BIOACTIVE SUBSTANCE COMPOSITION, SERUM-FREE MEDIUM COMPRISING THE COMPOSITION, AND USES THEREOF

Abstract

The invention provides a bioactive substance composition, a serum-free medium comprising the composition and the uses thereof. The bioactive substance composition is used for serum-free medium and/or composition and the preparation thereof; The serum-free medium and/or composition can be used for primary culture and secondary culture of cells and/or tissues. The cells are selected from any one or more of tendon and/or ligament derived cells, chondrocytes, meniscus stem cells, mesenchymal stem cells, skeleton stem cells, and muscle stem cells. The tissue is the musculoskeletal system tissue. The bioactive substance composition and/or serum-free medium and/or the composition can be used to prepare drugs for tissue and/or organ injury treatment; The tissue or organ injury is selected from the tissue or organ injury of the musculoskeletal system.

Claims

1. A bioactive substance composition, wherein the bioactive substance composition comprises fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, selenite, wherein the mass-volume concentration range ratio of each component is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=1-50:1-50:1-40:1-11:10-5000:10-100000:10-300000:1-25000:1-25:1-25000:1-25; preferably, fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=5-40:5-40:2-30:1-8:500-4000:1000-90000:1000-200000:10-15000:1-15:2-15000:1-15; more preferably, fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=10-30:10-30:3-20:2-5:1000-2000:10000-80000:2000-80000:100-5000:2-7:7-10000:2-7; preferably, the mass-volume concentration of the transferrin in the bioactive substance composition is 0.1-300 μg/ml, and the mass ratio is 0.00001%-0.03%; preferably, the mass-volume concentration of the transferrin in the bioactive substance composition is 1-200 μg/ml, and the mass ratio is 0.0001%-0.02%; more preferably, the mass-volume concentration of the transferrin in the bioactive substance composition is 1-150 μg/ml, and the mass ratio is 0.0001%-0.015%; preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 0.01-50 μg/ml, and the mass ratio is 0.000001%-0.005%; preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 0.1-g/ml, and the mass ratio is 0.00001%-0.003%; more preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 1-20 μg/ml, and the mass ratio is 0.0001%-0.002%; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 0.1-50 ng/ml, and the mass ratio is 0.00000001%-0.000005%; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 1-30 ng/ml, and the mass ratio is 0.0000001%-0.000003%; more preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 2-20 ng/ml, and the mass ratio is 0.0000002%-0.000002%.

2. The bioactive substance composition according to claim 1, wherein the fibroblast growth factor in the bioactive substance composition is selected from any one or more of FGF-basic, FGF1, FGF2, FGF4, FGF7, FGF10, FGF18, and fibroblast growth factor synthetic peptides; preferably, the mass-volume concentration of the fibroblast growth factor in the bioactive substance composition is 1-100 ng/ml, and the mass ratio is 0.0000001%-0.00001%; preferably, the mass-volume concentration of the fibroblast growth factor in the bioactive substance composition is 5-70 ng/ml, and the mass ratio is 0.0000005%-0.000007%; more preferably, the mass-volume concentration of the fibroblast growth factor in the bioactive substance composition is 10-40 ng/ml, and the mass ratio is 0.000001%-0.000004%.

3. The bioactive substance composition according to claim 1, wherein the platelet-derived growth factor in the bioactive substance composition is selected from any one or more of PDGF-AA, PDGF-AB, PDGF-BB, synthetic peptides of platelet-derived growth factor; Preferably, the mass-volume concentration of the platelet-derived factor in the bioactive substance composition is 1-100 ng/ml, accounting for 0.0000001%-0.00001% by mass; preferably, the mass-volume concentration of the platelet-derived factor in the bioactive substance composition is 5-70 ng/ml, accounting for 0.0000005%-0.000007% by mass; more preferably, the mass-volume concentration of the platelet-derived factor in the bioactive substance composition is 10-40 ng/ml, accounting for 0.000001%-0.000004% by mass.

4. The bioactive substance composition according to claim 1, wherein the transforming growth factor-β in the bioactive substance composition is selected from any one or more of TGF-β1, TGF-β2, TGF-β3, and transforming growth factor-β synthetic peptides; preferably, the mass-volume concentration of the transforming growth factor-β in the bioactive substance composition is 0.1-80 ng/ml, accounting for 0.00000001%-0.000008% by mass; preferably, the mass-volume concentration of the transforming growth factor-β in the bioactive substance composition is 2-50 ng/ml, accounting for 0.0000002%-0.000005% by mass; more preferably, the mass-volume concentration of the transforming growth factor-β in the bioactive substance composition is 5-25 ng/ml, accounting for 0.0000005%-0.0000025% by mass.

5. The bioactive substance composition according to claim 1, wherein the glucocorticoid in the bioactive substance composition is selected from any one or more of dexamethasone or its salt, dexamethasone solvent, hydrocortisone or its salt, cortisone acetate, cortisone acetate or its salt, methylprednisone sodium succinate, prednisone, betamethasone, betamethasone valerate, beclomethasone propionate, prednisolone acetate, prednisolone acetate, or prednisolone; preferably, the molar concentration of the glucocorticoid in the bioactive substance composition is 0.1-90 nM, accounting for 0.0000000039%-0.00000354% by mass; preferably, the molar concentration of the glucocorticoid in the bioactive substance composition is 1-50 nM, accounting for 0.000000039%-0.00000197% by mass; more preferably, the molar concentration of the glucocorticoid in the bioactive substance composition is 1-20 nM, accounting for 0.000000039%-0.000000785% by mass.

6. The bioactive substance composition according to claim 1, wherein the heparin or its salt in the bioactive substance composition is selected from any one or more of heparin, heparin sodium and heparin calcium; preferably, the mass volume concentration of the heparin or its salt in the bioactive substance composition is 0.1-10 μg/ml, accounting for 0.00001%-0.001% by mass; preferably, the mass volume concentration of the heparin or its salt in the bioactive substance composition is 0.5-8 μg/ml, accounting for 0.00005%-0.0008% by mass; more preferably, the mass volume concentration of the heparin or its salt in the bioactive substance composition is 1-5 μg/ml, accounting for 0.0001%-0.0005% by mass.

7. The bioactive substance composition according to claim 1, wherein the vitamin C or its derivatives in the bioactive substance composition are selected from any one or more of Vitamin C (i.e. ascorbic acid), ascorbic acid glucoside, ethyl vitamin C, 3-o-ethyl ascorbic acid, magnesium phosphate of vitamin C, sodium phosphate of vitamin C, L-ascorbic acid 2-phosphate sesquimagnesium salt complex, vitamin C tetraisopalmitate, ascorbic acid palmitate, ascorbic acid 2-phosphate 6-palmitate, esterified vitamin C, other solvates of ascorbic acid; preferably, the mass volume concentration of the vitamin C or its derivatives in the bioactive substance composition is 0.1-100 μg/ml, accounting for 0.00001%-0.01% by mass; preferably, the mass volume concentration of the vitamin C or its derivatives in the bioactive substance composition is 1-100 μg/ml, accounting for 0.0001%-0.01% by mass; more preferably, the mass volume concentration of the vitamin C or its derivatives in the bioactive substance composition is 10-80 μg/ml, accounting for 0.001%-0.008% by mass.

8. The bioactive substance composition according to claim 1, wherein the putrescine or its salt in the bioactive substance composition is selected from any one or more of putrescine and putrescine dihydrochloride; the mass volume concentration of putrescine or its salt in the bioactive substance composition is 0.01-50 μg/ml, accounting for 0.000001%-0.005% by mass; preferably, the mass volume concentration of putrescine or its salt in the bioactive substance composition is 0.1-40 μg/ml, accounting for 0.00001%-0.004% by mass; more preferably, the mass volume concentration of putrescine or its salt in the bioactive substance composition is 1-30 μg/ml, accounting for 0.0001%-0.003% by mass.

9. The bioactive substance composition according to claim 1, wherein the selenite in the bioactive substance composition is a water-soluble selenite; preferably, the selenite is sodium selenite; the mass volume concentration of the selenite in the bioactive substance composition is 0.1-50 ng/ml, accounting for 0.00000001%-0.000005% by mass; preferably, the mass volume concentration of the selenite in the bioactive substance composition is 1-30 ng/ml, accounting for 0.0000001%-0.000003% by mass; more preferably, the mass volume concentration of the selenite in the bioactive substance composition is 2-20 ng/ml, accounting for 0.0000002%-0.000002% by mass.

10. A method for preparing the bioactive substance composition, wherein the preparation of the bioactive substance composition comprises the following steps: mixing fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt and selenite in proportion; the addition order of each component is not in order; the mass volume concentration range of each component is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=1-50:1-50:1-40:1-11:10-5000:10-100000:10-300000:1-25000:1-25:1-25000:1-25; preferably, fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=5-40:5-40:2-30:1-8:500-4000:1000-90000:1000-200000:10-15000:1-15:2-15000:1-15; more preferably, fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=10-30:10-30:3-20:2-5:1000-2000:10000-80000:2000-80000:100-5000:2-7:7-10000:2-7; preferably, the mass-volume concentration of the transferrin in the bioactive substance composition is 0.1-300 μg/ml, and the mass ratio is 0.00001%-0.03%; preferably, the mass-volume concentration of the transferrin in the bioactive substance composition is 1-200 μg/ml, and the mass ratio is 0.0001%-0.02%; more preferably, the mass-volume concentration of the transferrin in the bioactive substance composition is 1-150 μg/ml, and the mass ratio is 0.0001%-0.015%; preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 0.01-50 μg/ml, and the mass ratio is 0.000001%-0.005%; preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 0.1-30 μg/ml, and the mass ratio is 0.00001%-0.003%; more preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 1-20 μg/ml, and the mass ratio is 0.0001%-0.002%; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 0.1-50 ng/ml, and the mass ratio is 0.00000001%-0.000005%; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 1-30 ng/ml, and the mass ratio is 0.0000001%-0.000003%; more preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 2-20 ng/ml, and the mass ratio is 0.0000002%-0.000002%; preferably, the temperature for the mixing is 0-37° C.

11. A serum-free medium, wherein the serum-free medium comprises a basic culture medium and additional components comprising a bioactive substance composition described in claim 1; the serum-free medium is a complete serum-free medium; preferably, the culture refers to primary culture and secondary culture of cells and/or tissues; more preferably, the culture refers to maintaining the proliferation and phenotype of cells and/or tissues, or enhancing the proliferation and phenotype of cells and/or tissues; preferably, the cell common characteristics and cell-specific phenotypes of the cells cultured in the serum-free medium all reach their respective pass lines, and the total cell score reaches more than 60 points.

12. The serum-free medium according to claim 11, wherein the basic medium is selected from any one or more of DMEM low sugar medium, DMEM high sugar medium, DMEM/F12 medium, F12 medium, F10 medium, MEM medium, BEM medium, RPMI 1640 medium, Media 199 medium, IMDM medium, mTesR medium and E8 medium.

13. A composition, wherein the composition comprises at least one bioactive component and at least one additive, and the bioactive component is selected from any of the bioactive substance compositions of claim 1; preferably, the cell common characteristics and cell-specific phenotypes of the cells cultured by the composition all reach their respective pass lines, and the total cell score reaches more than 60 points; preferably, the cell common characteristics and cell-specific phenotypes of the cells cultured by the composition all reach their respective pass lines, and the total cell score reaches more than 80 points; preferably, the cell common characteristics and cell-specific phenotypes of the cells cultured by the composition all reach their respective pass lines, and the total cell score reaches more than 90 points; more preferably, the cell common characteristics and cell-specific phenotypes of the cells cultured by the composition all reach their respective pass lines, and the total cell score reaches more than 100 points.

14. The composition according to claim 13, wherein the additive is selected from any one or more of cell culture additives, growth factors, small molecule drugs, hormones, vitamins, wall promoting substances, macromolecular proteins, synthetic peptides, amino acids, lipids, enzymes, carbohydrate, pH regulating substances, trace elements and antibiotics; the cell culture additive comprises one or more of B27 cell culture additive, N2 cell culture additive, chemically defined lipid concentrate, ITS, and fatty acid additive; More preferably, calculated by the total volume of the composition, the concentration of the cell culture additive in the composition is 0.1-5×; more preferably, based on the total volume of the composition, the concentration of the cell culture additive in the composition is 0.5-2×; the growth factor comprises one or more of vascular endothelial growth factor, vascular endothelial growth factor synthetic peptide, epidermal growth factor, epidermal growth factor synthetic peptide, insulin-like growth factor, insulin-like growth factor synthetic peptide, nerve growth factor, nerve growth factor synthetic peptide, colony stimulating factor, colony stimulating factor synthetic peptide, growth hormone release inhibiting factor, growth hormone release inhibiting factor synthetic peptide; the mass volume concentration of the growth factor is 1-100 ng/ml; preferably, the mass volume concentration of the growth factor is 1-50 ng/ml; more preferably, the mass volume concentration of the growth factor is 5-40 ng/ml; preferably, the small molecule drug is selected from GSK3 inhibitor; the GSK3 inhibitor is selected from CHIR99021; preferably, the molar concentration of the small molecule drug is 0.1-10 μM; more preferably, the molar concentration of the small molecule drug is 0.1-5 μM; preferably, the amino acid is selected from any one or more of nonessential amino acids, L-glutamic acid and L-glutamine; more preferably, the molar concentration of the amino acid is 0.01-4 mM; preferably, the carbohydrate is sodium pyruvate; more preferably, the mass volume concentration of the carbohydrate is 0.01-2 mM; preferably, the pH maintaining agent is selected from any one or more of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) and L-glyceryl phosphate disodium salt water complexes; more preferably, the molar concentration of the pH maintaining agent is 1-20 mM; preferably, the adhesion promoting substance is selected from any one or more of laminin, fibronectin, vitronectin, collagen, gelatin and the synthetic peptide of the adhesion promoting substance; preferably, the mass volume concentration range of laminin is 0.1-100 μg/ml; preferably, the mass volume concentration range of fibronectin is 0.1-200 μg/ml; preferably, the mass volume concentration range of vitronectin is 0.1-100 μg/ml; preferably, the mass volume concentration range of collagen is 0.1-100 μg/ml; preferably, the mass volume concentration range of gelatin is 0.1-100 μg/ml; preferably, the mass volume concentration range of synthetic peptides of the adhesion promoting substance is 0.1-100 μg/ml; preferably, the antibiotic is selected from any one or more of penicillin, streptomycin and gentamicin; more preferably, the mass volume concentration range of the antibiotic is 50-100 μg/mL.

15. A usage of the bioactive substance composition of claim 1, wherein the use is selected from the culture of cells and/or tissues, or the use in the preparation of tissue and/or organ injury treatment drugs; preferably, the cells are selected from any one or more of tendon and/or ligament derived cells, mesenchymal stem cells, meniscal stem cells, chondrocytes, skeletal stem cells, and muscle stem cells; preferably, the tissue is the tissue derived from the musculoskeletal system; preferably, the tissue derived from the musculoskeletal system is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, fat tissue and muscle tissue; preferably, the tissue and/or organ injury is the tissue and/or organ injury of the musculoskeletal system; preferably, the tissue and/or organ injury of the musculoskeletal system is selected from at least one of tendon and/or ligament injury, cartilage injury, bone injury, muscle injury, skin injury, and blood vessel injury.

16. A cell or tissue culture method, wherein the culture method comprises the step of contacting cells and/or tissues with a serum-free medium and/or a composition; the serum-free medium is the serum-free medium as described in claim 11; preferably, the culture method is selected from suspension culture method and adherent culture method; preferably, the adherent culture method is selected from the method of coating the surface of culture carriers by adhesion promoting substance, and the method of adding the adhesion promoting substance to the culture medium; preferably, the method of coating the surface of culture carriers by adhesion promoting substance comprises the following steps: 1) Treating the culture carrier with the adhesion promoting substance, preferably, the culture carrier is selected from at least one of the pore plate, culture dish, culture bottle, microcarrier, microsphere, microarray and bioactive material; 2) Inoculate cells and/or tissues into the culture carriers treated in step 1); 3) Add to the serum-free medium and/or the composition for culture; more preferably, the method of adding the adhesion promoting substance to the culture medium comprises the following steps: 1) inoculating cells and/or tissues into a culture carrier, preferably, the culture carrier is selected from at least one of the pore plates, culture dishes, culture bottles, microcarriers, microspheres, microarrays, and bioactive materials; 2) Add the adhesion promoting substance directly to the serum-free medium and/or the composition, and then add it to the culture carrier in step 1) for cell culture; preferably, the suspension culture method comprises the following steps: 1) Inoculate cells and/or tissues into low-adhesive or non-adhesive culture well plates, culture dishes, culture flasks, other culture carriers, cell dynamic culture bioreactors; 2) Add the serum-free medium and/or composition for culture; preferably, the cells are selected from any one or more of tendon and/or ligament derived cells, mesenchymal stem cells, meniscal stem cells, chondrocytes, skeletal stem cells, and muscle stem cells; preferably, the tissue is the tissue derived from the musculoskeletal system; preferably, the tissue derived from the musculoskeletal system is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, fat tissue and muscle tissue; preferably, the adhesion promoting substance is selected from any one or more of laminin, fibronectin, vitronectin, collagen, gelatin and the synthetic peptide of the adhesion promoting substance; preferably, the synthetic peptide of the adhesion promoting substance is a synthetic polypeptide, oligopeptide or amino acid sequence that can replace the adhesion promoting substance to promote cell adhesion, including any one or more of laminin synthetic peptide, fibronectin synthetic peptide, fibronectin synthetic peptide, RGD (Arg Gly Asp) peptide, KRSR (Lys Arg Ser Arg) peptide; preferably, the concentration range of laminin is 0.1-100 μg/ml, and/or the concentration range of fibronectin is 0.1-200 μg/ml, and/or fibronectin 0.1-100 μg/ml, and/or the concentration range of collagen is 0.1-100 mg/ml, and/or the concentration of gelatin is 0.1-100 mg/ml; preferably, the concentration range of laminin synthetic peptide is 0.1-100 μg/ml, and/or the concentration range of the fibronectin synthetic peptide is 0.1-200 μg/ml, and/or hyaluronan synthetic peptide 0.1-100 μg/ml, and/or the RGD (Arg-Gly-Asp) peptide concentration range is 50-1000 mg/ml, and/or the KRSR (Lys-Arg-Ser-Arg) peptide concentration range is 50-1000 mg/ml.

17. A cell and/or tissue, wherein the cell and/or tissue are obtained by culturing in the serum-free medium and/or the composition; the serum-free medium is prepared by the method of claim 11; preferably, the cells are selected from any one or more of tendon and/or ligament derived cells, mesenchymal stem cells, meniscal stem cells, chondrocytes, bone stem cells, and muscle stem cells; preferably, the tissue is the tissue derived from the musculoskeletal system; preferably, the tissue derived from the musculoskeletal system is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, fat tissue and muscle tissue; preferably, the scores of each single item in the cell common features and cell-specific phenotypes of the cells reach their respective pass lines, and the total score of the cells reaches more than 60 points; preferably, the scores of each single item in the cell common features and cell-specific phenotypes of the cells reach their respective pass lines, and the total score of the cells reaches more than 80 points; preferably, the scores of each single item in the cell common features and cell-specific phenotypes of the cells reach their respective pass lines, and the total score of the cells reaches more than 90 points; more preferably, the scores of each single item in the cell common features and cell-specific phenotypes of the cells reach their respective pass lines, and the total score of the cells reaches more than 100 points.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0130] FIG. 1 is the GPF fluorescence image of Scx-GFP tendon stem cells cultured by the existing culture technology and the statistical results of the Scx+ positive rate, showing that the prior art leads to the loss of the Scx phenotype, which is a specific marker of tendon lineage of tendon-derived cells (Biomaterials 2018; 172: 66-82).

[0131] FIG. 2 shows the detection results of DCN content in tendon-derived cells cultured with the existing culture technology, indicating that the existing culture technology has led to the loss of the DCN phenotype, which is a specific marker of tendon-derived cells, tendon lineage (Tissue Eng 2006; 12(7): 1843-9).

[0132] FIG. 3 is a transmission electron microscope image of the collagen transverse interface between the tendon and normal tendon tissue formed by the repair of tendon-derived cells cultured by the existing culture technology. The results show that the tissue repaired by cultured cells in the prior art is composed of a large amount of small collagen, which is much smaller than the diameter of normal tendon collagen.

[0133] FIG. 4 is a CT image of a tendon repaired by tendon-derived cells cultured in the prior culture technology, indicating that the tendon regenerated by cells cultured in the prior art has ectopic calcification and repair failure (STEM CELLS 2016; 34:1083-1096).

[0134] FIG. 5 is a broken line diagram of the number of cell proliferation in the prior art using insulin-like growth factor 1 and transforming growth factor R 3 to replace FBS for tenocyte culture, where the number of cell proliferation in the growth factor group is only ⅓ of that of the serum control group (Cells Tissues Organs 2013; 197:27-36).

[0135] FIG. 6 is a bar graph showing the collagen content of tendon cell culture using insulin-like growth factor 1 and transforming growth factor R 3 in place of FBS in the prior art. The collagen formation of the best combination of growth factor group is only ½ of that of the serum control group (Cells Tissues Organs 2013; 197:27-36).

[0136] FIG. 7 is a growth morphology diagram of primary tendon stem cells (P0) cultured in a serum-free medium in Example 1 of the present invention under an inverted microscope (4 times).

[0137] FIG. 8 is a diagram showing the growth morphology of tendon stem cells P1-P6 in the serum-free culture experimental group of Example 1 and the serum control group of Comparative Example 1 under an inverted microscope (4 times).

[0138] FIG. 9 is a diagram showing the growth morphology of tendon stem cells P3 in the serum-free culture experimental group of Example 1 and the serum control group of Comparative Example 1 under an inverted microscope (20 times).

[0139] FIG. 10 is a graph showing the multiplication of the cell proliferation multiples of each generation of tendon stem cells P1-P6 in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0140] FIG. 11 is a bar graph of the doubling time of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0141] FIG. 12 is a statistical diagram of the diameter of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0142] FIG. 13 is a diagram showing the karyotype analysis of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0143] FIG. 14 is a diagram showing the results of Mycoplasma detection in the serum-free medium, serum and Mycoplasma positive control in Example 1 of the present invention.

[0144] FIG. 15 shows the results of the ALP staining experiment of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 to detect the osteogenic ability of the present invention.

[0145] FIG. 16 shows the results of Alcian Blue staining of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 to test the cartilage ability of the present invention.

[0146] FIG. 17 is the results of oil red O staining of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention to detect the fat-forming ability.

[0147] FIG. 18 is a bar graph showing the relative expression of the tendon stem cells SCX, Nestin, TNMD and tendon-related genes in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0148] FIG. 19 is a diagram showing the results of Sirius scarlet staining of the tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention after 14 days of induction.

[0149] FIG. 20 is a diagram showing the results of the tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0150] FIG. 21 is a diagram showing the relative quantitative results of the ectopic formation of tendon tissue Scx, Nestin, Col1a1, and TNMD tendon line genes in nude mice in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0151] FIG. 22 is a graph showing the results of HE staining and Masson staining of tendon ectopic formation of tendon stem cells in nude mice in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0152] FIG. 23 is an immunofluorescence graph of the expression of ectopic tendon tissue Scx and Col1 tendon-related proteins in nude mice of the tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0153] FIG. 24 is a graph showing the results of HE staining and Masson staining of rat in situ patellar tendon repair samples of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0154] FIG. 25 is an immunofluorescence graph of the Nestin tendon-associated protein expression of the rat in situ patellar tendon repair samples of the tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0155] FIG. 26 is a diagram of the growth morphology of human ligament stem cells in the serum-free culture experiment group of Example 2 and the serum control group of Comparative Example 1 under an inverted microscope (20 times) of the present invention.

[0156] FIG. 27 is a diagram of the growth morphology of tendon stem cells in the serum-free culture experimental group of Example 3 and the serum control group of Comparative Example 1 under an inverted microscope (20 times) of the present invention.

[0157] FIG. 28 is the growth morphology diagram of human adipose-derived stem cells in the serum-free culture experimental group of Example 4 and the serum control group of Comparative Example 1 under an inverted microscope (20 times).

[0158] FIG. 29 shows the growth morphology of tendon stem cells under an inverted microscope (20 times) in the serum-free culture experimental group of Example 5, the serum-free control group of the comparative example 1, the commercialized Biological Industries MSC serum-free medium (BI SFM) group of comparative example 2, the commercialized Gibco MSC serum-free culture (ST SFM) group of the comparative example 3.

[0159] FIG. 30 shows a bar graph of the relative expression of genes related to tendon stem cells in the serum-free medium (SFM) group of Example 5, the serum-free medium (SCM) group of Comparative Example 1 and the commercialized bio-industrial MSC serum-free medium (BI SFM) in Comparative Example 2.

[0160] FIG. 31 is a three-dimensional growth morphology diagram of tendon stem cells in the serum-free culture experimental group of Example 6 and the serum control group of Comparative Example 1 under an inverted fluorescence microscope (10 times) of the present invention.

[0161] FIG. 32 is a three-dimensional growth morphology diagram of tendon stem cells in the serum-free culture experimental group of Example 7 and the serum control group of Comparative Example 1 of the present invention under an inverted fluorescence microscope (10 times).

[0162] FIG. 33 is a growth morphology diagram of human mesenchymal stem cells cultured in the serum-free culture experimental group in Example 8 and in Comparative Example 4 (SFM-Ctrl4) under an inverted microscope (4 times) of the present invention.

[0163] FIG. 34 is a bar graph of the cell proliferation ratio in the serum-free culture experimental group of Example 8 and in Comparative Example 4 (SFM-Ctrl4) after culturing human mesenchymal stem cells for 3 days.

[0164] FIG. 35 is a bar graph of the doubling time of human mesenchymal stem cells cultured in the serum-free culture experimental group in Example 8 and in Comparative Example 4 (SFM-Ctrl4) of the present invention.

[0165] FIG. 36 is a three-dimensional growth morphology diagram of human chondrocytes in the serum-free culture experimental group of Example 9 under an inverted fluorescence microscope (4 times).

[0166] FIG. 37 is a diagram of the cell growth morphology of human bone stem cells in the serum-free culture experimental group of Example 10 and the serum control group of Comparative Example 1 under an inverted fluorescence microscope (20 times) of the present invention.

[0167] FIG. 38 is a growth morphology diagram of tendon stem cells in the control medium containing only B27 cell culture additives in Comparative Example 5 under an inverted microscope (20 times).

[0168] FIG. 39 is the growth morphology of tendon stem cells in the serum-free medium of the comparative example 6 under the inverted microscope (4 times).

[0169] FIG. 40 is the growth morphology of tendon stem cells in the serum-free medium of the comparative example 7 under the inverted microscope (4 times).

[0170] FIG. 41 is the growth morphology of tendon stem cells in the serum-free medium of the comparative example 8 under the inverted microscope (4 times).

[0171] Table 1 shows the cell viability in each example and comparative example.

[0172] Table 2 shows the expression of cell surface markers in each example and comparative example.

[0173] Table 3 shows the statistics of cell clone formation ability in each example and comparative example.

[0174] Table 4 shows the percentage of Nestin+ cells cultured in each Example and Comparative Example.

[0175] Table 5 shows the scores of cells cultured in each example and comparative example.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0176] The present invention will be described in detail below in conjunction with specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit this discovery in any form. It should be pointed out that for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention.

[0177] These all belong to the protection scope of the present invention.

Example 1

[0178] In this example, two groups of culture media were prepared, namely: serum-free medium and serum control culture medium, and the cultured cells were tendon stem cells (hTSPCs) extracted from human normal tendon tissue. The following are detailed experiments and tests step:

[0179] Medium Configuration

[0180] Serum-Free Medium (SFM)

[0181] The serum-free medium includes a basal medium and additional components. The basal medium is selected from DMEM/F12 medium, and every 500 mL of DMEM/F12 medium is supplemented with 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of Streptomyces. The additional components are added so that the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite:epidermal growth factor:CHIR99021=10:10:5:2:1000:25000:2000:500:2:500:2:10:251. At the same time, the medium also contains 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 1 mM sodium pyruvate, and 1×B27 cell culture additive.

[0182] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00001 FGF2 20 ng/ml PDGF-BB 20 ng/ml TGF-β 3 10 ng/ml Dexamethasone 10 nM Heparin Sodium 2 μg/ml Vitamin C 50 μg/ml Transferrin 4 μg/ml Insulin 1 μg/ml Progesterone 4 ng/ml Putrescine 1 μg/ml Sodium selenite 4 ng/ml Epidermal growth factor 20 ng/ml CHIR99021 1 μM Non-essential amino acid 1 μM L-glutamic acid 2 mM Sodium pyruvate 1 mM B27 Cell Culture Additive 1X

Comparative Example 1

[0183] Serum Control Medium (SCM)

[0184] The serum control medium is selected from DMEM low-sugar medium, and every 500 mL of DMEM low-sugar medium is supplemented with 55 mL of fetal bovine serum, 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin.

Experimental Example 1 of Biological Activity

[0185] Cultured Cell Processing

[0186] Primary Cell Isolation and Culture

[0187] Take the tendons and put them in a petri dish with 10% PS in PBS, 5% PS, 2% PS, and 1% PS in PBS for 1 min each for sterilization. Add the tendon tissue to the 0.2% collagenase solution, cut it into a paste, and make up the digestive juice to 10 ml. Immerse the tendon in the digestion medium, as evenly as possible, and place the medium in a 37° C. incubator. Blow away the tendons every hour until they are basically digested. Centrifuge the digested cell suspension at 1200 rpm for 5 min, discard the supernatant, and add it to a 10 cm culture dish coated with fibronectin, add 10 ml of SFM and place it in a 37° C., 5% CO2 cell incubator for culture. Change the solution once every 3-5 days. After the cells have adhered to 80-90%, perform cell digestion and passage for follow-up experiments, and freeze the excess cells.

[0188] Secondary Cell Culture

[0189] P1-P6 generation tendon stem cells were seeded at a density of about 9×10{circumflex over ( )}3/cm2 in well plates, dishes or flasks coated with 80 μg/ml fibronectin. Serum-free medium and serum were used, respectively. Culture the cells in the control medium and place them in a 37° C., 5% CO2 cell incubator. Change the medium every 3 days until it is almost full, and perform cell photographs, cell counts, gene expression detection, and three-line differentiation, immunofluorescence and other experiments.

[0190] Analysis of Results

[0191] Cell Morphology Observation

[0192] In the process of the above-mentioned cultured cells, the growth and morphological changes of tendon stem cells in the serum-free culture experimental group and the serum-culture control group were observed with an inverted phase contrast microscope, and photographed and recorded through a microscope.

[0193] As shown in FIG. 7, under a 4× microscope, the primary cultured tendon stem cells cultured in serum-free culture grew into a single colony. The cells grew vigorously, were uniform in size, had a bright and abundant cytoplasm, and adhered well, indicating that the serum-free medium supported the primary culture of cells.

[0194] As shown in FIG. 8, 4× microscope showed the growth of P1-P6 generation cells when they were basically overgrown. The results showed that the tendon stem cells cultured in SFM (serum-free medium) grew vigorously, and the cell proliferation was significantly better than the SCM (serum medium) group.

[0195] As shown in FIG. 9, under a 20× microscope, compared with the SCM (serum medium) group, tendon stem cells cultured in SFM (serum-free medium) have more uniform cell morphology and size, rich cytoplasm, good adherence, and a larger number of cells.

[0196] Cell Count and Cell Proliferation Multiple Analysis

[0197] P1-P6 generation tendon stem cells were inoculated into a 10 cm culture dish coated with fibronectin at a density of about 9×10{circumflex over ( )}3/cm2, and the cells were cultured in serum-free medium and serum control medium until they were almost full. At this time, discard the culture medium, wash once with 1λPBS, trypsinize, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, resuspend the pellet in 1 ml culture medium, and mix. The cell count uses trypan blue counting method. The cell suspension and 0.4% trypan blue solution were mixed 9:1 (final concentration of trypan blue 0.04%).

[0198] Cell count automatic technique:aspirate 20 μl of cell suspension and use a cell count to automatically count.

[0199] Manual counting method of hemocytometer: Pipette the cell suspension into the hemocytometer, observe and count under a microscope, the counting method is: (total number of cells in four large cells/4)×10.sup.4×dilution factor=cell number of cell suspension/mL.

[0200] If the cell membrane is intact and the cell is not stained with trypan blue, it is a normal cell; if the cell membrane is incomplete or ruptured, the trypan blue dye enters the cell and the cell turns blue, which is a necrotic cell.

[0201] Statistic cell viability: living cell rate (%)=total number of live cells/(total number of live cells+total number of dead cells)×100%.

[0202] Record the number of days of cell culture, the number of harvested cells, and the diameter of the cells at harvest, and draw related maps.

[0203] As shown in FIG. 10, the cell proliferation of the SFM (serum-free medium) group was 1.8×10{circumflex over ( )}5 times, while the SCM (serum medium) group only expanded by 40 times.

[0204] As a result, the cell proliferation rate of the SFM (serum-free medium) group was 4500 times that of the SCM group, and the proliferation rate of tendon stem cells in the SFM (serum-free medium) group was significantly higher than that of the SCM (serum medium) group.

[0205] As shown in Table 1, the viability of tendon stem cells in the SFM (serum-free medium) group was significantly higher than that of the SCM (serum medium) group.

[0206] Cell Doubling Time Analysis

[0207] P1-P6 generation tendon stem cells were inoculated into a 10 cm culture dish coated with fibronectin at a density of about 9×10{circumflex over ( )}3/cm2, and the cells were cultured in serum-free medium and serum control medium until they were almost overgrown. At this time, record the number of cell culture days, and calculate the cell doubling time according to the formula DT=t*[lg2/(lgNt−lgNo)]) (t is the culture time, No is the number of cells recorded for the first time, and Nt is the number of cells after t time), draw the relevant map.

[0208] As shown in FIG. 11, the doubling time of tendon stem cells in the experimental group SFM (serum-free medium) was less than 30 hours, and was significantly lower than the SCM (serum medium) control group. The doubling time of the SCM group was 4.2 times that of the SFM group. The shorter the doubling time, the faster the cell proliferation, that is, the proliferation rate of SFM tendon stem cells in the experimental group was significantly faster than that of SCM control group. This shows that the serum-free medium can effectively replace the role of serum, and its ability to promote cell proliferation is significantly better than that of serum.

[0209] Cell Size Comparison

[0210] Plant tendon stem cells at a density of about 9×10{circumflex over ( )}3/cm2 in a well-coated 10 cm petri dish. Cultivate the cells under serum-free and serum-free conditions until they are almost full, discard the culture medium, wash once with 1λPBS, trypsinize, centrifuge at 1200 rpm, 5 minutes, discard the supernatant, resuspend the pellet in 1 ml culture medium, mix well, and aspirate Use a cell count to count 20 μl of cell suspension, record the cell diameter at harvest, and draw a bar graph.

[0211] As shown in FIG. 12, the cell diameter of the tendon stem cells harvested from the SFM experimental group was significantly smaller than that of the SCM control group. This shows that the SFM compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0212] Karyotype Analysis

[0213] Plant tendon stem cells at a density of about 9×10{circumflex over ( )}3/cm2 in a coated 10 cm petri dish, culture them under serum-free and serum-free conditions until they are almost fully grown, then send the cells to a genetic diagnosis company for karyotyping analyze.

[0214] As shown in FIG. 13, the results show that the tendon stem cells of the SFM experimental group and SCM control group are normal (the ratio of normal karyotype cells is greater than 90%). This indicates that the cells cultured in the serum-free medium are normal cells without karyotype mutation.

[0215] Mycoplasma Detection

[0216] Take the supernatant of the SFM group cell culture for 3 days and FBS, Mycoplasma positive control, and use the one-step rapid Mycoplasma detection kit for Mycoplasma detection. The main principle is that if the cell culture is contaminated by Mycoplasma, the conservative sequence of Mycoplasma DNA will be amplified in large quantities and quickly, so that the reaction solution will change from blue-purple to sky blue. The result can be distinguished by the naked eye without electrophoresis.

[0217] As shown in FIG. 14, the results showed that the SFM group detection reagent was original blue-purple, the Mycoplasma positive control was sky blue, and the FBS group detection color was slightly sky blue. This result indicates that the serum-free medium of the invention is free of Mycoplasma contamination and the cultured cells are safe, and the batch of FBS has slight Mycoplasma contamination, which also indicates that the addition of FBS to culture cells has a great risk of Mycoplasma contamination. The serum-free medium of the invention can avoid this risk.

[0218] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0219] Plant the P3 or P5 tendon stem cells at a density of about 9×10{circumflex over ( )}3/cm2 in a coated 10 cm petri dish, culture them under serum-free and serum-free conditions until they are almost full, discard the medium, and wash once with 1λPBS, trypsin digestion, centrifugation for 5 minutes, discard the supernatant, resuspend the pellet in the blocking solution, and block for 30 minutes, then separate the stem cells to stain the surface of the stem cells. After CD146, CD105, CD90, CD44, CD34, CD18 and other flow-type direct-labeled antibodies are stained for 30 minutes, add 1λPBS to mix and centrifuge to wash twice, add 500 ul PBS to resuspend on the machine to mix, and analyze the CD mark expression.

[0220] As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of tendon stem cells, and CD34 and CD18 are negative expression markers of tendon stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%, and it was more in line with the characteristics of tendon stem cells than the SCM control group. This shows that compared with SFM and SCM, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0221] Clone Forming Ability Test

[0222] P3 or P5 generation tendon stem cells were seeded in a 6 cm culture dish at a density of 100 cells/culture dish, in triplicate, cultured in serum-free and serum-free conditions for 10-12 days, stained with 1% crystal violet, several diameters >2 mm clone count.

[0223] As shown in Table 3, the results show that the clone formation ability of the SFM experimental group is significantly better than that of the SCM control group. This indicates that the serum-free medium is more consistent with the features of stem cells than the serum-free medium.

[0224] Three-Line Differentiation Ability Test

[0225] Take the P3-P5 generation serum-free medium and serum control medium to culture the cells and pass them to the well plates for osteoinduction, cartilage induction and fat induction respectively. The specific methods are as follows: The method and identification of directed differentiation into osteoblasts: collect and culture tendon stem cells, and then inoculate the digested cells in a 24-well plate at 1×10.sup.4 cells/cm2. After most of the cells adhere to the wall, the supernatant is removed and replaced. Use high-sugar DMEM culture medium containing 10% FBS, add the osteoinduction system and change the medium every 3 days for 14 days. Qualitatively using alkaline phosphatase (ALP) kit and alizarin red staining (ARS) to quantitatively detect the ability of cells to induce ossification. Use DAPI to label cell nuclei; then use 5% SDS-hydrochloric acid solution to elute Alizarin Red, read the microplate reader to obtain the OD value. The difference in the ratio of the OD value to the number of cells represents the difference in the quantitative osteogenic ability of different cells.

[0226] The method and identification of directional induction of chondrocyte differentiation: collect and culture TSPCs cells, digest the cells, drop the cells into the center of the 12-well plate at a concentration of 2×105 TSPCs/10 ul, and place them in the incubator until the cells adhere to the wall, add cartilage induction solution, change the whole solution every 2-3 days, fix it for Aclian blue staining after 2 weeks.

[0227] Methods and identification of directed differentiation into adipocytes: collect and culture TSPCs cells, and inoculate the digested cells in a 24-well plate at 1×104 cells/cm2. After most of the cells adhere to the wall, switch to high-glycemic DMEM medium containing 10% FBS and add the fat induction system. After maintaining for 2 weeks, observe the formation of intracellular fat droplets under a microscope, stained with oil red O (Oli redo). Use isopropanol to elute the red color, and read it under the microplate reader to obtain the value of its fat-forming ability (X±SD).

[0228] As shown in FIG. 15-17, the results show that the bone differentiation ability and chondrogenic differentiation ability of the SFM experiment is significantly better than the SCM control group. The lipid-forming ability of SFM and The SCM group is comparable. This indicates that the serum-free medium has a stronger ability to differentiate into three lines of cells cultured in the serum-free medium compared with the serum-containing control.

[0229] qPCR Detection of Gene Expression

[0230] Plant tendon stem cells in a well-coated 12-well plate, and culture them in serum-free medium and serum control medium until the 5th day, discard the medium, wash once with 1λPBS, and transfer the cells to the 12-well plate. Add 500 ul RNA cell lysate to the well, use RNA extraction kit to extract the cellular RNA, reverse transcribed into cDNA, and then load the sample on the machine for Qpcr to detect the relative expression of tendon-related genes in the cell. Analyze the results with the group as the abscissa and the relative gene expression as the ordinate, and draw a histogram of the relative expression of the gene.

[0231] As shown in FIG. 18, comparing SFM and SCM control group, SCX, nestin, and TNMD tendon-related genes are significantly higher in the serum-free experimental group. Therefore, it indicates that the SFM is more conducive to the maintenance of tendon lineage phenotype of tendon stem cells than SCM.

[0232] In Vitro Tendon Differentiation Ability Test

[0233] Take the P3-P5 generation serum-free medium and serum control medium to culture the cells in a 12-well plate at 4×10{circumflex over ( )}4 cells/well, with three multiple wells in each group, respectively in serum-free medium and serum control medium. When cultured in serum-free medium and serum control medium until they are almost overgrown, replace the tendon induction medium, change the medium once every 2-3 days. One to two weeks after the induction, the cells were stained with Sirius scarlet, and the cell sheets were rolled into a transmission electron microscope to evaluate the formation of collagen.

[0234] As shown in FIG. 19 by Sirius Scarlet staining, compared with the SFM control group, the amount of collagen formation in the SFM experimental group was significantly increased. It shows that under the condition of tendon line induction, the tendon stem cells cultured in the serum-free medium have stronger tendon line differentiation ability than the tendon stem cells in the serum medium.

[0235] As shown in FIG. 20, the results of transmission electron microscopy showed that compared with the control group of SFM (serum-free medium), the amount of collagen formed in the experimental group of SFM (serum-free medium) was significantly increased, and the diameter of the collagen formed was significantly larger than that in the control group. It shows that under the condition of tendon line induction, the tendon stem cells cultured in the serum-free medium have stronger tendon line differentiation ability than the tendon stem cells in the serum medium.

[0236] Flow Analysis to Detect Nestin Expression

[0237] Plant the P3 or P5 tendon stem cells at a density of about 9×10{circumflex over ( )}3/cm2 in a coated 10 cm petri dish, culture them under serum-free and serum-free conditions until they are almost full, discard the medium, wash once with 1λPBS, digest with trypsin, centrifuge for 5 minutes, discard the supernatant, resuspend the pellet in the blocking solution, and block for 30 minutes, break the membrane, separate the tube for Nestin staining of stem cells, after 30 minutes, add 1λPBS to mix and centrifuge to wash twice, add 500 ul PBS to resuspend and mix on the machine. Finally, analyze the expression of Nestin.

[0238] As shown in Table 4, the results show that the Nestin positive marker expression in the SFM experimental group is greater than 30%, while the Nestin positive marker expression in the SCM control group of the comparative example 1 is less than 5%. This shows that the phenotype of the cell tendon line cultured in the SFM experimental group is significantly higher than that of the SCM control group.

[0239] In Vivo Tendon Formation Ability Test

[0240] Take the serum-free medium and the serum control medium to culture the P5 generation cells at a density of about 9×10{circumflex over ( )}3/cm2 and inoculate them in a coated 10 cm petri dish, and cultivate them in the serum-free medium and the serum control medium respectively. When it is basically overgrown, it is cultured with tendon induction medium, and the medium is changed once every 2-3 days. Two weeks after induction, they were rolled into a cell sheet and implanted under the skin of the back of nude mice. The samples were collected two weeks later. The tendon formation of the implanted cells was evaluated by HE staining, masson staining, immunofluorescence staining, qPCR and other experiments.

[0241] As shown in FIG. 21, the QPCR results showed that compared with SCM control group, the expression of tendon-related genes such as SCX, Nestin, TNMD, COL1A1, etc., was significantly increased in the serum-free experimental group. It shows that the tendon stem cells cultured in the serum-free medium have stronger ability to differentiate tendon lineage, and the tendon tissue formed in the body is more mature.

[0242] As shown in FIG. 22, the histological results showed that compared with the SFM control group, the collagen in the tendon tissue formed in the SFM experimental group is arranged more neatly and densely, indicating that the tendon stem cells cultured in the serum-free medium have a stronger ability to form tendons in vivo.

[0243] As shown in FIG. 23, the immunofluorescence results showed that compared with the SFM (serum-free medium) control group, the expression of tendon-related proteins such as SCX and COL1A1 were significantly increased in the serum-free experimental group. The serum-free medium is more conducive to the differentiation of tendon stem cells into tendon lineage to form tendon tissue than serum medium.

[0244] Evaluation of In Situ Tendon Repair Ability In Vivo

[0245] Take the serum-free medium and the serum control medium to culture the P5 generation cells at a density of about 9×10{circumflex over ( )}3/cm2 and inoculate them in a coated 10 cm petri dish, change the medium once every 2-3 days, respectively in the serum-free medium and the serum control medium. After being cultured in serum-free medium and serum control medium until they are almost fully grown, the cells are digested into single cells, mixed with fibrin gel to form a gel, and then implanted into the local defect of the rat patellar tendon. After four or eight weeks, samples will be collected, and the formation of tendon of implanted cells will be evaluated by HE staining, masson staining, immunofluorescence staining and other experiments.

[0246] As shown in FIG. 24, the histological results show that compared with the SCM group, tendon tissue collagen in the SFM experimental group is more orderly and denser, indicating that the tendon stem cells cultured in the serum-free medium have a stronger ability to repair tendon in situ in vivo.

[0247] As shown in FIG. 25, the immunofluorescence results showed that compared with the SCM group, the expression of the tendon-related protein Nestin was significantly increased in the serum-free experimental group. Therefore, it indicates that the serum-free medium is more conducive to the differentiation of tendon stem cells into tendon lineage to form tendon tissue than serum medium.

Example 1 of Cell Score Calculation

[0248] According to the results of “Analysis of Cell Doubling Time” in Example 1, the doubling time of the cells cultured in the serum-free medium in Example 1 is less than 30 hours, so the cell proliferation rate is scored as 30 points. From the results of Example 1 “Detection of stem cell surface marker expression by flow cytometry”, it can be seen that the positive marker expression of cells cultured in serum-free medium is greater than 95%, and the negative marker expression is less than 1%, which is higher than that of SCM control. The cells in SFM group are more in line with the characteristics of tendon stem cells, indicating that the expression of surface markers on the cell stem cells cultured in the serum-free medium in Example 1 is increased. From the analysis result of “Clonogenic Ability Determination” in Example 1, it can be seen that the clonal forming ability of the SFM experimental group (25 cells/well) is significantly better than that of the SCM control group (12 cells/well). According to the analysis results of the “Triline Differentiation Ability Test” in Example 1, it can be seen that the bone differentiation ability and chondrogenic differentiation ability of the SFM experiment is significantly better than the SCM control group, and lipid-forming ability of SFM is equivalent to that of SCM group. This indicates that the serum-free medium has a stronger ability to differentiate into three lines of cells cultured in the serum-free medium compared with the serum-containing control. Based on the above results, it can be seen that using the conventional serum medium cultured cells in the prior art as a control, the surface marker expression, cloning ability and triline differentiation ability of the stem cells obtained by the serum-free medium culture of Example 1 are all improved. Therefore, the score for stem cell phenotype is 20 points.

[0249] It can be seen from the “karyotype analysis” result of Example 1 that the karyotype of the cells cultured in the serum-free medium of Example 1 is normal. It can be seen from the results of “Mycoplasma Detection” in Example 1 that the serum-free medium of Example 1 is free of Mycoplasma contamination, and the cultured cells are safe, and this batch of FBS has slight Mycoplasma contamination. In combination with the serum-free medium of the present invention, it is a completely serum-free medium, which can realize cell primary culture and secondary culture. Moreover, no serum participates in the whole process, so there is no serum residue. In summary, the karyotype of the cells obtained by the serum-free medium culture in Example 1 is normal, there is no serum residue, and no Mycoplasma contamination, so the safety score is 10 points.

[0250] From the results of “qPCR detection of gene expression” in Example 1, compared with the SCM control group, the SCX, Nestin, and TNMD tendon-related genes of cells cultured in serum-free medium were all significantly highly expressed in the serum-free experimental group. It can be seen from the results of Example 1 “Detection of Nestin Expression by Flow Cytometry” that the Nestin positive rate of cells cultured in the serum-free medium of Example 1 can reach 94%. And the “in vitro tendon differentiation ability test” results also show that the collagen-forming ability of the cells cultured in the serum-free medium of Example 1 is significantly higher than that of the serum control group. To sum up, the phenotype and differentiation ability of tendon line obtained by the serum-free medium of Example 1 were significantly improved compared with the serum control group. In addition, the three tendon line related genes of SCX, Nestin and TNMD were highly expressed, and the positive rate of Nestin was greater than 90%. Therefore, the score for tendon phenotype and tendon differentiation ability is 20 points.

[0251] From the results of Example 1 “In vivo tendon formation ability detection” and “In vivo in situ tendon repair ability evaluation”, the histological results show that compared with the serum control group (SCM group), the collagen of tendon tissue formed by repairing cells cultured in serum-free medium is arranged more neatly and densely, without bone, cartilage, muscle and other non-tendon tissues, which is closer to normal tissue morphology. Therefore, the ability to repair tendons and/or ligaments in the body is scored 20 points.

[0252] In summary, the total score of the cells obtained by the serum-free culture in Example 1 is 100 points, and at the same time, it meets the cell quantity and quality requirements for clinical cell therapy of tendon and or ligament injuries.

Example 2

[0253] In this embodiment, two groups of culture media are prepared, namely: serum-free medium and serum control culture medium, and the cultured cells are ligament stem cells obtained by separation and culture of human ligament tissue. The following are detailed experiments and detection steps:

[0254] Medium Configuration

[0255] Serum-Free Medium (SFM)

[0256] The serum-free medium includes a basal medium and additional components; the basal medium is selected from F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin are added to every 500 mL of F12 medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivatives:transferrin:insulin:progesterone:putrescine or its salt:selenite=15:10:6:2:1500:25000:75000:3000:4:7:4.

[0257] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00002 FGF-basic 30 ng/ml PDGF-AA 10 ng/ml PDGF-BB 10 ng/ml TGF-β 1 3 ng/ml TGF-β 3 3 ng/ml Dexamethasone 10 nM Heparin sodium 3 μg/ml Vitamin C 50 μg/ml Transferrin 150 μg/ml Insulin 6 μg/ml Progesterone 8 ng/ml Putrescine 14 μg/ml Sodium selenite 8 ng/ml

Example 2 Experimental Example of Biological Activity

[0258] Cultured Cell Processing

[0259] P3-P6 generation human ligament stem cells were seeded into wells coated with 100 μg/ml laminin, 200 μg/ml fibronectin and 100 ug/ml vitronectin at a density of about 9×10{circumflex over ( )}3/cm.sup.2 in a plate or a petri dish. Culture the cells in a serum-free medium and a serum control medium respectively, and place them in a 37° C., 5% CO2 cell incubator. Change the medium every 3 days until it is almost full, and take a photo of the cells to observe the cell growth condition.

[0260] Analysis of Results

[0261] Cell Morphology Observation

[0262] The method is the same as in Example 1. As shown in FIG. 26, the 20× microscope showed the cell morphology when the cells were basically overgrown. The results showed that compared with the SCM group, the human ligament stem cells cultured in SFM, human ligament stem cells cultured in SFM (serum-free medium) have more uniform morphology and size, rich cytoplasm, good adhesion, and a larger number.

[0263] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0264] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of human ligament stem cells, and CD34 and CD18 are negative expression of human ligament stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%. Moreover, the cells in the SFM experimental group were more consistent than that of the SCM (serum-free medium) control group. This indicates that compared with SFM and SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0265] Clone Forming Ability Test

[0266] The method is the same as in Example 1. As shown in Table 3, the results showed that the clone formation ability of the SFM (serum-free medium) experimental group were significantly better than that of the SCM (serum medium) control group. This indicates that the serum-free medium is more in line with the features of stem cells than the serum-free medium.

Example 3

[0267] In this example, two groups of culture media were prepared, namely: serum-free medium and serum control culture medium, and the cultured cells were tendon stem cells (hTSPCs) extracted from human normal tendon tissue. The following are detailed experiments and tests step:

[0268] Medium Configuration

[0269] Serum-Free Medium (SFM)

[0270] The serum-free medium includes a basal medium and additional components; the basal medium is selected from F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin are added to every 500 mL of F12 medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivatives:transferrin:insulin:progesterone:putrescine or its salt:selenite=50:50:40:11:1000:100000:300,000:25000:25:25000:25. At the same time, the medium also contains 0.1 mM non-essential amino acids, 0.1 mM L-glutamic acid, 0.1 mM sodium pyruvate, and 0.1× B27 cell culture additive.

[0271] Further, the concentration of each component of the serum-free medium is as follows:

TABLE-US-00003 FGF2 20 ng/ml FGF4 20 ng/ml FGF18 10 ng/ml PDGF-AA 25 ng/ml PDGF-AB 25 ng/ml TGF-β 1 20 ng/ml TGF-β 2 20 ng/ml Dexamethasone 15 nM Hydrocortisone 14 nM Heparin 1 μg/ml Vitamin C 50 μg/ml Vitamin C sodium phosphate 50 μg/ml Transferrin 300 μg/ml Insulin 25 μg/ml Progesterone 25 ng/ml Putrescine 15 μg/ml Putrescine dihydrochloride 10 μg/ml Sodium selenite 25 ng/ml Non-essential amino acid 0.1 mM L-glutamic acid 0.1 mM Sodium pyruvate 0.1 mM B27 Cell Culture Additive 0.1X

Example 3 Experimental Example of Biological Activity

[0272] Cultured Cell Processing

[0273] P3-P6 generation tendon stem cells were inoculated into 40 mg/ml gelatin-coated well plates, petri dishes or flasks at a density of about 9×10{circumflex over ( )}3/cm2. Culture the cells in a serum-free medium and a serum control medium respectively, and place them in a 37° C., 5% CO2 cell incubator, change the medium every 2-3 days, cultivate until the cells are basically full, and take pictures to observe cell growth.

[0274] Analysis of Results

[0275] Cell Morphology Observation

[0276] The method is the same as in Example 1. As shown in FIG. 27, under a 20× microscope, the cell morphology when the cells were basically overgrowth was shown. The results showed that compared with SCM group, the tendon stem cells cultured in SFM grew vigorously, and the cell proliferation was significantly better than that of the SCM group. The cell morphology and size were more uniform, the cytoplasm was transparent and abundant, and the adherence was good

[0277] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0278] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of tendon stem cells, and CD34 and CD18 are negative expression markers of tendon stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%. Moreover, the cells in SFM experimental group were more in line with the characteristics of tendon stem cells than the cells in SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0279] Clone Forming Ability Test

[0280] The method is the same as in Example 1. As shown in Table 3, the results show that the clone formation ability of the SFM experimental group is significantly better than that of the SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

Example 4

[0281] In this example, two groups of culture media were prepared, namely: a serum-free medium and a serum control medium, and the cultured cells were adipose-derived stem cells (ADSCs) extracted from human fat. The following are detailed experiments and detection steps:

[0282] Medium Configuration

[0283] Serum-Free Medium (SFM)

[0284] The serum-free medium includes a basal medium and additional components; the basal medium is selected from F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin are added to every 500 mL of F12 medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferiron Protein:insulin:progesterone:putrescine or its salt:selenite=1:1:1:1:10:10:10:1:1:1:1. At the same time, the medium also contains 1 mM non-essential amino acids, 4 mM L-glutamic acid, 2 mM sodium pyruvate, 2× B27 cell culture additive, 1 μg/ml vitronectin, 1 μg/ml fibronectin, 1 μg/ml laminin.

TABLE-US-00004 bFGF 10 ng/ml PDGF-AA 10 ng/ml TGF-β 2 10 ng/ml Dexamethasone 25 nM Heparin sodium 0.1 μg/ml Vitamin C 0.1 μg/ml Transferrin 0.1 μg/ml Insulin 0.01 μg/ml Progesterone 10 ng/ml Putrescine 0.01 μg/ml Sodium selenite 10 ng/ml Non-essential amino acid 1 mM L-glutamic acid 4 mM Sodium pyruvate 2 mM B27 Cell Culture Additive 2X Vitronectin 1 μg/ml Fibronectin 1 μg/ml Laminin 1 μg/ml

Experimental Example 4 of Biological Activity

[0285] Cultured Cell Processing

[0286] P3-P6 generation adipose-derived stem cells were seeded into a well plate, petri dish or flask at a density of about 5×10{circumflex over ( )}3/cm2, and the cells were cultured with serum-free medium and serum control medium, respectively. The cells were cultured in a 37° C., 5% CO2 cell incubator, and the medium was changed every 3 days until the cells were basically overgrown, and the cells were photographed to observe the growth of the cells.

[0287] Analysis of Results

[0288] Cell Morphology Observation

[0289] The method is the same as in Example 1. As shown in FIG. 28, a 20× microscope shows the cell morphology when the cells are basically overgrown. The adipose-derived stem cells cultured in SFM grew vigorously, and the cell proliferation was significantly better than that of the SCM group. In addition, the adipose-derived stem cells cultured in SFM have a more uniform cell morphology and size, a translucent and rich cytoplasm, and a good adherence to the wall.

[0290] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0291] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of tendon stem cells, and CD34 and CD18 are negative expression markers of tendon stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%. Moreover, the cells in SFM experimental group were more in line with the characteristics of tendon stem cells than the cells in SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0292] Clone Forming Ability Test

[0293] The method is the same as in Example 1. As shown in Table 3, the results show that the clone formation ability of the SFM experimental group is significantly better than that of the SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

Example 5

[0294] In this example, four groups of culture media were prepared, namely: serum-free medium, serum control medium, commercial Biological Industries MSC serum-free medium, commercial Gibco MSC serum-free culture, and the cultured cells were human Tendon stem cells (hTSPCs) extracted from normal tendon tissues, the following are the detailed experiments and detection procedures:

[0295] Medium Configuration

[0296] Serum-Free Medium (SFM)

[0297] The serum-free medium includes a basal medium and additional components; the basal medium is selected from F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin are added to every 500 mL of F12 medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite salt:epidermal growth factor:CHIR99021=30:30:5:2:2000:80000:50000:100:5:100:5:10:1004. At the same time, the medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, and 1× B27 cell culture additive.

[0298] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00005 FGF2 30 ng/ml PDGF-BB 30 ng/ml TGF-β 3 5 ng/ml Dexamethasone 5 nM Heparin sodium 2 μg/ml Vitamin C 80 μg/ml Transferrin 50 μg/ml Insulin 0.1 μg/ml Progesterone 5 ng/ml Putrescine 0.1 μg/ml Sodium selenite 5 ng/ml Epidermal growth factor 10 ng/ml CHIR99021 2 μM Non-essential amino acid 0.1 mM L-glutamic acid 1 mM Sodium pyruvate 0.5 mM B27 Cell Culture Additive 1X

Comparative Example 2

[0299] Commercialized Biological Industries MSC Serum-Free Medium (BI SFM):

[0300] MSC NutriStem®XF basal medium (medium):MSC nutrient stem Cell®XF supplement (additive):penicillin and streptomycin=500 ml:3 ml:5 ml.

Comparative Example 3

[0301] Commercialized Gibco MSC serum-free culture (ST SFM, or StemPro SFM):StemPro® MSC SFM Supplement CTS™:StemPro® MSC SFM Basal Medium CTS™:L-glutamine or GlutaMAX™-I CTS™=15 ml:84 ml:1 ml, of which the final concentration of L-glutamine or GlutaMAX™-I CTS™ is 2 mM.

Experimental Example 5 of Biological Activity

[0302] Cultured Cell Processing

[0303] Tendon stem cells of P3-P6 generation were inoculated into 5 μg/ml laminin-coated well plates, petri dishes or flasks at a density of about 9×10{circumflex over ( )}3/cm2, and cultured with serum-free medium, serum control medium, commercial Biological Industries MSC serum-free medium, commercial Gibco MSC serum-free medium. The cells were cultured in a 37° C., 5% CO2 cell incubator, and the medium was changed every 2-3 days. Cell photographs, qPCR and other experiments were performed to observe cell growth and tendon gene expression.

[0304] Analysis of Results

[0305] Cell Morphology Observation

[0306] The method is the same as in Example 1. As shown in FIG. 29, under a 20× microscope, the cell morphology when the cells were basically overgrowth was shown. The results showed that the tendon stem cells cultured in SFM (serum-free medium group) grew vigorously, while the proliferation of the cells in the BI SFM and SCM groups was slower. The cells cultured in ST SFM group has poor cell growth, basically does not proliferate, and produces a lot of cell secretion impurities. Therefore, the cell proliferation of the SFM group was significantly better than that of the SCM (serum medium group) group, the BI SFM group and the ST SFM group, indicating that the serum-free medium is more suitable for tendon stem cells than the two commercial serum-free medium and serum control medium. The commercialized Gibco MSC serum-free culture (ST SFM) is completely unsuitable for the proliferation of tendon stem cells in vitro.

[0307] QPCR Detection of Gene Expression in Tendon Lines

[0308] The method is the same as in Example 1. As shown in FIG. 30, serum-free medium (SFM), serum-free medium (SCM), and commercial Biological Industries MSC serum-free medium (BI SFM) are compared. SCX, Nestin, THBS4, TNMD these tendon-related genes were significantly high expressed in the SFM (serum-free medium) group, but the expression in the BI SFM commercial medium was lower, and there was no difference from the serum control group. Therefore, it shows that the serum-free medium is more conducive to the maintenance of tendon lineage phenotype of tendon stem cells than serum control medium and commercial BI MSC serum-free medium.

[0309] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0310] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of tendon stem cells, and CD34 and CD18 are negative expression markers of tendon stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%. Moreover, the cells in SFM experimental group were more in line with the characteristics of tendon stem cells than the cells in SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0311] Clone Forming Ability Test

[0312] The method is the same as in Example 1. As shown in Table 3, the results showed that the clone formation ability of the SFM (serum-free medium) experimental group was significantly better than that of the SCM (serum medium) control group. Thus, it shows that compared with serum-free medium, the cells cultured in serum-free medium are more in line with the features of stem cells.

Example 6

[0313] In this example, two groups of culture media were prepared, namely: serum-free medium and serum control culture medium, and the cultured cells were tendon stem cells (Scx-GFP mTSPCs) extracted from the normal tendon tissue of Scx-GFP mice.

[0314] The following is the detailed experiment and detection steps:

[0315] Medium Configuration

[0316] Serum-Free Medium (SFM)

[0317] The serum-free medium includes the basal medium and additional components; the basal medium is selected from F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin are added to every 500 mL of F12 medium.

[0318] Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=20:20:16:7:2000:4000:2000:10000:10:10000:10. At the same time, the medium also contains 0.01 mM non-essential amino acids, 0.01 mM L-glutamic acid, 0.01 mM sodium pyruvate, and 0.2× B27 cell culture additive. Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00006 FGF-basic 100 ng/ml PDGF-AA 100 ng/ml TGF-β 1 80 ng/ml Dexamethasone 90 nM Heparin sodium 10 μg/ml Vitamin C 20 μg/ml Transferrin 10 μg/ml Insulin 50 μg/ml Progesterone 50 ng/ml Putrescine 50 μg/ml Sodium selenite 50 ng/ml Non-essential amino acid 0.01 mM L-glutamic acid 0.01 mM Sodium pyruvate 0.01 mM B27 Cell Culture Additive 0.2X

Experimental Example 6 of Biological Activity

[0319] Cultured Cell Processing

[0320] Tendon stem cells of P3-P6 generation were seeded in a low-adhesion 6-well plate at a density of about 5×10{circumflex over ( )}4/cm2, and the cells were cultured in serum-free medium and serum control medium, 2 ml per well, 3 replicate wells per group. The cells were cultured in a cell incubator at 37° C. and 5% CO2, the medium was changed every 2-3 days, and experiments such as cell photographing were performed to observe cell growth and tendon gene expression.

[0321] Analysis of Results

[0322] Cell Morphology Observation

[0323] The method is the same as in Example 1. As shown in FIG. 31, under a 10× microscope, the cell morphology when the cells are basically overgrowth is shown. The results showed that the three-dimensional cell spheres formed by Scx-GFPmTSPCs cultured in SFM (serum-free medium group) were larger than those of SCM control group, indicating that the cell proliferation of SFM group was significantly better than that of SCM group. At the same time, SFM (serum-free medium group) cultured Scx-GFP mTSPCs formed three-dimensional cell spheres with stronger GFP fluorescence intensity, indicating that the serum-free medium is more suitable for the maintenance of the SCX phenotype of tendon stem cells than the serum control medium. The results show that our medium also supports the three-dimensional culture of cells.

[0324] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0325] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of tendon stem cells, and CD34 and CD18 are negative expression markers of tendon stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%. Moreover, the cells in SFM experimental group were more in line with the characteristics of tendon stem cells than the cells in SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0326] Clone Forming Ability Test

[0327] The method is the same as in Example 1. As shown in Table 3, the results showed that the clone formation ability of the SFM (serum-free medium) experimental group was significantly better than that of the SCM (serum medium) control group. Thus, it shows that compared with serum-free medium, the cells cultured in serum-free medium are more in line with the features of stem cells.

Example 7

[0328] In this example, two groups of culture media were prepared, namely: serum-free medium and serum control culture medium, and the cultured cells were tendon stem cells (Scx-GFP mTSPCs) extracted from the normal tendon tissue of Scx-GFP mice.

[0329] The following is the detailed experiment and detection steps:

[0330] Medium Configuration

[0331] Serum-Free Medium (SFM)

[0332] The serum-free medium includes the basal medium and additional components; the basal medium is selected from F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin are added to every 500 mL of F12 medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite:epidermal growth factor:CHIR99021=50:50:5:2:5000:100000:5000:5000:5:5000:5:1500:2510. At the same time, the medium also contains 0.01 mM non-essential amino acids, 0.01 mM L-glutamic acid, 0.01 mM sodium pyruvate, and 2× B27 cell culture additive.

[0333] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00007 FGF2 1 ng/ml PDGF-BB 1 ng/ml TGF-β 2 0.1 ng/ml Dexamethasone 0.1 nM Heparin calcium 0.1 μg/ml Vitamin C 2 μg/ml Transferrin 0.1 μg/ml Insulin 0.1 μg/ml Progesterone 0.1 ng/ml Putrescine dihydrochloride 0.1 μg/ml Sodium selenite 0.1 ng/ml Epidermal growth factor 30 ng/ml CHIR99021 0.1 μM Non-essential amino acid 0.01 mM L-glutamic acid 0.01 mM Sodium pyruvate 0.01 mM B27 Cell Culture Additive 2X

Experimental Example 7 of Biological Activity

[0334] Cultured Cell Processing

[0335] Tendon stem cells of P3-P6 generation were seeded in a low-adhesion 6-well plate at a density of about 1×10{circumflex over ( )}5/cm2, and the cells were cultured in serum-free medium and serum control medium, 2 ml per well, 3 replicate wells per group. The cells were cultured in a cell incubator at 37° C. and 5% CO2, the medium was changed every 2-3 days, and experiments such as cell photographing were performed to observe cell growth and tendon gene expression.

[0336] Analysis of Results

[0337] Cell Morphology Observation

[0338] The method is the same as in Example 1. As shown in FIG. 32, under a 10× microscope, the cell morphology when the cells are basically overgrowth is shown. The results showed that the three-dimensional cell spheres formed by Scx-GFPmTSPCs cultured in SFM (serum-free medium group) were larger than those of SCM control group, indicating that the cell proliferation of SFM group was significantly better than that of SCM group. At the same time, SFM (serum-free medium group) cultured Scx-GFP mTSPCs formed three-dimensional cell spheres with stronger GFP fluorescence intensity, indicating that the serum-free medium is more suitable for the maintenance of the SCX phenotype of tendon stem cells than the serum control medium. The results show that our medium also supports the three-dimensional culture of cells.

[0339] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0340] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of tendon stem cells, and CD34 and CD18 are negative expression markers of tendon stem cells. The results showed that the positive marker expression of the SFM experimental group was greater than 95%, and the negative marker expression was less than 1%. Moreover, the cells in SFM experimental group were more in line with the characteristics of tendon stem cells than the cells in SCM control group. Therefore, compared with the SCM control, the cells cultured in the serum-free medium are more in line with the features of stem cells.

[0341] Clone Forming Ability Test

[0342] The method is the same as in Example 1. As shown in Table 3, the results showed that the clone formation ability of the SFM (serum-free medium) experimental group was significantly better than that of the SCM (serum medium) control group. Thus, it shows that compared with serum-free medium, the cells cultured in serum-free medium are more in line with the features of stem cells.

Example 8

[0343] In this example, two groups of culture media were prepared, namely: serum-free medium and serum-free medium for stem cells described in Chinese Patent (CN111206017A). The cultured cells are human mesenchymal stem cells. The following are detailed experiments and detection procedures:

[0344] Medium Configuration

[0345] The serum-free medium includes a basal medium and additional components; the basal medium is selected from DMEM low-sugar medium, and 1 mmol of HEPES is added to every 500 mL of DMEM low-sugar medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite:epidermal growth factor:CHIR99021=40:15:5:2:3000:20000:50000:100:5:100:5:20:1004. At the same time, the medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, 1× B27 cell culture additive, 3 μg/ml vitronectin synthetic peptide, 3 μg/ml fibronectin synthetic peptide.

[0346] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00008 FGF-basic 40 ng/ml PDGF-BB 15 ng/ml TGF-β 3 5 ng/ml Dexamethasone 5 nM Heparin sodium 3 μg/ml Vitamin C 20 μg/ml Transferrin 50 μg/ml Insulin 0.1 μg/ml Progesterone 5 ng/ml Putrescine 0.1 μg/ml Sodium selenite 5 ng/ml Epidermal growth factor 20 ng/ml CHIR99021 2 μM Non-essential amino acid 0.1 mM L-glutamic acid 1 mM Sodium pyruvate 0.5 mM B27 Cell Culture Additive 1X Vitronectin synthetic peptide 3 μg/ml Fibronectin synthetic peptide 3 μg/ml Penicillin (10000 U) 5 mmoL Streptomycin (10000 U) 5 mmoL

Comparative Example 4

[0347] Serum-free medium for stem cells described in Chinese Patent (CN111206017A): Add the following components to every 500 mL of DMEM low-sugar basal medium (GIBCO), and make the concentration of the added components in the serum-free medium:

TABLE-US-00009 Recombinant human serum albumin 20 mg/mL Recombinant human PDGF-AA 20 ng/mL Recombinant human PDGF-BB 20 ng/mL Recombinant human bFGF 5 ng/mL Recombinant human TGF-β 1 5 ng/mL Recombinant human EGF 20 ng/mL Recombinant IGF 20 ng/mL Recombinant human fibronectin 5 μg/mL Heparin 5 μg/mL Lipid concentrate 0.1% (v/v) Recombinant human insulin 2 μg/mL Transferrin 1 μg/mL Sodium selenite 1 ng/mL Galactose 20 mM L-glutamine 292 mg/L Putrescine 50 μM Progesterone 20 nM Hydrocortisone 100 nM Vitamin C 200 μM Vitamin A 50 μM Sodium bicarbonate 20.5 mM Penicillin (10000 U) 5 mmoL Streptomycin (10000 U) 5 mmoL

Experimental Example 8 of Biological Activity

[0348] Cultured Cell Processing

[0349] Human mesenchymal stem cells of P3-P6 generation were inoculated into orifice plates or petri dishes at a density of about 5×10{circumflex over ( )}3/cm2, cultured in serum-free medium of Example 8 and Comparative Example 4, respectively. The cells were cultured in a cell incubator at 37° C. and 5% CO2. The medium was changed every 2-3 days, and experiments such as cell photographing were performed to observe cell growth and tendon gene expression.

[0350] Analysis of Results

[0351] Cell Morphology Observation

[0352] The method is the same as in Example 1. As shown in FIG. 33, the cell morphology of cells grown for 3 days under the 4× microscope. The results showed that the number of human mesenchymal stem cells cultured in SFM (serum-free medium group) in the same field of view under the same conditions was significantly more than that of comparative example 4 serum-free control group. In addition, the cells in the SFM group had a more uniform morphology, with smaller cells, more transparent and abundant cytoplasm, and better adhesion, indicating that the cell proliferation of the SFM group was significantly better than that of the comparative 4 serum-free control group.

[0353] Cell Count and Cell Proliferation Multiple Analysis

[0354] The method is the same as in Example 1. As shown in FIG. 34, the number of cells obtained in the serum-free control group of Comparative Example 4 is 1. The proliferation rate of human mesenchymal stem cells in the medium) group was significantly higher than that of the control group without serum.

[0355] Cell Doubling Time Analysis

[0356] The method is the same as in Example 1. As shown in FIG. 35, the doubling time of human mesenchymal stem cells in the SFM (serum-free medium) of Example 8 was significantly lower than that of SFM-Ctrl4 (comparative example 4 serum-free control group). The doubling time of the SFM-Ctrl4 group was 1.52 times that of the SFM group. The shorter the doubling time, the faster the cell proliferation, that is, the proliferation rate of human mesenchymal stem cells in SFM group was significantly faster than that of SFM-Ctrl4. This indicates that the serum-free medium composed of biologically active substances of the present invention has a significantly better ability to promote cell proliferation than the stem cell serum-free medium described in the Chinese patent (202010104684. 5).

[0357] Flow Cytometry to Detect the Expression of Stem Cell Surface Markers

[0358] The method is the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of human mesenchymal stem cells, and CD34 and CD18 are negative expression markers of human mesenchymal stem cells. The results showed that the expression of positive markers of SFM in the two groups was greater than 95%. The negative marker expression of human mesenchymal stem cells cultured in the SFM (serum-free medium) of Example 8 was less than 1%, and the negative marker expression of the cells cultured with SFM-Ctrl4 (comparative example 4 serum-free control group) was greater than 1%. It shows that the characteristics of the serum-free medium for culturing cell stem cells composed of biologically active substances of the present invention are slightly better than those of the serum-free medium for stem cells described in the Chinese patent (202010104684. 5).

[0359] Clone Forming Ability Test

[0360] The method is the same as in Example 1. As shown in Table 3, the results showed that the clone formation ability of the SFM (serum-free medium) experimental group of Example 8 was significantly better than that of the SFM-Ctrl4 (comparative example 4 serum-free control group). This shows that the characteristics of the serum-free medium composed of biologically active substances of the present invention for culturing cell stem cells are better than those described in the Chinese patent (202010104684. 5).

[0361] In summary, the Chinese patent (CN111206017A) discloses a stem cell serum-free medium and its application. From the experimental data provided by the invention patent and the comparative experiment conducted by the invention patent, it is found that the evaluation from the multi-angles of cell morphology, cell proliferation rate and cell doubling time shows that the proliferation rate of stem cells cultivated in this patent is significantly slower than that of the serum-free medium prepared by the bioactive substance composition of the patent of the present invention (FIGS. 32-34). Moreover, the patent does not provide sufficient evidence to prove that its published medium can be used for primary culture. It does not prove the safety of its cultured cells, and there is no in vivo animal experiment to prove that its cultured cells can be used for tissue engineering and injury repair. It cannot prove that its cultured cells can be used for clinical cell therapy.

Example 9

[0362] In this example, a serum-free medium was prepared, and the cultured cells were human chondrocytes. The following are detailed experiments and detection steps:

[0363] Medium Configuration

[0364] The serum-free medium includes a basal medium and additional components; the basal medium is selected from DMEM low-sugar medium, and 1 mmol of HEPES is added to every 500 mL of DMEM low-sugar medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite:epidermal growth factor:CHIR99021=40:40:3:2:5000:10000:1000:10:1:2:1:20:1004. At the same time, the medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, and 1× B27 cell culture additive.

[0365] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00010 FGF2 synthetic peptide 40 ng/ml PDGF-BB synthetic peptide 40 ng/ml TGF-β 3 synthetic peptide 3 ng/ml Dexamethasone 5 nM Heparin sodium 5 μg/ml Vitamin C 10 μg/ml Transferrin 1 μg/ml Insulin 0.01 μg/ml Progesterone 1 ng/ml Putrescine 2 ng/ml Sodium selenite 1 ng/ml Epidermal growth factor synthetic peptide 20 ng/ml CHIR99021 2 μM Non-essential amino acid 0.1 mM L-glutamic acid 1 mM Sodium pyruvate 0.5 mM B27 Cell Culture Additive 1X Penicillin (10000 U) 5 mmoL Streptomycin (10000 U) 5 mmoL

Experimental Example 9 of Biological Activity

[0366] Cultured Cell Processing

[0367] Human chondrocytes of P3-P6 generation were inoculated into a common 10 cm petri dish at a density of about 1×10{circumflex over ( )}4/cm2 in a serum-free medium in a 37° C., 5% CO2 cell incubator. The medium was changed every 2-3 days, and the cells were subjected to cell photography and other experiments to observe the cell growth.

[0368] Analysis of Results

[0369] Cell Morphology Observation

[0370] The method is the same as in Example 1. As shown in FIG. 36, 4× microscope shows that human chondrocytes cultured in SFM (serum-free medium group) can form large three-dimensional cell spheres well. In addition, the diameter of the spheres showed a tendency to increase, indicating that the serum-free medium is suitable for the culture of chondrocytes. At the same time, this result shows that our medium also supports the three-dimensional culture of cells.

[0371] Cell Count and Cell Proliferation Multiple Analysis

[0372] The method is the same as in Example 1. The cell count results showed that the number of harvested cells was 4.29×10{circumflex over ( )}6, and the total amount of initial cell inoculation was 5.5×10{circumflex over ( )}5. Therefore, the cell proliferation was 7.8 times. This result shows that the serum-free medium composed of biologically active substances of the present invention is suitable for chondrocytes in vitro expansion culture.

Example 10

[0373] In this example, two groups of culture media were prepared, namely a serum-free medium and a serum control culture medium. The cultured cells were human skeletal stem cells. The following are detailed experiments and detection procedures:

[0374] Medium Configuration

[0375] The serum-free medium includes a basal medium and additional components; the basal medium is selected from BEM medium, and 1 mmol of HEPES is added to every 500 mL of BEM medium. Moreover, additional components were added to the medium, and the ratio of the concentration range of each component in the serum-free medium is: fibroblast growth factor synthetic peptide:platelet-derived growth factor synthetic peptide:transforming growth factor-β synthetic peptide:glucocorticoid:heparin or its salt:vitamin C or its derivatives:transferrin:insulin:progesterone:putrescine or its salt:selenite:epidermal growth factor synthetic peptide:CHIR99021=30:30:20:5:2000:80,000:80,000:5000:7:10000:7:20:1004. At the same time, the medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, 1× B27 cell culture additive, 0.1 μg/ml vitronectin synthetic peptide, 0.1 μg/ml fibronectin synthetic peptide, 0.1 μg/ml laminin synthetic peptide.

[0376] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00011 FGF-basic 20 ng/ml FGF1 synthetic peptide 10 ng/ml PDGF-AA 20 ng/ml PDGF-AB synthetic peptide 10 ng/ml TGF-β3 10 ng/ml TGF-β 1 synthetic peptide 10 ng/ml Dexamethasone 12.5 nM Heparin sodium 2 μg/ml Vitamin C 80 μg/ml Transferrin 80 μg/ml Insulin 5 μg/ml Progesterone 7 ng/ml Putrescine 10 μg/ml Sodium selenite 7 ng/ml Epidermal growth factor 20 ng/ml CHIR99021 2 μM Non-essential amino acid 0.1 nM L-glutamine 1 mM Sodium pyruvate 0.5 mM B27 Cell Culture Additive 1X Vitronectin synthetic peptide 0.1 μg/ml Fibronectin synthetic peptide 0.1 μg/ml Laminin synthetic peptide 0.1 μg/ml Penicillin (10000 U) 5 mmol Streptomycin (10000 U) 5 mmoL

Experimental Example 10 of Biological Activity

[0377] Cultured Cell Processing

[0378] Human skeletal stem cells of P3-P6 generation were seeded into 500 mg/ml RGD (Arg-Gly-Asp) peptide and 200 mg/ml KRSR (Lys-Arg-Ser-Arg) peptide coated 10 cm petri dish. The cells were cultured in a serum-free medium in a cell incubator at 37° C. and 5% CO2, and the medium was changed every 2-3 days. Experiments such as taking pictures of the cells were performed to observe the growth of the cells.

[0379] Analysis of Results

[0380] Cell Morphology Observation

[0381] The method is the same as in Example 1. As shown in FIG. 37, observation under the 20× microscope showed that the human bone stem cells cultured in SFM (serum-free medium) grew vigorously, and the cell proliferation was significantly better than that of the SCM (serum medium) group. In addition, the human skeletal stem cells cultured in SFM (serum-free medium) had a more uniform cell morphology and size, a transparent and abundant cytoplasm, and a good adherence to the wall. This result indicates that the composition composed of biologically active substances of the present invention is suitable for the expansion and culture of bone stem cells in vitro.

[0382] Clone Forming Ability Test

[0383] The method is the same as in Example 1. As shown in Table 3, the results show that the clone formation ability of the SFM (serum-free medium) experimental group of Example 10 is significantly better than that of the SCM serum control group. Therefore, it is shown that the features of the stem cells of the skeletal stem cells cultured in the serum-free medium composed of the biologically active substance of the present invention are better than those of the skeletal stem cells cultured in the serum control group.

Comparative Example 5

[0384] Medium Containing Only B27 Cell Culture Additives (B27)

[0385] The medium containing only B27 cell culture additives was selected from DMEM/F12 medium, and 5 mmol of HEPES, 10000 U of penicillin and 10000 U of streptomycin were added to every 500 mL of DMEM/F12 medium. In addition, the additional components were added, and the concentration of the additional components in the serum-free medium were:

TABLE-US-00012 Non-essential amino acid 0.1 mM L-glutamic acid 2 mM Sodium pyruvate 1 mM B27 Cell Culture Additive 1X

Experimental Example 5 of Biological Activity

[0386] Cultured Cell Processing

[0387] Tendon stem cells of P3-P6 generation were inoculated into a 12-well plate coated with 20 mg/ml type I collagen at a density of about 9×10{circumflex over ( )}3/cm2. Culture the cells with serum-free medium, B27 control medium and serum control medium. 1 ml per well, 3 multiple wells per group. Place the cells in a 37° C., 5% CO2 cell incubator, change the medium every 3 days, culture for 5 days, and take photos of the cells to observe the cell growth.

[0388] Analysis of Results

[0389] Cell Morphology Observation

[0390] The method is the same as in Example 1. As shown in FIG. 38, under a 20× microscope, the growth of the cells after 5 days of culture was displayed. The results showed that the cells in group B27 did not proliferate, indicating that the cell culture additives alone cannot effectively proliferate the cells.

Comparative Example 6

[0391] In this comparative example, a set of serum-free media disclosed in the paper (Chinese Journal of Experimental Surgery. 2014. 31(2): 395-398) was configured, and the cultured cells were tendon stem cells (hTSPCs) extracted from human normal tendon tissue. The following is the detailed experiment and detection steps:

[0392] Medium Configuration

[0393] Comparative Serum-Free Medium

[0394] The serum-free medium includes a basal medium and additional components; the basal medium is selected from α-MEM medium, and every 500 mL of α-MEM medium is added with 25 μg IGF-1 and 5 ug TGF-β3, namely 50 μg/L IGF-1 and 10 μg/L TGF-β3.

Comparative Example 6 of Biological Activity

[0395] Cultured Cell Processing

[0396] The primary culture of the cultured cells was carried out with serum culture medium.

[0397] After passage, culture in the comparative medium was carried out. Other methods are the same as in Example 1.

[0398] Analysis of Results

[0399] Cell Morphology Observation

[0400] The method is the same as in Example 1. As shown in FIG. 39, the growth of the cells after 5 days of culture under the 4× microscope showed that the tendon stem cells cultured in the serum-free medium of the comparative example showed slow proliferation. This result indicates that the medium of this paper cannot maintain cell proliferation. At the same time, since the cultured cells were derived from the serum medium, there was serum residue, and the safety was 0 points. Therefore, this comparative example shows that the primary culture is a serum culture medium, and the subsequent secondary culture is a serum-free culture. The cells obtained cannot meet the requirements of the number and quality of clinical treatment cells.

Comparative Example 7

[0401] In this comparative example, a set of serum-free medium with a concentration far beyond the concentration range of the existing biologically active substances was configured, and the cultured cells were tendon stem cells (hTSPCs) extracted from human normal tendon tissue. The following are detailed experiments and detection procedures:

[0402] Medium Configuration

[0403] Comparative Serum-Free Medium

[0404] The serum-free medium includes a basal medium and additional components; the basal medium is selected from DMEM/F12 medium, and every 500 mL of DMEM/F12 medium was supplemented with 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin. In addition, the additional components were added, and the concentration of the additional components in the serum-free medium were: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=203:120:100:120:13000:200000:400,000:100000:100:100000:100. At the same time, the medium also contains 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 1 mM sodium pyruvate, and 6× B27 cell culture additives.

TABLE-US-00013 FGF-basic 203 ng/ml PDGF-AA 120 ng/ml TGF-β 1 100 ng/ml Dexamethasone 300 nM Heparin sodium 200 μg/ml Transferrin 400 μg/ml Insulin 100 μg/ml Progesterone 100 ng/ml Putrescine 100 μg/ml Sodium selenite 100 ng/ml Non-essential amino acid 0.1 mM L-glutamic acid 2 mM Sodium pyruvate 1 mM B27 Cell Culture Additive 6X

[0405] Comparative Example 6 of Biological Activity

[0406] Cultured Cell Processing

[0407] Tendon stem cells of P3-P6 generation were inoculated into a 12-well plate coated with 20 mg/ml type I collagen at a density of about 9×10{circumflex over ( )}3/cm2. Serum-free medium, B27 control medium and serum control were used respectively. Cultivate the cells, 1 ml per well, 3 replicate wells per group, and place them in a 37° C., 5% CO2 cell incubator. Change the medium every 3 days for 5 days, and take photos of the cells to observe the cell growth.

[0408] Analysis of Results

[0409] Cell Morphology Observation

[0410] The method is the same as in Example 40. As shown in FIG. 39, the growth of the cells after 5 days of culture was displayed under a 4× microscope. The results showed that the tendon stem cell cells cultured in the serum-free medium of the comparative example did not proliferate or even died. The concentration range of each component of the medium is unique.

Comparative Example 8

[0411] The medium prepared in this comparative example was not added with fibroblast growth factor, and other conditions were the same as in Example 2.

[0412] Analysis of Results

[0413] Cell Morphology Observation

[0414] The method is the same as in Example 1. As shown in FIG. 41, the growth of the cells after 5 days of culture was shown under a 4× microscope. The results showed that the cultured tendon stem cells in the serum-free medium of this comparative example did not proliferate. This result indicates that the components of the biologically active composition and serum-free medium developed by the present invention are necessary for its function, and the uniqueness of each component of the bioactive substance composition of the present invention.

Comparative Example 9

[0415] The medium prepared in this comparative example did not add transforming growth factor-β, but added 5 ng/ml epidermal growth factor, and other conditions were the same as in Example 2.

[0416] Analysis of Results

[0417] The cell culture effect of the comparative example shows that cell proliferation is slowed down and the phenotype maintenance ability is significantly reduced, indicating that the components of the biologically active composition developed by the present invention are necessary for its function and cannot be replaced by other components, indicating the uniqueness of each component of the bioactive substance composition of the present invention.

Example 11

[0418] A set of serum-free medium was prepared in this example and the cultured cells were tendon stem cells (Scx-GFP mTSPCs) extracted from the normal tendon tissue of Scx-GFP mice. The following are the detailed experiments and detection steps:

[0419] Medium Configuration

[0420] The serum-free medium includes a basal medium and additional components; the basal medium is selected from DMEM/F12 medium, and every 500 mL of DMEM/F12 medium was supplemented with 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin. In addition, the additional components were added, and the concentration of the additional components in the serum-free medium were: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=5:5:2:1:4000:90000:200000:15000:15:15000:15. At the same time, the medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamine, 0.5 mM sodium pyruvate, 1×B27 cell culture supplement.

[0421] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00014 FGF-basic 5 ng/ml PDGF-AA 5 ng/ml TGF-β3 2 ng/ml Dexamethasone 2.5 nM Heparin sodium 4 μg/ml Vitamin C 90 μg/ml Transferrin 200 μg/ml Insulin 15 μg/ml Progesterone 15 ng/ml Putrescine 15 μg/ml Sodium selenite 15 ng/ml Non-essential amino acid 0.1 mM L-glutamine 1 mM Sodium pyruvate 0.5 mM B27 Cell Culture Additive 1X Penicillin (10000 U) 5 mmoL Streptomycin (10000 U) 5 mmoL

Experimental Example 11 of Biological Activity

[0422] The treatment of cultured cells was the same as in Example 6.

[0423] Analysis of Results

[0424] The cell culture effect of this example was similar to that of Example 6. It shows that the culture medium of the present invention supports the culture of mouse tendon stem cells and also supports cell suspension culture.

Example 12

[0425] In this embodiment, a set of serum-free medium was prepared, and the cultured cells were ligament stem cells obtained by separation and culture of human ligament tissue.

[0426] The following are detailed experiments and detection steps:

[0427] Medium Configuration

[0428] Serum-Free Medium (SFM)

[0429] The serum-free medium includes a basal medium and additional components; the basal medium is selected from F10 medium, and every 500 mL of F10 medium was supplemented with 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin. In addition, the additional components were added, and the concentration of the additional components in the serum-free medium were: fibroblast growth factor:platelet-derived growth factor:transforming growth factor-β:glucocorticoid:heparin or its salt:vitamin C or its derivative:transferrin:insulin:progesterone:putrescine or its salt:selenite=26:15:30:8:500:1000:75000:3000:4:7:4. At the same time, the medium also contains 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 1 mM sodium pyruvate, 1× B27 cell culture additive, and 2 μg/ml vitronectin.

[0430] Further, the concentration of each component in the serum-free medium is as follows:

TABLE-US-00015 FGF-basic 26 ng/ml PDGF-BB 15 ng/ml TGF-β 2 30 ng/ml Dexamethasone 20 nM Heparin sodium 0.5 μg/ml Vitamin C 1 μg/ml Transferrin 75 μg/ml Insulin 3 μg/ml Progesterone 4 ng/ml Putrescine 7 μg/ml Sodium selenite 4 ng/ml Non-essential amino acid 0.1 mM L-glutamic acid 2 mM Sodium pyruvate 1 mM B27 Cell Culture Additive 1X Vitronectin 2 ug/ml Penicillin (10000 U) 5 mmoL Streptomycin (10000 U) 5 mmoL

Experimental Example 12 of Biological Activity

[0431] The treatment of cultured cells is the same as in Example 2

[0432] Analysis of Results

[0433] The cell culture effect of this example is similar to that of example 2. It shows that the culture medium of the present invention supports the cultivation of human ligament stem cells.

TABLE-US-00016 TABLE 1 The viability of cultured cells in different examples and comparative examples Cell viability Example 1 98% Example 2 95.4%.sup.  Example 3 90.3%.sup.  Example 4 92% Example 5 97.6%.sup.  Example 6 88% Example 7 91% Example 8 95.4%.sup.  Example 9 92% Example 10 94.5%.sup.  Example 11 90.6%.sup.  Example 12 93% Comparative 78% Example 1 Comparative 90% Example 2 Comparative 65% Example 3 Comparative 87.4%.sup.  Example 4 Comparative 40% Example 5 Comparative 72% Example 6 Comparative 0 Example 7 Comparative 71% Example 8 Comparative 69.5%.sup.  Example 9

TABLE-US-00017 TABLE 2 The results of flow cytometry indicate the expression of CD markers on the surface of cells cultured in different examples and comparative examples CD105 CD90 CD44 CD34 CD18 Example 1 98.9% 99.9% 99.9% 0.4% 0.1% Example 2 98.7% 99.8% 99.5% 0.3% 0.3% Example 3 98.5% 99.5% 99.4% 0.1% 0.3% Example 4 99.3% 99.8% 99.7% 0.5% 0.4% Example 5 99.5% 99.7% 99.2% 0.3% 0.1% Example 6 98.1% 97.3% 97.5% 0.6% 0.6% Example 7 98.3% 96.8% 97.2% 0.8% .sup. 1% Example 8 99.8% 99.2% 98.6% 0.8% 0.6% Example 11 97.9% 98.2% 97.4% 0.2% 0.5% Example 12 98.9% 98.8% 99.2% 0.7% 0.9% Comparative 97.2% 95.5% 96.5% 2.8% .sup. 1% Example 1 Comparative 95.5% 92.2% 96.3% 2.4% .sup. 2% Example 2 Comparative \ \ \ \ \ Example 3 Comparative 99.6% 98.7% 95.4% 1.1% 1.6% Example 4 Comparative \ \ \ \ \ Example 5 Comparative \ \ \ \ \ Example 6 Comparative \ \ \ \ \ Example 7 Comparative \ \ \ \ \ Example 8 Comparative \ \ \ \ \ Example 9 Note: \ Means that the cells did not proliferate in this medium, and the amount of cells is too small to be detected.

TABLE-US-00018 TABLE 3 CFU experiment results show the clone formation ability of P3 generation cell tendon line in different examples and comparative examples. Number of single clones formed in each well/piece Example 1 25 Example 2 19 Example 3 20 Example 4 17 Example 5 23 Example 6 15 Example 7 13 Example 8 23 Example 10 21 Example 11 16 Example 12 20 Comparative 12 Example 1 Comparative 6 Example 2 Comparative 0 Example 3 Comparative 12 Example 4 Comparative 0 Example 5 Comparative 0 Example 6 Comparative 0 Example 7 Comparative 0 Example 8 Comparative 4 Example 9

TABLE-US-00019 TABLE 4 Percentage of Nestin+ Cells Cultured in Each Example and Comparative Example Nestin + TSPC(%) Example 1 94 Example 2 90 Example 3 78 Example 4 72 Example 5 92 Example 6 35 Example 7 60 Example 8 92 Example 10 86 Example 11 62 Example 12 71 Comparative 5 Example 1 Comparative 3 Example 2 Comparative 0 Example 3 Comparative 5 Example 4 Comparative 0 Example 5 Comparative 10 Example 6 Comparative 0 Example 7 Comparative 5 Example 8 Comparative 2 Example 9

TABLE-US-00020 TABLE 5 Scoring of cells cultured in each example and comparative example 4. Tendon phenotype 5. The in vivo and tendon ability to 1. Proliferation 2. Stem cell differentiation repair tendon Total rate phenotype 3. Security ability injuries score Example 1 30 20 10 20 20 100 Example 2 20 20 10 20 20 90 Example 3 25 15 10 15 20 85 Example 4 20 15 10 15 20 80 Example 5 30 20 10 20 20 100 Example 6 15 15 10 10 15 65 Example 7 10 10 10 15 15 60 Example 8 30 20 10 20 20 100 Example 10 30 20 10 20 0 80 Example 11 15 15 10 15 15 70 Example 12 20 20 10 15 20 85 Comparative 5 10 0 0 0 15 Example 1 Comparative 25 10 10 0 0 45 Example 2 Comparative 0 0 10 0 0 10 Example 3 Comparative 25 15 0 0 0 40 Example 4 Comparative 0 0 10 0 0 10 Example 5 Comparative 0 0 0 5 0 5 Example 6 Comparative 0 0 10 0 0 10 Example 7 Comparative 0 0 10 0 0 10 Example 8 Comparative 5 0 10 0 0 15 Example 9

[0434] The comparison of each example with Comparative Example 1 shows that the serum-free medium and/or composition described in this application is a completely serum-free medium, which can completely replace the serum-containing medium, realize the primary culture and secondary culture of cells, and realize the rapid proliferation of cells in vitro and the maintenance or improvement of phenotype.

[0435] The comparison between Example 1 and Comparative Example 1 shows that the cells cultured in the serum-free medium and/or composition described in this application proliferate rapidly and the phenotype is significantly increased. These cells cultured in vitro with good proliferation and phenotype are still powerful when transplanted into the body, and can realize tissue injury repair. The tissue injury repair effect of the cell in Example 1 is significantly better than that of Comparative Example 1 cells obtained from the control group cultured in serum-containing medium. It shows that the cells obtained by the serum-free medium and/or composition culture of the present application have strong functions, and can quickly participate in the regeneration of injured parts and repair tissue injury when implanted in the body. The tissue or organ injury is selected from musculoskeletal system tissue or organ injury; preferably, the musculoskeletal system tissue or organ injury is selected from at least one of tendon and/or ligament injury, cartilage injury, bone injury, muscle injury, skin injury, blood vessel injury.

[0436] The cells cultured in Examples 6, 7, and 11 are of animal origin, and the cells cultured in other examples are of human origin, indicating that the serum-free medium and/or composition described in this application can achieve in vitro culture of cells of human or animal origin. At the same time, Examples 6 and 7 are three-dimensional suspension culture, and other examples are adherent culture, indicating that the serum-free medium and/or composition described in this application can not only carry out suspension culture of cells, but also carry out adherent culture of cells.

[0437] The results of comparison between Example 5 and Comparative Examples 2 and 3 (common MSC commercial serum-free medium) show that the cell proliferation ability, stem cell phenotype and tendinoid phenotype of the serum-free medium and/or composition described in this application are significantly better than that of Comparative Examples 2 and 3. It shows that the serum-free medium and/or composition described in this application are more suitable for in vitro culture of cells and maintenance/improvement of phenotype.

[0438] The experimental results of Comparative Example 7 show that the use concentration range of the biologically active substance and the serum-free medium developed by the present invention is unique, and the concentration range beyond the concentration range is not effective. The experimental results of Comparative Examples 8 and 9 indicate that the biologically active composition and the serum-free medium developed by the present invention are necessary for their functions and cannot be replaced. It illustrates the uniqueness of the composition and concentration of each component of the bioactive substance composition of the present invention.

[0439] In summary, this application adjusts the serum-free medium used in the process of in vitro expansion of stem cells by adjusting the basal medium, bioactive substance composition, additives and their content, and/or the bioactive substance composition of the composition. The additives and their contents are verified using different cell cultures, and a serum-free medium and/or composition that can improve the proliferation ability and phenotype of cells in vitro is obtained. The cells are selected from any one or more of cells derived from tendons and/or ligaments, mesenchymal stem cells, meniscal stem cells, chondrocytes, skeletal stem cells, and muscle stem cells. Preferably, the cell common features and cell-specific phenotypes of the cells cultured in the serum-free medium and/or the composition have individual scores of each individual item reaching their respective passing lines and the total score reaching 60 points or more. Preferably, the cell common features and cell-specific phenotypes of the cells cultured in the serum-free medium reach the respective passing line and the total score reaches 80 points or more. Preferably, the cell common features and the cell-specific phenotype of the cells cultured in the serum-free medium have a single score of each individual item reaching their respective passing line and a total score of 90 points or more.

[0440] It can be seen from the various examples and comparative examples that the serum-free medium and/or composition described in this application can achieve in vitro expansion and phenotype maintenance of tendon and/or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells. These cells are the main cell members of the musculoskeletal system and play an important role in the formation and function of the musculoskeletal system.

[0441] For example, tendon is composed of two major components: tendon-derived cells and collagen matrix. Tendon-derived cells are the only cell member of tendon tissue and play a major role in the development, homeostasis maintenance, and injury repair of tendon tissue, and the collagen matrix in tendon is also formed by the secretion of tendon-derived cells. The serum-free medium and/or composition described in the present application realize the in vitro proliferation and phenotype maintenance of tendon stem cells, and therefore, the in vitro culture and functional maintenance of tendon tissue can also be realized. Therefore, the serum-free medium and/or composition described in this application can be used for in vitro culture and functional maintenance of tissues derived from the exercise system. Preferably, the exercise system tissue is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, adipose tissue, muscle tissue. At the same time, it can be seen from the various examples that the serum-free medium and composition can promote the in vitro expansion of cells and the maintenance or improvement of phenotype, and the core components of these two substances are biologically active substances. It also shows that the bioactive substance composition has super activity and can be used to prepare cell culture reagents. The bioactive substance composition, the serum-free medium, and the composition utilize each component to simulate the complex microenvironment of cell growth in vivo to realize cell culture in vitro. In the process of tissue injury, the microenvironment of the tissue injury site is destroyed, resulting in slow tissue regeneration/repair. Injecting and/or smearing the bioactive substance composition and/or serum-free medium and/or the composition at the injury site can rapidly remodel the microenvironment of the injury site in the body, and accelerate injury repair and tissue regeneration. Therefore, the bioactive substance composition and/or serum-free medium and/or composition described in the present application have application in the preparation of a medicine for the treatment of tissue and/or organ injury. The tissue or organ injury is selected from musculoskeletal system tissue or organ injury; preferably, the musculoskeletal system tissue or organ injury is selected from at least one of tendon and/or ligament injury, cartilage injury, bone injury, muscle injury, skin injury, blood vessel injury.