1. A serum-free medium comprising a basal medium and additional components, wherein the additional components comprise a JNK pathway inhibitor, an ERK1/2 pathway inhibitor, a P38MAPK pathway inhibitor and aminoethanol, the JNK pathway inhibitor comprises SP600125, the ERK1/2 pathway inhibitor comprises PD0325901, and the P38MAPK pathway inhibitor comprises at least one of SB202190, SB203580 and BIRB 796.

2. The culture medium of claim 1, wherein the basal medium comprises DMEM/F12, a-MEM, DMEM, or IMDM;

preferably, the concentrations of the SP600125, PD0325901, P38MAPK pathway inhibitor and aminoethanol are 1-10uM, 1-10uM, 1-10uM and 0-10mg/L, respectively;

preferably, the concentrations of the SP600125, PD0325901, P38MAPK pathway inhibitor and aminoethanol are 1-5uM, 1-5uM and 0-5mg/L, respectively;

preferably, the P38MAPK pathway inhibitor is 1-5uM of SB 202190;

preferably, the P38MAPK pathway inhibitor is 1-10uM of SB 203580;

preferably, the P38MAPK pathway inhibitor is 1-2uM BIRB 796.

3. The culture medium of claim 1, wherein the additional ingredients further comprise L-glutamine, sodium bicarbonate, hepes, insulin, transferrin, albumin, polyvinyl alcohol, 2-mercaptoethanol, lipids, amino acids, vitamins, trace elements, pluronic F-68, hydrocortisone, vitamins, cohesin, progesterone, putrescine, heparin, EGF, b-FGF, PDGF-BB, IGF-1, GM-CSF, TGF- β 1 and r-hGH, dexamethasone, NEAA, sodium selenite, gama-aminobutyric acid, collagen, ethanolamine, estradiol, reduced glutathione, 5-hydroxytryptamine, linoleic acid, lecithin, sodium pyruvate, thyroxine;

preferably, the additive components comprise 1-5mM of L-glutamine, 2-5 g/L, hepes 1-5mM of sodium bicarbonate, 1-10mg/L of insulin, 5-20mg/L of transferrin, 1-5g/L of albumin, 0-1 w/v% of polyvinyl alcohol, 50-60nM of 2-mercaptoethanol, 0.1-1mg/L of lipid, 1-10g/L of amino acid, 1-5mg/L of trace element, 500mg/L of pluronic F-68100, 1-50mg/L of hydrocortisone, 1-80mg/L of vitamin, 1-5mg/L of cohesive amine, 10-20mg/L of progesterone, 1-10mg/L of putrescine, 1-10IU/mL of heparin, EGF 1-10mg/L, b-FGF 1-10mg/L, PDGF-BB 1-10mg/L, IGF-11-10 mg/L, GM-CSF 1-10mg/L, TGF-beta 11-10 mg/L and r-hGH 2-4 ug/L, dexamethasone 2-50nM, NEAA 0-500mg/L, sodium selenite 1-50mg/L, gamma-aminobutyric acid 0-100mg/L, collagen 0.1-50mg/L, ethanolamine 20-50nM, estradiol 1-5mg/L, reduced glutathione 2.5-5mg/L, 5-hydroxytryptamine 20-35 ug/L, linoleic acid 2.5-5ug/L, oleic acid 1-3ug/L, 1-3ug/L of lecithin, 100-200 mg/L of sodium pyruvate and 80-90 nmol/L of thyroxine.

4. The culture medium according to claim 3, wherein the additive component comprises L-glutamine 1-4mM, sodium bicarbonate 2-4.5 g/L, hepes 1-4.5mM, insulin 3-10mg/L, transferrin 8-20mg/L, albumin 1-4.5g/L, polyvinyl alcohol 0-0.8 w/v%, 2-mercaptoethanol 50-58nM, lipid 0.1-0.8mg/L, amino acid 1-8g/L, trace element 1-4mg/L, pluronic F-68100-400 mg/L, hydrocortisone 1-40mg/L, vitamin 1-75mg/L, bondable amine 1-4mg/L, progesterone 10-18mg/L, progesterone, 1-8mg/L putrescine, 1-8IU/mL heparin, 1-8mg/L, b-FGF 1-8mg/L, PDGF-BB 1-8mg/L, IGF-11-8 mg/L, GM-CSF 1-8mg/L, TGF-beta 11-8 mg/L r-hGH 2-3.8 ug/L, 2-40nM dexamethasone, 0-400mg/L NEAA, 1-40mg/L sodium selenite, 0-80mg/L gamma-aminobutyric acid, 0.1-40mg/L collagen, 20-40nM ethanolamine, 1-4mg/L estradiol, 2.5-4.5mg/L glutathione, 20-31 ug/L5-hydroxytryptamine, Linoleic acid 2.5-4.5ug/L, oleic acid 1-2.8ug/L, lecithin 1-2.8ug/L, sodium pyruvate 100-180 mg/L, and thyroxine 80-88 nmol/L.

5. The culture medium according to claim 3, wherein the additive component comprises 1-3mM of L-glutamine, 2-4 g/L, hepes 1mM of sodium bicarbonate, 5-10mg/L of insulin, 10-20mg/L of transferrin, 1-3g/L of albumin, 0-0.5 w/v% of polyvinyl alcohol, 50-55nM of 2-mercaptoethanol, 0.1-0.5mg/L of lipid, 1-5g/L of amino acid, 1-3mg/L of trace element, 300mg/L of pluronic F-68100-, 1-20mg/L of hydrocortisone, 1-70mg/L of vitamin, 1-3mg/L of cohesive amine, 10-15mg/L of progesterone, 1-5mg/L putrescine, 3-8IU/mL heparin, 1-6mg/L, b-FGF 1-6mg/L, PDGF-BB 1-6mg/L, IGF-11-5 mg/L, GM-CSF 1-6mg/L, TGF-beta 11-6 mg/L r-hGH 2-3 ug/L, 2-30nM dexamethasone, 0-300mg/L NEAA, 1-30mg/L sodium selenite, 0-30mg/L gamma-aminobutyric acid, 0.1-20mg/L collagen, 20-30nM ethanolamine, 1-3mg/L estradiol, 2.5-4mg/L glutathione, 25-31 ug/L5-hydroxytryptamine, Linoleic acid 2.5-4ug/L, oleic acid 1-2.5ug/L, lecithin 1-2.5ug/L, sodium pyruvate 120-180 mg/L, and thyroxine 80-85 nmol/L.

6. The culture medium of claim 3, wherein the insulin is recombinant human insulin;

preferably, the transferrin is recombinant human transferrin;

preferably, the albumin is recombinant human albumin;

preferably, the vitamins include vitamin C, vitamin E, vitamin B12;

preferably, the vitamins include vitamin C1-50mg/L, vitamin E10-35mg/L, vitamin B1210-35 mg/L;

preferably, the vitamins include vitamin C1-20mg/L, vitamin E15-35mg/L, vitamin B1215-35 mg/L.

7. Use of a culture medium according to any one of claims 1 to 6 for culturing cells;

preferably, the cells comprise stem cells;

preferably, the stem cells include totipotent stem cells and pluripotent stem cells;

preferably, the stem cells comprise mesenchymal stem cells and induced pluripotent stem cells;

preferably, the cells obtained by the culture medium are cultured to highly express ABCB5, IDO-1, CD106 and PD-L1;

preferably, the culture medium cultures the obtained cells to produce exosomes with high expression of SCF and low expression of IL-6 and MCP-3.

8. A method of culturing cells using the medium according to any one of claims 1 to 6;

preferably, the cells comprise stem cells;

preferably, the stem cells include totipotent stem cells and pluripotent stem cells;

preferably, the stem cells include mesenchymal stem cells and induced pluripotent stem cells.

9. Cells obtained by the method of claim 8;

preferably, the cells comprise stem cells;

preferably, the stem cells include totipotent stem cells and pluripotent stem cells;

preferably, the stem cells include mesenchymal stem cells and induced pluripotent stem cells.

10. Use of a cell according to claim 8 in the preparation of a medicament for the treatment of a disease;

preferably, the disease includes wound repair, immune disease;

preferably, the cells comprise stem cells;

preferably, the stem cells include totipotent stem cells and pluripotent stem cells;

preferably, the stem cells comprise mesenchymal stem cells and induced pluripotent stem cells;

preferably, the wound is a skin lesion;

preferably, the skin injury is a wound of a diabetic patient;

preferably, the agent for treating immune diseases comprises an agent that inhibits T cell proliferation.

Background

Stem cells can be classified into totipotent stem cells, pluripotent stem cells, progenitor cells, and the like according to differentiation ability, and can be classified into placental stem cells, adipose-derived stem cells, hematopoietic stem cells, and the like according to source. Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, are derived from early-developing mesoderm, belong to pluripotent stem cells, and are originally found in bone marrow, so that MSCs are increasingly concerned by people due to the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. For example, under in vivo or in vitro specific induction conditions, mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes. At present, the mesenchymal stem cells are clinically applied to the aspects of solving various blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, repair of partial resection injury of knee joint meniscus, autoimmune diseases and the like, and make a major breakthrough, thereby saving the lives of more patients. In addition, mesenchymal stem cells have long-term development prospects in nervous system repair and more aspects.

Although the present animal experiments and partial clinical researches find that the MSC has better treatment effect on promoting tissue repair and regulating abnormal immune response, a plurality of problems in MSC application are not solved, firstly, the large-scale in vitro amplification is the premise of realizing the clinical treatment of the MSC, but the present in vitro amplification system of the MSC does not reach the agreement: according to the different kinds of culture medium, the MSC amplification system can be divided into a culture system containing fetal calf serum and a culture system which has definite components, no animal-derived components and no serum.

The culture system based on fetal calf serum is proved to be the most effective culture system of MSC cells at present, the MSC cells show excellent proliferation capacity and differentiation capacity under the system and have good clinical treatment potential, but the fetal calf serum has the main problem that no specific index is used for evaluating the serum quality, so the quality of the fetal calf serum in different producing areas and batches is different, the quality of the cultured cells is also different, and researches prove that the 'aging' phenomenon of the cells can be generated in advance when the MSC cells are cultured by using inferior serum, and the phenomena that the appearance of the cells is changed into a non-spindle shape, telomerase is gradually shortened, the cell proliferation capacity is reduced, and the differentiation potential of the cells to osteocytes, chondrocytes, adipocytes and the like is also obviously weakened. Therefore, more and more researchers currently culture the MSC cells by using a culture system which is clear in components, free of animal-derived components and free of serum, and the serum-free system can support the long-term culture of the MSC cells from the current results, but the serum-free system still has the defects compared with a fetal bovine serum system, and the main problems are slow cell proliferation and differential differentiation capacity. The most ideal serum substitute at present is human platelet lysate, which can not only avoid the possibility of xenovirus pollution caused by fetal calf serum, but also completely replace serum to promote the in vitro culture of MSC, but the source of the platelet lysate is the main problem existing at present, and the high price greatly limits the application scale of the platelet lysate.

Disclosure of Invention

In some embodiments, the invention provides a serum-free medium comprising a basal medium and additional components including a JNK pathway inhibitor including, but not limited to, SP600125, an ERK1/2 pathway inhibitor including, but not limited to, PD0325901, a P38MAPK pathway inhibitor including at least one of SB202190, SB203580, and bibb 796, an ERK1/2 pathway inhibitor, a P38MAPK pathway inhibitor, and aminoethanol.

In some embodiments, the basal medium comprises DMEM/F12, a-MEM, DMEM, or IMDM.

In some embodiments, the basal medium may also be Knock out DMEM/F12.

In some embodiments, the concentrations of the SP600125, PD0325901, P38MAPK pathway inhibitor and aminoethanol are 1-10uM, and 0-10mg/L, respectively.

In some embodiments, the concentrations of the SP600125, PD0325901, P38MAPK pathway inhibitor, and aminoethanol are 1-5uM, and 0-5mg/L, respectively.

In some embodiments, the P38MAPK pathway inhibitor is 1-5uM of SB 202190.

In some embodiments, the P38MAPK pathway inhibitor is 1-10uM of SB 203580.

In some embodiments, the P38MAPK pathway inhibitor is 1-2uM of BIRB 796.

In some embodiments, the additional ingredient further comprises L-glutamine, sodium bicarbonate, hepes, insulin, transferrin, albumin, polyvinyl alcohol, 2-mercaptoethanol, lipids, amino acids, vitamins, trace elements, pluronic F-68, hydrocortisone, vitamins, cohesive amines, progesterone, putrescine, heparin, EGF, b-FGF, PDGF-BB, IGF-1, GM-CSF, TGF-. beta.1 and r-hGH, dexamethasone, NEAA, sodium selenite, gama-aminobutyric acid, collagen, ethanolamine, estradiol, reduced glutathione, 5-hydroxytryptamine, linoleic acid, lecithin, sodium pyruvate, thyroxine.

In some embodiments, the additive component comprises 1-5mM of L-glutamine, 2-5 g/L, hepes 1-5mM of sodium bicarbonate, 1-10mg/L of insulin, 5-20mg/L of transferrin, 1-5g/L of albumin, 0-1 w/w% of polyvinyl alcohol, 50-60nM of 2-mercaptoethanol, 0.1-1mg/L of lipid, 1-10g/L of amino acid, 1-5mg/L of trace element, 500mg/L of pluronic F-68100, 1-50mg/L of hydrocortisone, 1-80mg/L of vitamin, 1-5mg/L of cohesive amine, 10-20mg/L of progesterone, 1-10mg/L of putrescine, 1-10IU/mL heparin, 1-10mg/L, b-FGF 1-10mg/L, PDGF-BB 1-10mg/L, IGF-11-10 mg/L, GM-CSF 1-10mg/L, TGF-beta 11-10 mg/L r-hGH 2-4 ug/L dexamethasone 2-50nM, 0-500mg/L NEAA, 1-50mg/L sodium selenite, 0-100mg/L gamma-aminobutyric acid, 0.1-50mg/L collagen, 20-50nM ethanolamine, 1-5mg/L estradiol, 2.5-5mg/L reduced glutathione, 20-35 ug/L5-hydroxytryptamine, 2.5-5ug/L linoleic acid, Oleic acid 1-3ug/L, lecithin 1-3ug/L, sodium pyruvate 100-200 mg/L, and thyroxine 80-90 nmol/L.

In some embodiments, the additive component comprises 1-4mM of L-glutamine, 2-4.5 g/L, hepes 1-4.5mM of sodium bicarbonate, 3-10mg/L of insulin, 8-20mg/L of transferrin, 1-4.5g/L of albumin, 0-0.8 w/w% of polyvinyl alcohol, 50-58nM of 2-mercaptoethanol, 0.1-0.8mg/L of lipid, 1-8g/L of amino acid, 1-4mg/L of trace element, 400mg/L of pluronic F-68100-, 1-40mg/L of hydrocortisone, 1-75mg/L of vitamin, 1-4mg/L of bonded amine, 10-18mg/L of progesterone, 1-8mg/L of putrescine, 1-8IU/mL heparin, 1-8mg/L, b-FGF 1-8mg/L, PDGF-BB 1-8mg/L, IGF-11-8 mg/L, GM-CSF 1-8mg/L, TGF-beta 11-8 mg/L and r-hGH 2-3.8 ug/L dexamethasone 2-40nM, NEAA0-400mg/L, sodium selenite 1-40mg/L, gamma-aminobutyric acid 0-80mg/L, collagen 0.1-40mg/L, ethanolamine 20-40nM, estradiol 1-4mg/L, glutathione reduced form 2.5-4.5mg/L, 5-hydroxytryptamine 20-31 ug/L, linoleic acid 2.5-4.5ug/L, Oleic acid 1-2.8ug/L, lecithin 1-2.8ug/L, sodium pyruvate 100-180 mg/L, and thyroxine 80-88 nmol/L.

In some embodiments, the additive component comprises 1-3mM of L-glutamine, 2-4 g/L, hepes 1-4mM of sodium bicarbonate, 5-10mg/L of insulin, 10-20mg/L of transferrin, 1-3g/L of albumin, 0-0.5 w/w% of polyvinyl alcohol, 50-55nM of 2-mercaptoethanol, 0.1-0.5mg/L of lipid, 1-5g/L of amino acid, 1-3mg/L of trace element, 300mg/L of pluronic F-68100, 1-20mg/L of hydrocortisone, 1-70mg/L of vitamin, 1-3mg/L of cohesive amine, 10-15mg/L of progesterone, 1-5mg/L of putrescine, Heparin 3-8IU/mL, EGF 1-6mg/L, b-FGF 1-6mg/L, PDGF-BB 1-6mg/L, IGF-11-5 mg/L, GM-CSF 1-6mg/L, TGF-beta 11-6 mg/L and r-hGH 2-3 ug/L, dexamethasone 2-30nM, NEAA0-300mg/L, sodium selenite 1-30mg/L, gamma-aminobutyric acid 0-30mg/L, collagen 0.1-20mg/L, ethanolamine 20-30nM, estradiol 1-3mg/L, reduced glutathione 2.5-4mg/L, 5-hydroxytryptamine 25-31 ug/L, linoleic acid 2.5-4ug/L, Oleic acid 1-2.5ug/L, lecithin 1-2.5ug/L, sodium pyruvate 120-180 mg/L, and thyroxine 80-85 nmol/L.

In some embodiments, the insulin is recombinant human insulin.

In some embodiments, the transferrin is recombinant human transferrin.

In some embodiments, the albumin is recombinant human albumin.

In some embodiments, the vitamins include vitamin C, vitamin E, vitamin B12.

In some embodiments, the vitamins include vitamin C1-50mg/L, vitamin E10-35mg/L, vitamin B1210-35 mg/L.

In some embodiments, the vitamins include vitamin C1-20mg/L, vitamin E15-35mg/L, vitamin B1215-35 mg/L.

In some embodiments, the invention provides the use of said medium for culturing cells.

In some embodiments, the cells comprise stem cells.

In some embodiments, the stem cells include totipotent stem cells and pluripotent stem cells.

In some embodiments, the stem cells comprise mesenchymal stem cells and induced pluripotent stem cells.

The serum-free culture medium provided by the invention does not contain FBS (fetal bovine serum), does not contain heterologous animal-derived components, reduces the probability of cell pollution, and simultaneously plays a role in promoting the expansion of MSC (mesenchymal stem cell).

In some embodiments, the serum-free medium for culturing the mesenchymal stem cells is obtained by researching various growth factors and various components and using amounts, and realizes the in vitro culture of the mesenchymal stem cells in a serum-free manner. The cells obtained by the culture medium are in accordance with the properties of conventional MSCs, such as the forms and cell surface molecules are consistent with the conventional MSCs, the cells have better differentiation capacity (including chondrogenic differentiation, adipogenic differentiation and osteogenic differentiation) and the like, and the cells are negative in telomerase activity, negative in soft agar clone formation test and very good in safety. In the serum-free culture medium system, the mesenchymal stem cells can realize adherent growth and effectively improve the amplification capacity of the mesenchymal stem cells, so that the culture system without exogenous serum is more favorable for the stem cells to be safely applied to clinical transplantation.

In some embodiments, the mesenchymal stem cells cultured in the serum-free culture medium have higher expression of inflammatory factors such as SCF, lower expression of inflammatory factors such as IL-6 and MCP-3, and are suggested to have stronger immune function compared with the mesenchymal stem cells cultured in the serum.

In some embodiments, the mesenchymal stem cells obtained by the serum-free culture medium culture have better repairing effect on the diabetic skin injury compared with the mesenchymal stem cells cultured by the serum.

In addition, UC-MSC cells highly express multiple factors such as ABCB5, IDO-1, CD106, PD-L1 and the like, and are superior to cells harvested by other culture media in the market. Has good practical significance in treating some specific diseases.

In some embodiments, the cells obtained from the culture medium are highly expressing ABCB5, IDO-1, CD106, PD-L1.

In some embodiments, the culture medium cultures the resulting cells to produce exosomes expressing high SCF and low IL-6, MCP-3.

In some embodiments, the invention provides a method of culturing cells using the medium.

In some embodiments, the cells comprise stem cells.

In some embodiments, the stem cells include totipotent stem cells and pluripotent stem cells.

In some embodiments, the stem cells comprise mesenchymal stem cells and induced pluripotent stem cells.

In some embodiments, the invention provides cells obtained by the methods described herein.

In some embodiments, the cells comprise stem cells.

In some embodiments, the stem cells include totipotent stem cells and pluripotent stem cells.

In some embodiments, the stem cells comprise mesenchymal stem cells and induced pluripotent stem cells.

In some embodiments, the invention provides the use of the cell in the preparation of a medicament for the treatment of a disease.

In some embodiments, the disease comprises wound repair, immune disease.

In some embodiments, the cells comprise stem cells.

In some embodiments, the stem cells include totipotent stem cells and pluripotent stem cells.

In some embodiments, the stem cells comprise mesenchymal stem cells and induced pluripotent stem cells.

In some embodiments, the wound is a skin lesion.

In some embodiments, the skin injury is a wound in a diabetic patient.

Drawings

FIG. 1 is a microscopic view of UC-MSC cells.

FIG. 2 shows the molecular flow assay result of UC-MSC cell surface.

FIG. 3 is a flowchart of the UC-MSC immunosuppressive function assay.

FIG. 4 shows the result of the UC-MSC immunosuppressive function assay.

FIG. 5 shows the results of the differentiation of UC-MSC into chondrocytes.

FIG. 6 shows the results of the UC-MSC adipogenic and osteogenic differentiation assays.

FIG. 7 shows the results of UC-MSC cell cycle assays.

FIG. 8 shows UC-MSC telomerase activity assay results.

FIG. 9 shows the results of UC-MSC soft agar colony formation assay.

FIG. 10 shows the UC-MSC karyotype detection results.

FIG. 11 shows the results of Western blot analysis for the expression levels of various inflammatory factors in UC-MSC P7 generation cells.

FIG. 12 is the bioinformatic statistics of UC-MSC extracellular exosomes differential inflammatory factor cultured in the present medium and 10% FBS medium, A is the biological functional classification of differential inflammatory factor; b is the signal path analysis result of the differential inflammatory factor

FIG. 13 shows the result of the repairing effect of UC-MSC cells on the skin damage of diabetic rats.

FIG. 14 is a comparison of growth rates of cells cultured in medium supplemented with and without small molecule compounds.

FIG. 15 is a graph of population doubling time of UC-MSCs in culture.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

EXAMPLE 1 serum-free Medium formulation

Serum-free medium formulation 1 is shown in table 1.

TABLE 1 serum-free Medium formulation

The preparation method of the serum-free medium formula 1 comprises the following steps: 1mL of the 47-component 1000X stock solution with the above serial numbers 2 to 48 was added to the above DMEM/F12 basal medium to prepare 1L of serum-free medium. The final concentrations of the individual components in serum-free medium are shown in table 1.

Example 2 serum-free Medium formulation

Serum-free medium formulation 2 is shown in table 2.

TABLE 2 serum-free Medium formulation

The preparation method of the serum-free medium formula 2 is the same as that of example 1.

EXAMPLE 3 serum-free Medium formulation

Serum-free medium formulation 3 is shown in table 3.

TABLE 3 serum-free Medium formulation

The preparation method of the serum-free medium formula 3 is the same as that of example 1.

EXAMPLE 4 serum-free Medium formulation

Serum-free Medium formulation 4 was the same as in example 1 except that the basal medium was a-MEM. The serum-free medium was prepared as in example 1.

Example 5MSC isolation and culture

1. After the placenta of the pregnant woman is delivered, cutting off an umbilical cord from a part close to the placenta, putting the umbilical cord into a sterilized PBS liquid containing double antibodies, and quickly bringing back to a laboratory;

2. taking the umbilical cord tissue back under aseptic conditions, washing the surface of the umbilical cord tissue with PBS (phosphate buffer solution) to remove blood stains, putting the umbilical cord tissue into 75% alcohol for 3 minutes for sterilization, and washing the umbilical cord with PBS for 3 times to remove residual alcohol;

3. cutting off about 1cm of tissue at each end of the umbilical cord, discarding, cutting the rest umbilical cord into about 2-3cm of tissue, placing into a dish containing PBS, repeatedly washing umbilical cord tissue, and removing blood in umbilical vein and umbilical artery blood vessel;

4. taking a section of umbilical cord tissue to another plate, removing the amnion and three blood vessels by using forceps, and placing the rest Wharton's jelly into another plate;

5. separating all umbilical cord tissues;

6. cutting Wharton's jelly into 1-2mm in diameter, adding 1% (w/v) collagenase II, 1% (w/v) collagenase IV, 1% (w/v) hyaluronidase, 1% (w/v) DNase, 0.05% pancreatin, and digesting at 37 deg.C for 30-60 min;

7. filtering the liquid completely digested by enzyme with a100 um cell sieve, and discarding the tissues which are not completely digested;

8. centrifuging and filtering the liquid at 1000rpm/min for 10 minutes, and discarding the supernatant;

9. the precipitated cells were resuspended in the medium and cultured in a10 cm dish.

The whole experimental procedure took about 4-5 hours (one umbilical cord isolated). Cells isolated from a section of 20cm umbilical cord tissue can be divided into 5 10cm dishes,

example 6 identification of UC-MSC

1. Cell morphology observation

When the cells isolated in example 5 were inoculated into the medium described in example 1, adherent cells were observed 24h after inoculation, about 7 days, and the results are shown in fig. 1, and microscopic observation shows that the UC-MSC cells are typically spindle-shaped, grow in an adherent manner, and the cell growth state is normal. And the cells are full and can be subcultured or frozen for storage.

2. UC-MSC cell surface molecule identification

The cells isolated in example 5 were seeded in the medium described in example 1 and subcultured.

Detecting a characteristic marker of the UC-MSC cell by using a flow cytometer, and confirming that the UC-MSC cells of P5 and P8 generations express CD73, CD90 and CD105 (more than or equal to 95 percent) protein according to the result; does not express CD14, CD34, CD45 and CD79a, and HLA (less than or equal to 5 percent) meets the MSC standard.

3. Immunosuppressive function assay

The serum-free medium used in this example was, as shown in example 1, the number of cell generations used for recovery was P3.

The formula of the complete culture medium for T cell culture comprises the following components: RPMI 1640 medium containing 10% FBS.

The generation number of PBMC is primary.

Complete medium for T cell + MSC co-culture refers to RPMI 1640 medium containing 10% FBS.

Treating UC-MSC cells with mitomycin C to make UC-MSC cells unable to continue proliferation and division, culturing with CFSE labeled PBMC (primary isolated) at a certain ratio for 4 days, and detecting the inhibition effect of UC-MSC cells on T cell proliferation in PBMC.

The experimental flow chart is shown in fig. 3, and the detection results are shown in fig. 4 and table 4.

TABLE 4

Comparison of data	T cell independent culture group	T cell and MSC coculture group
			Primary seeded cells	0.50x106	0.50x106T cells and 0.30x106Co-culture of individual MSCs
Proliferation fold of T cells	2.2 times of	0.33 times of

The results show that UC-MSC and CFSE labeled T cells are cultured for five days, the T cells of the single culture group are greatly expanded, and the T cells of the co-culture group are not expanded, which indicates that the UC-MSC cells can inhibit the proliferation of the T cells.

4. Detection of differentiation-inducing ability

(1) Differentiation of UC-MSC into chondrocytes

The UC-MSC used for cartilage differentiation was the P3 th generation UC-MSC obtained by using the isolation and culture of example 5, and the culture medium used for the culture was the serum-free medium of example 1.

1) Suction 2.5X 10⁵UC-MSC cells were transferred to appropriate culture tubes and the cells were washed.

2) Resuspend the cells in differentiation medium, centrifuge at 150g for 5min at room temperature, discard the supernatant, resuspend the cell pellet again in differentiation medium (Gibco)^TMA1007101), centrifuge at room temperature of 150g for 5 minutes, and discard the supernatant.

3) Resuspending the cells in differentiation medium and adjusting the cell density to 5X 10⁵Cells per ml.

4) 0.5 ml (2.5X 10)⁵Cells) were added to a 15ml polypropylene tube and centrifuged at 150g for 5 minutes at room temperature.

5) Placing the polypropylene tube in an incubator, releasing half of the tube cover to allow gas exchange, at 37 deg.C, 5% CO₂The culture was carried out for 24 hours. Do not scatter the cells.

6) The culture medium is changed every 2-3 days, and the liquid change amount is 0.5 ml each time. Care was taken not to scatter cells during the exchange.

7) After changing the liquid, flicking the culture tube wall to suspend the cell mass, putting the culture tube back into the incubator again for culture, and half sending the tube cover to exchange gas.

8) The culture was continued for 14-28 days, and cell pellets were collected, fixed with 4% formaldehyde, and stained with an Alcian blue.

The observation results are shown in FIG. 5, which are the results of 21 days of differentiation of UC-MSC into cartilage induced by toluidine blue staining.

(2) Differentiation of UC-MSC cells into adipocytes

The UC-MSCs used for adipogenic differentiation were the UC-MSCs isolated and cultured using example 5, and the culture medium used for the culture was the serum-free medium of example 1. 1) Placing UC-MSC cells at 37 deg.C and 5% CO₂Cultured in an incubator.

2) When the cell fusion degree reaches 80% -90%, digesting with 0.25% Trypsin-0.04% EDTA.

3) The digested mesenchymal stem cells are arranged according to 2 x10⁴cells/cm₂The cell density of (a) was seeded in six-well plates, and 2mL of complete medium (serum-free medium described in example 1) was added per well.

4) The cells were incubated at 37 ℃ with 5% CO₂The incubator of (2) for cultivation.

5) Fluid was changed every third day until cell confluence reached 100% or over-confluency.

6) Carefully aspirate the mesenchymal stem cell complete medium and add 2mL of OriCellTM human umbilical cord mesenchymal stem cell adipogenic induction differentiation medium A (Guangzhou Seisakusho Co.) to the six-well plate.

7) After 3 days of induction, the solution A in the six-well plate was aspirated, and 2mL of OriCellTM human umbilical mesenchymal stem cell adipogenic differentiation medium solution B was added.

8) After 24h, the solution B was aspirated and replaced with solution A for induction.

9) After the alternating action of solution A and solution B for 3-5 times (12-20 days), the culture is continued for 4-7 days with solution B until the lipid droplets become large enough and round. During the maintenance culture period of the solution B, fresh solution B is required to be replaced every 2-3 days.

10) After the adipogenic induction differentiation is finished, the mesenchymal stem cell adipogenic induction differentiation culture medium in the six-well plate is sucked away and washed 1-2 times by 1 XPBS. 2mL of 4% neutral formaldehyde solution was added to each well and fixed for 30 min.

11) The neutral formaldehyde solution was aspirated and washed 2 times with 1 × PBS. Adding 1mL of oil red O dye working solution into each hole for dyeing for 30min (working solution preparation method: oil red O stock solution: distilled water: 3:2, mixing uniformly and filtering with neutral filter paper).

12) The oil red O stain was aspirated and washed 2-3 times with 1 XPBS.

13) The plates were placed under a microscope to observe the effect of adipogenic staining. The results are shown in FIG. 6, which is an experimental result 21 days after adipogenesis induction.

(3) Differentiation of UC-MSC into osteoblasts

The UC-MSC for osteogenic differentiation was the UC-MSC isolated and cultured using example 4, and the culture medium used for the culture was the serum-free medium of example 1.

1) Placing UC-MSC cells at 37 deg.C and 5% CO₂Cultured in an incubator.

2) When the cell fusion degree reaches 80-90%, digestion is carried out by using 0.25% Trypsin-0.04% EDTA.

3) The digested human umbilical cord mesenchymal stem cells are processed according to 2 x10⁴cells/cm²The cell density of (a) was seeded in six-well plates previously coated with 0.1% gelatin, and 2mL of complete medium was added per well. .

4) The cells were incubated at 37 ℃ with 5% CO₂The incubator of (2) for cultivation.

5) When the cell confluence reaches 60% -70%, carefully remove the complete medium from the wells, and add 2mL OriCellTM human umbilical mesenchymal stem cell osteogenic induction differentiation complete medium to the six-well plate (Guangzhou Seisakusho).

6) Changing fresh OriCellTM human umbilical mesenchymal stem cells every 3 days to obtain complete medium for osteogenic induced differentiation (preheating to 37 ℃ before use)

7) After 2-4 weeks of induction, cells were stained with alizarin red, depending on morphological changes and growth.

The results are shown in fig. 6, which is an experimental result 28 days after osteogenic induction.

(4) Cell cycle assay

The UC-MSC used in the cell cycle assay was the UC-MSC isolated and cultured in example 5, and the culture medium used in the culture was the serum-free medium of example 2.

(1) Normally culturing UC-MSC cells, sucking and discarding supernatant when the confluence degree of the cells reaches about 50%, and cleaning the cells twice by DPBS;

(2) adding 1 mL/hole pancreatin digestive juice, placing in a 37 ℃ incubator for digestion for 3min, and taking out to terminate digestion when observing that cells can float into single cells under a mirror;

(3) add 2ml of medium, shake the plate to dislodge all cells from the bottom of the dish, aspirate the cell suspension into a 15ml EP tube, and centrifuge at 200g for 5 minutes.

(4) The supernatant was aspirated off, and the resuspended cells were gently pipetted by adding 1mL of DPBS (calcium and magnesium free) and counted.

(5) According to the counting result, suck 1x10⁶Putting the cell suspension with the corresponding volume of the cells into a 1.5mL EP tube, adding DPBS (without calcium and magnesium) to 1mL, gently blowing, beating and uniformly mixing, and centrifuging for 15-20 seconds by using a palm centrifuge.

(6) Removing supernatant, flicking tube bottom to disperse cell precipitate, adding 1ml pre-cooled 70% ethanol solution, gently blowing, mixing, fixing at 4 deg.C overnight, or storing at 4 deg.C for 5 days.

(7) Centrifuging the fixed cells for 10min at 500 Xg with a desk temperature-controlled low-speed centrifuge, removing the supernatant, adding 1ml of DPBS (without calcium and magnesium) for re-suspension and mixing, and centrifuging for 10min at 500 Xg with the desk temperature-controlled low-speed centrifuge again.

(8) The supernatant was aspirated off, the cell pellet was dispersed at the bottom of the vial by flicking, 500ul of PI staining working solution was added, and incubation was carried out at room temperature for 30 minutes in the dark

(9) After the incubation, the cells were analyzed by flow cytometry, and 3 ten thousand cells were collected.

(10) The target cell population was circled to exclude cell debris and set at gate P1.

(11) A biparametric plot was created and analyzed in P1 on the ordinate PE-H and on the abscissa PE-A to exclude adherent cells, and a gate P2 was set.

(12) A new histogram was created and analyzed in P2, wherein the highest peak on the left of the peak position of the cell population was G1, the peak position at twice the corresponding value was G2, and the middle was S phase.

As shown in FIG. 7, it can be seen from FIG. 7 that the UC-MSC cell division at the P3 generation is more vigorous than that at the P8 generation.

The left panel of FIG. 7 shows the results of P3 generation cells, and the right panel shows the results of P8 generation cells.

7. Telomerase activity detection

The UC-MSC used for detecting telomerase activity was the 3 rd generation UC-MSC isolated and cultured in example 5, and the culture medium used for the culture was the serum-free medium of example 1.

(1) Normally culturing UC-MSC cells, sucking and discarding supernatant when the confluence degree of the cells reaches about 50%, and cleaning the cells twice by DPBS;

(2) adding 1 mL/hole pancreatin digestive juice, placing in a 37 ℃ incubator for digestion for 3min, and taking out to terminate digestion when observing that cells can float into single cells under a mirror;

(3) add 2ml of medium, shake the plate to dislodge all cells from the bottom of the dish, aspirate the cell suspension into a 15ml EP tube, and centrifuge at 200g for 5 minutes.

(4) The supernatant was aspirated off, and the resuspended cells were gently pipetted by adding 1mL of DPBS (calcium and magnesium free) and counted.

(5) According to the counting result, 1X10 aspirates⁶The cell suspension is put into a 1.5mL EP tube, added with DPBS (without calcium and magnesium) to 1mL, gently blown and beaten evenly, and centrifuged for 15-20 seconds by using a palm centrifuge.

(6) The supernatant was aspirated off, 200. mu.l CHAPS Lysis B. mu.ffer was added immediately to resuspend the cells, and the cell suspension was placed on ice for 30min and centrifuged at 12000 Xg, 4 ℃ for 20min using a tabletop temperature-controlled low-speed centrifuge.

(7) Sucking 160 mu of supernatant, subpackaging 25 mu of supernatant into each tube, immediately quick-freezing on dry ice, and storing at-80 ℃; one tube was left for protein quantification (bradford method, guaranteed to contain < 1.5. mu.g protein per reaction).

(8) Subsequent experiments were performed by diluting the samples to 0.1. mu.g/. mu.l with CHAPS Lysis B. mu.ffer based on protein concentration measurements.

(9) The reaction was prepared according to the experimental requirements, multiplied by the desired number of reactions, as shown in the following Table 5.

TABLE 5

5×Reaction Mix	2 μ l/PCR reaction
		Taq Polymerase	0.16. mu.l/PCR reaction
Nuclease Free Water	7.04. mu.l/PCR reaction

The plates were closed and mounted using a multi-well plate centrifuge for 10 seconds.

(10) The prepared 384-well reaction plate was placed in a fluorescent quantitative PCR instrument and the qPCR reaction was performed according to the following procedure.

TABLE 6

The experimental result is shown in fig. 8, the UC-MSC cell telomerase activity is negative, which represents its particularly good safety (no tumorigenicity).

8. Soft agar colony formation assay

The UC-MSCs used in the soft agar colony formation assay were the obtained 3 rd generation UC-MSCs obtained by isolation and culture in example 4, and the culture medium used in the culture was the serum-free medium of example 1.

The principle is as follows: the cell seeding survival rate only indicates the number of cells attached after seeding, but the cells attached do not necessarily have to be each capable of proliferating and forming a clone. While the cells forming the clone must be adherent and viable. The clonogenic rate reflects two important traits, cell population dependence and proliferative capacity. Because of different cell biological properties, the cell clone formation rate is very different, the clone formation rate of general primary culture cells is weak, and the continuous cell line is strong; the clone formation rate of the diploid cell is low, and the transformed cell line is strong; the normal cell clone formation rate is weak, and the tumor cell is strong. And the clone formation rate has a certain relation with the inoculation density, when the clone formation rate is measured, the inoculated cells must be dispersed into single cell suspension, and the single cell suspension is directly inoculated into a dish for a week and is checked at any time, and the culture is stopped when the cells form the clone.

The method comprises the following basic steps:

(1) taking cells in logarithmic growth phase, digesting with 0.25% trypsin and gently blowing to make them into single cells, counting living cells, adjusting cell density to 1 × 10 with DMEM culture solution containing 20% fetal calf serum⁶cell/L. Then, gradient multiple dilution is carried out according to the experimental requirements.

(2) Low melting point agarose solutions of 1.2% and 0.7% were prepared separately from distilled water and maintained at 40 ℃ after autoclaving without coagulation.

(3) Mixing 1.2% agarose and 2 × DMEM medium (containing 2 × antibiotics and 20% calf serum) according to a ratio of 1:1, pouring 3mL of mixed solution into a plate with the diameter of 6cm (adding 7-10 mL into a plate with the diameter of 10 cm), cooling and solidifying, and placing the mixed solution into a CO2 incubator for later use as bottom-layer agar.

(4) After 0.7% agarose and 2 × DMEM medium were mixed in a sterile tube at a ratio of 1:1, 0.2mL of cell suspension was added to the tube, mixed well and poured into a plate with a 1.2% agarose base to form a layer of diagarose. After the upper agar is solidified, put in 5% CO at 37 DEG C₂Culturing in an incubator for 10-14 days.

(5) The plate was placed under an inverted microscope and the cell clone number was observed. The formation rate is calculated.

Soft agar culture methods are commonly used to detect tumor cells and transformed cell lines. When the agar is mixed with the cells in the test, the temperature of the agar should not exceed 40 ℃. The density of seeded cells does not exceed 35 cells per square centimeter, typically 1000 cells are seeded on a 6cm dish. Normal cells were unable to proliferate in suspension and were not suitable for soft agar colony formation assays.

As shown in FIG. 9 and Table 7, UC-MSC cells did not form colonies in soft agar medium.

TABLE 7

Name (R)	Number of clones of Day12	Number of clones of Day21
			UC-MSC	0	0
Hela	195	300

9. Karyotype detection

The UC-MSC used for karyotyping was the 3 rd generation UC-MSC isolated and cultured in example 4, and the culture medium used for culturing was the serum-free medium of example 1.

(1) When the confluency of the cells in the 6-well plate reaches 60%, colchicine with the concentration of 50ng/ml is added, and the treatment is carried out for 2h at 37 ℃.

(2) Cells were digested into single cells, harvested by centrifugation at 1200rpm for 5min, and washed once with PBS.

(3) Preheating hypotonic KCl at 37 ℃, transferring the hypotonic KCl heavy suspension cells into a 15ml centrifuge tube, adding the hypotonic KCl to supplement to 10ml, and treating for 20-40min at 37 ℃.

(4) Adding methanol: glacial acetic acid (3:1) stationary solution, centrifugation at 1000rpm for 10min, supernatant removal, and 1ml of supernatant retention.

(5) Gently blowing and beating the suspension, slowly adding the stationary liquid while shaking until the centrifugal tube is full, and centrifuging at 1500rpm for 5 min.

(6) Gently blowing and beating the suspension, slowly adding the stationary liquid while shaking until the centrifugal tube is full, and standing for 15 min.

(7)1500rpm, 5min, centrifuged and the supernatant discarded.

(8) The fixative was added slowly with shaking until the tube was full and allowed to stand overnight at 4 ℃.

(9) The supernatant was discarded the next day, and the slides pre-cooled to-20 ℃ were removed and dropped immediately.

(10) Drying at 75 deg.C for 1-2 h.

(11) Staining slides were numbered and then stained with pancreatin, saline stop digestion, Giemsa stain (5ml Giemsa +45ml PBS), and dried at room temperature.

(12) Scanning and analyzing, namely uploading the slide to a come card scanning machine for scanning and then carrying out chromosome karyotype analysis.

The results are shown in FIG. 10, G shows band, 20 dividing phases are counted, 5 are analyzed by karyotype, no chromosome number or structural abnormality is found, and the UC-MSC karyotype is normal and is 46, XX.

10. Detection of multiple inflammatory factors

The UC-MSC used for the detection of various inflammatory factors was P7 generation UC-MSC obtained by using the isolation and culture of example 5, and the culture medium used for the culture was the serum-free medium of example 1.

The detection method comprises the following steps: and (3) after the cell growth exceeds 95%, discarding the old culture medium, washing with PBS for 3 times, digesting the MSC cells, centrifuging, discarding the supernatant, lysing the cells by using RIPAbuffer, centrifuging at a high speed, and sucking the protein supernatant to prepare Western blot.

The expression levels of various inflammatory factors of P7 UC-MSC cells are detected by a Western blot method, and the result is shown in figure 11, and the UC-MSC cells harvested by the culture medium have high expression of various factors such as ABCB5, IDO-1, CD106, PD-L1 and the like, and are better than the cells harvested by other culture media in the market.

In FIG. 11, the culture medium corresponding to each band is:

strip 1: 10% FBS + DMEM/F12

Strip 2: 10% FBS + a-MEM

The strip 3: gibco^TM，A1033201

The strip 4: culture Medium formulation of inventive example 1

The strip 5: StemCell MesenCult mesenchymal stem cell culture medium (Cat No. 05448)

Strip 6: 2% ultroser g + DMEM/F12

11. Detection of inflammatory factors in exosomes

(1) Complete drying of slide chips

And taking the slide chip out of the box, after the slide chip is balanced for 1 hour at room temperature, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying the chip for 1 to 2 hours at room temperature.

(2) Sealing and incubation

1) Adding 100 mul of 1 Xconfining liquid into each chip hole, and incubating for 1h on a shaking bed at room temperature to avoid generating bubbles;

2) the blocking solution was removed and 60. mu.l of sample, one sample in an array, was added to each well and incubated overnight at 4 ℃ with shaking. (samples were loaded at 250 ug/ml.)

3) The slide was washed with a Thermo Scientific Wellwash Versa chip washer in two steps, first with 1 XWash solution I, 200. mu.l of 1 XWash solution I per well, 7 times with 5s shaking each time, with high shaking intensity, diluted with deionized water to 20 XWash solution I. Then, the washing is carried out by changing to 1 Xwashing liquid II channel, 200 mul of 1 Xwashing liquid II is washed for 3 times, each time the washing is carried out for 5s, the shaking intensity is selected to be high, and 20 Xwashing liquid II is diluted by deionized water.

4) Preparing biotin-labeled antibody, quickly centrifuging small tubes of the biotin-labeled antibody, adding 300 mul of 1 × confining liquid into each small tube, and uniformly mixing. 70ul of biotin-labeled antibody was added to each well and shaken at room temperature for 2 hours.

5) Cleaning, synchronous step 3).

6) Add 70. mu.l of fluorescent agent-streptavidin diluted 1500 times into each well (1.5 ml of blocking solution is added into a small tube of fluorescent agent-streptavidin after rapid centrifugation), stick the slide on a sealing strip, and then wrap the slide with aluminum foil paper and shake and incubate the slide for 2 hours at room temperature in the dark. (this step may also be carried out overnight at 4 ℃ C.)

7) Cleaning and synchronizing with step 3.

(3) Fluorescence detection

1) Scanning the signal with a laser scanner Innoscan 300 using Cy3 or green channel (excitation frequency 532nm)

Instrument model InnoScan 300Microarray Scanner; the manufacturer is Innopsys; the producing area: parc d' Activies ActiveStre; 31390 Carbonne-France; scanning parameters are as follows: WaveLengh: 532 nm; resolution: 10 μm

2) Data analysis was performed using data analysis software of AAH-CYT-G5.

Wherein the sample is:

experimental groups: the p5 th generation UC-MSC obtained from the isolation and culture of example 5, the UC-MSC obtained from the culture in the serum-free medium of example 1 is used for extracting the obtained exosome by using the conventional exosome separation and extraction steps;

control group: the difference from the experimental group was that the medium was DMEM/F12 medium containing 10% FBS.

The results are shown in FIGS. 12A-B, which are tabulated: by usingThe result of comparative analysis of the UC-MSC cell-derived exosome obtained by culturing the culture medium of the embodiment 1 of the invention and the UC-MSC cell-derived exosome obtained by culturing the culture medium of 10% FBS by using a Human Cytokine Antibody Microarray slide protein chip shows that the UC-MSC cell-derived exosome obtained by using the culture medium of the invention has high expression of SCF, low expression of inflammatory factors such as IL-6, MCP-3 and the like, and the protein chip has stronger immune function.

TABLE 8

Name (R)	Ratio of
		SCF	5.4244604
MCP-3	0.2601518
		IL-6	0.2076086

Remarking: the ratio described in table 8 is the ratio of the expression amount of the corresponding factor in UC-MSC cell-derived exosomes obtained from the inventive culture medium to the expression amount of the corresponding factor in UC-MSC cell-derived exosomes obtained from the 10% FBS culture medium.

12. Repairing effect of UC-MSC cells on skin injury of diabetic rats

(1) Construction of diabetic rat model

Seven-week-old ZDF female rats are purchased from Beijing Wintolite Hua, and are fed with high-fat feed (Shanghai Yongli) after being fed with conventional feed for one week, the circadian rhythm is kept for 12h indoors, the temperature is 25 +/-2 ℃, food and water are taken freely, the blood sugar value of the rats is detected every week, the weight change is recorded until the fasting blood sugar value is stably maintained at more than 11.1mmol/L, and a diabetic rat model is successfully constructed.

(2) Wound repair

Diabetic rats successfully modeled were randomly grouped: PBS dried group, ordinary UC-MSC (DMEM/F12 medium containing 10% FBS), UC-MSC obtained from the culture medium of the present invention (culture medium of example 1 of the present invention).

A square wound of 1.5X 1.5cm2 size was created on the back of each group of rats, and 200. mu.l PBS-resuspended 1X10 were injected subcutaneously at four sides of the wound at fixed points⁶UC-MSCs (10% FBS), 200. mu.l PBS resuspended 1X10⁶UC-MSCs (culture medium of example 1 of the invention), wound area on the back of rats is measured every other day, and wound healing curves are drawn.

The results are shown in fig. 13, the subcutaneous dry prognosis is performed on the back injury part of the diabetic rat, the area of the back wound of the rat is measured every other day, and the healing rate of the skin of the back injury of the diabetic rat in the UC-MSC intervention group obtained by calculating the minimal medium on the 3 rd day is 29.9%, which is 15.9% higher than that of the UC-MSC intervention group cultured by the common medium and 2.4% higher than that of the PBS control group; the healing of the skin lesion on the back of diabetic rats in the UC-MSC intervention group obtained by the culture medium can be observed better than that of the other two groups by naked eyes by the day 15, and the healing rate of the skin lesion on the back of each group of rats is measured to reach 90%.

Remarking: the generation number of the UC-MSC obtained by culturing the common UC-MSC and the culture medium of the invention is P5.

Example 7

On the basis of the formulation in table 1 of example 1, the culture medium without adding the "small molecule compound" described in table 1 and the culture medium in table 1 of example 1 were cultured with UC-MSC according to the method described in example 5, and the growth of the cells was as shown in fig. 14.

The growth rate of UC-MSC cells in basal medium without added compound was slightly lower than that of complete medium.

FIG. 15 shows the results of UC-MSC culture using the media formulations in Table 2 of example 2.