium is replaced until the cell fusion degree reaches more than 80%, removing old culture medium, cleaning cells with D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution to dilute, centrifuging, and re-suspending cell sediment with the primary full culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in step (3), the Wharton's jelly is cut into 0.25cm 2 Small blocks of size.
According to the fourth aspect of the invention, the primary umbilical cord mesenchymal stem cells are inoculated into the culture flask according to the specified tissue amount in the step (4), namely 1.5g of the Wharton's jelly tissue block obtained in the step (3) is weighed and inoculated into a T225 culture flask.
In the step (4), 15ml of primary complete culture medium is added to each bottle, the mixture is fully and uniformly mixed, the tissue blocks are uniformly paved on the bottle bottom, and the mixture is cultured in a CO2 incubator.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in step (4), CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in step (5), the culture is continued until the 3 rd d is supplemented with 10ml of the primary complete medium.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in step (5), the culture is continued until 5d is supplemented with 10ml of the primary complete medium.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in step (5), 5ml of the recombinant pancreatin solution is added per bottle to digest the cells for 2min.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in the step (5), 25ml of the D-hanks solution is added per bottle for dilution.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein in step (5), are centrifuged at 100xg for 10min.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein the D-Hanks fluid is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000ml. For example, the preparation method is as follows: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein the primary complete medium is formulated with DMEM-F12 medium as a matrix and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.
The primary umbilical cord mesenchymal stem cells according to the fourth aspect of the present invention, wherein the primary complete medium is replaced with a primary supplemental medium. The method according to the first aspect of the invention, wherein the primary supplemental medium is formulated with DMEM-F12 medium as a substrate and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose.
Further, the fifth aspect of the present invention provides a passaging complete medium for passaging umbilical cord mesenchymal stem cells, which is prepared by using a DMEM-F12 medium as a substrate and comprises: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
The passaging complete medium according to the fifth aspect of the present invention, which is used for the isolated culture and the passaging of umbilical cord mesenchymal stem cells, comprises (a) two stages of isolated culture of primary umbilical cord mesenchymal stem cells and (b) passaging of umbilical cord mesenchymal stem cells, wherein
(a) The stage of isolated culture of primary umbilical cord mesenchymal stem cells comprises the following steps:
(a1) Treating umbilical cord samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(a2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections by using surgical scissors, the small sections are placed in a plate, repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(a3) Cutting off tissues, peeling off the epidermis, removing arteries and veins to obtain the tissue of the Whatman's jelly, cutting the Whatman's jelly into small blocks, and weighing;
(a4) Inoculating the tissue into a culture flask according to a specified tissue amount, adding a primary complete culture medium, fully and uniformly mixing to ensure that the tissue blocks are evenly paved on the bottom of the flask, and culturing in a CO2 incubator;
(a5) Culturing until the 3 rd full culture medium is supplemented, continuously culturing until the 5 th full culture medium is supplemented, continuously culturing until the 7 th full culture medium is replaced until the cell fusion degree reaches more than 80%, removing old culture medium, cleaning cells with D-hanks solution, adding recombinant pancreatin solution to digest the cells, allowing the cells to fall off, adding D-hanks solution for dilution, centrifuging, and re-suspending cell sediment with the primary full culture medium to obtain primary umbilical cord mesenchymal stem cells (namely P0 generation);
(b) The stage of subculturing umbilical cord mesenchymal stem cells comprises the following steps:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Use of a serum-free medium according to the sixth aspect of the invention for isolating umbilical cord mesenchymal stem cells, the serum-free medium (which may also be referred to as primary supplemental medium) being formulated with DMEM-F12 medium as a matrix and comprising: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose.
Use of a serum-free medium according to the seventh aspect of the invention for passaging umbilical cord mesenchymal stem cells, the serum-free medium (which may also be referred to as passaging complete medium) being formulated with DMEM-F12 medium as a matrix and comprising: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
In the present invention, "10≡6" represents the 6 th power of 10 when representing the number of cells; in the present invention, "cm 2" represents square centimeters when representing the culture area; other cases involving "<" > symbols, all have similar meanings; the meaning of this symbol is also well known in the art.
Of the various operating steps described above, although specific steps are described herein as being distinguished in some details or language description from those described in the preparation examples of the detailed description section below, those skilled in the art can readily generalize the method steps described above based on the detailed disclosure of the invention as a whole.
Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
In the present invention, the term "umbilical cord mesenchymal stem cells" refers to mesenchymal stem cells derived from umbilical cord. Thus, in the context of the present invention, and in particular in relation to the present invention, the term "umbilical cord mesenchymal stem cells" may be used interchangeably with "umbilical cord stem cells", "mesenchymal stem cells", unless explicitly indicated otherwise.
In the present invention, the term "PBS buffer" or "PBS" refers to phosphate buffer. The general formulation and method of formulation of the PBS used in the context of the present invention and their general properties such as pH or pH range are well known to those skilled in the art, and these PBS buffers are typically commercially available pre-formulations (or pre-powders), e.g., PBS used in the field of the present invention is typically a commercial buffer of pH7.4 (±0.1), e.g., hyClone brand PBS buffer; the typical PBS buffer composition used in this field includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and the composition of the PBS used in this invention is that described herein unless otherwise specified.
The method provided by the invention is used for separating and culturing umbilical cord mesenchymal stem cells, and the obtained umbilical cord mesenchymal stem cells have very high activity and very high yield. The methods of the present invention exhibit excellent technical effects as described in one or more aspects herein.
Drawings
Fig. 1: microscopic cell morphology of primary mesenchymal stem cells (100×).
Fig. 2: primary cell directional differentiation potential, a) adipogenic control, B) adipogenic induction; c) Osteogenic control, D) osteogenic induction; e) Cartilage forming control, F) cartilage forming induction.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
In the present invention, the DMEM-F12 medium formulation used in the test was composed as follows, unless otherwise specified: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL; preparing: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
In the present invention, the platelet lysate used in the test is readily available from the market, as not specifically described, and as used in the test herein is PLTGold Human Platelet Lysate from Sigma-Aldrich under the designation SCM151.
In the present invention, the type II collagenase used in the test is readily available from the market, as not specifically described, and used in the test herein is available from Gibco.
In the present invention, bFGF (basic fibroblast growth factor) used in the test is readily available from the market, as not specifically described herein, and is available from Sigma-Aldrich under the designation GF003.
In the present invention, EGF (epidermal growth factor) used in the test is readily available from the market, and as not specifically described, gibco, product number PHG0311L, was used in the test herein.
In the present invention, recombinant insulin used in the test is readily available commercially, as not specifically described, and is used in the test herein as available from Solarbio under the designation I8830.
In the present invention, the recombinant pancreatin solution used in the test is readily commercially available, and as not specifically described, the recombinant pancreatin solution of 2000u/ml concentration available from lanbo Kang Si company, cat No. RT2S01 was used in the test herein.
In the present invention, the D-Hanks solution used in the test had the following composition and preparation method, unless otherwise specified: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000ml; preparing: dissolving the materials with 1000ml, filtering with 0.22 μm microporous membrane, and sterilizing.
In the present invention, the primary complete medium used in the test was formulated with DMEM-F12 medium as a substrate and contained as follows, unless otherwise specified: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF.
In the present invention, the primary supplemental medium used in the test was formulated with DMEM-F12 medium as a substrate and included, unless specified otherwise: 0.8% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose.
In the specific experiments of the present invention, the obtained generation of stem cells were sampled, nucleated cells, i.e., MSC cells were counted using a sysmex hemocytometer, and cell viability was measured by trypan blue staining and sampled for microbial detection.
Example 1: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QA) from volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
After the above steps (1) to (5) are inoculated into T225 flasks at an amount of 1.5 g/flask of the mass of the hualong gel tissue per 1.5 g/flask (precisely converted into 1.5 g/flask in terms of the actual inoculum size), the number of nucleated cells obtained per flask (average of 5 repeated executions) is 0.618×10ζ6 (n=5) (this data may be referred to herein as the cell harvest amount), and the cell viability is 95.3% (n=5).
Example 1a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued to example 1;
(4) 1.5g (inoculum size 1.5 g/bottle, precision weighed tissue block) of the Whatman gel tissue block obtained in step (3) of example 1 was weighed and inoculated into a T225 flask, followed by addition of 15ml of primary supplemental mediumFully and evenly mixing, evenly paving the tissue blocks on the bottle bottom, and placing the mixture in a CO2 incubator (5 percent CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 1a after the steps (1) to (5) above were inoculated into T225 flasks at an amount of 1.5 g/flask of the mass of the hualong gel tissue per 1.5 g/flask (accurately converted into 1.5 g/flask at the actual inoculum size), the number of nucleated cells obtained per flask (average of 5 repeated executions) was 3.84×10≡6 (n=5) (this data may be referred to herein as the cell harvest amount), and the cell viability was 94.6% (n=5).
Example 1b: the procedure of example 1a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells. The primary umbilical cord mesenchymal stem cells of example 1b had a cell yield of 0.642×10≡6 (n=5) and a cell viability of 92.6% (n=5).
Example 1c: the procedure of example 1a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells. The primary umbilical cord mesenchymal stem cells of example 1b had a cell yield of 0.511×10≡6 (n=5) and a cell viability of 93.1% (n=5).
In this context, example 1a, example 1b, example 1c may also be referred to as auxiliary examples of example 1, while example 1 may be referred to as main examples, which may be referred to as example families with respect to each other.
The cell viability of example 1a, example 1b, example 1c was substantially the same as that of example 1, and all were in the range of 92 to 96%; however, the cell yield of example 1a was about 6.21 times that of example 1, the cell yield of example 1b was about 1.04 times that of example 1, and the cell yield of example 1c was about 0.83 times that of example 1; these unexpected findings indicate that the addition of small amounts of low-cost thioglycerol and fructose to primary complete medium not only allows primary stem cells to be obtained with substantially equivalent cell viability, but also, more encouraging, significant increases in cell harvest up to 4-fold, with substantially unchanged production costs. However, although the cell viability was not changed when only thioglycerol was added or only fructose was added, the cell yield was not increased, and even when only thioglycerol was additionally added, the cell yield was significantly reduced.
In terms of cell viability, the main examples 2 to 12 and their respective subsidiary examples a, b, c herein also present substantially the same results as those of example 1 and its subsidiary examples (examples 1a, 1b, 1 c) above, the cell viability was in the range of 92 to 96%, for example, the cell viability of the primary mesenchymal stem cells obtained in examples 2 and 2a, 2b, 2c were 94.7%, 93.2%, 95.3%, 92.8%, respectively.
In terms of cell harvest, the main examples 2-12 and their respective subsidiary a, b, c examples herein also exhibit substantially the same trend or even result as the above-described example 1 and its subsidiary examples (example 1a, example 1b, example 1 c), with the cell harvest of the main examples 2-12 each ranging from (0.577-0.653) x 10-6, the cell harvest of the subsidiary examples of group a each being 5.83-7.06 times their respective main examples, the cell harvest of the subsidiary examples of group b each being 0.93-1.21 times their respective main examples, and the cell harvest of the subsidiary examples of group c each being 0.77-0.86 times their respective main examples; for example, the cell harvest amounts (n=5) of example 2, example 2a, example 2b, example 2c are 0.627X10-6, 3.73X10-6 (5.95 times), 0.644X 10-6 (1.03 times), 0.517X 10-6 (0.82 times), respectively.
The primary umbilical cord mesenchymal stem cells obtained in examples 1-12 and their respective subsidiary examples a, b, c of the main examples herein were examined and were all normal in cell morphology, and immunophenotyping revealed that each primary umbilical cord mesenchymal stem cell was positive for CD73, CD90, CD105 (all greater than 98%, for example, the stem cell obtained in example 1 was greater than 99.5% for CD 73), negative for CD19, CD11b, CD31, CD45, HLADR, CD34 (all less than 2%, for example, the stem cell obtained in example 1 was less than 0.24% for CD 19).
Example 2: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QB) of volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 2a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued to example 2;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed tissue block) of the Whatman gel tissue block obtained in step (3) of example 2 was weighed and inoculated to T225 culture flask, adding 15ml primary supplementary culture medium, mixing, spreading the tissue block on the bottom of flask, and placing into CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 2b: the procedure of example 2a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 2c: the procedure of example 2a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 3: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donated by volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet (sample QC);
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 3a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued to example 3;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of the example 3 is weighed and inoculated into a T225 culture flask, 15ml of primary supplementary culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is uniformly paved on the bottom of the flask, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 3b: the procedure was as in example 3a, except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 3c: the procedure of example 3a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 4: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QD) from volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 4a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 4;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of example 4 was weighed and inoculated into a T225 flask, 15ml of primary supplementary medium was added, and the mixture was thoroughly mixed to uniformly spread the tissue block on the bottom of the flask, and placed in a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 4b: the procedure of example 4a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 4c: the procedure of example 4a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 5: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QE) of volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 5a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 5;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of example 5 is weighed and inoculated into a T225 culture flask, 15ml of primary supplementary culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is uniformly paved on the bottom of the flask, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 5b: the procedure of example 5a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 5c: the procedure of example 5a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 6: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QF) from volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting off tissue, peeling off epidermis, removing artery andvein, obtaining tissue of Whatman's jelly, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 6a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 6;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of the example 6 is weighed and inoculated into a T225 culture flask, 15ml of primary supplementary culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is uniformly paved on the bottom of the flask, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 6b: the procedure of example 6a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 6c: the procedure of example 6a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 7: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QG) of volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 7a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 7;
(4) Weighing the Whatman gum tissue block 1 obtained in the step (3) of example 75g (inoculum size 1.5 g/bottle, precisely weighing tissue block) were inoculated into T225 flask, and then 15ml of primary supplemental medium was added, thoroughly mixed, the tissue block was evenly spread on the flask bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 7b: the procedure of example 7a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 7c: the procedure of example 7a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 8: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QH) of volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 ，37At C, saturated humidity);
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 8a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 8;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of example 8 is weighed and inoculated into a T225 culture flask, 15ml of primary supplementary culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is uniformly paved on the bottom of the flask, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 8b: the procedure of example 8a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 8c: the procedure of example 8a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 9: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donated by volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet (sample QI);
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 9a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 9;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of the example 9 is weighed and inoculated into a T225 culture flask, 15ml of primary supplementary culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is uniformly paved on the bottom of the flask, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 9b: the procedure of example 9a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 9c: the procedure of example 9a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 10: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QJ) from volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 10a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 10;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of example 10 was weighed and inoculated into a T225 flask, 15ml of primary supplementary medium was added, and the mixture was thoroughly mixed to uniformly spread the tissue block on the bottom of the flask, and placed in a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 10b: the procedure was as in example 10a, except that no thioglycerol was added to the primary supplemental medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.
Example 10c: the procedure of example 10a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 11: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QK) from volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 11a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 11;
(4) 1.5g (inoculum size 1.5 g/bottle, precision weighing tissue block) of the Whatman gum tissue block obtained in the step (3) of the example 11 is weighed and inoculated into a T225 culture bottle, 15ml of primary supplementary culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 11b: the procedure of example 11a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 11c: the procedure of example 11a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 12: isolated culture of primary umbilical cord mesenchymal stem cells
(1) Handling umbilical cord donations (sample QL) from volunteers transported to laboratory via cold chain at 2-8 ℃ in biosafety cabinet;
(2) D-Hanks is sufficiently cleaned to remove surface blood stains, an umbilical cord is sheared into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks;
(3) Cutting tissue, peeling off epidermis, removing artery and vein to obtain Whatman's jelly tissue, cutting Whatman's jelly into 0.25cm pieces 2 Small blocks with the size are weighed;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighing tissue block) of the Whatman gum tissue block obtained in the step (3) is weighed and inoculated into a T225 culture bottle, 15ml of primary complete culture medium is added, and the mixture is fully and uniformly mixed, so that the tissue block is evenly paved on the bottle bottom, and placed into a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary complete culture medium, continuously culturing until the 7 th day is completely changed to a cell fusion degree of (10-11D) above 80%, removing old culture medium, cleaning cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks solution into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 12a: isolated culture of primary umbilical cord mesenchymal stem cells
The operations and materials of steps (1) to (3) are continued in example 12;
(4) 1.5g (inoculum size 1.5 g/bottle, precisely weighed) of the Whatman gel tissue block obtained in the step (3) of example 12 was weighed and inoculated into a T225 flask, 15ml of primary supplementary medium was added, and the mixture was thoroughly mixed to uniformly spread the tissue block on the bottom of the flask, and placed in a CO2 incubator (5% CO) 2 37 ℃, saturated humidity) in the culture;
(5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced with liquid until the cell fusion degree reaches more than 80 percent (10-11D), removing old culture medium, cleaning cells with D-hanks liquid, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min to enable the cells to fall off, adding 25ml of D-hanks liquid into each bottle to dilute, centrifuging for 10min at 100xg, and re-suspending cell sediment with the primary supplementary culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).
Example 12b: the procedure of example 12a was followed except that no thioglycerol was added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
Example 12c: the procedure of example 12a was followed except that fructose was not added to the primary supplemental medium, and isolated culture was performed to obtain primary umbilical cord mesenchymal stem cells.
The passaging complete medium used in examples 21 to 28 below was formulated with DMEM-F12 medium as a substrate and contained: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
Example 21: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 1a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 22: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 2a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 23: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 3a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 24: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 4a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 25: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 5a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 26: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 6a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 27: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 7a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
Example 28: subculture of umbilical cord mesenchymal stem cells
Subculturing was performed with the P0 generation umbilical cord mesenchymal stem cells obtained in example 8a, and the procedure was as follows:
(b1) Inoculating primary (namely P0 generation) mesenchymal stem cells according to the density of 5000/cm < 2 >, adding the primary (namely P0 generation) mesenchymal stem cells into a T225 bottle, supplementing a passage complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ C., saturated humidity) for culture until the cell fusion degree reaches 70-80% (generally reached on day 3), discarding the old culture medium, washing the cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle for dilution, merging all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using the passage complete culture medium to obtain P1 generation mesenchymal stem cells [ then sampling can be carried out for cell counting and activity rate measurement ];
(b2) Inoculating the P1 generation mesenchymal stem cells into a T225 bottle according to the density of 5000/cm < 2 >, supplementing the passaging complete culture medium to 45ml, placing the mixture into a CO2 incubator (5% CO2, 37 ℃ and saturated humidity), and obtaining the P2 generation mesenchymal stem cells by referring to a P1 passaging culture method; and then continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain the mesenchymal stem cells of each generation.
The cell viability of the P1-P10 generation cells obtained in examples 21 to 28 was examined by trypan blue staining, and as a result, the cell viability of the P1-P10 generation cells obtained in example 21 was in the range of 90-96%, the cell viability of the P1-P10 generation cells obtained in example 22 was in the range of 91-95%, the cell viability of the P1-P10 generation cells obtained in example 23 was in the range of 89-94%, the cell viability of the P1-P10 generation cells obtained in example 24 was in the range of 89-95%, the cell viability of the P1-P10 generation cells obtained in example 25 was in the range of 88-97%, the cell viability of the P1-P10 generation cells obtained in example 26 was in the range of 90-96%, the cell viability of the P1-P10 generation cells obtained in example 27 was in the range of 90-94%, the cell viability of the P1-P10 generation cells obtained in example 28 was in the range of 88-94%, the cell viability of the P1-P10 generation cells obtained in example 24 was in the range of 88-94%, the cell viability of the P1-P10 generation cells obtained in example 25 was in the range of 21.92%, the cell viability of the P1-P10 generation cell viability was in the example 1.10% was in the range of 3.93.93%, the cell viability of the P1-3.93.93.93.93% was in the cell% was in the range of 3.93.93%.
Example 31: subculture of adipose-derived mesenchymal stem cells
Subculturing of adipose-derived mesenchymal stem cells was performed with reference to example 21, except that the amount of thioglycerol added to the total culture medium for the passage used was 0.1%, obtaining P1 to P10-generation adipose-derived mesenchymal stem cells, and the cell viability of these cells was examined by trypan blue staining, and as a result, the cell viability of P1 to P6-generation cells was all in the range of 81 to 94% and decreased with increasing passage times, for example, the cell viability of P1 and P6-generation was 93.4% and 81.3%, respectively; the cell viability of the P7-P10 generation cells continues to decrease and is in the range of 59-78% and decreases with increasing passage, e.g., 77.4% and 59.3% for the P7 and P10 generation, respectively. The results of this example demonstrate that the addition of higher amounts of thioglycerol to passaged complete medium has a detrimental effect on cell viability.
Example 32: subculture of umbilical cord mesenchymal stem cells
The umbilical cord mesenchymal stem cells were subcultured with reference to example 21 except that thioglycerol was not added to the passaging complete medium used to obtain P1 to P10 generation umbilical cord mesenchymal stem cells, and the cell viability of these cells was examined by trypan blue staining, and as a result, the cell viability of P1 to P10 generation cells was in the range of 63 to 92% and decreased with increasing passage, for example, the cell viability of P2 generation and P8 generation were 88.6% and 67.4%, respectively. The results of this example demonstrate that the absence of thioglycerol added to passaging complete medium has a detrimental effect on cell viability.
Example 33: subculture of umbilical cord mesenchymal stem cells
The umbilical cord mesenchymal stem cells were subcultured with reference to example 21 except that fructose was not added to the total subculture medium used to obtain P1 to P10 generation umbilical cord mesenchymal stem cells, and the cell viability of these cells was examined by trypan blue staining, and as a result, the cell viability of P1 to P10 generation cells was in the range of 67 to 94% and decreased with increasing passage, for example, the cell viability of P2 generation and P8 generation were 90.4% and 74.4%, respectively. The results of this example show that the absence of fructose added to the passaged complete medium has a detrimental effect on cell viability.
Test example 1: detection of mesenchymal stem cells
Typical characteristics of mesenchymal stem cells include: the cells are in a spindle shape and grow on the wall under a microscope, and the flow cytometry identifies that all of CD73, CD90 and CD105 are positive, all of CD19, CD11b, CD31, CD45, HLADR and CD34 are negative, and the directional differentiation potential test shows that the cells are in bone, cartilage and fat differentiation potential.
The mesenchymal stem cells prepared in examples 1 to 12 of the present invention and their respective subsidiary examples a, b, c were examined using methods well known in the art, and as a result: all mesenchymal stem cells exhibited spindle shape and were grown on wall (e.g., the cell morphology under microscope of P0 generation mesenchymal stem cells obtained in example 1a is shown in fig. 1), all of CD73, CD90 and CD105 of all mesenchymal stem cells were greater than 98% (e.g., cd73=99.1%, cd90=98.7%, cd105=99.6%) of all of the mesenchymal stem cells, all of CD19, CD11b, CD31, CD45, HLADR, CD34 of all of the mesenchymal stem cells were less than 2% (e.g., cd19=0.11%, cd1b=0.23%, cd31=0.07%, cd45=0.12%, hladr=0.08%, cd34=0.14%) of all of the mesenchymal stem cells were shown to have differentiation potential of bone, cartilage, lipid (e.g., the differentiation potential of bone, cartilage, lipid, and lipid, as shown in fig. 2 of one of the mesenchymal stem cell batches obtained in example 1 a).
In addition, 8P 10 generation cells obtained in examples 21 to 28 of the present application were examined by a method known in the art, and as a result: all mesenchymal stem cells showed fusiform and adherent growth, CD73, CD90 and CD105 of 8 cells were greater than 98% (e.g. cd73=99.1%, cd90=98.7%, cd105=99.4%) of the P10 generation mesenchymal stem cells of example 21, CD19, CD11b, CD31, CD45, HLADR, CD34 of 8 cells were less than 2% (e.g. cd19=0.36, cd11b=0.42%, cd31=0.18%, cd45=0.14%, hladr=0.46%, cd34=0.35%) of the P10 generation mesenchymal stem cells of example 21), and the directional differentiation potential test showed that all mesenchymal stem cells had osteogenic, chondrogenic, adipogenic differentiation potential (not shown).
The above-described embodiments are merely preferred embodiments for fully explaining the present application, and the scope of the present application is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present application, and are intended to be within the scope of the present application. The protection scope of the application is subject to the claims.

Claims (21)

1. A method of subculturing umbilical cord mesenchymal stem cells comprising the steps of:
(b1) According to 5000/cm 2 Density inoculating primary P0 generation mesenchymal stem cells in T225 bottle, supplementing passage complete culture medium to 45ml, and placing 5% CO 2 CO at 37 ℃ and saturated humidity 2 When the cell fusion degree reaches 70-80% in the incubator, discarding the old culture medium, washing cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle to dilute, combining all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using a passage complete culture medium to obtain P1 generation mesenchymal stem cells;
(b2) According to 5000/cm 2 Density the P1 generation mesenchymal stem cells were inoculated in T225 flask, passaged complete medium was supplemented to 45ml, and 5% CO was placed 2 CO at 37 ℃ and saturated humidity 2 Obtaining P2 generation mesenchymal stem cells by referring to a P1 subculture method in an incubator; continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain mesenchymal stem cells of each generation;
the subculture complete medium is prepared by taking DMEM-F12 medium as a matrix and adding: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
2. The method of claim 1, wherein the D-Hanks liquid is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH 2 PO 4 Na of 0.08g 2 HPO 4 .12H 2 O, 0.35g NaHCO 3 Water to 1000ml.
3. The method according to claim 2, wherein the D-Hanks liquid is formulated as follows: dissolving each material with 1000ml, and filtering and sterilizing by a microporous filter membrane with the size of 0.22 mu m.
4. The method according to claim 1, wherein the DMEM-F12 medium is formulated as follows: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL.
5. A method for isolating and subculturing umbilical cord mesenchymal stem cells, comprising (a) two stages of isolating and culturing primary umbilical cord mesenchymal stem cells and (b) subculturing umbilical cord mesenchymal stem cells, wherein
(a) The stage of isolated culture of primary umbilical cord mesenchymal stem cells comprises the following steps:
(a1) Treating umbilical cord samples transported to a laboratory via a cold chain at 2-8 ℃ in a biosafety cabinet;
(a2) The D-Hanks liquid is sufficiently cleaned to remove surface blood stains, the umbilical cord is sheared into small sections by using surgical scissors, the small sections are placed in a plate, repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the small sections are cleaned by the D-Hanks liquid;
(a3) Cutting off tissues, peeling off the epidermis, removing arteries and veins to obtain the tissue of the Whatman's jelly, cutting the Whatman's jelly into small blocks, and weighing;
(a4) Inoculating into culture flask according to specified tissue amount, adding primary supplementary culture medium, mixing thoroughly to make tissue block uniformly spread on bottom of flask, placing CO 2 Culturing in an incubator;
(a5) Culturing until the 3 rd day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 5 th day is supplemented with 10ml of primary supplementary culture medium, continuously culturing until the 7 th day is completely replaced, removing old culture medium until the cell fusion degree reaches more than 80%, cleaning cells by using D-hanks solution, adding recombinant pancreatin solution to digest the cells to enable the cells to fall off, adding D-hanks solution to dilute, centrifuging, and re-suspending cell precipitation by using the primary supplementary culture medium to obtain primary umbilical cord mesenchymal stem cells, namely P0 generation;
(b) The stage of subculturing umbilical cord mesenchymal stem cells comprises the following steps:
(b1) According to 5000/cm 2 Density inoculating primary P0 generation mesenchymal stem cells in T225 bottle, supplementing passage complete culture medium to 45ml, and placing 5% CO 2 CO at 37 ℃ and saturated humidity 2 When the cell fusion degree reaches 70-80% in the incubator, discarding the old culture medium, washing cells with D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle to digest the cells for 2min so as to enable the cells to fall off, adding 15ml of D-hanks solution into each bottle to dilute, combining all cell suspensions into a 50ml centrifuge tube, centrifuging, and re-suspending cell sediment by using a passage complete culture medium to obtain P1 generation mesenchymal stem cells;
(b2) According to 5000/cm 2 Density the P1 generation mesenchymal stem cells were inoculated in T225 flask, passaged complete medium was supplemented to 45ml, and 5% CO was placed 2 CO at 37 ℃ and saturated humidity 2 Obtaining P2 generation mesenchymal stem cells by referring to a P1 subculture method in an incubator; continuously passaging the cells to the generation P10 by the method of the generation P1 and the generation P2 to obtain mesenchymal stem cells of each generation;
wherein,
the primary supplementary culture medium is prepared by taking DMEM-F12 culture medium as a matrix and adding: 0.8% platelet lysate, 1% human serum albumin, 2 μg/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF, 0.1% thioglycerol, 1% fructose;
The subculture complete medium is prepared by taking DMEM-F12 medium as a matrix and adding: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
6. The method according to claim 5, wherein in the step (a 3), the Whatman's jelly is cut into 0.25cm pieces 2 Small blocks of size.
7. The method according to claim 5, wherein in the step (a 4), the step of inoculating the culture flask with a predetermined amount of tissue is performed by weighing 1.5g of the mass of Whatman's jelly tissue obtained in the step (3) and inoculating the culture flask with T225.
8. The method according to claim 5, wherein in step (a 4), 15ml of primary supplemental medium is added per flask, and the mixture is thoroughly mixed to uniformly spread the tissue mass on the bottom of the flask, and placed in CO 2 Culturing in an incubator.
9. The method according to claim 5, wherein in step (a 4), CO 2 The conditions for culturing in the incubator are: 5% CO 2 Saturated humidity at 37 ℃.
10. The method according to claim 5, wherein in step (a 5), the cells are digested by adding 5ml of the recombinant pancreatin solution to each bottle for 2min.
11. The method according to claim 5, wherein in step (a 5), 25ml of D-hanks solution is added to each bottle for dilution.
12. The method according to claim 5, wherein in step (a 5), the centrifugation is performed at 100Xg for 10min.
13. According to claimThe method of claim 5, wherein the formula of the D-Hanks liquid comprises the following components: 8.0g NaCl, 0.4g KCl, 0.06g KH 2 PO 4 Na of 0.08g 2 HPO 4 .12H 2 O, 0.35g NaHCO 3 Water to 1000ml.
14. The method according to claim 5, wherein the DMEM-F12 medium is formulated as follows: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL.
15. The method according to claim 5, further comprising performing cell morphology detection and immunophenotyping of the primary umbilical cord mesenchymal stem cells obtained by the isolated culture.
16. The method according to claim 15, wherein said immunophenotyping is the detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34.
17. The method according to claim 16, wherein the primary umbilical cord mesenchymal stem cells obtained are positive for CD73, CD90, CD105, and negative for CD19, CD11b, CD31, CD45, HLADR, CD 34.
18. A passaging complete medium for passaging umbilical cord mesenchymal stem cells, which is prepared by taking a DMEM-F12 medium as a matrix and adding: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
19. The passaging complete medium of claim 18, wherein the DMEM-F12 medium formulation consists of: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL.
20. Use of serum-free passaging complete medium for passaging umbilical cord mesenchymal stem cells, the passaging complete medium is prepared by taking DMEM-F12 medium as a matrix and adding: 2% platelet lysate, 1% human serum albumin, 2. Mu.g/ml recombinant insulin, 10ng/ml EGF, 15ng/ml bFGF, 0.035% thioglycerol, 1% fructose.
21. The use of claim 20, wherein the DMEM-F12 medium formulation is composed as follows: anhydrous calcium chloride 116.6mg, L-leucine 59.05mg, linoleic acid 0.042mg, cupric sulfate pentahydrate 0.0013mg, L-lysine hydrochloride 91.25mg, lipoic acid 0.105mg, ferric nitrate nonahydrate 0.05mg, L-methionine 17.24mg, phenol red 8.1mg, ferrous sulfate heptahydrate 0.417mg, L-phenylalanine 35.48mg, 1, 4-butanediamine dihydrochloride 0.081mg, potassium chloride 311.8mg, L-serine 26.25mg, sodium pyruvate 55mg, magnesium chloride 28.64mg, L-threonine 53.45mg, vitamin H0.0035mg, anhydrous magnesium sulfate 48.84mg, L-alanine 4.45mg, calcium D-pantothenate 2.24mg, sodium chloride 7000mg, L-asparagine 7.5mg, choline chloride 8.98mg, anhydrous sodium dihydrogen phosphate 54.35mg, L-aspartic acid 6.65mg folic acid 2.65mg, disodium hydrogen phosphate 71.02mg, L-cysteine hydrochloride 17.56mg, i-inositol 12.6mg, zinc sulfate heptahydrate 0.432mg, L-glutamic acid 7.35mg, nicotinamide 2.02mg, L-arginine hydrochloride 147.5mg, L-proline 17.25mg, pyridoxal hydrochloride 2mg, L-cystine hydrochloride 31.29mg, L-tryptophan 9.02mg, pyridoxine hydrochloride 0.031mg, L-glutamine 365mg, L-tyrosine 38.4mg, riboflavin 0.219mg, glycine 18.75mg, L-valine 52.85mg, thiamine hydrochloride 2.17mg, L-histidine hydrochloride 31.48mg, D-glucose 3151mg, thymidine 0.365mg, L-isoleucine 54.47mg, hypoxanthine 2mg, vitamin B12 0.68mg, and water in an appropriate amount to 1000mL.
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