CN112481202A

CN112481202A - Method for serum-free separation culture of umbilical cord mesenchymal stem cells by using platelet lysate

Info

Publication number: CN112481202A
Application number: CN202011381203.1A
Authority: CN
Inventors: 肖海蓉
Original assignee: Shenzhen Boya Perception Pharmaceutical Co ltd
Current assignee: Shenzhen Boya Perception Pharmaceutical Co ltd
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-12
Anticipated expiration: 2040-11-30
Also published as: CN112481202B

Abstract

The invention relates to a method for culturing umbilical cord mesenchymal stem cells by serum-free separation of platelet lysate. The method comprises the following steps: processing the umbilical cord sample; fully cleaning to remove blood stain on the surface, cutting into small sections, removing residual blood clots, and cleaning; peeling off epidermis, removing artery and vein to obtain Wharton's jelly tissue, and cutting into small pieces; inoculating to a culture bottle, adding a primary complete culture medium, uniformly mixing, and culturing; removing old culture medium after the cell fusion degree reaches more than 80%, cleaning cells by using D-hanks liquid, adding recombinant pancreatin solution to digest cells to make the cells fall off, adding D-hanks liquid to dilute, centrifuging, and resuspending cell precipitates by using the culture medium. The invention also relates to a culture medium which is prepared by taking the DMEM-F12 culture medium as a matrix and comprises the following components: platelet lysate, human serum albumin, recombinant insulin, EGF, bFGF. The method of the present invention exhibits excellent technical effects as described in the specification.

Description

Method for serum-free separation culture 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 umbilical cord mesenchymal stem cells from an umbilical cord. The invention also relates to a culture medium and a related test solution used in the umbilical cord mesenchymal stem cell culture. When the umbilical cord mesenchymal stem cells are isolated and cultured by using the method, the excellent technical effect can be shown. In particular, the invention relates to methods of isolating cultured mesenchymal stem cells from umbilical cord using serum-free medium. In particular, platelet lysate is contained in the serum-free medium used in the isolated culture of the umbilical cord mesenchymal stem cells according to the present invention.

Background

Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow and a class of tissue stem cells derived from the mesoderm, which have the potential for multipotent differentiation and the ability to self-renew, have the ability to differentiate into various adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, hepatocytes, etc., under specific conditions in vivo and in vitro (Cap AI. mesenchymal stem cells. J Orthop Res.1991,9:641-650.Pittenger MF, Mackay AM, Beck, et al. multilineage patent of epithelial man stem cells. science.1999; 284:143 Across 147). Recent research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoietic support, and are easy to introduce and express exogenous genes. Therefore, the mesenchymal stem cells are not only seed cells in the construction of tissue engineering bone, cartilage and cardiac muscle and important carrier cells in gene therapy, but also have wide application prospect in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft-versus-host reaction. Mesenchymal stem cells have the characteristic of adherent growth in vitro, and by utilizing the characteristic, the mesenchymal stem cells are successfully separated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.

Stem cells are progenitors of human cells, and all cells in our body are derived from stem cells. When cells in the body age, die or the lesion denatures, stem cells grow and transform out of cells that can replace them. As seed cells, the compound is mainly used for treating various refractory diseases of tissue cells and organ injuries which cannot be naturally repaired by an organism clinically; as immunoregulatory cells, for the treatment of immune rejection and autoimmune diseases. Human mesenchymal stem cells are important members of a stem cell family, are derived from mesoderm in early development and belong to pluripotent stem cells, and are discovered in bone marrow initially, so that the human mesenchymal stem cells are increasingly concerned because of the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. Initial clinical studies were conducted in 1995 by Lazarus et al, who collected autologous MSCs of patients with hematological tumors in remission, cultured for 4-7 weeks in vitro for expansion, and then injected intravenously into patients, who were divided into 3 groups, administered different doses of MSCs, respectively, and no toxic side effects were observed after injection, suggesting that MSCs are safe and reliable for transplantation therapy. Then, clinical reports of autologous MSC are gradually increased, and the disease types comprise hematopoietic reconstruction after radiotherapy and chemotherapy, graft-versus-host disease (GVHD), heart system diseases and the like, and clinical intravenous infusion is proved to be safe and reliable in the reports.

The isolation culture and subculture process of mesenchymal stem cells are key steps related to the use safety of stem cells 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 documents, when mesenchymal stem cells are isolated and subcultured, the culture medium to be used is usually supplemented with serum, such as fetal bovine serum, particularly 10% fetal bovine serum, and for example, the mesenchymal stem cells are isolated and subcultured usually in MSC complete medium (which is DMEM-F12 medium containing 10% fetal bovine serum).

On the one hand, however, the cost of fetal bovine serum is rather high, which is disadvantageous for the culture of mesenchymal stem cells; on the other hand, the presence of fetal bovine serum as an exogenous material of animal origin in stem cells poses a potential risk to the safety of clinical use of the cells. Therefore, serum-free isolation and subculture of mesenchymal stem cells are of interest.

Umbilical Cord Mesenchymal Stem Cells (UCMSCs) are multifunctional Stem Cells existing in Umbilical cord tissues of newborn, can be differentiated into a plurality of tissue Cells, and have wide clinical application prospects. The application of the inactivated umbilical cord serum culture system can successfully amplify human umbilical cord mesenchymal stem cells, the cultured cells have the basic characteristics of the mesenchymal stem cells, and theoretical basis is provided for establishing a mesenchymal stem cell bank and clinical application, however, the yield of the method is quite limited. Umbilical cord Mesenchymal Stem Cells (MSCs) have a high differentiation potential and can be differentiated in multiple directions. It has wide clinical application prospect in the aspects of tissue engineering such as bones, cartilages, muscles, tendons, ligaments, nerves, livers, endothelia, cardiac muscles and the like. It has been reported that MSCs are separated from human umbilical cord, and the cell content and proliferation ability are superior to bone marrow MSCs, and the immunogenicity is lower than that of bone marrow MSCs, and the method has the advantages of easily available materials, no ethical dispute, and the like, and thus has been receiving more and more attention from researchers. The main uses of umbilical cord mesenchymal stem cells include: the medicine has stronger immunoregulation function, can be used for treating autoimmune diseases such as lupus erythematosus and scleroderma, reducing the immunological rejection reaction after cell or organ transplantation, and improving the success rate of cell or organ transplantation; the hematopoietic recovery function can be promoted, and compared with single hematopoietic stem cell transplantation, the mesenchymal stem cell and hematopoietic stem cell co-transplantation can remarkably improve the treatment effect of diseases such as leukemia, refractory anemia and the like; can repair damaged or diseased tissues and organs, and can be used for treating degenerative diseases of bone and muscle, cardiovascular and cerebrovascular diseases, liver diseases, brain and spinal nerve injury, senile dementia, etc. A typical isolated culture method of umbilical cord mesenchymal stem cells comprises the steps of taking a fresh healthy umbilical cord, washing the umbilical cord with PBS, removing blood vessels with scissors and forceps, peeling off Fahrenheit glue tissues in the umbilical cord mesenchymal stem cells, fully shearing the obtained tissues, adding alpha-MEM culture solution, and culturing in a 5% CO2 culture box at 37 ℃, wherein the culture solution contains 10% FBS, 100U/ml penicillin and 100U/ml streptomycin. After the umbilical cord tissue is cultured for 5-7 days, part of cells climb out from the periphery of the tissue block and are in a fine spindle shape, after one week, the cells begin to rapidly proliferate to form cell colonies with different sizes, and after the cells grow full, the cells are digested by 0.25% trypsin for passage.

The prior art discloses a plurality of culture methods related to the isolated culture of umbilical cord mesenchymal stem cells. For example, CN102965338A (application No. 2012105103791) discloses a method for extracting and culturing human umbilical cord mesenchymal stem cells. After being minced, the umbilical cord tissue is digested by collagenase I and then transferred into a culture flask containing a culture medium for continuous culture. The culture medium is low-sugar DMEM basic culture 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 of the invention can be used for culturing the human umbilical cord mesenchymal stem cells for a long time and maintaining the activity of the stem cells. The invention is believed to solve the problems of too rapid cell aging and differentiation in the conventional culture of human umbilical cord mesenchymal stem cells, and can obtain the human umbilical cord mesenchymal stem cells with the characteristics of stem cells for a long time.

CN103421739A (application No. 2013101962521) provides a simple and efficient method for separating umbilical cord mesenchymal stem cells, which comprises the following steps: isolating umbilical cord mesenchymal stem cells using a cannula and culturing the umbilical cord mesenchymal stem cells using trypLETM digestion and serum-free medium. The umbilical cord mesenchymal stem cells completely from a parent body can be obtained more quickly, and the damage to the cells is smaller; experiments prove that the method can protect the mesenchymal stem cells to the greatest extent, has higher activity rate and higher activity, and can more effectively perform adipogenic and osteogenic 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-triphosphate trisodium salt. The invention also provides application of the growth medium in culturing the 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 seeded in the growth medium as described above for culture. Through the technical scheme, the invention is believed to greatly improve the in-vitro amplification capacity of the umbilical cord mesenchymal stem cells.

CN105112365A (application number: 2015105051216) provides a serum-free culture medium for human umbilical cord mesenchymal stem cells, which belongs to the technical field of stem cells, and comprises a DMEM basic culture medium and also comprises the following components: recombinant human insulin, human serum albumin, transferrin, fibronectin, vitamin C, biotin, stem cell growth factor and stem cell factor. The serum-free culture medium for the human umbilical cord mesenchymal stem cells provided by the invention is free from culture bottle coating, is simple and convenient to operate, has a high cell proliferation rate, maintains good stem cell juvenile form and stem cell characteristics, has good cell induced differentiation potential, and reduces the cost.

CN105112360A (application No. 2015104231387) provides a mass culture method of umbilical cord mesenchymal stem cells, which comprises the following steps: obtaining the Wharton jelly of the umbilical cord; carrying out P0 subculture on the Wharton jelly of the umbilical cord until the cell fusion degree is 40-50%, and then carrying out P1 subculture until the cell fusion degree is 85-95%; inoculating and culturing the P1 generation cells in a plate culture medium, and separating suspension cells which are not adhered by the plate culture medium; the suspension cells were cultured in culture medium containing leukemia inhibitory factor and fibroblast-like growth factor for P2 passages. According to the tissue adherent culture method, the P0 cells with low early-stage multidirectional change are directly cultured to the P1 generation, and then the tissue culture plate is used for adhesion screening, so that the uniformity of the differentiation degree of the cells is kept; and then increasing the adherence performance of the cells in the culture of P2 generation by using leukemia inhibitory factor and fibroblast-like growth factor and inhibiting differentiation, thereby obtaining the umbilical cord mesenchymal stem cells with lower and consistent differentiation degree and stronger adherence capability and favorable collection.

CN105602893A (2015109956392) discloses a method for serum-free culture of umbilical cord mesenchymal stem cells, comprising the following steps: (1) preparing a coating solution 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 Walton's gum, shearing, and adding into the step (1); (3) adding a serum-free culture medium into the obtained product in the step (2); (4) culturing in a 5% carbon dioxide incubator at 37 deg.C for 10-12 days to obtain mesenchymal stem cells; (5) washing with phosphate buffer solution and digesting with Triple enzyme; (6) adding phosphate buffer solution to stop digestion; (7) collecting mesenchymal stem cell supernatant; (8) centrifuging; (9) collecting the precipitate to obtain the required mesenchymal stem cells. According to the method disclosed by the invention, the mesenchymal cells can be passaged for 20 generations, the characteristics of the mesenchymal stem cells are maintained, diseases can be treated, and the method has an application prospect.

CN106399237A (application number: 2016110843258) discloses a primary isolation method of umbilical cord mesenchymal stem cells. Obtaining the Wharton's jelly from an umbilical cord, mixing the Wharton's jelly with a DMEM/F12 culture medium containing 20% FBS, crisscross grinding in a mortar for 30-50 rounds, pausing for 15s every 10 rounds, and collecting the Wharton's jelly again; and (3) resuspending the ground Wharton's jelly by using a culture medium, filtering, and culturing the filtrate in the culture medium until mesenchymal stem cells are obtained. According to the invention, the specific grinding mode and frequency are used for replacing the scissors in the tissue block adherent culture method to cut, so that the aim of rapidly processing the umbilical cord tissue is achieved, the appearance time of the creeping-out cells is obviously shortened, the multiplication capacity of the passage cells is not influenced, and the primary separation efficiency of the umbilical cord mesenchymal stem cells is integrally improved.

CN107653226A (application number: 2017111313893) relates to a method for separating and culturing human umbilical cord mesenchymal stem cells, which comprises the following steps: taking an in-vitro umbilical cord of a fetus born by a full-term cesarean section, and placing the umbilical cord into sterile tissue preservation solution for preservation; flushing with normal saline for several times to remove residual blood stain; removing two umbilical veins and two umbilical arteries of the tissue block by using a pair of forceps with teeth, and completely cutting the huatong glue into pieces by using sterile scissors; transferring the cut Huatong glue into a culture medium, oscillating and centrifuging; transferring the centrifuged pellet fraction to a cell culture flask; culturing for 5-6 days, allowing part of cells to climb out from the small tissue blocks, replacing the culture medium every 3 days, and continuously culturing; the cell fusion degree reaches more than 80% at about 14 days, and the cells grow in a vortex manner; then, sufficient mesenchymal stem cells can be obtained every 3 days. The invention is believed to be easier and higher in stem cell purity and yield than traditional tissue block methods.

Although the prior art discloses some culture methods such as those described above in relation to umbilical cord mesenchymal stem cells, these methods often require the use of a medium containing a relatively high concentration of fetal bovine serum.

It is still expected to provide a new method for isolated culture or even subculture of mesenchymal stem cells, and in particular to provide a new method without fetal calf serum for isolated culture or even subculture of mesenchymal stem cells.

Disclosure of Invention

The invention aims to provide a novel method for preparing umbilical cord mesenchymal stem cells, in particular to provide a method suitable for separating and obtaining umbilical cord mesenchymal stem cells from umbilical cords, and the method is expected to show the characteristics of high cell yield, high cell viability and the like and/or other excellent performances. The present inventors have surprisingly found that the technical effects of one or more aspects as described herein can be obtained using the process of the present invention. The present invention has been completed based on this finding.

To this end, the invention provides in a first aspect a method for isolated culture of primary umbilical cord mesenchymal stem cells, comprising the steps of:

(1) treating the umbilical cord sample transported to a laboratory through a cold chain at 2-8 ℃ in a biosafety cabinet;

(2) D-Hanks is fully cleaned to remove surface bloodiness, the umbilical cord is cut into small sections by using a surgical scissors, the small sections are placed in a plate, the small sections are repeatedly squeezed by using surgical forceps, residual blood clots in tissues are removed, and the umbilical cord is cleaned by using the D-Hanks;

(3) cutting tissue, peeling off epidermis, removing artery and vein to obtain tissue of Wharton jelly, cutting Wharton jelly into small pieces, and weighing;

(4) inoculating the tissue to a culture bottle according to a specified tissue quantity, adding a primary complete culture medium, fully and uniformly mixing to enable tissue blocks to be uniformly spread on the bottom of the bottle, and culturing in a CO2 culture box;

(5) and (3) culturing until the complete culture medium is supplemented in the 3 rd step, continuously culturing until the primary complete culture medium is supplemented in the 5 th step, continuously culturing until the liquid is completely changed in the 7 th step, removing the old culture medium after the cell fusion degree reaches more than 80%, cleaning the cells by using D-hanks liquid, adding recombinant pancreatin solution to digest the cells to make the cells fall off, adding the D-hanks liquid to dilute, centrifuging, and re-suspending the cell precipitate by using the primary complete 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 the step (3), the gordonia gel is cut into 0.25cm²Small pieces of size.

The method according to the first aspect of the present invention, wherein the step (4) of inoculating the tissue mass into the culture flask according to the predetermined tissue mass means that 1.5g of the tissue mass of Wharton jelly obtained in the step (3) is weighed and inoculated into a T225 culture flask.

The method according to the first aspect of the present invention, wherein in step (4), 15ml of primary complete culture medium is added to each bottle, the mixture is thoroughly mixed, the tissue blocks are evenly spread on the bottom of the bottle, and the bottle is placed in a CO2 incubator for culture.

The process according to the first aspect of the present invention, wherein in the step (4), CO₂The conditions for the culture in the incubator were: 5% CO₂，37℃，And (4) saturated humidity.

The method according to the first aspect of the present invention, wherein in step (5), the culture is continued until 3d supplemented with 10ml of primary complete medium.

The method according to the first aspect of the present invention, wherein in step (5), the culture is continued until 5d supplemented with 10ml of primary complete medium.

The method according to the first aspect of the present invention, wherein in the step (5), 5ml of the recombinant pancreatin solution is added per vial for digesting the cells for 2 min.

The process according to the first aspect of the present invention, wherein in step (5), 25ml of D-hanks solution is added per bottle for dilution.

The method according to the first aspect of the present invention, wherein in step (5), the centrifugation is carried out at 100Xg for 10 min.

The method according to the first 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 1000 ml. For example, the preparation method is as follows: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

The method according to the first aspect of the invention, wherein said primary complete medium is prepared 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 method according to the first aspect of the invention, wherein said primary complete medium is replaced by primary supplemented medium. The method according to the first aspect of the invention, wherein said primary supplementary medium is formulated in DMEM-F12 medium as a medium 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 present invention, wherein said DMEM-F12 medium formula consists of: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, 0.432mg of zinc sulfate heptahydrate, 7.35mg of L-glutamic acid, 2.02mg of nicotinamide, 147.5mg of L-arginine hydrochloride, 17.25mg of L-proline, 2mg of pyridoxal hydrochloride, 31.29mg of L-cystine hydrochloride, 9.02mg of L-tryptophan, 0.031mg of pyridoxine hydrochloride, 365mg of L-glutamine, 38.4mg of L-tyrosine, 0.219mg of riboflavin, 18.75mg of glycine, 52.85mg of L-valine, 2.17mg of thiamine hydrochloride, 31.48mg of L-histidine hydrochloride, 3151mg of D-glucose, 0.365mg of thymidine, 54.47mg of L-isoleucine, 2mg of hypoxanthine, 0.68mg of vitamin B12 and adding a proper amount of water to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

The method according to the first aspect of the present invention, further comprising detecting the primary umbilical cord mesenchymal stem cells obtained by isolated culture. For example, detection of cell morphology and/or immunophenotypic identification. In one embodiment, said immunophenotypic identification refers to detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary umbilical cord mesenchymal stem cells obtained by the method are positive in CD73, CD90 and CD105 (all more than 98%), and negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%).

Further, the second aspect of the present invention provides a culture medium for isolated culture of mesenchymal stem cells, which may also be referred to as primary complete culture medium, prepared by using 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.

The medium according to the second aspect of the present invention is prepared with DMEM-F12 medium as a medium 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 culture medium according to the second aspect of the present invention, wherein the isolated culture of mesenchymal stem cells is carried out according to a method comprising the steps of:

The culture medium according to the second aspect of the present invention, wherein in the step (3) of isolated culture of mesenchymal stem cells, Wharton's jelly is cut into 0.25cm²Small pieces of size.

The culture medium according to the second aspect of the present invention, wherein in the step (4) of isolated culture of mesenchymal stem cells, the step of inoculating into the culture flask according to the specified tissue quantity means that 1.5g of the tissue pieces of Wharton jelly obtained in the step (3) are weighed and inoculated into a T225 culture flask.

The culture medium according to the second aspect of the present invention, wherein in the step (4) of mesenchymal stem cell isolation culture, 15ml of primary complete culture medium is added into each bottle, the mixture is fully mixed, so that the tissue mass is evenly spread on the bottom of the bottle, and the bottle is placed in a CO2 incubator for culture.

The medium according to the second aspect of the present invention, wherein in the step (4) of isolated culture of mesenchymal stem cells, CO₂The conditions for the culture in the incubator were: 5% CO₂At 37 deg.C, saturated humidity.

The culture medium according to the second aspect of the present invention, wherein in the step (5) of isolated culture of mesenchymal stem cells, the culture is continued until the 3 rd supplement with 10ml of primary complete medium.

The culture medium according to the second aspect of the present invention, wherein in the step (5) of isolated culture of mesenchymal stem cells, the culture is continued until 5d supplemented with 10ml of primary complete medium.

The medium according to the second aspect of the present 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 2 min.

The culture medium according to the second aspect of the present invention, wherein in the step (5) of isolated culture of mesenchymal stem cells, 25ml of D-hanks solution is added per bottle for dilution.

The medium according to the second aspect of the present invention, wherein the mesenchymal stem cells are centrifuged at 100Xg for 10min in the step (5) of the isolated culture.

The culture medium according to the second aspect of the present invention, wherein the mesenchymal stem cell isolation culture is the D-Hanks liquid with the following formulation: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000 ml. For example, the preparation method is as follows: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

The medium according to the second aspect of the present invention, wherein the DMEM-F12 medium formula consists of: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, 0.432mg of zinc sulfate heptahydrate, 7.35mg of L-glutamic acid, 2.02mg of nicotinamide, 147.5mg of L-arginine hydrochloride, 17.25mg of L-proline, 2mg of pyridoxal hydrochloride, 31.29mg of L-cystine hydrochloride, 9.02mg of L-tryptophan, 0.031mg of pyridoxine hydrochloride, 365mg of L-glutamine, 38.4mg of L-tyrosine, 0.219mg of riboflavin, 18.75mg of glycine, 52.85mg of L-valine, 2.17mg of thiamine hydrochloride, 31.48mg of L-histidine hydrochloride, 3151mg of D-glucose, 0.365mg of thymidine, 54.47mg of L-isoleucine, 2mg of hypoxanthine, 0.68mg of vitamin B12 and adding a proper amount of water to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

The culture medium according to the second aspect of the present invention, wherein the step of isolated culture of mesenchymal stem cells further comprises detecting the primary umbilical cord mesenchymal stem cells obtained from the isolated culture. For example, detection of cell morphology and/or immunophenotypic identification. In one embodiment, said immunophenotypic identification refers to detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34. The primary umbilical cord mesenchymal stem cells obtained by the method are positive in CD73, CD90 and CD105 (all more than 98%), and negative in CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%).

Further, the third aspect of the present invention provides a primary umbilical cord mesenchymal stem cell, which is positive for CD73, CD90 and CD105 and negative for CD19, CD11b, CD31, CD45, HLADR and CD34, and is isolated and cultured according to the following steps:

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein in step (3), Wharton's jelly is cut into 0.25cm²Small pieces of size.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein the step (4) of inoculating into the culture flask according to the specified tissue amount means that 1.5g of the tissue piece of Wharton jelly obtained in the step (3) is weighed and inoculated into a T225 culture flask.

According to the third aspect of the invention, in the step (4), 15ml of primary complete culture medium is added into each bottle, the mixture is fully mixed, the tissue blocks are evenly spread on the bottom of the bottle, and the bottle is placed in a CO2 incubator for culture.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein in step (4), the CO₂The conditions for the culture in the incubator were: 5% CO₂At 37 deg.C, saturated humidity.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein in the step (5), the culture is continued until the 3 rd supplement with 10ml of primary complete medium.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein in the step (5), the culture is continued until the 5 th d supplemented with 10ml of primary complete medium.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein in the step (5), 5ml of the recombinant pancreatin solution is added per bottle for digesting the cells for 2 min.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the invention, wherein in the step (5), 25ml of D-hanks solution is added to each bottle for dilution.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein in step (5), the cells are centrifuged at 100Xg for 10 min.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the invention, wherein the formula of the D-Hanks liquid comprises the following components: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3, water to 1000 ml. For example, the preparation method is as follows: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

The primary umbilical cord mesenchymal stem cells according to the third aspect of the present invention, wherein the primary complete medium is prepared with DMEM-F12 medium as a medium 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 third aspect of the invention, wherein the primary complete medium is replaced with primary supplemented medium. The method according to the first aspect of the invention, wherein said primary supplementary medium is formulated in DMEM-F12 medium as a medium 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.

In the present invention, "10 ^ 6" indicates the power of 6 of 10 when indicating the number of cells; in the present invention, "cm ^ 2" represents a square centimeter when representing a 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 process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

In the present invention, the term "umbilical cord mesenchymal stem cells" refers to mesenchymal stem cells derived from the umbilical cord. Thus, in the present invention, and in particular in the context relating to the present invention, the term "umbilical cord mesenchymal stem cell" may be used interchangeably with "umbilical cord stem cell", "mesenchymal stem cell", unless otherwise specifically indicated.

In the present invention, the term "PBS buffer" or "PBS" refers to a phosphate buffer. The general formulation and formulation of the PBS used in the context of the present invention, as well as their general properties such as pH value or pH range, are well known to those skilled in the art and are typically commercially available pre-formulations (or powders), e.g. the PBS used in the field of the present invention is typically a commercial buffer at pH7.4(± 0.1), e.g. HyClone brand PBS buffer; in the present invention, the composition of PBS buffer solution in the classical application of the art includes 137mM sodium chloride, 2.7nM potassium chloride and 10mM phosphate, and PBS used in the present invention has the same composition as that in the present invention, unless otherwise specified.

The umbilical cord mesenchymal stem cells are isolated and cultured by the method, and the obtained umbilical cord mesenchymal stem cells have very high survival rate and very high yield. The present methods exhibit superior technical effects in one or more aspects as described herein.

Drawings

FIG. 1: microscopic cell morphology of mesenchymal stem cells (100 ×).

FIG. 2: cell directed differentiation potential, A) adipogenic control, B) adipogenic induction; C) osteogenic control, D) osteogenic induction; E) chondrogenic control, F) chondrogenic 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. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

In the present invention, the DMEM-F12 medium used in the experiments had the following formulation, unless otherwise specified: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, 0.432mg of zinc sulfate heptahydrate, 7.35mg of L-glutamic acid, 2.02mg of nicotinamide, 147.5mg of L-arginine hydrochloride, 17.25mg of L-proline, 2mg of pyridoxal hydrochloride, 31.29mg of L-cystine hydrochloride, 9.02mg of L-tryptophan, 0.031mg of pyridoxine hydrochloride, 365mg of L-glutamine, 38.4mg of L-tyrosine, 0.219mg of riboflavin, 18.75mg of glycine, 52.85mg of L-valine, 2.17mg of thiamine hydrochloride, 31.48mg of L-histidine hydrochloride, 3151mg of D-glucose, 0.365mg of thymidine, 54.47mg of L-isoleucine, 2mg of hypoxanthine, 0.68mg of vitamin B12 and adding a proper amount of water to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

In the present invention, Platelet lysates used in the experiments can be readily purchased from the market, and as not specifically indicated herein, PLTGold Human Platelet Lysate from Sigma-Aldrich, having the product number SCM151, was used in the experiments.

In the present invention, collagenase type II used in the experiments can be easily obtained from the market, and as not specifically mentioned, used in the experiments herein is from Gibco.

In the present invention, bFGF (basic fibroblast growth factor) used in the experiment can be easily purchased from the market, and as not specifically mentioned, it is purchased from Sigma-Aldrich under the trade name GF003 in the experiment herein.

In the present invention, EGF (epidermal growth factor) used in the test is readily available from the market, and as not specifically mentioned herein, it is used in the test from Gibco under the reference PHG 0311L.

In the present invention, recombinant insulin used in the test can be easily obtained from the market, and as not specifically mentioned, it is used in the test herein that it is obtained from Solarbio and its product number is I8830.

In the present invention, the recombinant pancreatin solution used in the test can be easily obtained from the market, and as not specifically mentioned, the recombinant pancreatin solution of 2000u/ml concentration available from Rambox corporation, cat # RT2S01, is used in the test herein.

In the present invention, the D-Hanks solution used in the test was formulated and prepared as follows, unless otherwise specified: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3 and water to 1000 ml; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

In the present invention, the primary complete medium used in the experiments was prepared with DMEM-F12 medium as a medium and contained, 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 supplement medium used in the experiments was prepared with DMEM-F12 medium as a medium and contained, if not specifically stated: 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 a specific experiment of the present invention, the prepared stem cells of a certain generation were sampled, nucleated cells, i.e., MSC cells were counted using a sysmex hemocytometer, cell viability was detected by trypan blue staining, and the samples were taken for microbial detection.

Example 1: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via cold chain at 2-8 ℃ were processed in a biosafety cabinet (sample QA);

(2) D-Hanks are fully cleaned to remove surface bloodiness, the umbilical cord is cut into small sections with the length of 3cm by using surgical scissors, the small sections are placed in a 100mm flat dish, the umbilical cord is repeatedly extruded by using surgical forceps, residual blood clots in tissues are removed, and the umbilical cord is cleaned by using D-Hanks;

(3) cutting tissue, peeling epidermis, removing artery and vein to obtain tissue of Wharton jelly, and cutting Wharton jelly into 0.25cm²Weighing small blocks of different sizes;

(4) weighing 1.5g of the tissue block of the gordonia gum obtained in the step (3) (the inoculation amount is 1.5 g/bottle, the tissue block is precisely weighed) and inoculating the tissue block to a T225 culture bottle, adding 15ml of primary complete culture medium, fully and uniformly mixing to enable the tissue block to be uniformly spread on the bottom of the bottle, and placing the bottle in a CO2 incubator (5% CO)₂At 37 ℃, saturated humidity);

(5) and (3) supplementing 10ml of primary complete culture medium to the 3 rd stage for continuous culture, supplementing 10ml of primary complete culture medium to the 5 th stage for continuous culture, completely replacing the culture solution to the 7 th stage, removing the old culture medium after the cell fusion degree reaches more than 80 percent (10-11D), cleaning the cells by using D-hanks solution, adding 5ml of recombinant pancreatin solution into each bottle for digesting the cells for 2min to make the cells fall off, adding 25ml of D-hanks solution into each bottle for dilution, centrifuging for 10min at 100Xg, and re-suspending the cell sediment by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).

The above steps (1) to (5) after inoculating the T225 flasks in step (4) with the amount of each 1.5 g/flask of the gordonia gum tissue piece (exactly converted to 1.5 g/flask from the actual inoculation amount), the number of nucleated cells obtained per flask (average of 5 repetitions) was 0.618 × 10^6(n ═ 5) (herein, this data may be referred to as cell harvest amount), and the cell viability was 95.3% (n ═ 5).

Example 1 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) are continued in example 1;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 1 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread on the bottom of the bottle uniformly, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

(5) and (3) supplementing 10ml of primary supplement culture medium to the 3 rd stage, continuously culturing, supplementing 10ml of primary supplement culture medium to the 5 th stage, continuously culturing, completely replacing the culture solution to the 7 th stage, removing the old culture medium after the cell fusion degree reaches more than 80 percent (10-11D), cleaning the cells by using 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 25ml of D-hanks solution into each bottle to dilute the cells, centrifuging the solution for 10min at 100Xg, and re-suspending the cell sediment by using the primary supplement culture medium to obtain the primary umbilical cord mesenchymal stem cells (namely P0 generation).

Example 1a after the above-described steps (1) to (5) were inoculated into T225 flasks in step (4) in an amount of 1.5 g/flask of gordonia gum tissue mass (exactly converted to 1.5 g/flask from the actual inoculum amount), the number of nucleated cells (average of 5 repetitions) obtained per flask was 3.84 × 10^6(n ═ 5) (herein, this data may be referred to as cell harvest amount), and the cell viability was 94.6% (n ═ 5).

Example 1 b: primary umbilical cord mesenchymal stem cells were obtained as in example 1a, except that no thioglycerol was added to the primary supplement medium and isolated for culture. The cell harvest of primary umbilical cord mesenchymal stem cells of example 1b was 0.642 x 10^6(n ═ 5) and the cell viability was 92.6% (n ═ 5).

Example 1 c: the procedure of example 1a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells. The cell harvest of primary umbilical cord mesenchymal stem cells of example 1b was 0.511 × 10^6(n ═ 5) and the cell viability was 93.1% (n ═ 5).

In this document, the embodiment 1a, the embodiment 1b and the embodiment 1c can also be referred to as an auxiliary example of the embodiment 1, while the embodiment 1 can be referred to as a main example, and they can be referred to as an example family.

Compared with the example 1, the cell viability rates of the example 1a, the example 1b and the example 1c are basically the same and are all in the range of 92-96%; however, the cell harvest was about 6.21 times that of example 1a, about 1.04 times that of example 1b, and about 0.83 times that of example 1c for example 1a for example 1 b; these unexpected findings indicate that adding a small amount of cheap thioglycerol and fructose in the primary complete culture medium can not only obtain primary stem cells with substantially equivalent cell viability rate, but also promote a significant increase in cell yield of up to 4 times, and a significant increase in cell yield with substantially unchanged production cost. However, when only thioglycerol or only fructose is added, although the cell viability rate is not changed, the cell yield is not increased, and even when only thioglycerol is additionally added, the cell yield is obviously reduced.

In terms of cell viability, the main examples 2-12 and their respective auxiliary a, b, c examples also show substantially the same results as the above example 1 and its auxiliary examples (example 1a, example 1b, example 1c), and the cell viability is in the range of 92-96%, for example, the cell viability of the primary mesenchymal stem cells obtained in example 2 and the fourth of example 2a, example 2b, example 2c is 94.7%, 93.2%, 95.3%, 92.8%, respectively.

In terms of cell harvest, the main examples 2-12 and their respective auxiliary examples a, b, c also show substantially the same trend and even result as the above-mentioned example 1 and its auxiliary examples (example 1a, example 1b, example 1c), the cell harvest of the main examples 2-12 is within (0.577-0.653) × 10^6, the cell harvest of the group a auxiliary examples is 5.83-7.06 times that of their respective main examples, the cell harvest of the group b auxiliary examples is 0.93-1.21 times that of their respective main examples, and the cell harvest of the group c auxiliary examples is 0.77-0.86 times that of their respective main examples; for example, the cell harvest yields (n ═ 5) of examples 2, 2a, 2b and 2c were 0.627 × 10^6, 3.73 × 10^6(5.95 times), 0.644 × 10^6(1.03 times) and 0.517 × 10^6(0.82 times), respectively.

The primary placental mesenchymal stem cells obtained from examples 1-12 and their respective appendant a, b and c of the main examples herein were tested to determine that the morphology of the cells was normal, and immunophenotyping showed that each primary placental mesenchymal stem cell was positive for CD73, CD90 and CD105 (all greater than 98%, for example, greater than 99.5% for CD73 obtained from example 1), and negative for CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%, for example, less than 0.24% for CD19 obtained from example 1).

Example 2: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors (sample QB) transported to the laboratory via the 2-8 ℃ cold chain were processed in a biosafety cabinet;

(4) weighing the Huatong obtained in the step (3)Inoculating 1.5g of gelatin tissue block (inoculum size 1.5 g/bottle, precisely weighing tissue block) into T225 culture bottle, adding 15ml of primary complete culture medium, mixing, spreading the tissue block uniformly on the bottom of the bottle, and placing in CO2 incubator (5% CO)₂At 37 ℃, saturated humidity);

Example 2 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 2;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 2 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 2 b: primary umbilical cord mesenchymal stem cells were obtained as in example 2a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 2 c: the procedure of example 2a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 3: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the 2-8 ℃ cold chain (sample QC) were processed in a biosafety cabinet;

Example 3 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) are continued in example 3;

(4) weighing 1.5g of the tissue mass of gordonia gum obtained in step (3) of example 3 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating the tissue mass into a T225 culture bottle, adding 15ml of primary supplement culture medium, fully and uniformly mixing to enable the tissue mass to be uniformly spread on the bottom of the bottle, and placing the bottle in a CO2 incubator(5％CO₂At 37 ℃, saturated humidity);

Example 3 b: primary umbilical cord mesenchymal stem cells were obtained as in example 3a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 3 c: the procedure of example 3a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 4: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in biosafety cabinets (sample QD);

Example 4 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 4;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 4 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 4 b: the procedure of example 4a was followed except that thioglycerol was not added to the primary supplement medium and isolated for culture to obtain primary umbilical cord mesenchymal stem cells.

Example 4 c: the procedure of example 4a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 5: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords (sample QE) from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in a biosafety cabinet;

Example 5 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 5;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 5 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 5 b: primary umbilical cord mesenchymal stem cells were obtained as in example 5a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 5 c: the procedure of example 5a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 6: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in a biosafety cabinet (sample QF);

Example 6 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 6;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 6 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 6 b: primary umbilical cord mesenchymal stem cells were obtained as in example 6a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 6 c: the procedure of example 6a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 7: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in biosafety cabinets (sample QG);

Example 7 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 7;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 7 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 7 b: primary umbilical cord mesenchymal stem cells were obtained as in example 7a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 7 c: the procedure of example 7a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 8: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in biosafety cabinets (sample QH);

Example 8 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 8;

(4) 1.5g of the tissue piece of Wharton's jelly obtained in step (3) of example 8 (inoculum size 1.5 g/bottle, precision-weighed tissue piece) was weighed and inoculated into a T225 flask, and 15ml of primary supplement medium was added thereto, followed by well-distributed addition of the culture mediumMixing, spreading the tissue blocks uniformly on the bottom of the bottle, and placing in a CO2 incubator (5% CO)₂At 37 ℃, saturated humidity);

Example 8 b: the procedure of example 8a was followed except that thioglycerol was not added to the primary supplement medium and isolated for culture to obtain primary umbilical cord mesenchymal stem cells.

Example 8 c: the procedure of example 8a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 9: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in a biosafety cabinet (sample QI);

Example 9 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued from example 9;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 9 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 9 b: primary umbilical cord mesenchymal stem cells were obtained as in example 9a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 9 c: the procedure of example 9a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 10: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in biosafety cabinets (sample QJ);

Example 10 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued in example 10;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 10 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 10 b: primary umbilical cord mesenchymal stem cells were obtained as in example 10a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 10 c: the procedure of example 10a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 11: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors (sample QK) transported to the laboratory via the cold chain at 2-8 ℃ were processed in a biosafety cabinet;

Example 11 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials of the steps (1) to (3) are continued from the example 11;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 11 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 11 b: primary umbilical cord mesenchymal stem cells were obtained as in example 11a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 11 c: the procedure of example 11a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

Example 12: isolation culture of primary umbilical cord mesenchymal stem cells

(1) Umbilical cords from volunteer donors transported to the laboratory via the cold chain at 2-8 ℃ were processed in biosafety cabinets (sample QL);

(3) cutting tissue, peeling epidermis, removing artery and vein to obtain Wharton jelly tissue,cutting Wharton's jelly into 0.25cm pieces²Weighing small blocks of different sizes;

Example 12 a: isolation culture of primary umbilical cord mesenchymal stem cells

The operations and materials from step (1) to step (3) were continued from example 12;

(4) weighing 1.5g of the tissue mass of Wharton's jelly obtained in step (3) of example 12 (the inoculum size is 1.5 g/bottle, the tissue mass is precisely weighed) and inoculating to a T225 culture bottle, adding 15ml of primary supplement culture medium, mixing well to make the tissue mass spread evenly on the bottom of the bottle, placing in a CO2 incubator (5% CO 2)₂At 37 ℃, saturated humidity);

Example 12 b: primary umbilical cord mesenchymal stem cells were obtained as in example 12a, except that no thioglycerol was added to the primary supplement medium and isolated for culture.

Example 12 c: the procedure of example 12a was followed except that fructose was not added to the primary supplemented medium, and isolated and cultured to obtain primary umbilical cord mesenchymal stem cells.

The subculture of the mesenchymal stem cells of generation P0 prepared in examples 1 to 12 and their respective attached a, b and c shows that all cells can be continuously passaged to more than P10 generation, the cells maintain stable continuous proliferation capacity, and the cell viability of each generation is stable to more than 90% in the continuous passage process, for example, the cell viability of a batch of mesenchymal stem cells of generation P0 obtained in example 1a is stable to 93.3% when the batch of mesenchymal stem cells of generation P10 is continuously passaged.

Test example 1: detection of mesenchymal stem cells

Typical characteristics of mesenchymal stem cells include: microscopic fusiform and adherent growth, flow cytometry identification shows that CD73, CD90 and CD105 are all positive and CD19, CD11b, CD31, CD45, HLADR and CD34 are all negative, and a directional differentiation potential test shows that the cells present the differentiation potential of osteogenesis, chondrogenesis and adipogenesis.

The mesenchymal stem cells prepared in the embodiments 1 to 12 and the auxiliary a, b and c examples thereof are detected by using a method known in the field, and the results are as follows: all mesenchymal stem cells exhibited spindle and adherent growth (e.g., microscopic cell morphology of the mesenchymal stem cells of generation P0 obtained in example 1a is shown in fig. 1), 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 a batch of the mesenchymal stem cells of generation P0 obtained in example 1 a), CD19, CD11b, CD31, CD45 of all mesenchymal stem cells, HLADR and CD34 are less than 2% (e.g., CD19 of the batch of the P0-generation mesenchymal stem cells obtained in example 1a is 0.11%, CD11b is 0.23%, CD31 is 0.07%, CD45 is 0.12%, HLADR is 0.08%, and CD34 is 0.14%), and the directed differentiation potential test shows that all the mesenchymal stem cells have osteogenic, chondrogenic and adipogenic differentiation potential (e.g., the differentiation potential results of the batch of the P0-generation mesenchymal stem cells obtained in example 1a are shown in fig. 2).

The above-described embodiments are merely preferred embodiments for fully illustrating the present application, and the scope of the present application is not limited thereto. The equivalent substitution or change made by the person skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of this application is subject to the claims.

Claims

1. The method for separating and culturing the primary umbilical cord mesenchymal stem cells comprises the following steps:

(5) and (4) culturing until the 3 rd supplemented complete culture medium is continuously cultured, continuing culturing until the 5 th supplemented primary complete culture medium is continuously cultured, completely replacing the culture solution until the 7 th cell fusion degree reaches more than 80%, removing the old culture medium, cleaning the cells by using D-hanks solution, adding recombinant pancreatin solution to digest the cells to make the cells fall off, adding the D-hanks solution to dilute, centrifuging, and re-suspending the cell precipitate by using the primary complete culture medium to obtain the primary umbilical cord mesenchymal stem cells.

2. The method according to claim 1, wherein in the step (3), the gordonia gel is cut into 0.25cm²Small pieces of size.

3. The method according to claim 1, wherein the step (4) of inoculating the culture flask with the predetermined amount of the tissue is a step of weighing the starch obtained in the step (3)Inoculating 1.5g of the gelatin tissue block to a T225 culture bottle; or, in the step (4), adding 15ml of primary complete culture medium into each bottle, fully and uniformly mixing, uniformly spreading the tissue blocks at the bottom of the bottle, and culturing in a CO2 incubator; alternatively, in step (4), CO₂The conditions for the culture in the incubator were: 5% CO₂At 37 deg.C, saturated humidity.

4. The method according to claim 1, wherein in step (5), the culture is continued until 3d supplemented with 10ml of primary complete medium; or, in the step (5), the culture is continued until the 5 th day is supplemented with 10ml of primary complete culture medium; or, in the step (5), 5ml of recombinant pancreatin solution is added into each bottle to digest the cells for 2 min; or, in the step (5), adding 25ml of D-hanks liquid into each bottle for dilution; alternatively, in step (5), centrifugation is carried out at 100Xg for 10 min.

5. The method of claim 1 wherein the D-Hanks liquid is formulated as follows: 8.0g NaCl, 0.4g KCl, 0.06g KH2PO4, 0.08g Na2HPO4.12H2O, 0.35g NaHCO3 and water to 1000 ml; for example, the preparation method is as follows: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization.

6. The method according to claim 1, wherein said primary complete medium is formulated with DMEM-F12 medium as a matrix and comprises: 0.8% platelet lysate, 1% human serum albumin, 2 μ g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF; or, the culture medium is prepared by taking DMEM-F12 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.

7. The method according to claim 1, wherein said DMEM-F12 medium formula consists of: 116.6mg of anhydrous calcium chloride, 59.05mg of L-leucine, 0.042mg of linoleic acid, 0.0013mg of copper sulfate pentahydrate, 91.25mg of L-lysine hydrochloride, 0.105mg of lipoic acid, 0.05mg of ferric nitrate nonahydrate, 17.24mg of L-methionine, 8.1mg of phenol red, 0.417mg of ferrous sulfate heptahydrate, 35.48mg of L-phenylalanine, 0.081mg of 1, 4-butanediamine dihydrochloride, 311.8mg of potassium chloride, 26.25mg of L-serine, 55mg of sodium pyruvate, 28.64mg of magnesium chloride, 53.45mg of L-threonine, 0.0035mg of vitamin H, 48.84mg of anhydrous magnesium sulfate, 4.45mg of L-alanine, 2.24mg of D-calcium pantothenate, 7000mg of sodium chloride, 7.5mg of L-asparagine, 8.98mg of choline chloride, 54.35mg of anhydrous sodium dihydrogen phosphate, 6.65mg of L-aspartic acid, 2.65mg of L-cysteine, 56.52 mg of L-disodium hydrogen phosphate, 17.12 mg of L-17 mg of inositol phosphate, 6.52 mg of L-asparagine, 0.432mg of zinc sulfate heptahydrate, 7.35mg of L-glutamic acid, 2.02mg of nicotinamide, 147.5mg of L-arginine hydrochloride, 17.25mg of L-proline, 2mg of pyridoxal hydrochloride, 31.29mg of L-cystine hydrochloride, 9.02mg of L-tryptophan, 0.031mg of pyridoxine hydrochloride, 365mg of L-glutamine, 38.4mg of L-tyrosine, 0.219mg of riboflavin, 18.75mg of glycine, 52.85mg of L-valine, 2.17mg of thiamine hydrochloride, 31.48mg of L-histidine hydrochloride, 3151mg of D-glucose, 0.365mg of thymidine, 54.47mg of L-isoleucine, 2mg of hypoxanthine, 0.68mg of vitamin B12 and adding a proper amount of water to 1000 mL; preparation: dissolving the materials in 1000ml, and filtering with 0.22 μm microporous membrane for sterilization; or, the method also comprises the step of detecting the primary umbilical cord mesenchymal stem cells obtained by separation culture; alternatively, detection of cellular morphology and/or immunophenotypic identification; alternatively, said immunophenotypic identification refers to detection of CD73, CD90, CD105 and CD19, CD11b, CD31, CD45, HLADR, CD 34; alternatively, the obtained primary umbilical cord mesenchymal stem cells are positive in all of CD73, CD90 and CD105 (all greater than 98%), and negative in all of CD19, CD11b, CD31, CD45, HLADR and CD34 (all less than 2%).

8. A culture medium for isolated culture of mesenchymal stem cells, which may also be referred to as primary complete culture medium, is prepared with DMEM-F12 medium as a matrix and comprises: 0.8% platelet lysate, 1% human serum albumin, 2 μ g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml bFGF; or, the culture medium is prepared by taking DMEM-F12 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.

9. A primary umbilical cord mesenchymal stem cell which is positive for CD73, CD90 and CD105 and negative for CD19, CD11b, CD31, CD45, HLADR and CD34 is obtained by the following steps:

10. The primary umbilical cord mesenchymal stem cells according to claim 9, 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 μ g/ml recombinant insulin, 15ng/ml EGF, 25ng/ml Bfgf; for example, it is prepared with DMEM-F12 medium as a medium and comprises: 0.8% platelet lysate, 1% human serum albumin, 2. mu.g/ml recombinant insulin, 0.1% thioglycerol, 1% fructose, 15ng/ml EGF, 25ng/ml bFGF.