Serum-free culture medium and application thereof

Abstract

The invention provides a serum-free culture medium and application thereof, the serum-free culture medium comprises bioactive substances and a basic culture medium, does not contain B-27 cell culture additives, and is simple in preparation method and high in safety; the bioactive substances comprise vitamin C or a derivative thereof, heparin or a salt thereof, a transforming growth factor-beta, a fibroblast growth factor and a platelet-derived growth factor, and the basic culture medium is a commercialized mesenchymal stem cell serum-free culture medium with definite components. The serum-free culture medium is simple in preparation method and high in safety, the primary culture proliferation effect of the tendon-derived mesenchymal stem cells can be improved, and the tendon differentiation capacity and the osteogenesis, cartilage formation and adipogenesis differentiation capacity of the mesenchymal stem cells can be improved. The types and concentrations of the five bioactive factors and the type of the basic culture medium are screened and optimized, the effect of the serum-free culture medium is further improved, and a prospect is provided for clinical treatment of motor system injury.

Description

Technical Field

[0001] The present invention belongs to the field of stem cell culture, and more particularly, relates to a serum-free medium and its application. Background Art

[0002] Mesenchymal stem cells, also known as multipotent stromal cells, abbreviated as MSCs, are a type of pluripotent stem cells belonging to the mesoderm. They mainly exist in connective tissues and organ interstitium. Their sources include tissues such as bone marrow, umbilical cord, adipose tissue, mucosa, bone, muscle, tendon, lung, liver, pancreas, etc., as well as amniotic fluid, amnion, placenta, etc. They are cells with self-renewal ability and can differentiate into various tissues such as tendon, adipose tissue, bone, and cartilage under appropriate conditions. They include, but are not limited to, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose stem cells, mucosal mesenchymal stem cells, tendon mesenchymal stem cells, amniotic fluid mesenchymal stem cells, amnion mesenchymal stem cells, placenta mesenchymal stem cells, etc.

[0003] Currently, the media for culturing mesenchymal stem cells basically contain fetal bovine serum (FBS), which can provide the hormones and growth factors necessary for cell adhesion and proliferation during growth. Fetal bovine serum is derived from fetal bovine blood, has a complex composition, and may carry the risk of being contaminated by pathogens; cells cultured with fetal bovine serum may cause immune rejection reactions in patients due to the residual xenoproteins if used for clinical cell therapy; moreover, fetal bovine serum needs to be stored at -20 °C, and there are problems such as large differences between batches. Therefore, media containing fetal bovine serum are not recommended for culturing cells for clinical applications. Commercial serum-free media have a clear composition, can enable cells to adhere and grow normally and have good amplification effects without using fetal bovine serum, are convenient to prepare by adding additives to the basal medium, and are widely used in the culturing of cells for stem cell therapy products. However, they have poor effects on maintaining the stem cell phenotype. Therefore, they lack specificity for seed cells and indications and cannot exert the special advantages of seed cells from different sources. In a serum-free culture system, due to the lack of various nutritional components and growth factors provided by serum, the growth and survival of cells will be affected to a certain extent. At this time, adding B-27 cell culture additive can supplement the necessary nutrients, promote the growth and proliferation of mesenchymal stem cells, and maintain the good state of cells. However, the B-27 cell culture additive is expensive. For example, Gibco 50×B-27 TM additive (17504044) costs 18,357 yuan per 100 mL, and this additive occupies a large market share by a few brands (such as Thermo Fisher Scientific), showing a monopolistic tendency.

[0004] A serum-free medium for human mesenchymal stem cells is disclosed in CN117511861A, which can improve the proliferation ability of human umbilical cord mesenchymal stem cells. However, the amplification efficiency of the mesenchymal stem cells in primary culture is low, and the number of stem cells can only reach 1×10 7 order of magnitude when passaged to P4 generation; A serum-free medium for large-scale culture of human umbilical cord mesenchymal stem cells is disclosed in CN 109370985 A. The number of human umbilical cord mesenchymal stem cells in primary culture with this medium can only reach 1×10 5 order of magnitude. If more cells are to be obtained, subculture must be carried out. CN117448268A discloses a serum-free medium for dental pulp mesenchymal stem cells and its culture method, and this serum-free culture is only applicable to the subculture of dental pulp mesenchymal stem cells. The amplification efficiency of the above three serum-free media for mesenchymal stem cells in primary culture of stem cells is low, and they are only applicable to the subculture of stem cells, not applicable to the rapid isolation, purification and rapid amplification of primary stem cells; In addition, the above three disclosed documents do not state that these three serum-free media can endow the corresponding mesenchymal stem cells with the advantages of specific-direction treatment after culturing the mesenchymal stem cells.

[0005] Therefore, there is an urgent need for a serum-free medium to solve the above problems. Summary of the Invention

[0006] Aiming at the difficulties in the prior art, the present invention provides a serum-free medium and its application. The serum-free medium includes bioactive substances and a basal medium, and does not contain B-27 cell culture additive. The preparation method is simple and has high safety. The bioactive substances include vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor, and the basal medium is a commercially available serum-free medium with defined components for mesenchymal stem cells. The serum-free medium of the present invention has a simple preparation method and high safety. It can not only improve the proliferation effect of tendon-derived mesenchymal stem cells in primary culture, and the number of primary cultured cells can reach 5×10 7 , but also significantly improve the tendon lineage differentiation ability of the mesenchymal stem cells and maintain their osteogenic, chondrogenic and adipogenic differentiation abilities. The mesenchymal stem cells can be directly induced to differentiate into tendon stem cells, osteocytes, chondrocytes and adipocytes without subculture after primary culture. The present invention also screens and optimizes the types and concentrations of five bioactive factors and the type of the basal medium to further improve the effect of the serum-free medium, providing a prospect for clinical treatment of sports system injuries.

[0007] On the one hand, a serum-free medium, comprising bioactive substances and a basal medium; the bioactive substances include any one or more of vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor; the serum-free medium does not contain B-27 serum-free additive.

[0008] The basal medium is one or more of Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro, Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium MAX, Huakan 3D FloTrix Mesenchymal Stem Cell Serum-Free Medium, and Youkang Mesenchymal Stem Cell Serum-Free Medium.

[0009] The research team of the present invention found that adding different combinations of bioactive compositions to commercial mesenchymal stem cell serum-free media can not only improve the proliferation effect of primary culture of tendon-derived mesenchymal stem cells, but also improve the differentiation ability of the cultured mesenchymal stem cells. At the same time, it was also found that there is no need to add B-27 serum-free additive to the serum-free medium. Therefore, the present invention provides a serum-free medium, which is simple to prepare, has high safety and low cost. It can be prepared by simply adding vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor to commercial mesenchymal stem cell serum-free media. It can not only expand the number of tendon-derived mesenchymal stem cells by primary culture to 5×10 7 , but also significantly improve the tendon lineage differentiation ability of the cultured tendon-derived mesenchymal stem cells and maintain their osteogenic, chondrogenic, and adipogenic differentiation abilities. The tendon-derived mesenchymal stem cells after primary culture with the serum-free medium of the present invention can be directly used for induced culture. The cultured mesenchymal stem cells can be successfully differentiated into tendon stem cells, osteocytes, chondrocytes, and adipocytes after being induced by the induction medium; the serum-free medium provided by the present invention can also be used for the primary culture of umbilical cord-derived mesenchymal stem cells, improving the primary culture proliferation effect and pluripotency of umbilical cord-derived mesenchymal stem cells.

[0010] The research team of the present invention has previously applied for "A Bioactive Substance Composition, a Serum-Free Medium Containing the Composition and Its Use", with the patent number CN113692282A. Among them, the serum-free medium containing the bioactive substance composition enables the primary culture and subculture of various sources of stem cells in vitro, and its ability to promote cell proliferation and phenotype maintenance is superior to that of the fetal bovine serum-containing medium. The serum-free medium containing the bioactive substance composition is particularly suitable for the completely serum-free culture of tendon- and / or ligament-derived stem cells in vitro, enabling tendon- and / or ligament-derived stem cells to have a fast proliferation rate, a short cell doubling time, and the ability to maintain the tendon lineage ability and trilineage differentiation (osteogenic differentiation, chondrogenic differentiation, adipogenic differentiation) ability and other stem cell phenotypes of the cells. However, the number of tendon- and / or ligament-derived stem cells after primary culture is low, and the effect of directly using them for induction in the induction medium to differentiate into tendon stem cells, osteoblasts, chondrocytes, and adipocytes is poor. Because the serum-free medium alone cannot enable stem cells to adapt to the in vitro growth environment by only primary culturing tendon- and / or ligament-derived stem cells, the proliferation ability of stem cells is limited. When primary cultured stem cells are used for induction and differentiation, due to the relatively small number of cells, the cell subsets with high differentiation potential are relatively insufficient. These cells with high differentiation ability play a key role in the formation of functional stem cells during the induction process. The insufficient number of them will lead to poor overall induction effect, making it difficult to produce a sufficient number and quality of functional stem cells, resulting in poor induction and differentiation effect. Therefore, the stem cells need to be subcultured to adapt to the in vitro growth environment and achieve the purpose of rapid proliferation, realizing the exponential growth of cell number and improving the induction and differentiation effect. However, if the same primary culture time is used, the cell number of subcultured to passage 2 (P2) generation reaches only 1.9×10 7 , and the B-27 serum-free additive must be added when culturing cells in primary culture and subculture, increasing the culture cost. The present invention provides a new serum-free medium that can enable tendon-derived mesenchymal stem cells to quickly adapt to the in vitro growth environment during primary culture without adding the B-27 serum-free additive, and the number of primary cell proliferation can reach 5×10 7 , which is much higher than the cell proliferation effect achieved by culturing tendon- and / or ligament-derived stem cells subcultured to P2 generation with the serum-free medium previously applied by the research team of the present invention. Moreover, the tendon-derived mesenchymal stem cells primary cultured with the serum-free medium provided by the present invention can be directly used for induction and successfully differentiated into tendon stem cells, osteoblasts, chondrocytes, and adipocytes without subculture, and the differentiation effect is better.

[0011] Furthermore, the vitamin C or its derivatives include any one or more of vitamin C, ascorbyl glucoside, ethyl vitamin C, 3-o-ethyl ascorbic acid, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, L-ascorbic acid 2-phosphate sesquimagnesium hydrate, tetra-isopalmitoyl ascorbate, ascorbyl palmitate, L-ascorbic acid-2-phosphate-6-palmitate, esterified vitamin C, and solvates of ascorbic acid; the heparin or its salts include any one or more of heparin, sodium heparin, calcium heparin, and heparan sulfate; the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, TGF-β3, and synthetic peptides of transforming growth factor-β.

[0012] The vitamin C or its derivatives refer to vitamin C (also known as ascorbic acid), salts of vitamin C, and solvates of vitamin C, which have antioxidant effects, can inhibit cell senescence, and promote cell growth and phenotype maintenance; in addition, the vitamin C or its derivatives enhance the cartilage repair ability of mesenchymal stem cells through metabolic regulation, showing its promoting effect in the specific differentiation direction of stem cells.

[0013] The heparin or its salts are strongly acidic and are a natural anticoagulant substance in animals. It can inhibit the conversion of prothrombin to thrombin, and thus inhibit the formation of fibrin from fibrinogen. In stem cell culture, heparin can prevent stem cell aggregation, protect cells, and ensure the stability and activity of cells during the culture process; secondly, the heparin or its salts provide a suitable growth environment for stem cells and directly promote the growth of stem cells; in addition, the heparin or its salts can specifically induce the differentiation of mesenchymal stem cells into osteoblasts and other directions.

[0014] The transforming growth factor-β (TGF-β) belongs to the multifunctional cytokines of the transforming growth factor superfamily and has the functions of regulating stem cell growth and differentiation and maintaining the stem cell phenotype, which helps to maintain the stability and pluripotency of stem cells.

[0015] Furthermore, the fibroblast growth factor includes any one or more of FGF-basic, FGF-1, FGF-4, FGF-7, FGF-10, FGF-18, and synthetic peptides of fibroblast growth factor; the platelet-derived growth factor includes platelet growth factor and vascular endothelial cell factor; the platelet growth factor includes any one or more of PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD, placental growth factor, VEGF-41, VEGF-B, VEGF-C, VEGF-D, and synthetic peptides of platelet-derived growth factor.

[0016] Fibroblast growth factor (FGF), also known as heparin-binding growth factor, mainly includes two categories: acidic and basic, which refer to a class of active proteins or polypeptides that can promote cell growth. Fibroblast growth factor plays a crucial role in stem cell culture, mainly including promoting stem cell proliferation, maintaining the undifferentiated state of stem cells, regulating stem cell differentiation, and participating in the regulation of stem cell signal transduction. Different types of fibroblast growth factors can induce stem cells to differentiate into specific cell types. For example, some studies have shown that FGF-4 can significantly promote the proliferation of bone marrow stromal stem cells and induce their differentiation into ligaments or tendons at specific concentrations (DOI: 10.7666 / d.d090536, DOI: 10.7666 / d.d090536).

[0017] Platelet derived growth factor (PDGF) is an important cytokine that regulates the proliferation and differentiation of stem cells. In specific physiological and pathological processes, PDGF can induce stem cells to differentiate into specific cell types. For example, some studies have shown that rat bone marrow mesenchymal stem cells can be induced to differentiate into functional osteoblasts by PDGF-BB (Wei Qin, Zhang Xue, Ma Lei, et al. Platelet-derived growth factor BB induces the differentiation of rat bone marrow mesenchymal stem cells into osteoblasts [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(19): 2953-2957.).

[0018] Furthermore, the vitamin C or its derivative is vitamin C or L-ascorbic acid 2-phosphate sesquimagnesium hydrate; the heparin or its salt is heparin or heparin sodium; the transforming growth factor-β is any one or more of TGF-β1, TGF-β2, TGF-β3; the fibroblast growth factor is any one or more of FGF-basic, FGF-7; the platelet-derived growth factor is any one or more of PDGF-AA, PDGF-BB.

[0019] Furthermore, the mass ratio of the bioactive substance to the basal medium is (0.302-190.2):100000000.

[0020] Furthermore, the final added concentration of the vitamin C or its derivative is 0.1 - 100 μg / ml, the final added concentration of the heparin or its salt is 0.1 - 10 μg / ml, the final added concentration of the transforming growth factor-β is 0.1 - 80 ng / ml, the final added concentration of the fibroblast growth factor is 1 - 100 ng / ml, and the final added concentration of the platelet-derived growth factor is 1 - 100 ng / ml.

[0021] In some ways, the effects of serum-free media containing different concentrations of the 5 bioactive substances on the proliferation and differentiation ability of primary cultured tendon-derived mesenchymal stem cells were compared through single-variable experiments. The results showed that within the above concentration ranges, the 5 bioactive substances could expand the number of mesenchymal stem cells to 1×10 7 and above and maintain a cell viability of more than 90%, and simultaneously have the ability of tendon lineage differentiation and trilineage differentiation ability of osteogenic, chondrogenic, and adipogenic differentiation.

[0022] Furthermore, the basal medium is Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro.

[0023] In some ways, by comparing the primary culture proliferation and differentiation effects of tendon-derived mesenchymal stem cells cultured in different commercial mesenchymal stem cell serum-free media and DMEM low-glucose medium, the experimental results showed that the mesenchymal stem cells cultured in the medium containing Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro had the strongest proliferation and differentiation ability.

[0024] On the other hand, the present invention provides a method for culturing mesenchymal stem cells, which uses the above-mentioned serum-free medium to culture mesenchymal stem cells.

[0025] On yet another aspect, the present invention provides a use of a bioactive substance composition for preparing a medium for improving the primary culture effect of mesenchymal stem cells. The medium does not contain B-27 serum-free additive; the bioactive substance composition includes vitamin C or its derivative, heparin or its salt, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor; the vitamin C or its derivative includes any one of vitamin C and L-ascorbic acid 2-phosphate sesquimagnesium hydrate, the heparin or its salt includes any one of heparin and sodium heparin, the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, and TGF-β3, the fibroblast growth factor includes any one or more of FGF-basic and FGF-7, the platelet-derived growth factor includes any one or more of PDGF-AA and PDGF-BB; the medium includes a basal medium.

[0026] The basal medium is one or more of Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro, Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium MAX, Huakan 3D FloTrix Mesenchymal Stem Cell Serum-Free Medium, and Youkang Mesenchymal Stem Cell Serum-Free Medium.

[0027] In some methods, primary culture is carried out by an improved primary culture method. Comparing the effects of the serum-free medium provided by the present invention, the serum-free medium provided in CN113692282A previously applied by the team of the present invention, and the medium containing fetal bovine serum on the proliferation ability of primary cultured tendon-derived mesenchymal stem cells, the results show that the number of cells in the primary culture of mesenchymal stem cells with the serum-free medium provided by the present invention is three orders of magnitude higher than that of the serum-free medium provided in CN113692282A and two orders of magnitude higher than that of the medium containing fetal bovine serum.

[0028] On the other hand, the use of a bioactive substance composition for preparing a medium for enhancing the proliferation and differentiation ability of mesenchymal stem cells, wherein the medium does not contain B-27 serum-free additive; the bioactive substance composition includes vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor; the vitamin C or its derivatives include any one of vitamin C and L-ascorbic acid 2-phosphate sesquimagnesium hydrate, the heparin or its salts include any one of heparin and heparin sodium, the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, and TGF-β3, the fibroblast growth factor includes any one or more of FGF-basic and FGF-7, the platelet-derived growth factor includes any one or more of PDGF-AA and PDGF-BB; the medium includes a basal medium.

[0029] The basal medium is one or more of Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro, Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium MAX, Huakan 3D FloTrix Mesenchymal Stem Cell Serum-Free Medium, and Youkang Mesenchymal Stem Cell Serum-Free Medium.

[0030] In some ways, the serum-free medium provided by the present invention, the serum-free medium provided in CN113692282A previously applied by the team of the present invention, and the fetal bovine serum-containing medium are respectively used for primary culture of tendon-derived mesenchymal stem cells, and then the relative expression levels of SCX, THBS4, and NES genes of the cultured mesenchymal stem cells are detected. The results show that the relative expression levels of SCX, THBS4, and NES genes of the mesenchymal stem cells cultured in the serum-free medium provided by the present invention are significantly higher than those of the other two groups. Moreover, the high relative expression level of the SCX gene indicates that the mesenchymal stem cells cultured in the serum-free medium of the present invention have stronger tendon differentiation potential, while the high relative expression level of the NES gene indicates that the mesenchymal stem cells also have pluripotency and can differentiate into other functional stem cells, such as osteogenic, chondrogenic, and adipogenic differentiation.

[0031] In some ways, the tendon-derived mesenchymal stem cells primary-cultured in the serum-free medium of the present invention are further induced by a tendon lineage induction medium and stained with Sirius red. The staining results prove that the induced mesenchymal stem cells express a large amount of collagen fibers, indicating that they have differentiated into tendon stem cells.

[0032] The tendon stem cells can be used to treat sports system injuries. For example, in injuries such as Achilles tendon rupture, by transplanting tendon stem cells or using growth factors secreted by them, etc., the healing of the Achilles tendon can be accelerated and the healing quality can be improved.

[0033] In some ways, the tendon-derived mesenchymal stem cells primary-cultured in the serum-free medium of the present invention are further induced by an osteogenic induction medium and stained with ALP, ARS, and DAPI respectively. The staining results prove that the induced mesenchymal stem cells have active proliferation, high ALP content, and high calcium salt content, indicating that they have begun to differentiate into osteoblasts and are in the stage of osteoblast precursor cells or early osteoblasts.

[0034] The osteoblasts can be used to promote bone regeneration. For large segmental bone defects caused by reasons such as trauma and tumor resection, autologous bone transplantation is the main current treatment method, but there are problems such as limited donor bone sources and donor site complications. Osteoblasts can be implanted into the bone defect site by direct injection or in combination with a carrier to promote the regeneration of bone tissue and reduce the dependence on autologous bone transplantation.

[0035] In some ways, the tendon-derived mesenchymal stem cells primary-cultured in the serum-free medium of the present invention are further induced by a chondrogenic induction medium and stained with Alcian blue and Safranin O respectively. The staining results prove that the induced mesenchymal stem cells have higher contents of components such as acidic mucopolysaccharides and glycosaminoglycans in the intracellular or extracellular matrix, as well as higher contents of acidic glycosaminoglycan components such as proteoglycans, indicating that they have differentiated into chondrocytes.

[0036] The chondrocytes can be used to promote cartilage repair. For diseases such as articular cartilage injury, the chondrocytes can be implanted into the injury site, and these chondrocytes can proliferate and secrete cartilage matrix to promote the repair of the injured cartilage.

[0037] In some methods, the tendon-derived mesenchymal stem cells cultured in the serum-free medium of the present invention are further induced by an adipogenic induction medium and stained with Oil Red O. The staining results prove that a large amount of lipids are synthesized and accumulated in the induced mesenchymal stem cells, indicating that they have differentiated into adipocytes.

[0038] The adipocytes can be used for the regeneration and repair of adipose tissue. Adipocytes have endocrine functions and can secrete various adipokines such as leptin and adiponectin. These factors are crucial in regulating the body's energy metabolism, insulin sensitivity, etc., and can be used as seed cells for cell therapy to treat some diseases related to abnormal fat metabolism.

[0039] In some methods, the tendon-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are primary cultured using the serum-free medium provided by the present invention, and then the cell number, cell viability, immunophenotype, and relative expression levels of SCX, THBS4, and NES genes of the two different sources of mesenchymal stem cells cultured are detected. The results show that the serum-free medium provided by the present invention can make the primary culture of the two sources of mesenchymal stem cells conform to the immunophenotype, and the cell quantity reaches 1×10 7 and maintain a high cell viability. However, the results of the relative expression levels of the three genes show that the serum-free medium provided by the present invention is particularly suitable for the culture of tendon-derived mesenchymal stem cells and can significantly improve their tendon lineage differentiation ability.

[0040] The present invention has the following beneficial effects:

[0041] (1) Provide a serum-free medium, the serum-free medium includes bioactive substances and a basal medium, and does not contain B-27 serum-free additive. The bioactive substances include vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor, and the basal medium is a commercial serum-free medium;

[0042] (2) The preparation method of the serum-free medium of the present invention is simple and can be prepared by simply adding bioactive substances to the basal medium. It can not only improve the primary culture proliferation ability of tendon-derived mesenchymal stem cells, and the primary culture cell number can reach 5×10 7 but also improve the differentiation ability of the cultured mesenchymal stem cells. The mesenchymal stem cells can be directly induced to differentiate into tendon stem cells, osteocytes, chondrocytes, and adipocytes without subculture after primary culture;

[0043] (3) The present invention also screens and optimizes the types and concentrations of the five bioactive factors and the type of the basal medium, further improving the effect of the serum-free medium and providing a prospect for clinical treatment of sports system injuries;

[0044] (4) The serum-free medium provided by the present invention can also be used for the primary culture of umbilical cord-derived mesenchymal stem cells, improving the proliferation effect and differentiation ability of the primary cultured umbilical cord-derived mesenchymal stem cells. DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the growth morphology diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 in Example 1 on the 5th day. The blocky substances in the figure are the tissue microparticles remaining when the cells are separated from the tendon tissue by the digestive solution;

[0046] Figure 2 It is the growth morphology diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 in Example 1 on the 10th day;

[0047] Figure 3 It is the growth morphology diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 in Example 1 on the 14th day;

[0048] Figure 4 It is the growth morphology diagram of the mesenchymal stem cells primary cultured with the fetal bovine serum-containing medium combination 3 in Example 1 on the 6th day. The blocky substances in the figure are the tissue microparticles remaining when the cells are separated from the tendon tissue by the digestive solution;

[0049] Figure 5 It is the growth morphology diagram of the mesenchymal stem cells primary cultured with the fetal bovine serum-containing medium combination 3 in Example 1 on the 13th day;

[0050] Figure 6 It is the NES protein fluorescence staining diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 and the fetal bovine serum-containing medium combination 3 in Example 1;

[0051] Figure 7 It is the Sirius red staining diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 in Example 1 after being induced by the tendon lineage induction medium;

[0052] Figure 8 It is the ALP staining diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 in Example 1 after being induced by the osteogenic induction medium;

[0053] Figure 9 It is the ARS staining diagram of the mesenchymal stem cells primary cultured with the serum-free medium combination 1 in Example 1 after being induced by the osteogenic induction medium;

[0054] Figure 10DAPI fluorescence staining image of mesenchymal stem cells primary cultured with serum-free medium combination 1 in Example 1 after induction with osteogenic induction medium;

[0055] Figure 11 Alcian blue staining image of mesenchymal stem cells primary cultured with serum-free medium combination 1 in Example 1 after induction with chondrogenic induction medium;

[0056] Figure 12 Safranin O staining image of mesenchymal stem cells primary cultured with serum-free medium combination 1 in Example 1 after induction with chondrogenic induction medium;

[0057] Figure 13 Oil Red O staining image of mesenchymal stem cells primary cultured with serum-free medium combination 1 in Example 1 after induction with adipogenic induction medium;

[0058] Figure 14 Growth morphology image of tendon-derived mesenchymal stem cells primary cultured with medium combination 1 in Example 2 at the time of harvest after 14 days;

[0059] Figure 15 Growth morphology image of tendon-derived mesenchymal stem cells primary cultured with medium combination 2 in Example 2 at the time of harvest after 14 days;

[0060] Figure 16 Growth morphology image of tendon-derived mesenchymal stem cells cultured with medium combination 3 in Example 2 at the time of harvest at passage P2;

[0061] Figure 17 Growth morphology image of tendon-derived mesenchymal stem cells cultured with medium combination 4 in Example 2 at the time of harvest at passage P2;

[0062] Figure 18 Growth morphology of tendon-derived mesenchymal stem cells primary cultured with medium combination 5 containing fetal bovine serum at the time of harvest after 14 days;

[0063] Figure 19 Growth morphology image of umbilical cord-derived mesenchymal stem cells primary cultured with serum-free medium in Example 5 on the fourth day of culture;

[0064] Figure 20 Growth morphology image of umbilical cord-derived mesenchymal stem cells primary cultured with medium containing fetal bovine serum in Example 5 on the sixth day of culture. Detailed implementation manners

[0065] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0066] Example 1: Effects of Serum-Free Medium and Fetal Bovine Serum-Containing Medium on Primary Culture of Tendon-Derived Mesenchymal Stem Cells

[0067] In this example, the effects of the serum-free medium provided by the present invention and the fetal bovine serum-containing medium on the primary culture of tendon-derived mesenchymal stem cells were compared.

[0068] 1. Preparation of media

[0069] 1) Preparation of the serum-free medium of the present invention, the specific steps are as follows:

[0070] Preparation of vitamin C or its derivative solution: Add 100 mg of vitamin C or its derivative to 20 mL of PBS buffer solution for dissolution, the concentration of the vitamin C or its derivative solution is 5 mg / mL, and filter with a 0.22 μm sterile filter membrane;

[0071] Preparation of heparin or its salt solution: Add 10 mg of heparin or its salt to 1 mL of PBS-5% trehalose buffer solution for dissolution, the concentration of heparin or its salt is 10 mg / mL;

[0072] Preparation of platelet-derived growth factor (PDGF) solution: Add 50 μg of platelet-derived growth factor to 5 mL of PBS buffer solution for dissolution, the concentration of the platelet-derived growth factor solution is 10 μg / mL;

[0073] Preparation of fibroblast growth factor (FGF) solution: Add 500 μg of fibroblast growth factor to 5 mL of PBS buffer solution for dissolution, the concentration of the fibroblast growth factor solution is 100 μg / mL;

[0074] Preparation of transforming growth factor-β (TGF-β) solution: Add 50 μg of transforming growth factor-β to 5 mL of PBS buffer solution for dissolution, the concentration of the transforming growth factor-β solution is 10 μg / mL.

[0075] Add the above 5 kinds of bioactive factor solutions to the basal medium in sequence according to the specified concentrations, and there is no order for the addition of each component. The basal medium is 500 mL of Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium (Pro Zhongke Ruiji RGM1051), and its accompanying additives are added.

[0076] 2) Preparation of the serum-free medium provided in Example 1 of CN113692282A: Add the basal medium and the provided bioactive substance composition provided in Example 1 of CN113692282A to the final concentration.

[0077] 3) Preparation of the fetal bovine serum-containing medium

[0078] Preparation method: Add fetal bovine serum to the conventional low-glucose DMEM medium. Add 55 mL of fetal bovine serum to every 500 mL of low-glucose DMEM medium.

[0079] Set the following 3 groups of media respectively, as shown in Table 1 below. Medium combination 1 is the serum-free medium provided by the present invention, combination 2 is the serum-free medium provided in Example 1 of CN113692282A, and combination 3 is the medium containing fetal bovine serum.

[0080] Table 1. Serum-free media with different combinations and medium containing fetal bovine serum

[0081]

[0082] 2. Primary culture method of tendon-derived mesenchymal stem cells

[0083] Primary culture method 1 of tendon-derived mesenchymal stem cells: Weigh the tendon tissue and rinse it in PBS buffer containing 10%, 5%, 2%, and 1% P / S double antibiotics for 1 min in sequence. Then transfer the tendon tissue to a 10 cm culture dish, add 0.5 mL of DMEM digestive solution containing 2% collagenase and 1% P / S double antibiotics, cut the tendon tissue into pieces of 1 mm×1 mm size, add 7.5 mL of digestive solution again and mix well, and place it in a 37 °C, 5% carbon dioxide incubator for digestion for about 4 h. Stir and mix the digestive solution every 1 h until the tissue digestive solution is clear and not viscous. Transfer the digestive solution to a 15 mL centrifuge tube and centrifuge it at 2500 rpm for 15 min. Discard the supernatant. Resuspend the precipitate with the media in Table 1 and divide it equally into multiple 15 cm culture dishes. Each dish corresponds to 100 mg of tissue dissociation precipitate, and the culture system is 10 mL / dish. Place it in a 37 °C, 5% carbon dioxide incubator for static culture. Perform a medium change operation on the fifth day of culture. Transfer the culture supernatant to a centrifuge tube, centrifuge it at 2500 rpm for 10 min, discard the supernatant, resuspend the precipitate with the corresponding medium in Table 1 and divide it equally back into the culture dishes, and place it in a 37 °C, 5% carbon dioxide incubator for static culture. On the 10th day of culture, perform a replating operation on the culture dishes. Remove the culture supernatant, wash the bottom of the dish with 8 mL of PBS buffer, discard the PBS buffer, add 2 mL of mild digestive enzyme to each dish, place it in a 37 °C incubator for digestion for about 2 min, observe the cell morphology under the microscope until it is round and plump, then add 7 mL of culture supernatant to each dish, disperse the cells and transfer them to a centrifuge tube, wash each culture dish with 5 mL of PBS buffer and transfer it to the centrifuge tube, centrifuge it at 1200 rpm for 5 min, discard the supernatant, resuspend the precipitate with the corresponding medium in Table 1 and evenly seed it back into the original culture dishes. The culture system is 20 mL / dish. Place it in a 37 °C, 5% carbon dioxide incubator for static culture. Harvest on the 14th day.

[0084] It should be noted that the reseeding operation in the above-mentioned first culture method is not subculture, because the reseeding operation only involves simply treating the cultured cells with digestive enzymes and then inoculating them into the original culture dish, and the total number of cells before and after the reseeding operation does not change.

[0085] The second primary culture method for tendon-derived mesenchymal stem cells: Weigh the tendon tissue and rinse it successively in PBS buffer containing 10%, 5%, 2%, and 1% P / S double antibiotics for 1 minute. Subsequently, transfer the tendon tissue to a 10 cm culture dish, add 0.5 mL of DMEM digestive solution containing 2% collagenase and 1% P / S double antibiotics, cut the tendon tissue into pieces of 1 mm × 1 mm size, add another 7.5 mL of digestive solution and mix well, and place it in a 37 °C, 5% carbon dioxide incubator for digestion for about 4 hours. Stir and mix the digestive solution every 1 hour until the tissue digestive solution becomes clear and not viscous. Transfer the digestive solution to a 15 mL centrifuge tube and centrifuge it at 2500 rpm for 15 minutes. Discard the supernatant, resuspend the precipitate with the medium in Table 1 and evenly divide it into multiple 15 cm culture dishes, with 100 mg of tissue dissociation precipitate corresponding to each dish. The culture system is 20 mL / dish. Place it in a 37 °C, 5% carbon dioxide incubator for static culture, and change the medium once every 5 days with the corresponding medium in Table 1. Fix and harvest on the 14th day.

[0086] 3. Effects of different combinations of serum-free media and fetal bovine serum-containing media on the primary culture of tendon-derived mesenchymal stem cells

[0087] The primary culture of tendon-derived mesenchymal stem cells was carried out for the 3 groups of media in Table 1 respectively by the above-mentioned first and second culture methods.

[0088] During the culture process, an inverted microscope was used to observe the growth status and morphological changes of mesenchymal stem cells, and records were taken by microscope photography. As Figures 1 - 3 shown, the growth morphology diagrams of tendon-derived mesenchymal stem cells cultured by the first primary culture method with serum-free medium combination 1 (the present invention) on the 5th, 10th, and 14th days of culture are presented. The results show that the cultured tendon-derived mesenchymal stem cells can maintain good adherent properties, have abundant cytoplasm, and present a spindle-shaped cell morphology. Although the proliferation is relatively slow from the 1st to the 10th day, the proliferation rate accelerates after 10 days. As Figures 4 - 5 shown, the growth morphology diagrams of tendon-derived mesenchymal stem cells cultured by the second primary culture method with fetal bovine serum-containing medium combination 3 on the 6th and 13th days of culture are presented. The cultured tendon-derived mesenchymal stem cells also maintain good adherent properties, have abundant cytoplasm, and present a spindle-shaped cell morphology, and the proliferation rate is fast from the 1st to the 10th day, but the proliferation rate is slow after 10 days.

[0089] Furthermore, the cell numbers and viability rates of the tendon-derived mesenchymal stem cells cultured from the above three groups of culture media by different primary culture methods were detected after harvesting, and the results are shown in Table 2 below.

[0090] Table 2. Proliferation effects of different culture media and different culture methods on the primary culture of tendon-derived mesenchymal stem cells

[0091]

[0092] According to the data analysis in Table 2, by comparing the cell numbers and viability rates of tendon-derived mesenchymal stem cells cultured from different culture medium combinations by two different primary culture methods, the difference lies in that one of the primary culture methods added a re-plating operation. There was no significant difference in the cell numbers of culture medium combinations 2 and 3 cultured by method 1 compared to method 2, indicating that the primary culture proliferation effects of the serum-free medium and the bovine serum-containing medium provided in Example 1 of CN113692282A for the primary culture of tendon-derived mesenchymal stem cells were limited. However, the number of tendon-derived mesenchymal stem cells cultured from culture medium combination 1 (the present invention) by primary culture method 1 was significantly higher than that by primary culture method 2, indicating that the number of tendon-derived mesenchymal stem cells cultured from culture medium combination 1 (the present invention) by primary culture method 2 did not reach the proliferation upper limit. Because the re-plating operation was not added in method 2, resulting in an excessive density of cloning centers and a contact inhibition effect, the growth rate of central cells was limited after 10 days of culture, thus reducing the harvest yield. By adding a re-plating operation to culture medium combination 1 (the present invention) by primary culture method 1 on the tenth day of culturing tendon-derived mesenchymal stem cells, the proliferation rate of tendon-derived mesenchymal stem cells can be faster after 10 days of culture, and the cell volume decreases. More cells can be produced with the same culture area, reaching a cell quantity level of 1×10 7 . In addition, using method 1 and method 2, the mesenchymal stem cells primary-cultured from combination 1 (the present invention) medium were three orders of magnitude and two orders of magnitude higher than those from combination 2 medium respectively, indicating that the serum-free medium provided in Example 1 of CN113692282A was not suitable for the primary culture and amplification of stem cells, while the combination 1 serum-free medium provided by the present invention was suitable for the primary culture, isolation, purification, and rapid amplification of stem cells.

[0093] In addition, the basal medium of Medium Combination 2 is DMEM low-glucose medium or F12 medium, and the rest of the bioactive substances need to be prepared by oneself. Besides the five bioactive components including vitamin C or its salts, heparin or its salts, PDGF, FGF, and TGF-β, it also includes components such as hormones, other growth factors, and non-essential amino acids. As a result, some bioactive components that promote cell proliferation are not added, so the cell proliferation effect is limited. However, for Medium Combination 1 provided by the present invention, only five bioactive components, namely vitamin C or its salts, heparin or its salts, PDGF, FGF, and TGF-β, need to be added to Zhongkeruiji Mesenchymal Stem Cell Serum-Free Medium Pro without adding other components. The other bio-nutritional active substances contained in Zhongkeruiji Mesenchymal Stem Cell Serum-Free Medium Pro are more comprehensive than those in Medium Combination 2. At the same time, the types and concentrations of the five bioactive components, namely vitamin C or its salts, heparin or its salts, PDGF, FGF, and TGF-β, in Combination 1 are different from those in Combination 2. Therefore, Medium Combination 1 is more suitable for the proliferation culture of tendon-derived mesenchymal stem cells.

[0094] Furthermore, the tendon-derived mesenchymal stem cells cultured by the first primary culture method with the above Medium Combinations 1-3 were subcultured to the P5 generation, and the cell numbers and survival rates of the P1-P5 generations were detected. The cell numbers and survival rates of the P2 and P5 generations are shown in Table 3 below.

[0095] Table 3. Cell numbers and survival rates of tendon-derived mesenchymal stem cells cultured to the P2 and P5 generations with different media by the first primary culture method

[0096]

[0097] According to the data analysis in Table 3, when the primary tendon-derived mesenchymal stem cells cultured with Medium Combinations 1-3 were subcultured to the P5 generation, the stem cell amplification quantity increased further. When the stem cells were cultured with Medium Combination 1 (the present invention) to the P2 generation, the cell number was at the order of magnitude of 1×10 9 two orders of magnitude higher than that of Medium Combination 2. When cultured to the P5 generation, the cell number increased to the order of magnitude of 1×10 14 four orders of magnitude higher than that of Medium Combination 2. Combining the results of the first primary culture method in Table 2 above, it shows that the stem cells cultured with Medium Combination 1 provided by the present invention only in terms of the primary cell number (5.7×10 7 ) reached and exceeded the proliferation effect that the stem cells cultured with Medium Combination 2 in Example 1 of CN113692282A could achieve after being subcultured to the P2 generation (1.9×10 7 ).

[0098] In summary, the serum-free medium provided by the present invention can significantly improve the proliferation effect of tendon-derived mesenchymal stem cells only through primary culture.

[0099] 3. Effects of Different Combinatorial Media on the Differentiation Ability of Tendon-Derived Mesenchymal Stem Cells

[0100] Furthermore, a testing company was commissioned to perform flow cytometry on the surface markers of tendon-derived mesenchymal stem cells cultured by the above-mentioned medium combinations 1-3 through the primary culture method and the P2 culture method. The test results are shown in Table 4 below, and the reference intervals for the test indicators are shown in Table 5 below.

[0101] Table 4. Detection Results of the Immunophenotypes of Tendon-Derived Mesenchymal Stem Cells Cultured in Different Media

[0102]

[0103] Table 5. Reference Intervals for the Detection of Immunophenotypes of Tendon-Derived Mesenchymal Stem Cells

[0104]

[0105] According to the data analysis in Tables 4-5, the tendon-derived mesenchymal stem cells cultured by the serum-free medium combinations 1-2 in the primary culture and the P2 culture both meet the immunophenotype requirements of mesenchymal stem cells and meet the safety indicators. However, the immunophenotype CD34 of the tendon-derived mesenchymal stem cells cultured by the fetal bovine serum-containing medium 3 in the primary culture and the P2 culture does not meet the requirements; CD73 is highly expressed on the surface of mesenchymal stem cells, CD105 and CD90 are highly expressed on the surfaces of both mesenchymal stem cells and tendon stem cells, CD14, CD19, and HLA-DR are lowly expressed or not expressed on the surface of mesenchymal stem cells, and CD45 and CD34 are lowly expressed or not expressed on the surface of tendon stem cells. The detection results of the surface antigens of the mesenchymal stem cells cultured by the serum-free medium combinations 1-2 in the primary culture and the P2 culture show that the expression levels of CD73, CD105, and CD90 are all ≥95%, and the expression levels of CD45, CD34, CD14, CD19, and HLA-DR are all ≤2%, indicating that the cultured cells meet the mesenchymal stem cell phenotype and have the potential to differentiate into tendon stem cells.

[0106] 3.1 Detection of the Tendon Lineage Differentiation Ability of Tendon-Derived Mesenchymal Stem Cells Cultured in Different Serum-Free Media

[0107] The relative expression levels of the SCX, NES, and THBS4 genes of the tendon-derived mesenchymal stem cells cultured by the medium combinations 1-3 in the primary culture and the P2 culture in Table 4 above were detected respectively. The specific detection method is as follows: 1×10 5Cells were added, and after adding 2 mL of culture medium, they were placed in a carbon dioxide incubator at 37 °C and 5% CO2 concentration and cultured until the cells grew confluently. The culture medium in the culture dish was discarded, and 1 mL of Trizol was added to each well to cover the cell surface. 0.2 mL of chloroform was added to the sample, and it was shaken and mixed well, then left standing for 10 min, and centrifuged at 12,000×g at 4 °C for 15 min. The supernatant was transferred to a new 1.5 mL RNase-free EP tube, an equal volume of isopropanol was added to the supernatant, and it was inverted up and down to mix well, left standing at room temperature for 5 min, and centrifuged at 12,000×g at 4 °C for 15 min. The supernatant was discarded, 0.5 mL of 75% ethanol was added to wash the precipitate, and it was centrifuged at 12,000×g at 4 °C for 5 min. After discarding the supernatant, it was air-dried, 10 μL of DEPC water was added, and the RNA was dissolved at 4 °C. After standing for 30 min, it was vortexed and centrifuged, and 1 μL was taken to measure the concentration. The sample was processed according to the instructions using the Toyobo reverse transcription kit. After incubating at 65 °C for 5 min, it was quickly placed on ice. The amount of sample to be added for 1000 ng of RNA in a 10 μL system was calculated. 2 μL of reverse transcription reagent was added to each group, and double-distilled water was added to make up 10 μL. The reverse transcription program was obtained using a thermal cycler, and the reverse transcription program is shown in Table 6 below. The qPCR experiment was performed using the takara TB Green dye-based quantification kit, and the qPCR program is shown in Table 7 below. The expression levels of the NES, SCX, and THBS4 genes were detected. The 10 μL system included 0.2 μL of cDNA template, 4.6 μL of double-distilled water, 0.2 μL of upstream and downstream primers for the corresponding gene sequence, and 5 μL of 2×TB Green Mix. The primer sequences are shown in Table 8 below. The results were analyzed using 2-ΔΔCt as the relative expression level of the gene. Taking the relative expression level of the gene of tendon-derived mesenchymal stem cells cultured in the fetal bovine serum-containing medium combination 3 as the unit "1", the calculation results are shown in Table 9 below.

[0108] Table 6, Reverse Transcription Program

[0109]

[0110] Table 7, qPCR Program

[0111]

[0112] Table 8, qPCR Primer Sequences

[0113]

[0114] Table 9, Results of Relative Expression Levels of SCX, NES, and THBS4 Genes

[0115]

[0116] According to the data analysis in Table 9, the relative expression levels of SCX, NES, and THBS4 genes in tendon-derived mesenchymal stem cells cultured in serum-free medium combination 1 (the present invention) at the primary and P2 generations are higher than those in tendon-derived mesenchymal stem cells cultured in medium combinations 2-3 at the primary and P2 generations. This indicates that compared with the serum-free medium provided in Example 1 of CN113692282A, the tendon-derived mesenchymal stem cells cultured only at the primary generation with the serum-free medium provided by the present invention can significantly increase the relative expression levels of NES, SCX, and THBS4, especially the SCX gene. Among them, the relative expression levels of NES, SCX, and THBS4 in the stem cells cultured only at the primary generation with combination 1 (the present invention) medium are comparable to those of the P2-generation stem cells cultured with combination 2 medium. The expression of the SCX gene is related to the differentiation of mesenchymal stem cells into cells related to the musculoskeletal system such as tendons and ligaments; the THBS4 gene is involved in regulating the synthesis and deposition of the extracellular matrix, providing a suitable microenvironment for the differentiation of mesenchymal stem cells into tendon stem cells and promoting the differentiation process; while the expression of the NES gene indicates that the mesenchymal stem cells are still in an undifferentiated state and have the ability of self-renewal. The higher relative expression levels of the SCX gene and THBS4 gene in the tendon-derived mesenchymal stem cells cultured at the primary generation with the serum-free medium of the present invention indicate that they have greater potential to differentiate into tendon stem cells. At the same time, the higher relative expression level of the NES gene indicates that the tendon-derived mesenchymal stem cells also have stronger pluripotency and can differentiate into other functional stem cells, such as osteogenic, chondrogenic, and adipogenic differentiation.

[0117] Further, the NES protein expression level in tendon-derived mesenchymal stem cells cultured by primary culture method 1 with the above serum-free medium combination 1 (the present invention) and fetal bovine serum-containing medium combination 3 was detected. The specific method is as follows: Use an eight-chamber culture. Inoculate 1×10 4 cells in each culture well. After adding 200 μL of medium to each well, place it in a carbon dioxide incubator at 37 °C and 5% CO2 concentration for 48 h; remove the medium, wash 3 times with 200 μL of PBS, add 200 μL of 4% paraformaldehyde and fix at room temperature for 20-30 min, and wash 3 times with PBS; add 200 μL of permeabilizing agent and permeabilize for 10 min, and wash 3 times with PBS; add 100 μL of blocking solution and block at room temperature for 30 min; remove the blocking solution, add 50 μL of primary antibody, and incubate overnight at 4 °C in a wet box; warm up at room temperature for 30 min, wash the sample 3 times with PBS, add 100 μL of secondary antibody, incubate at room temperature in the dark for 1 h, and then wash 3 times with PBS; treat with 200 μL of DAPI-containing mounting medium, store at 4 °C in a wet box, and take pictures of the sample using a confocal fluorescence microscope. The detection results are as Figure 6As shown, it is demonstrated that the tendon-derived mesenchymal stem cells cultured in the serum-free medium combination 1 express more NES protein than those cultured in the fetal bovine serum-containing medium combination 3, and have stronger pluripotency, indicating that the mesenchymal stem cells can differentiate into multiple cell types, such as osteoblasts, chondrocytes, adipocytes, etc., under appropriate conditions.

[0118] 3.2 Verification that the tendon-derived mesenchymal stem cells cultured with the serum-free medium combination 1 by the primary culture method 1 have the ability to differentiate into tendon lineage

[0119] Furthermore, to verify whether the tendon-derived mesenchymal stem cells cultured with the above-mentioned serum-free medium combination 1 (the present invention) by the primary culture method 1 can differentiate into tendon stem cells, the specific method is as follows: The suspension of tendon-derived mesenchymal stem cells primary-cultured with the serum-free medium combination 1 was seeded into a 12-well plate at a dose of 5×10 4 cells / well, and 1 mL of complete medium was added to each well. The cells were cultured until they were almost confluent. The medium was discarded in the control group, and the staining step was carried out and photographed for record; the medium was discarded in the experimental group, and it was replaced with tendon lineage induction medium, and the medium was changed every three days. After culturing for about 2 weeks, the medium was aspirated, rinsed once with PBS, fixed with 70% alcohol for 30 min, rinsed three times with double-distilled water, 500 μL of Sirius red staining solution was added for staining for about 15 min, quickly washed twice with double-distilled water, the gross image was scanned, and photographed under a 10× field of view of a microscope, with 3 fields of view in each group, the staining situation was observed, and it was stored with double-distilled water. The staining results are as Figure 7 shown, showing that the tendon-derived mesenchymal stem cells cultured with the serum-free medium combination 1 have deeper Sirius red staining, indicating that the tendon-derived mesenchymal stem cells express a large amount of collagen fibers, suggesting that the induced mesenchymal stem cells have differentiated into tendon stem cells.

[0120] 3.3 Verification that the tendon-derived mesenchymal stem cells cultured with the serum-free medium combination 1 by the primary culture method 1 have the ability to differentiate into three lineages

[0121] It has been verified in the above 3.2 that the tendon-derived mesenchymal stem cells of the serum-free medium combination 1 (the present invention) of the present invention cultured by the primary culture method 1 can differentiate into tendon stem cells. However, the gene detection results in 3.1 prove that the tendon-derived mesenchymal stem cells also have pluripotency. The following experiments will verify whether the tendon-derived mesenchymal stem cells also have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes, and detect the osteogenic, chondrogenic, and adipogenic differentiation abilities of the mesenchymal stem cells according to the following experimental methods.

[0122] 1) The method for detecting osteogenic differentiation ability is as follows: The suspension of tendon-derived mesenchymal stem cells cultured with the serum-free medium combination 1 by the primary culture method 1 was seeded at 1×10 4Seed the cells at a dosage of [number] cells / well in a 24-well plate, and add 0.5 mL of osteogenic induction medium to each well. Incubate for 12 h until the cells adhere to the well surface. For the control group, discard the medium, perform the staining procedure, and take photos for record; for the experimental group, discard the medium and replace it with osteogenic induction medium, and change the medium every three days. For ALP staining, after one week of induction culture, discard the medium, add 500 μL of 4% paraformaldehyde per well, fix for 20 min, wash three times with PBS for 5 min each time, add 500 μL of ALP staining working solution to each well, ensure that the working solution fully covers the sample, incubate at room temperature, observe every 10 minutes. If the experimental group has developed color and there is a significant difference from the control group, stop the staining, wash twice with distilled water to terminate the staining reaction, blot dry the distilled water, and quickly scan the gross image using a scanner; for ARS staining, stop culturing the cells until obvious calcium precipitation appears, which takes about two weeks of induction culture. Discard the medium, add 500 μL of 4% paraformaldehyde per well, fix for 10 min, wash three times with double-distilled water, add 500 μL of 2% ARS solution with pH = 4.2 to each well, incubate at room temperature for at least 30 min, add distilled water to rinse three times, blot dry the distilled water, quickly scan the gross image using a scanner, then add 500 μL of PBS buffer to each well, take photos under a 10× field of view of a microscope, with three fields of view for each group, and observe the staining situation; for DAPI fluorescence staining, add 300 μL of DAPI staining solution (prepared at a ratio of 1:8000) to each well and stain for 20 minutes, then wash three times with double-distilled water for 5 min each time, add 500 μL of double-distilled water to each well, take photos under a 10× field of view of a microscope, with three fields of view for each group, and observe the staining situation.

[0123] The results of ALP staining are as Figure 8 shown, indicating that the tendon-derived mesenchymal stem cells cultured by primary culture method 1 with serum-free medium combination 1 showed deeper ALP staining after induction, suggesting a higher ALP content, indicating that the induced mesenchymal stem cells have differentiated into osteoblasts and may have reached the stage of osteoblast precursor cells or early osteoblasts.

[0124] The results of ARS staining are as Figure 9 shown, indicating that the tendon-derived mesenchymal stem cells cultured by primary culture method 1 with serum-free medium combination 1 showed deeper ARS staining after induction, suggesting a higher calcium salt content, indicating that the induced mesenchymal stem cells have started to differentiate into osteoblasts and the cells are actively undergoing mineralization activities.

[0125] The results of DAPI fluorescence staining are as Figure 10 shown, indicating that the mesenchymal stem cells cultured by primary culture method 1 with serum-free medium combination 1 showed higher DAPI fluorescence intensity after induction, suggesting active proliferation of mesenchymal stem cells.

[0126] 2) The method for detecting the chondrogenic differentiation ability is as follows: The tendon-derived mesenchymal stem cell suspension cultured by the primary culture method 1 with the serum-free medium combination 1 was seeded into a 12-well plate at a dose of 2.5×10 5 cells / 10 μL / well. Pay attention to keeping the humidity. After culturing for 3 - 4 h, the cells adhered to the wall. The negative control group was directly stained. 1 mL of chondrogenic induction medium was added to each well of the experimental group. The medium was changed every 3 days. After culturing for 14 days, the induction medium was discarded, and Alcian blue or Safranin O was used for staining and photographing. For Alcian blue staining, after culturing for 14 days, the medium was discarded, and the cells were rinsed 3 times with PBS buffer for 5 min each time, fixed with 1 mL of 4% paraformaldehyde at room temperature for 30 min, rinsed 3 times with PBS, rinsed with 1 mL of 0.1 N HCl solution for 5 min, incubated with 1 mL of Alcian blue staining solution overnight, rinsed 3 times with 0.1 N HCL for 5 min each time to remove the background, the gross image was scanned, and photographed under a microscope at 10× magnification. The staining situation was observed in 3 fields of view for each group; For Safranin O staining, after culturing for 14 days, the medium was discarded, and the cells were rinsed 3 times with PBS buffer for 5 min each time, fixed with 1 mL of 4% paraformaldehyde at room temperature for 30 min, rinsed 3 times with PBS, stained with 1 mL of Safranin O staining solution for 3 min, decolorized with 95% ethanol, and photographed under a microscope.

[0127] The results of Alcian blue staining are as Figure 11 shown, showing that the tendon-derived mesenchymal stem cells cultured by the primary culture method 1 with the serum-free medium combination 1 had deeper Alcian blue staining after induction, indicating that the content of acidic mucopolysaccharides, glycosaminoglycans and other components in the intracellular or extracellular matrix of the induced mesenchymal stem cells was higher, indicating that the mesenchymal stem cells had differentiated into chondrocytes.

[0128] The results of Safranin O staining are as Figure 12 shown, showing that the tendon-derived mesenchymal stem cells cultured by the primary culture method 1 with the serum-free medium combination 1 had deeper Alcian blue staining after induction, indicating that the content of acidic glycosaminoglycans such as proteoglycans in the induced mesenchymal stem cells was higher, indicating that the mesenchymal stem cells had differentiated into chondrocytes.

[0129] 3) The method for detecting the adipogenic differentiation ability is as follows: The tendon-derived mesenchymal stem cell suspension cultured by the primary culture method 1 with the serum-free medium combination 1 was seeded into a 12-well plate at a dose of 5×10 4Seed the cells at a dose of [number] per well in a 24-well plate, and add 0.5 mL of adipogenic induction medium to each well. Incubate for 12 h until the cells adhere to the wall. For the control group, discard the medium, perform the staining step, and take pictures for record; for the experimental group, discard the medium and replace it with adipogenic induction medium, and change the medium every three days. Culture for about 2 weeks until obvious lipid droplets appear and then stop the culture. If there are no obvious lipid droplets, the culture can be continued for 3 weeks before staining and taking pictures. The preparation of Oil Red O staining working solution is as follows: Mix the Oil Red O stock solution and double-distilled water at a ratio of 3:2, filter with filter paper, and let it stand at room temperature for 10 min. Use it up within a few hours after dilution. The steps of Oil Red O staining are as follows: For the cells in the experimental group, aspirate the medium, rinse once with PBS, use 500 μL / well of 4% paraformaldehyde to fix for 30 min, then rinse twice with PBS, add 500 μL of Oil Red O staining working solution to stain for about 30 min, observe in real time, quickly decolorize with 75% alcohol, and add 500 μL of PBS. Take pictures under a 20× field of view of the microscope, with 3 fields for each group, and observe the staining situation.

[0130] The results of Oil Red O staining are as Figure 13 shown, indicating that the tendon-derived mesenchymal stem cells cultured by the primary culture method 1 with the serum-free medium combination 1 showed deeper Oil Red O staining after induction, suggesting that a large amount of lipids were synthesized and accumulated in the induced mesenchymal stem cells, indicating that the mesenchymal stem cells had differentiated into adipocytes.

[0131] 3.4 Detect the tendon lineage differentiation ability and trilineage differentiation ability of tendon-derived mesenchymal stem cells cultured with serum-free medium combination 2 at the primary and P2 generations

[0132] 1) Detect the tendon lineage differentiation ability of tendon-derived mesenchymal stem cells cultured with serum-free medium combination 2 at the primary and P2 generations respectively, and the detection method refers to the method described in 3.2 of this example. The results of Sirius red staining showed that the staining of tendon-derived mesenchymal stem cells in primary culture was lighter than that of tendon-derived mesenchymal stem cells in P2 generation culture, indicating that tendon-derived mesenchymal stem cells in primary culture did not express a large amount of collagen fibers after induction and had a poor differentiation effect into tendon stem cells. However, the Sirius red staining of the cells in P2 generation culture after induction was not as deep as that of the cells in primary culture with serum-free medium combination 1 in 3.2.

[0133] 2) Detect the osteogenic differentiation ability of tendon-derived mesenchymal stem cells cultured in serum-free medium combination 2 at the primary and P2 generations respectively. The detection method refers to the method described in 3.3 of this example. The results of ALP, ARS, and DAPI fluorescence staining showed that the staining of tendon-derived mesenchymal stem cells cultured at the primary generation was lighter than that of tendon-derived mesenchymal stem cells cultured at the P2 generation, indicating that the tendon-derived mesenchymal stem cells cultured at the primary generation had a poor effect of differentiating into osteoblasts after induction, the calcium salt content in the cells was low after induction, and the cell proliferation was not active. At the same time, the ALP, ARS, and DAPI fluorescence staining of the P2-generation cultured cells after induction was also lighter than that of the primary-cultured cells of serum-free medium combination 1 in 3.3.

[0134] 3) Detect the chondrogenic differentiation ability of tendon-derived mesenchymal stem cells cultured in serum-free medium combination 2 at the primary and P2 generations respectively. The detection method refers to the method described in 3.3 of this example. The results of Alcian blue and safranin O staining showed that the staining of tendon-derived mesenchymal stem cells cultured at the primary generation was lighter than that of tendon-derived mesenchymal stem cells cultured at the P2 generation, indicating that the content of acidic mucopolysaccharides, glycosaminoglycans, and other components such as proteoglycans in the intracellular or extracellular matrix of tendon-derived mesenchymal stem cells cultured at the primary generation was relatively low after induction, indicating that the tendon-derived mesenchymal stem cells cultured at the primary generation had a poor ability to differentiate into chondrocytes. At the same time, the Alcian blue and safranin O staining of the P2-generation cultured cells after induction was also lighter than that of the primary-cultured cells of serum-free medium combination 1 in 3.3.

[0135] 4) Detect the adipogenic differentiation ability of tendon-derived mesenchymal stem cells cultured in serum-free medium combination 2 at the primary and P2 generations respectively. The detection method refers to the method described in 3.3 of this example. The results of Oil Red O staining showed that the staining of tendon-derived mesenchymal stem cells cultured at the primary generation was lighter than that of tendon-derived mesenchymal stem cells cultured at the P2 generation, indicating that only a small amount of lipids were synthesized and accumulated in the cells of tendon-derived mesenchymal stem cells cultured at the primary generation after induction, indicating that the tendon-derived mesenchymal stem cells cultured at the primary generation had a poor ability to differentiate into adipocytes. At the same time, the Oil Red O staining of the P2-generation cultured cells after induction was also lighter than that of the primary-cultured cells of serum-free medium combination 1 in 3.3.

[0136] The above experimental results prove that directly using the tendon-derived mesenchymal stem cells primary cultured in the culture medium combination 2 provided in CN113692282A for tendon lineage differentiation and trilineage differentiation will result in poor cell differentiation effects, and subculture is necessary to improve the differentiation effects of tendon-derived mesenchymal stem cells. However, the culture medium combination 1 provided by the present invention can achieve and exceed the tendon lineage differentiation effect and trilineage differentiation effect that can only be achieved after inducing the P2 generation of tendon-derived mesenchymal stem cells cultured in the culture medium combination 2 by only inducing the tendon-derived mesenchymal stem cells in primary culture, and the tendon-derived mesenchymal stem cells are successfully differentiated into tendon stem cells, osteoblasts, chondrocytes and adipocytes after being induced by different induction culture media.

[0137] Example 2. Effect of B-27 serum-free additive on culturing mesenchymal stem cells in serum-free medium

[0138] This example explores the effect of adding B-27 serum-free additive to the serum-free culture medium combination 1 in Example 1 on the proliferation ability and differentiation ability of tendon-derived mesenchymal stem cells in primary culture, and makes a comparison with the serum-free culture medium in CN113692282A previously applied by the research team of the present invention.

[0139] Set up the following five groups of culture media as shown in Table 10 below.

[0140] Table 10. Serum-free culture medium combinations with or without B-27 serum-free additive

[0141]

[0142] The above culture medium combinations 1-2 and 5 were used to culture tendon-derived mesenchymal stem cells according to the primary culture method 1 in Example 1, and the proliferation ability and differentiation ability of tendon-derived mesenchymal stem cells in primary culture were detected. The detection method refers to Example 1; the culture medium combinations 3-4 were first used to culture tendon-derived mesenchymal stem cells according to the primary culture method 1 in Example 1, and then subcultured to the P2 generation, and the proliferation ability and differentiation ability of the P2 generation of tendon-derived mesenchymal stem cells were detected. The detection method refers to Example 1.

[0143] Figure 14 It is the growth morphology of stem cells when the tendon-derived mesenchymal stem cells are primary cultured in the culture medium of combination 1 (the present invention) until harvested at 14 days. Figure 15 It is the growth morphology of stem cells when the tendon-derived mesenchymal stem cells are primary cultured in the culture medium of combination 2 until harvested at 14 days. In comparison, the stem cells cultured in combination 1 (the present invention) are evenly distributed and have a good growth morphology, while the addition of 1×B-27 serum-free additive in the culture medium of combination 2 causes local over-aggregation or sparsity of the cultured stem cells, and the cell growth morphology is not good. Figure 16Growth morphology of tendon-derived mesenchymal stem cells cultured in Medium 3 at the time of harvest at passage P2 Figure 17 Growth morphology of tendon-derived mesenchymal stem cells cultured in Medium 4 at the time of harvest at passage P2. In comparison, there is no significant difference in the growth morphology of stem cells cultured in the two media, but the stem cells cultured in Medium 4 are sparser than those in Medium 3 due to the lack of 1× B-27 serum-free additive Figure 18 Growth morphology of tendon-derived mesenchymal stem cells at the time of harvest after 14 days of primary culture in Medium 5 containing fetal bovine serum, as a control

[0144] The detection results of the proliferation number and cell viability of tendon-derived mesenchymal stem cells are shown in Table 11 below, the detection results of immunophenotype are shown in Table 12 below, and the detection results of the relative expression levels of SCX, NES, and THBS4 genes are shown in Table 13 below. The expression levels of the three genes of tendon-derived mesenchymal stem cells cultured in Medium 5 with fetal bovine serum are used as the unit "1".

[0145] Table 11. Effect of the presence or absence of B-27 serum-free additive in serum-free medium on stem cell proliferation

[0146]

[0147] Table 12. Detection results of immunophenotype of stem cells cultured in serum-free medium with or without B-27 serum-free additive

[0148]

[0149] Table 13. Detection results of relative expression levels of genes of stem cells cultured in serum-free medium with or without B-27 serum-free additive

[0150]

[0151] According to the results analysis in Table 11, there is a significant difference in the cell numbers of tendon-derived mesenchymal stem cells at passage P2 cultured in the two groups of serum-free media, Medium 3 and Medium 4. The absence of 1× B-27 serum-free additive in Medium 4 significantly reduces the number of cultured stem cells ( Figures 16 - 17 ), indicating that the serum-free medium provided in Example 1 of the previously filed CN113692282A by the present invention team must be added with 1× B-27 serum-free additive to improve the proliferation effect of tendon-derived mesenchymal stem cells during passage; while comparing the cell numbers of tendon-derived mesenchymal stem cells cultured in the two groups of serum-free media, Medium 1 and Medium 2, the number of stem cells cultured in Medium 1 (the present invention) is higher than that in Medium 2 ( Figures 14 - 15), indicating that adding 1×B-27 serum-free additive in combination 1 inhibited the primary proliferation of tendon-derived mesenchymal stem cells. It is speculated that there are overlaps between various growth factors and hormones in the 1×B-27 serum-free additive and certain components in the combination 1 medium, resulting in too high concentrations of certain components, nutrient imbalance, triggering cell metabolic disorders, and thus inhibiting the normal growth of cells. The number of cells cultured in the serum-free medium of combination 1 (the present invention) was significantly higher than that in combinations 3 and 4, indicating that the serum-free medium provided by the present invention, compared with the serum-free medium provided in Example 1 of the previous team of the present invention in CN113692282A, not only does not require the addition of 1×B-27 serum-free additive, but also can make the number of tendon-derived mesenchymal stem cells as high as 5.7×10 7 , exceeding the proliferation effect (1.9×10 7 ) that could only be achieved by culturing the previous medium to passage 2 (P2). The B-27 serum-free additive is expensive, and the serum-free medium provided by the present invention can reduce the cost of culturing tendon-derived mesenchymal stem cells.

[0152] According to the results analysis in Table 12, it shows that the tendon-derived mesenchymal stem cells cultured in the serum-free medium combinations 1-4 all meet the immunophenotype requirements of mesenchymal stem cells and comply with the safety indicators; and the detection results of mesenchymal stem cell surface antigens show that the expression levels of CD73, CD105, and CD90 are all ≥95%, and the expression levels of CD45, CD34, CD14, CD19, and HLA-DR are all ≤2%, indicating that the cells cultured in these four groups meet the mesenchymal stem cell phenotype and have the potential to differentiate into tendon stem cells.

[0153] According to the result analysis of Table 13, the relative expression levels of SCX, NES, and THBS4 genes in tendon-derived mesenchymal stem cells of passage 2 cultured in the serum-free medium of combination 4 were significantly lower than those in combination 3, indicating that adding 1× B-27 serum-free additive to the serum-free medium provided in Example 1 of the previously applied CN113692282A by the research team of the present invention can improve the differentiation ability of tendon-derived mesenchymal stem cells; while there was no significant difference in the relative expression levels of SCX, NES, and THBS4 genes in tendon-derived mesenchymal stem cells cultured in the primary culture of the serum-free medium of combination 1 (the present invention) and combination 2, indicating that adding 1× B-27 serum-free additive to the serum-free medium provided in the present invention only affects the amplification efficiency of primary cultured stem cells, but does not affect the relative expression levels of the three genes. The relative expression levels of SCX, NES, and THBS4 genes in primary tendon-derived mesenchymal stem cells cultured in the serum-free medium of combination 1 (the present invention) were comparable to those in tendon-derived mesenchymal stem cells of passage 2 cultured in the serum-free medium of combination 3, indicating that the serum-free medium provided in the present invention can achieve the relative expression levels of the three genes in passage 2 cells cultured in the serum-free medium provided in Example 1 of CN113692282A without adding 1× B-27 serum-free additive, further improving the tendon lineage differentiation ability and pluripotency of primary cultured tendon-derived mesenchymal stem cells.

[0154] Example 3. Effects of Different Basic Media on the Proliferation and Differentiation Abilities of Mesenchymal Stem Cells

[0155] 1. Effects of Serum-Free Media Containing Different Basic Media on the Proliferation Ability of Mesenchymal Stem Cells

[0156] The basic medium used in the serum-free medium combination 1 in Example 1 was the Serum-Free Medium Pro for Mesenchymal Stem Cells of Zhongke Ruiji. In this example, the Serum-Free Medium Pro for Mesenchymal Stem Cells of Zhongke Ruiji was replaced with different commercial serum-free media, and the effects of different media on the proliferation of tendon-derived mesenchymal stem cells were detected. A medium containing fetal bovine serum was used as a control, and the following 5 groups were set up:

[0157] Group 1: Serum-Free Medium Pro for Mesenchymal Stem Cells of Zhongke Ruiji (Zhongke Ruiji RGM1051);

[0158] Group 2: Serum-Free Medium MAX for Mesenchymal Stem Cells of Zhongke Ruiji (Zhongke Ruiji RGM1071);

[0159] Group 3: Serum-Free Medium for 3D FloTrix Mesenchymal Stem Cells of Huakan (Huakan Biology RMZ112-PYJ);

[0160] Group 4: Serum-Free Medium for Mesenchymal Stem Cells of Youkang (Youkang Biology NC0106);

[0161] Group 5: Fetal bovine serum-containing medium.

[0162] The effects of the above 5 groups of media on the proliferation ability of tendon-derived mesenchymal stem cells were detected. The primary culture method of mesenchymal stem cells referred to the primary culture method 1 in Example 1, and the method for detecting the cell number referred to Example 1. After harvesting on the 14th day of culture, the cell number and cell viability of the cells cultured in each medium were detected, and the results are shown in Table 14 below.

[0163] Table 14. Effects of different basal media on the primary culture of tendon-derived mesenchymal stem cells

[0164]

[0165] According to the data analysis in Table 14, the serum-free media containing different basal media in Groups 1-3 had stronger proliferation ability than the fetal bovine serum-containing medium in Group 5 for tendon-derived mesenchymal stem cells, while the proliferation effect of the medium in Group 4 was inferior to that of the medium in Group 5. Comparing the cell numbers after culturing in the media of Groups 1-3 for 14 days, Group 1 (Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro) had the strongest cell proliferation effect and was the preferred one. The basic components for maintaining cell growth in the above four commercial mesenchymal stem cell media should be similar, but the unknown components added in Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Medium Pro made its primary culture effect significantly better than the other three commercial media. It is speculated that substances such as human albumin and human platelet lysate may be added to the serum-free medium to improve the cell amplification effect.

[0166] 2. Effects of serum-free media containing different basal media on the differentiation ability of tendon-derived mesenchymal stem cells

[0167] Furthermore, the tendon-derived mesenchymal stem cells cultured in the 5 groups of media in Table 14 above were entrusted to a testing company for flow cytometry detection of the surface markers of the cultured mesenchymal stem cells. The detection results are shown in Table 15 below, and the reference intervals for the detection indicators are as shown in Table 5 in Example 1.

[0168] Table 15. Detection results of the immunophenotype of mesenchymal stem cells cultured in serum-free media containing different basal media

[0169]

[0170] According to the data analysis of Table 15, the tendon-derived mesenchymal stem cells cultured in the serum-free media of Groups 1-4 all meet the immunophenotype requirements of mesenchymal stem cells and comply with the safety indicators. CD73 is highly expressed on the surface of mesenchymal stem cells. CD105 and CD90 are highly expressed on the surfaces of both mesenchymal stem cells and tendon stem cells. CD14, CD19, and HLA-DR are lowly expressed or not expressed on the surface of mesenchymal stem cells. CD45 and CD34 are lowly expressed or not expressed on the surface of tendon stem cells. The detection results of the surface antigens of the tendon-derived mesenchymal stem cells cultured in the serum-free media of Groups 1-4 show that the expression levels of CD73, CD105, and CD90 are ≥95%, and the expression levels of CD45, CD34, CD14, CD19, and HLA-DR are ≤2%. This indicates that the tendon-derived cells cultured in the media of Groups 1-4 conform to the mesenchymal stem cell phenotype and have great potential to differentiate into tendon stem cells.

[0171] Furthermore, the tendon-derived mesenchymal stem cells cultured in these 5 groups of media were respectively detected for the expression of the tendon-related gene SCX and the expression of the NES and THBS4 genes. The detection method refers to Example 1. The relative expression levels of the three genes are shown in Table 16 below.

[0172] Table 16. Effects of Serum-Free Media Containing Different Basic Media on the Relative Gene Expression Levels of Mesenchymal Stem Cells

[0173]

[0174] According to the data analysis of Table 16, the relative expression levels of the SCX, NES, and THBS4 genes of the tendon-derived mesenchymal stem cells cultured in the serum-free media of Groups 1-4 containing different basic media are all higher than those of Group 5 with fetal bovine serum-containing medium. This indicates that the tendon-derived mesenchymal stem cells cultured in the media of Groups 1-4 all have the ability of tendon lineage differentiation and pluripotency. Among them, the relative expression levels of these three genes of the stem cells cultured in the medium of Group 1 are significantly higher than those of the other three groups, indicating that the tendon lineage differentiation ability and pluripotency of the stem cells cultured in the medium of Group 1 are stronger. Therefore, the preferred basic medium is the Serum-Free Medium Pro for Zhongke Ruiji Mesenchymal Stem Cells.

[0175] Example 4. Screening of Bioactive Substances

[0176] 1. Effects of Serum-Free Media Containing Different Bioactive Substances on the Culture of Mesenchymal Stem Cells

[0177] In this example, the serum-free medium combination 1 of Example 1 was used as the experimental object, and the types of its five bioactive substances were changed to explore the effects of different bioactive substances on the proliferation and differentiation abilities of tendon-derived mesenchymal stem cells. A fetal bovine serum-containing medium was used as a control, and 18 groups of media were set up as shown in Table 17 below.

[0178] Table 17. Serum-free media containing different bioactive substances

[0179]

[0180]

[0181] The above 18 groups of serum-free media were used for primary culture of tendon-derived mesenchymal stem cells, and cell proliferation was detected. The culture method referred to the first primary culture method in Example 1, and the detection method referred to Example 1. The detection results are shown in Table 18 below.

[0182] Table 18. Effects of serum-free media containing different bioactive substances on the proliferation ability of primary cultured mesenchymal stem cells

[0183]

[0184] According to the results analysis in Table 18, comparing the effects of serum-free media groups 1-7 on the primary culture of tendon-derived mesenchymal stem cells, in serum-free media groups 2-7, one bioactive substance in serum-free media group 1 was replaced respectively. The results showed that there was no significant difference in the cell number and cell viability of stem cells cultured in serum-free media groups 2-6 compared with serum-free media group 1, but the cell number of stem cells cultured in serum-free media group 1 was the highest.

[0185] Comparing the effects of serum-free media group 1 and serum-free media groups 8-12 on the primary culture of tendon-derived mesenchymal stem cells, it was shown that adding two platelet-derived growth factors, two fibroblast growth factors or two transforming growth factor-β to the serum-free media all had good proliferation effects on the primary culture of tendon-derived mesenchymal stem cells.

[0186] Comparing the effects of serum-free media group 1 and serum-free media groups 13-17 on the primary culture of tendon-derived mesenchymal stem cells, in serum-free media groups 13-17, one bioactive substance in serum-free media group 1 was missing respectively. The results showed that the cell number of stem cells cultured in serum-free media groups 13-17 decreased significantly compared with serum-free media group 1, indicating that the lack of any one of the five bioactive substances would lead to a decrease in the proliferation effect of primary cultured mesenchymal stem cells.

[0187] Furthermore, the immunophenotypes of tendon-derived mesenchymal stem cells primary cultured in 18 groups of media were detected. The detection method referred to Example 1. The immunophenotype detection results are shown in Table 19 below.

[0188] Table 19. Immunophenotype detection results of mesenchymal stem cells cultured in serum-free media containing different bioactive substances

[0189]

[0190] According to the data analysis in Table 19, the tendon-derived mesenchymal stem cells cultured in the serum-free medium of groups 1-17 all met the immunophenotype requirements of mesenchymal stem cells and conformed to the safety indicators. The detection results of the surface antigens of the mesenchymal stem cells cultured in the serum-free medium of groups 1-17 showed that the expression levels of CD73, CD105, and CD90 were all ≥95%, and the expression levels of CD45, CD34, CD14, CD19, and HLA-DR were all ≤2%, indicating that the cells cultured in the serum-free medium of groups 1-17 conformed to the mesenchymal stem cell phenotype and had good potential to differentiate into tendon stem cells.

[0191] Furthermore, the relative expression levels of SCX, NES, and THBS4 genes of the tendon-derived mesenchymal stem cells cultured in the primary culture of the 18 groups of serum-free medium were detected. The detection method referred to Example 1, and the detection results are shown in Table 20 below.

[0192] Table 20. Detection results of the relative expression levels of genes of mesenchymal stem cells cultured in serum-free medium containing different bioactive substances

[0193]

[0194] According to the data analysis in Table 20, by comparing the relative expression levels of SCX, THBS4, and NES genes of the tendon-derived mesenchymal stem cells cultured in the primary culture of the serum-free medium of group 1 and groups 13-17, the results showed that the relative expression levels of SCX, THBS4, and NES genes of the mesenchymal stem cells cultured in the primary culture of the serum-free medium of groups 13-17 decreased compared with those of group 1. Among them, the relative expression level of the SCX gene decreased most significantly, indicating that the lack of any one of the five bioactive substances in the serum-free medium of group 1 would lead to a decrease in the ability of the cultured mesenchymal stem cells to differentiate into tendon stem cells and pluripotency; by comparing the relative expression levels of SCX, THBS4, and NES genes of the tendon-derived mesenchymal stem cells cultured in the primary culture of the serum-free medium of group 1 and groups 2-7 and group 1 and groups 8-12, the relative expression levels of the three genes of the mesenchymal stem cells cultured in group 1 were the highest, indicating that the stem cells had stronger tendon lineage differentiation ability and pluripotency and were preferred; the relatively low expression level of the SCX gene in groups 10-12 may be because TGF-β1, TGF-β2, and TGF-β3 have similar functions and some of their receptors are shared. Therefore, when two of the three transforming growth factors are added to the medium, receptor competition and signal pathway antagonism may occur between the two transforming growth factors, resulting in a decrease in the relative expression level of the SCX gene.

[0195] 2. Effects of serum-free medium containing different concentrations of bioactive substances on the culture of mesenchymal stem cells

[0196] Further, the serum-free medium group 1 in the preferred solution in 1 was used as the experimental subject, and the concentrations of its five bioactive substances were changed to explore the effects of different concentrations on the proliferation and differentiation abilities of tendon-derived mesenchymal stem cells.

[0197] The following 22 different groups were set up as shown in Table 21 below.

[0198] Table 21. Serum-free medium containing bioactive substances at different concentrations

[0199]

[0200] 2.1 Effects of the concentration of bioactive substances on the proliferation ability of tendon-derived mesenchymal stem cells in primary culture

[0201] The 22 groups of media in Table 21 above were used for the primary culture of tendon-derived mesenchymal stem cells, and the proliferation ability of the cultured tendon-derived mesenchymal stem cells was detected. The culture method referred to the primary culture method 1 in Example 1, and the detection method referred to Example 1. The detection results are shown in Table 22 below.

[0202] Table 22. Effects of serum-free media containing bioactive substances at different concentrations on the primary culture proliferation of mesenchymal stem cells

[0203]

[0204] According to the data analysis in Table 22, the serum-free media containing bioactive substances at different concentrations in groups 1-21 were used for the primary culture of tendon-derived mesenchymal stem cells, and the cell number reached 1×10 7 or more and the cell viability was high, all above 90%, indicating that tendon-derived mesenchymal stem cells had good proliferation ability when cultured in the serum-free media containing bioactive substances at different concentrations in groups 1-21.

[0205] Further, the above vitamin C was replaced with magnesium L-ascorbate 2-phosphate sesquihydrate, heparin was replaced with heparin sodium, PDGF-BB was replaced with PDGF-AA or a combination of PDGF-BB and PDGF-AA, FGF-basic was replaced with FGF-7 or a combination of FGF-basic and FGF-7, and TGF-β3 was replaced with TGF-β1 or TGF-β2 to prepare the serum-free media at the above different concentrations. The tendon-derived mesenchymal stem cells cultured all had good proliferation ability, and the number of primary cultured cells could reach 1×10 7And maintain a high cell viability rate. Therefore, the final added concentration of vitamin C or its derivatives is 0.1 - 100 μg / ml, the final added concentration of heparin or its salt is 0.1 - 10 μg / ml, the final added concentration of transforming growth factor-β is 0.1 - 80 ng / ml, the final added concentration of fibroblast growth factor is 1 - 100 ng / ml, and the final added concentration of platelet-derived growth factor is 1 - 100 ng / ml.

[0206] 2.2 Effects of serum-free media containing different concentrations of bioactive substances on the differentiation ability of mesenchymal stem cells

[0207] Detect the immunophenotypes of tendon-derived mesenchymal stem cells primary cultured in 22 groups of serum-free media in Table 21. The detection method refers to Example 1, and the immunophenotype detection results are shown in Table 23 below.

[0208] Table 23. Detection results of immunophenotypes of mesenchymal stem cells cultured in serum-free media containing different concentrations of bioactive substances

[0209]

[0210] According to the data analysis in Table 23, the tendon-derived mesenchymal stem cells cultured in the serum-free media of groups 1 - 21 all meet the immunophenotype requirements of mesenchymal stem cells and comply with the safety indicators. The surface antigen detection results of the mesenchymal stem cells cultured in the serum-free media of groups 1 - 21 show that the expression levels of CD73, CD105, and CD90 are all ≥ 95%, and the expression levels of CD45, CD34, CD14, CD19, and HLA-DR are all ≤ 2%, indicating that the cells cultured in the serum-free media of groups 1 - 21 conform to the mesenchymal stem cell phenotype and have good potential to differentiate into tendon stem cells.

[0211] Furthermore, detect the relative expression levels of SCX, NES, and THBS4 genes of tendon-derived mesenchymal stem cells primary cultured in 22 groups of serum-free media. The detection method refers to Example 1, and the detection results of the relative expression levels of the three genes are shown in Table 24 below.

[0212] Table 24. Effects of serum-free media containing different concentrations of bioactive substances on the relative expression levels of genes in mesenchymal stem cells

[0213]

[0214] According to the result analysis of Table 24, by comparing the results of Group 1 with those of Groups 2-5, Groups 6-9, Groups 10-13, Groups 14-17, and Groups 18-21 respectively, the relative expression levels of the three genes SCX, THBS4, and NES are all very high. Among them, the expression levels of the SCX and NES genes in the medium of Group 1 are the highest. The above results indicate that when the final added concentration of vitamin C or its derivatives is in the range of 0.1-100 μg / ml, the final added concentration of heparin or its salts is in the range of 0.1-10 μg / ml, the final added concentration of transforming growth factor-β is in the range of 0.1-80 ng / ml, the final added concentration of fibroblast growth factor is in the range of 1-100 ng / ml, and the final added concentration of platelet-derived growth factor is in the range of 1-100 ng / ml, the relative expression levels of the three genes of tendon-derived mesenchymal stem cells can be significantly increased, and the stem cells have good tendon lineage differentiation ability and pluripotency.

[0215] Furthermore, when the above-mentioned vitamin C is replaced with L-ascorbic acid 2-phosphate sesquimagnesium hydrate, heparin is replaced with heparin sodium, PDGF-BB is replaced with PDGF-AA or a combination of PDGF-BB and PDGF-AA, FGF-basic is replaced with FGF-7 or a combination of FGF-basic and FGF-7, and TGF-β3 is replaced with TGF-β1 or TGF-β2, and ser-free media with the above different concentrations are formulated, the tendon-derived mesenchymal stem cells cultured all have good tendon lineage differentiation ability and pluripotency.

[0216] Example 5. Effects of Ser-free Medium on Primary Culture, Proliferation and Differentiation of Umbilical Cord-derived Mesenchymal Stem Cells

[0217] In Example 1, it was proved that the ser-free medium combination 1 provided by the present invention is particularly suitable for the primary culture of tendon-derived mesenchymal stem cells, which can not only improve the proliferation effect of primary culture of tendon-derived mesenchymal stem cells, but also improve their tendon differentiation ability and trilineage differentiation ability. This example explores whether the ser-free medium combination 1 can affect the primary culture proliferation effect and differentiation ability of mesenchymal stem cells from other sources. The specific method is as follows:

[0218] The umbilical cord was cultured with reference to the primary culture method 1 of Example 1 and using the ser-free medium combination 1 of Example 1, and the fetal bovine serum-containing medium of combination 3 in Example 1 was used as a control. When the cells were harvested, the proliferation effect, immunophenotype, and relative expression levels of the three genes SCX, THBS4, and NES of the cultured umbilical cord-derived mesenchymal stem cells were detected, and the detection methods refer to Example 1. The growth morphology of the umbilical cord-derived mesenchymal stem cells cultured with the ser-free medium of combination 1 in Example 1 on the fourth day of culture was as Figure 19As shown, it is shown that the cultured umbilical cord-derived mesenchymal stem cells can maintain good adherent properties, have abundant cytoplasm, present a spindle-shaped cell morphology, and have a dense cell population; the growth morphology of the umbilical cord-derived mesenchymal stem cells cultured in the medium containing fetal bovine serum on the sixth day of culture is as Figure 20 shown, showing that although the cultured umbilical cord-derived mesenchymal stem cells can also maintain good adherent properties and present a spindle-shaped cell morphology, the cell population is sparse. The detection results of the cell number and viability of the umbilical cord-derived mesenchymal stem cells at the time of harvest are shown in Table 25 below, and the immunophenotype detection results are shown in Table 26 below. The detection results of the relative expression levels of the three genes SCX, THBS4, and NES are shown in Table 27 below.

[0219] Table 25. Proliferation effects of umbilical cord-derived mesenchymal stem cells by primary culture in different media

[0220]

[0221] According to the data analysis in Table 25, the use of the serum-free medium provided by the present invention for primary culture of umbilical cord-derived mesenchymal stem cells can also make the cell number reach 1×10 7 , and the cell viability is high. However, compared with the cell number of the tendon-derived mesenchymal stem cells cultured in Example 1, it shows that the serum-free medium has a better proliferation effect on culturing tendon-derived mesenchymal stem cells.

[0222] Table 26. Immunophenotypes of umbilical cord-derived mesenchymal stem cells by primary culture in different media

[0223]

[0224] According to the data analysis in Table 26, the umbilical cord-derived mesenchymal stem cells primary cultured using the serum-free medium provided by the present invention all meet the immunophenotype requirements of mesenchymal stem cells and meet the safety indicators.

[0225] Table 27. Relative expression levels of three genes of umbilical cord-derived mesenchymal stem cells by primary culture in different media

[0226]

[0227] According to the data analysis in Table 27, the relative expression level of NES in umbilical cord-derived mesenchymal stem cells (UC-MSCs) primary cultured with the serum-free medium provided by the present invention is significantly higher than that of SCX and THBS4, indicating that the umbilical cord-derived mesenchymal stem cells have high self-renewal ability and multi-directional differentiation potential. However, comparing with the relative expression level data of SCX in tendon-derived mesenchymal stem cells (TD-MSCs) primary cultured with the above serum-free medium in Example 1, the relative expression level of SCX in the cultured umbilical cord-derived mesenchymal stem cells is significantly lower than that in tendon-derived mesenchymal stem cells, indicating that the cultured umbilical cord-derived mesenchymal stem cells have poor tendon lineage differentiation ability, which shows that the serum-free medium provided by the present invention is more suitable for the primary culture of tendon-derived mesenchymal stem cells and can significantly improve their tendon lineage differentiation ability.

[0228] In summary, the serum-free medium provided by the present invention can also be used for the primary culture of umbilical cord-derived mesenchymal stem cells to improve the proliferation effect and differentiation ability of primary culture.

[0229] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A serum-free medium, characterized in that, It includes bioactive substances and a basal medium; the bioactive substances include any one or more of vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor; the serum-free medium does not contain B-27 serum-free additive.

2. The serum-free medium according to claim 1, wherein The vitamin C or its derivatives include any one or more of vitamin C, ascorbyl glucoside, ethyl vitamin C, 3-o-ethyl ascorbic acid, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, L-ascorbic acid 2-phosphate sesquimagnesium hydrate, tetrahexyldecyl ascorbate, ascorbyl palmitate, L-ascorbic acid-2-phosphate-6-palmitate, esterified vitamin C, and solvates of ascorbic acid; the heparin or its salts include any one or more of heparin, sodium heparin, calcium heparin, and heparan sulfate; the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, TGF-β3, and synthetic peptides of transforming growth factor-β.

3. The serum-free medium according to claim 2, wherein The fibroblast growth factor includes any one or more of FGF-basic, FGF-1, FGF-4, FGF-7, FGF-10, FGF-18, and synthetic peptides of fibroblast growth factor; the platelet-derived growth factor includes platelet growth factor and vascular endothelial cell factor; the platelet growth factor includes any one or more of PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, PDGF-DD, placental growth factor, VEGF-41, VEGF-B, VEGF-C, VEGF-D, and synthetic peptides of platelet-derived growth factor.

4. The serum-free medium according to claim 3, wherein, The vitamin C or its derivatives are vitamin C or L-ascorbic acid 2-phosphate sesquimagnesium hydrate; the heparin or its salts are heparin or sodium heparin; the transforming growth factor-β is any one or more of TGF-β1, TGF-β2, and TGF-β3; the fibroblast growth factor is any one or more of FGF-basic and FGF-7; the platelet-derived growth factor is any one or more of PDGF-AA and PDGF-BB.

5. The serum-free medium according to claim 4, wherein, The mass ratio of the bioactive substances to the basal medium is (0.302 - 190.2):100000000.

6. The serum-free medium according to claim 5, wherein The final added concentration of the vitamin C or its derivatives is 0.1 - 100 μg / ml, the final added concentration of the heparin or its salts is 0.1 - 10 μg / ml, the final added concentration of the transforming growth factor-β is 0.1 - 80 ng / ml, the final added concentration of the fibroblast growth factor is 1 - 100 ng / ml, and the final added concentration of the platelet-derived growth factor is 1 - 100 ng / ml.

7. A method for culturing mesenchymal stem cells, characterized in that, Use the serum-free medium according to any one of claims 1 to 6 to culture mesenchymal stem cells.

8. Use of a composition of bioactive substances for preparing a culture medium for improving the primary culture effect of mesenchymal stem cells, characterized in that, The culture medium does not contain B-27 serum-free additive; the bioactive substance composition includes vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor; the vitamin C or its derivatives include any one of vitamin C and L-ascorbic acid 2-phosphate sesquimagnesium hydrate, the heparin or its salts include any one of heparin and sodium heparin, the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, and TGF-β3, the fibroblast growth factor includes any one or more of FGF-basic and FGF-7, the platelet-derived growth factor includes any one or more of PDGF-AA and PDGF-BB; the culture medium includes a basal medium.

9. Use of a composition of bioactive substances for preparing a culture medium for enhancing the proliferation and differentiation ability of mesenchymal stem cells, characterized in that, The culture medium does not contain B-27 serum-free additive; the bioactive substance composition includes vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor; the vitamin C or its derivatives include any one of vitamin C and L-ascorbic acid 2-phosphate sesquimagnesium hydrate, the heparin or its salts include any one of heparin and sodium heparin, the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, and TGF-β3, the fibroblast growth factor includes any one or more of FGF-basic and FGF-7, the platelet-derived growth factor includes any one or more of PDGF-AA and PDGF-BB; the culture medium includes a basal medium.

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