A serum-free culture medium and its application

Abstract

This invention provides a serum-free culture medium and its applications. The serum-free culture medium comprises bioactive substances and a basal culture medium, is free of B-27 cell culture additives, has a simple preparation method, and high safety. The bioactive substances include vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor. The basal culture medium is a commercially available serum-free mesenchymal stem cell culture medium with clearly defined components. This invention's serum-free culture medium has a simple preparation method and high safety, which can not only improve the primary culture proliferation effect of tendon-derived mesenchymal stem cells, but also enhance their tendon differentiation ability, as well as their osteogenic, chondrogenic, and adipogenic differentiation abilities. This invention also screens and optimizes the types and concentrations of the five bioactive factors and the type of the basal culture medium to further improve the effectiveness of the serum-free culture medium, providing prospects for the clinical treatment of musculoskeletal injuries.

Description

Technical Field

[0001] This invention belongs to the field of stem cell culture, and more specifically, relates to a serum-free culture medium and its application. Background Technology

[0002] Mesenchymal stem cells, also known as pluripotent stromal cells (MSCs), are a type of pluripotent stem cells belonging to the mesoderm. They are mainly found in connective tissues and organ stroma, and their sources include bone marrow, umbilical cord, adipose tissue, mucosa, bone, muscle, tendon, lung, liver, pancreas, amniotic fluid, amnion, and placenta. They are cells with self-renewal capacity and can differentiate into various tissues such as tendons, adipose tissue, bone, and cartilage under suitable 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, amniotic membrane mesenchymal stem cells, and placental mesenchymal stem cells.

[0003] Currently, most culture media for mesenchymal stem cells contain fetal bovine serum (FBS), which provides the hormones and growth factors necessary for cell adhesion and proliferation. However, FBS, derived from fetal bovine blood, has a complex composition and may be contaminated with pathogens. Cells cultured with FBS may cause immune rejection in patients due to residual foreign proteins if used for clinical cell therapy. Furthermore, FBS requires storage at -20°C, leading to significant batch-to-batch variations. Therefore, culture media containing FBS are not recommended for clinical cell culture. Commercially available serum-free culture media, with clearly defined components, enable normal cell adhesion and growth with good expansion without FBS. They are easy to prepare by adding additives to the basal medium and are widely used for culturing cells in stem cell therapy products. However, they are less effective at maintaining stem cell phenotypes and lack specificity for seed cells and indications, failing to leverage the unique advantages of seed cells from different sources. In serum-free culture systems, the lack of various nutrients and growth factors provided by serum can negatively impact cell growth and survival. Adding B-27 cell culture additive can replenish essential nutrients, promote the growth and proliferation of mesenchymal stem cells, and maintain optimal cell health. However, B-27 cell culture additive is expensive, such as Gibco 50×B-27. TM The additive (17504044) costs 18,357 yuan per 100 mL, and a few brands (such as Thermo Fisher Scientific) occupy a large market share of this additive, which shows a tendency to monopolize.

[0004] CN117511861A discloses a serum-free culture medium for human mesenchymal stem cells, which can enhance the proliferation capacity of human umbilical cord mesenchymal stem cells. However, the expansion efficiency of its primary cultured mesenchymal stem cells is low, and the number of stem cells can only reach 1×10⁴ after passage to P4. 7 Order of magnitude; CN 109370985 A discloses a serum-free culture medium for large-scale culture of human umbilical cord mesenchymal stem cells, but the number of primary cultured human umbilical cord mesenchymal stem cells in this medium can only reach 1×10⁻⁶. 5 The order of magnitude is too large; to obtain more cells, passage culture is necessary. CN117448268A discloses a serum-free culture medium for dental pulp mesenchymal stem cells and a method for culturing the same. This serum-free culture is only suitable for passage culture of dental pulp mesenchymal stem cells. The above three serum-free culture media for mesenchymal stem cells have low expansion efficiency in primary culture and are only suitable for passage culture of stem cells, not for rapid isolation, purification, and expansion of primary stem cells. Furthermore, none of the above three publications explain that these three serum-free culture media can give the corresponding mesenchymal stem cells a specific therapeutic advantage after culturing them.

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

[0006] To address the difficulties of existing technologies, this invention provides a serum-free culture medium and its applications. The serum-free culture medium comprises bioactive substances and a basal culture medium, contains no B-27 cell culture additives, has a simple preparation method, and high safety. The bioactive substances include vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor. The basal culture medium is a commercially available serum-free mesenchymal stem cell culture medium with clearly defined components. This invention's serum-free culture medium has a simple preparation method and high safety, and can not only improve the primary culture proliferation effect of tendon-derived mesenchymal stem cells, but also achieve a primary culture cell count of 5 × 10⁻⁶ cells / year. 7 Furthermore, it can significantly improve the tendon differentiation capacity of the mesenchymal stem cells and maintain their osteogenic, chondrogenic, and adipogenic differentiation capabilities. After primary culture, the mesenchymal stem cells can be directly induced to differentiate into tendon stem cells, osteocytes, chondrocytes, and adipocytes without passage culture. This invention also screens and optimizes the types and concentrations of five bioactive factors and the types of the basal culture medium to further improve the effectiveness of serum-free culture medium, providing prospects for the clinical treatment of musculoskeletal injuries.

[0007] On one hand, a serum-free culture medium includes a bioactive substance and a basal culture medium; the bioactive substance includes 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 culture medium does not contain B-27 serum-free additive.

[0008] The basal culture medium is one or more of the following: Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro, Zhongke Ruiji mesenchymal stem cell serum-free culture medium MAX, Huakan 3D FloTrix mesenchymal stem cell serum-free culture medium, and Youkang mesenchymal stem cell serum-free culture medium.

[0009] The team behind this invention discovered that adding different combinations of bioactive compositions to commercial serum-free mesenchymal stem cell (MSC) culture media can not only improve the proliferation of primary cultured tendon-derived MSCs but also enhance their differentiation capacity. Furthermore, they found that the serum-free culture medium does not require the addition of B-27 serum-free additive. Therefore, this invention provides a serum-free culture medium that is simple to prepare, highly safe, and low in cost. It can be prepared simply by adding vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor to commercial serum-free MSC culture media. This medium can not only expand the number of tendon-derived MSCs in primary culture to 5 × 10⁶ cells / year, but also... 7 Furthermore, it can significantly improve the tendon differentiation ability of cultured tendon-derived mesenchymal stem cells and maintain their ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Tendon-derived mesenchymal stem cells primary cultured using the serum-free medium of this invention can be directly used for induction culture. After induction with the induction medium, the cultured mesenchymal stem cells can successfully differentiate into tendon stem cells, osteoblasts, chondrocytes, and adipocytes. The serum-free medium provided by this invention can also be used for the primary culture of umbilical cord-derived mesenchymal stem cells, improving the proliferation effect and pluripotency of primary cultured umbilical cord-derived mesenchymal stem cells.

[0010] The invention team previously applied for a patent titled "A Bioactive Substance Composition, a Serum-Free Culture Medium Containing the Composition, and Its Uses," patent number CN113692282A. The serum-free culture medium containing the bioactive substance composition enables primary and passaged culture of stem cells from various sources in vitro. Its ability to promote cell proliferation and maintain phenotype is superior to that of culture medium containing fetal bovine serum. This serum-free culture medium is particularly suitable for completely serum-free in vitro culture of tendon and / or ligament-derived stem cells, resulting in rapid proliferation, short cell doubling time, and maintenance of tendinogenic capacity and trilineage differentiation (osteogenic, chondrogenic, and adipogenic) phenotypes. However, the number of tendon and / or ligament-derived stem cells after primary culture is low, and their effectiveness in differentiating into tendon stem cells, osteoblasts, chondrocytes, and adipocytes after direct induction induction is poor. Because the serum-free culture medium alone cannot adapt tendon and / or ligament-derived stem cells to the in vitro growth environment through primary culture, the stem cell proliferation capacity is limited. When primary cultured stem cells are used for induced differentiation, the number of cells is small, and the subpopulation of cells with high differentiation potential is relatively insufficient. These cells with high differentiation capacity play a key role in the formation of functional stem cells during the induction process. Their insufficient number leads to poor overall induction effect, making it difficult to produce a sufficient number and quality of functional stem cells, resulting in poor induced differentiation effect. Therefore, the stem cells need to be passaged to adapt to the in vitro growth environment and achieve rapid proliferation, realizing exponential growth in cell number and improving the induced differentiation effect. However, if the same primary culture time is used, the maximum number of cells reached in the P2 generation is only 1.9 × 10⁻⁶. 7 Furthermore, the aforementioned culture medium requires the addition of B-27 serum-free additive during primary and passaged cell culture, increasing culture costs. This invention provides a novel serum-free culture medium that eliminates the need for B-27 serum-free additive, allowing tendon-derived mesenchymal stem cells to rapidly adapt to the in vitro growth environment during primary culture, achieving a primary cell proliferation rate of 5 × 10⁶ cells / year. 7 This is far superior to the cell proliferation effect achieved by the team of this invention in the previous application of serum-free culture medium cultured tendon and / or ligament-derived stem cells that had to be passaged to the P2 generation. Furthermore, the primary cultured tendon-derived mesenchymal stem cells provided by this invention do not require passaged culture and can be directly used to induce and successfully differentiate into tendon stem cells, osteoblasts, chondrocytes and adipocytes, with better differentiation effect.

[0011] Further, the vitamin C or its derivatives include any one or more of vitamin C, ascorbate glucoside, ethyl vitamin C, 3-o-ethyl ascorbic acid, vitamin C magnesium phosphate, vitamin C sodium phosphate, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, vitamin C tetraisopalmitate, ascorbate 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, heparin sodium, heparin calcium, and heparin phospholipids; the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, TGF-β3, and transforming growth factor-β synthetic peptides.

[0012] The vitamin C or its derivatives refer to vitamin C (also known as ascorbic acid), vitamin C salts, and vitamin C solvates, which have antioxidant effects, can inhibit cell aging, and promote cell growth and phenotype maintenance. In addition, vitamin C or its derivatives enhance the cartilage repair capacity of mesenchymal stem cells through metabolic regulation, showing that it has a promoting effect on specific differentiation directions of stem cells.

[0013] Heparin or its salts are strongly acidic and are a natural anticoagulant in animals. They can inhibit the conversion of prothrombin into thrombin, thereby inhibiting the formation of fibrin from fibrinogen. In stem cell culture, heparin can prevent stem cell aggregation, protecting the cells and ensuring their stability and activity during culture. Secondly, heparin or its salts provide a suitable growth environment for stem cells, directly promoting their growth. In addition, heparin or its salts can specifically induce mesenchymal stem cells to differentiate into osteoblasts and other morphologies.

[0014] Transforming growth factor-β (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. It has the function of regulating stem cell growth and differentiation, maintaining stem cell phenotype, and helping to maintain the stability and pluripotency of stem cells.

[0015] Further, the fibroblast growth factor includes any one or more of FGF-basic, FGF-1, FGF-4, FGF-7, FGF-10, FGF-18, and fibroblast growth factor synthetic peptides; 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 platelet-derived growth factor synthetic peptides.

[0016] Fibroblast growth factor (FGF), also known as heparin-binding growth factor, mainly includes two categories: acidic and basic. It refers to a class of active proteins or polypeptides that promote cell growth. FGF plays a crucial role in stem cell culture, primarily by promoting stem cell proliferation, maintaining the undifferentiated state of stem cells, regulating stem cell differentiation, and participating in stem cell signaling regulation. Different types of FGF can induce stem cells to differentiate into specific cell types. For example, studies have shown that FGF-4 at specific concentrations can significantly promote the proliferation of bone marrow stromal stem cells and induce their differentiation into ligaments or tendons (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, studies have shown that rat bone marrow mesenchymal stem cells can differentiate into functional osteoblasts after being induced by PDGF-BB (Wei Qin, Zhang Xue, Ma Lei, et al. Platelet-derived growth factor BB induces differentiation of rat bone marrow mesenchymal stem cells into osteoblasts [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(19): 2953-2957.).

[0018] Further, the vitamin C or its derivative is vitamin C or L-ascorbic acid 2-phosphate sesquimagnesium salt 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, and TGF-β3; the fibroblast growth factor is any one or more of FGF-basic and FGF-7; and the platelet-derived growth factor is any one or more of PDGF-AA and PDGF-BB.

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

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

[0021] In some methods, single-variable experiments were conducted to compare the effects of serum-free culture media containing different concentrations of the five bioactive substances on the proliferation and differentiation capacity of primary cultured tendon-derived mesenchymal stem cells. The results showed that all five bioactive substances, within the aforementioned concentration range, could increase the number of mesenchymal stem cells to 1 × 10⁻⁶. 7 It has a cell viability of 90% or higher and also possesses the ability to differentiate into tendons, osteoblasts, chondrocytes, and adipocytes.

[0022] Furthermore, the basal culture medium is Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro.

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

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

[0025] In another aspect, the present invention provides the use of a bioactive substance composition for preparing a culture medium that improves the primary culture effect of mesenchymal stem cells, wherein the culture medium is free of B-27 and serum-free additives; the bioactive substance composition includes vitamin C or its derivatives, heparin or its salts, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor; the vitamin C or its derivatives include any one of vitamin C and L-ascorbic acid 2-phosphate sesquimagnesium salt 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; and the platelet-derived growth factor includes any one or more of PDGF-AA and PDGF-BB; the culture medium includes a basal culture medium.

[0026] The basal culture medium is one or more of the following: Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro, Zhongke Ruiji mesenchymal stem cell serum-free culture medium MAX, Huakan 3D FloTrix mesenchymal stem cell serum-free culture medium, and Youkang mesenchymal stem cell serum-free culture medium.

[0027] In some methods, primary culture is performed using an improved primary culture method. The effects of the serum-free culture medium provided by this invention, the serum-free culture medium provided in CN113692282A (a previous application filed by our team), and the culture medium containing fetal bovine serum on the proliferation capacity of primary cultured tendon-derived mesenchymal stem cells are compared. The results show that the serum-free culture medium provided by this invention produces three orders of magnitude more cells in primary culture of mesenchymal stem cells than the serum-free culture medium provided in CN113692282A, and two orders of magnitude more cells than the culture medium containing fetal bovine serum.

[0028] In another aspect, the use of a bioactive substance composition for preparing a culture medium to enhance the proliferation and differentiation capacity of mesenchymal stem cells, wherein the culture medium is free of B-27 serum-free additive; the bioactive substance composition includes vitamin C or a derivative thereof, heparin or a salt thereof, transforming growth factor-β, fibroblast growth factor, and platelet-derived growth factor; wherein the vitamin C or a derivative thereof includes any one of vitamin C and L-ascorbic acid 2-phosphate sesquimethyl hydrate, wherein the heparin or a salt thereof includes any one of heparin and heparin sodium, wherein the transforming growth factor-β includes any one or more of TGF-β1, TGF-β2, and TGF-β3, wherein the fibroblast growth factor includes any one or more of FGF-basic and FGF-7, and wherein the platelet-derived growth factor includes any one or more of PDGF-AA and PDGF-BB; and wherein the culture medium includes a basal culture medium.

[0029] The basal culture medium is one or more of the following: Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro, Zhongke Ruiji mesenchymal stem cell serum-free culture medium MAX, Huakan 3D FloTrix mesenchymal stem cell serum-free culture medium, and Youkang mesenchymal stem cell serum-free culture medium.

[0030] In some methods, primary culture of tendon-derived mesenchymal stem cells was performed using the serum-free culture medium provided by this invention, the serum-free culture medium provided in CN113692282A previously filed by the team of this invention, and a culture medium containing fetal bovine serum. The relative expression levels of SCX, THBS4, and NES genes in the cultured mesenchymal stem cells were then detected. The results showed that the relative expression levels of SCX, THBS4, and NES genes in the mesenchymal stem cells cultured in the serum-free culture medium provided by this invention were significantly higher than those in the other two groups. The high relative expression level of SCX gene indicates that the mesenchymal stem cells cultured in the serum-free culture medium of this invention have stronger tendon differentiation potential, while the high relative expression level of NES gene indicates that the mesenchymal stem cells also have pluripotency and can differentiate into other functional stem cells, such as osteoblasts, chondrocytes, and adipocytes.

[0031] In some methods, tendon-derived mesenchymal stem cells, which are primary cultured in the serum-free medium of the present invention, are induced again in the tendon induction medium and stained with Sirius red. The staining results show 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 injuries of the musculoskeletal system, such as Achilles tendon rupture. By transplanting tendon stem cells or utilizing the growth factors they secrete, the healing of the Achilles tendon can be accelerated and the quality of healing can be improved.

[0033] In some methods, tendon-derived mesenchymal stem cells, which are primary cultured in the serum-free medium of the present invention, are induced again in bone-inducing medium and stained with ALP, ARS, and DAPI, respectively. The staining results show 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 mentioned can be used to promote bone regeneration. For large bone defects caused by trauma, tumor resection, etc., autologous bone transplantation is currently the main treatment method, but it has problems such as limited bone donor sources and donor site complications. Osteoblasts can be implanted into the bone defect site by direct injection or after binding with a carrier, promoting bone tissue regeneration and reducing dependence on autologous bone transplantation.

[0035] In some methods, tendon-derived mesenchymal stem cells, which are primary cultured in the serum-free medium of the present invention, are induced again in chondrogenic medium and stained with Alcian blue and Safranin O, respectively. The staining results show that the induced mesenchymal stem cells have high levels of acidic mucopolysaccharides, glycosaminoglycans and other components in the intracellular or extracellular matrix, as well as high levels of acidic glycosaminoglycans 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 damage, chondrocytes can be implanted into the damaged area. These chondrocytes can proliferate and secrete cartilage matrix, promoting the repair of damaged cartilage.

[0037] In some methods, tendon-derived mesenchymal stem cells cultured in the serum-free medium of the present invention are induced again in adipose-induced medium and stained with Oil Red O. The staining results show that the induced mesenchymal stem cells synthesize and accumulate a large amount of lipids, indicating that they have differentiated into adipocytes.

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

[0039] In some methods, primary cultures of tendon-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells were performed using the serum-free culture medium provided by this invention. 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 were then detected. The results showed that the serum-free culture medium provided by this invention could ensure that the primary culture of mesenchymal stem cells from both sources conformed to the immunophenotype, with a cell count reaching 1×10⁻⁶. 7 While maintaining high cell viability, the relative expression levels of the three genes showed that the serum-free culture medium provided by this invention is particularly suitable for the culture of tendon-derived mesenchymal stem cells, and can significantly improve their tendon differentiation ability.

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

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

[0042] (2) The serum-free culture medium of the present invention is simple to prepare. It can be prepared simply by adding bioactive substances to the basic culture medium. It can not only improve the primary culture proliferation capacity of tendon-derived mesenchymal stem cells, but also achieve a primary culture cell count of 5×10⁻⁶. 7 Furthermore, it can enhance the differentiation capacity of cultured mesenchymal stem cells, which, after primary culture, can be directly induced to differentiate into tendon stem cells, osteocytes, chondrocytes, and adipocytes without passage culture.

[0043] (3) The present invention also screens and optimizes the types and concentrations of the five bioactive factors and the types of the basic culture medium to further improve the effect of serum-free culture medium and provide prospects for clinical treatment of musculoskeletal injuries.

[0044] (4) The serum-free culture medium provided by the present invention can also be used for primary culture of umbilical cord-derived mesenchymal stem cells, thereby improving the proliferation and differentiation capacity of primary cultured umbilical cord-derived mesenchymal stem cells. Attached Figure Description

[0045] Figure 1 This is a morphological image of primary cultured mesenchymal stem cells in serum-free culture medium combination 1 of Example 1 on day 5. The lumpy material in the image is tissue microparticles left over when the cells were separated from the tendon tissue by digestion solution.

[0046] Figure 2 This is a morphological image of primary cultured mesenchymal stem cells in serum-free culture medium combination 1 of Example 1 on day 10.

[0047] Figure 3 This is a morphological image of primary cultured mesenchymal stem cells in serum-free culture medium combination 1 of Example 1 on day 14.

[0048] Figure 4 This is a morphological diagram of primary cultured mesenchymal stem cells in Example 1 containing fetal bovine serum culture medium combination 3 on day 6. The lumpy material in the diagram is tissue microparticles left over when the cells were separated from the tendon tissue by digestion fluid.

[0049] Figure 5 This is a morphological image of primary cultured mesenchymal stem cells in Example 1 containing fetal bovine serum culture medium combination 3 on day 13.

[0050] Figure 6 The images show NES protein fluorescence staining of primary cultured mesenchymal stem cells from serum-free culture medium combination 1 and fetal bovine serum-containing culture medium combination 3 in Example 1.

[0051] Figure 7 This is a Sirius red staining image of primary cultured mesenchymal stem cells from Example 1, after induction with tendinoid induction medium;

[0052] Figure 8 This is an ALP staining image of primary cultured mesenchymal stem cells from Example 1 (serum-free culture medium combination 1) after induction with bone-inducing culture medium.

[0053] Figure 9 This is an ARS staining image of primary cultured mesenchymal stem cells from Example 1 (serum-free culture medium combination 1) after induction with bone-inducing culture medium.

[0054] Figure 10This is a DAPI fluorescence staining image of primary cultured mesenchymal stem cells from Example 1 (serum-free culture medium combination 1) after induction with bone-inducing culture medium.

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

[0056] Figure 12 This is a Safranin O staining image of primary cultured mesenchymal stem cells from Example 1, cultured in serum-free medium combination 1, after induction with chondrogenic medium.

[0057] Figure 13 This is an Oil Red O staining image of primary cultured mesenchymal stem cells from Example 1, prepared with adipose-induced culture medium, after induction.

[0058] Figure 14 This is a diagram showing the growth morphology of primary cultured tendon-derived mesenchymal stem cells in culture medium combination 1 of Example 2, harvested after 14 days.

[0059] Figure 15 This is a diagram showing the growth morphology of primary cultured tendon-derived mesenchymal stem cells in culture medium combination 2 of Example 2, harvested after 14 days.

[0060] Figure 16 This is a diagram showing the growth morphology of tendon-derived mesenchymal stem cells cultured in culture medium combination 3 in Example 2 until harvest at passage P2.

[0061] Figure 17 This is a diagram showing the growth morphology of tendon-derived mesenchymal stem cells cultured in culture medium combination 4 in Example 2 until harvest at passage P2.

[0062] Figure 18 The growth morphology of primary cultured tendon-derived mesenchymal stem cells in combination 5 of Example 2 containing fetal bovine serum at harvest time after 14 days;

[0063] Figure 19 This is a morphological image of umbilical cord-derived mesenchymal stem cells cultured in serum-free medium on the fourth day of culture in Example 5.

[0064] Figure 20 This is a morphological image of umbilical cord-derived mesenchymal stem cells cultured in primary culture medium containing fetal bovine serum on day 6 of Example 5. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below 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 culture medium and fetal bovine serum-containing culture medium on primary culture of tendon-derived mesenchymal stem cells.

[0067] This embodiment compares the effects of serum-free culture medium and fetal bovine serum-containing culture medium provided by the present invention on the primary culture of tendon-derived mesenchymal stem cells.

[0068] 1. Prepare the culture medium

[0069] 1) The specific steps for preparing the serum-free culture medium of this invention are as follows:

[0070] Preparation of vitamin C or its derivative solution: Dissolve 100 mg of vitamin C or its derivative in 20 mL of PBS buffer to obtain a vitamin C or its derivative solution concentration of 5 mg / mL, and filter using a 0.22 μm sterile filter membrane;

[0071] To prepare a solution of heparin or its salts: Dissolve 10 mg of heparin or its salts in 1 mL of PBS-5% trehalose buffer to achieve a concentration of 10 mg / mL.

[0072] Preparation of platelet-derived growth factor (PDGF) solution: Dissolve 50 μg of platelet-derived growth factor in 5 mL of PBS buffer to obtain a platelet-derived growth factor solution concentration of 10 μg / mL;

[0073] Prepare a fibroblast growth factor (FGF) solution: Dissolve 500 μg of fibroblast growth factor in 5 mL of PBS buffer to obtain a fibroblast growth factor solution concentration of 100 μg / mL.

[0074] To prepare the transforming growth factor-β (TGF-β) solution: Dissolve 50 μg of transforming growth factor-β in 5 mL of PBS buffer to obtain a TGF-β solution concentration of 10 μg / mL.

[0075] The above five bioactive factor solutions were added to the basal culture medium in sequence according to the specified concentrations. There was no order in which the components were added. The basal culture medium was 500 mL of Zhongke Ruiji mesenchymal stem cell serum-free culture medium (Pro Zhongke Ruiji RGM1051), with its accompanying additives added.

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

[0077] 3) Prepare culture medium containing fetal bovine serum

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

[0079] The following three groups of culture media were set up as shown in Table 1 below. Culture media combination 1 is the serum-free culture medium provided by the present invention, combination 2 is the serum-free culture medium provided in Example 1 of CN113692282A, and combination 3 is a culture medium containing fetal bovine serum.

[0080] Table 1. Different combinations of serum-free culture media and culture media containing fetal bovine serum

[0081]

[0082] 2. Primary culture methods for tendon-derived mesenchymal stem cells

[0083] Method 1 for primary culture of tendon-derived mesenchymal stem cells: After weighing the tendon tissue, wash it sequentially in PBS buffer containing 10%, 5%, 2%, and 1% P / S antibiotics for 1 min. Then, transfer the tendon tissue to a 10 cm culture dish, add 0.5 mL of DMEM digestion solution containing 2% collagenase and 1% P / S antibiotics, and cut the tendon tissue into 1 mm × 1 mm pieces. Add another 7.5 mL of digestion solution and mix well. Incubate at 37 ℃ and 5% CO2 for about 4 h, stirring and mixing the digestion solution every 1 h until the tissue digestion solution is clear and non-viscous. Transfer the digestion solution to a 15 mL centrifuge tube and centrifuge at 2500 rpm for 15 min. Discard the supernatant, resuspend the precipitate in the culture medium listed in Table 1, and aliquot it into multiple 15 cm culture dishes, with each dish containing 100 mg of tissue for dissociation and precipitation. The culture system is 10 mL / dish. Incubate at 37 ℃ and 5% CO2. On day 5 of culture, the medium was changed. The culture supernatant was transferred to centrifuge tubes and centrifuged at 2500 rpm for 10 min. The supernatant was discarded, and the pellet was resuspended in the corresponding medium in Table 1 and evenly distributed back into the original culture dishes. The dishes were then incubated at 37 ℃ with 5% CO2. On day 10 of culture, the culture dishes were reseeded. The culture supernatant was removed, and the bottom of the dishes was washed with 8 mL of PBS buffer. The PBS buffer was discarded, and 2 mL of mild digestive enzyme was added to each dish. The dishes were incubated at 37 ℃ for approximately 2 min. After observing the rounded cell morphology under a microscope, 7 mL of culture supernatant was added to each dish to disperse the cells. The cells were then transferred to centrifuge tubes, and each dish was washed with 5 mL of PBS buffer. The dishes were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. The pellet was resuspended in the corresponding medium in Table 1 and evenly distributed back into the original culture dishes. The culture volume was 20 mL / dish. The dishes were then incubated at 37 ℃ with 5% CO2. Harvesting was performed on day 14.

[0084] It should be noted that the replanting operation in the above-mentioned culture method one is not a subculture, because the replanting operation is simply to treat the cultured cells with a simple digestive enzyme and then inoculate them into the original culture dish. The total number of cells before and after the replanting operation does not change.

[0085] Method 2 for primary culture of tendon-derived mesenchymal stem cells: After weighing the tendon tissue, rinse it sequentially in PBS buffer containing 10%, 5%, 2%, and 1% P / S antibiotics for 1 min. Then, transfer the tendon tissue to a 10 cm culture dish, add 0.5 mL of DMEM digestion solution containing 2% collagenase and 1% P / S antibiotics, cut the tendon tissue into 1 mm × 1 mm pieces, add another 7.5 mL of digestion solution and mix well. Incubate at 37 ℃ and 5% CO2 for about 4 h, stirring and mixing the digestion solution every 1 h until the tissue digestion solution is clear and non-viscous. Transfer the digestion solution to 15 mL centrifuge tubes and centrifuge at 2500 rpm for 15 min. Discard the supernatant, resuspend the precipitate in the culture medium listed in Table 1, and aliquot it into multiple 15 cm culture dishes, with each dish containing 100 mg of tissue for dissociation and precipitation. The culture volume is 20 mL / dish. Incubate at 37 ℃ with 5% CO2, changing the medium every 5 days using the corresponding culture medium listed in Table 1. Harvest on day 14.

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

[0087] The three culture media in Table 1 were used for primary culture of tendon-derived mesenchymal stem cells using the culture methods described above, Method 1 and Method 2, respectively.

[0088] During the culture process, the growth and morphological changes of mesenchymal stem cells were observed using an inverted microscope, and the data were recorded by microscopic imaging. For example... Figures 1-3 The image shows the growth morphology of tendon-derived mesenchymal stem cells cultured using primary culture method one in serum-free culture medium combination 1 (of this invention) on days 5, 10, and 14 of culture. The results show that the cultured tendon-derived mesenchymal stem cells maintained good adherence, had abundant cytoplasm, and exhibited a spindle-shaped cell morphology. Although proliferation was relatively slow from days 1 to 10, the proliferation rate accelerated after day 10. Figures 4-5 The image shows the growth morphology of tendon-derived mesenchymal stem cells cultured using primary culture method 2 on days 6 and 13 of culture in culture medium combination 3 containing fetal bovine serum. Although the cultured tendon-derived mesenchymal stem cells maintained good adherence, had abundant cytoplasm, and exhibited a spindle-shaped cell morphology, and had a rapid proliferation rate from day 1 to day 10, the proliferation rate slowed down after day 10.

[0089] Furthermore, the number and viability of tendon-derived mesenchymal stem cells harvested from the three culture media cultured using different primary culture methods were detected, and the results are shown in Table 2 below.

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

[0091]

[0092] Based on the data analysis in Table 2, the number and viability of tendon-derived mesenchymal stem cells cultured using two different primary culture methods with different culture medium combinations were compared. The difference lies in the addition of a reseeding plate operation in one of the primary culture methods. There was no significant difference in the number of cells cultured using method one compared to method two for culture medium combinations 2 and 3, indicating that the primary culture proliferation effect of tendon-derived mesenchymal stem cells in the serum-free and bovine serum-containing culture media provided in Example 1 of CN113692282A was limited. However, the number of tendon-derived mesenchymal stem cells cultured using primary culture method one (of this invention) was significantly higher than that using primary culture method two. This indicates that the number of tendon-derived mesenchymal stem cells cultured using primary culture method two for culture medium combination 1 (of this invention) did not reach the upper limit of proliferation. This is because the reseeding plate operation was not added in method two, resulting in excessive clonal center density and contact inhibition. Therefore, the growth rate of central cells was limited after 10 days of culture, thus reducing the harvest yield. Culture medium combination 1 (in this invention) involves adding a reseeding plate to tendon-derived mesenchymal stem cells on the tenth day of primary culture. This allows for faster proliferation of tendon-derived mesenchymal stem cells after 10 days of culture, with reduced cell volume, enabling the production of more cells (up to 1 × 10⁻⁶) within the same culture area. 7 The number of cells was on the order of magnitude. Furthermore, using Method 1 and Method 2, the number of mesenchymal stem cells cultured in the primary culture medium of Combination 1 (the present invention) was three orders of magnitude and two orders of magnitude higher than that of Combination 2, respectively. This indicates that the serum-free culture medium provided in Example 1 of CN113692282A is not suitable for primary stem cell culture and expansion, while the serum-free culture medium of Combination 1 provided by the present invention is suitable for primary stem cell culture, isolation, purification, and rapid expansion.

[0093] In addition, the basal medium of culture medium combination 2 is DMEM low-glucose medium or F12 medium, and the remaining bioactive substances need to be prepared by themselves. In addition to the five bioactive components of vitamin C or its salt, heparin or its salt, PDGF, FGF, and TGF-β, it also includes hormones, other growth factors, non-essential amino acids, etc., which means that some active components that promote cell proliferation are not added, thus limiting the cell proliferation effect. In contrast, the culture medium combination 1 provided by this invention only requires the addition of the five bioactive components of vitamin C or its salt, heparin or its salt, PDGF, FGF, and TGF-β to Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro, without the need to add other components. The other bioactive nutrients contained in Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro are more comprehensive than those in combination 2. At the same time, the types and concentrations of the five bioactive components of vitamin C or its salt, heparin or its salt, PDGF, FGF, and TGF-β in combination 1 are different from those in combination 2. Therefore, the culture medium of combination 1 is more suitable for the proliferation culture of tendon-derived mesenchymal stem cells.

[0094] Furthermore, the tendon-derived mesenchymal stem cells cultured in the above culture media combinations 1-3 using primary culture method 1 were passaged to the P5 generation, and the number and viability of cells in the P1-P5 generations were detected. The number and viability of cells in the P2 and P5 generations are shown in Table 3 below.

[0095] Table 3. Cell number and viability at passages P2 and P5 of tendon-derived mesenchymal stem cells cultured in different culture media using primary culture method 1

[0096]

[0097] Based on the data analysis in Table 3, primary tendon-derived mesenchymal stem cells cultured in combination 1-3 culture media were passaged to passage P5, resulting in a further increase in the number of stem cells. Stem cells cultured in combination 1 (the present invention) culture medium to passage P2 had a cell count of 1 × 10⁻⁶. 9 The number of cells was orders of magnitude higher than that of combination 2 culture medium, and the cell number increased to 1×10⁵ by the P5 generation. 14 The number of primary cells cultured in the combined medium (combination 1) is on the order of magnitude higher than that in combination 2 medium, by four orders of magnitude. Combined with the results of primary culture method 1 in Table 2 above, this indicates that the number of primary cells cultured in the combined medium (combination 1) provided by this invention is only 5.7 × 10⁻⁶. 7 The culture medium for combination 2 provided in Example 1 of CN113692282A was used to passage stem cells to generation P2 (1.9 × 10⁻⁶). 7 The proliferation effect can only be achieved through this process.

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

[0099] 3. Effects of different culture medium combinations on the differentiation capacity of tendon-derived mesenchymal stem cells

[0100] Furthermore, a testing company was commissioned to perform flow cytometry analysis on the surface markers of tendon-derived mesenchymal stem cells cultured using primary and P2 generation culture methods 1-3 as described above. The test results are shown in Table 4 below, and the reference ranges for the test indicators are shown in Table 5 below.

[0101] Table 4. Immunophenotyping results of tendon-derived mesenchymal stem cells cultured in different culture media

[0102]

[0103] Table 5. Reference intervals for immunophenotypic detection of tendon-derived mesenchymal stem cells

[0104]

[0105] Based on the data analysis in Tables 4-5, the primary and P2 cultured tendon-derived mesenchymal stem cells in serum-free culture medium combinations 1-2 all met the immunophenotype requirements for mesenchymal stem cells and complied with safety indicators. However, the CD34 immunophenotype of the primary and P2 cultured tendon-derived mesenchymal stem cells in fetal bovine serum-containing culture medium combination 3 did not meet the requirements. CD73 was highly expressed on the surface of mesenchymal stem cells, CD105 and CD90 were highly expressed on the surface of both mesenchymal and tendon stem cells, CD14, CD19, and HLA-DR were lowly expressed or not expressed on the surface of mesenchymal stem cells, and CD45 and CD34 were lowly expressed or not expressed on the surface of tendon stem cells. The surface antigen detection results of mesenchymal stem cells cultured in serum-free culture medium combinations 1-2 (primary) and P2 (secondary) showed that the expression levels of CD73, CD105, and CD90 were all ≥95%, while the expression levels of CD45, CD34, CD14, CD19, and HLA-DR were all ≤2%. This indicates that the cultured cells conform to the phenotype of mesenchymal stem cells and have the potential to differentiate into tendon stem cells.

[0106] 3.1 Detection of tendon-derived mesenchymal stem cells cultured in different serum-free culture media to determine their tendon system differentiation capacity

[0107] The relative expression levels of SCX, NES, and THBS4 genes in primary and P2 generations of tendon-derived mesenchymal stem cells cultured in the culture medium combinations listed in Table 4 were detected. The specific detection method is as follows: 1 × 10⁶ cells / well were seeded per well of a 6-well plate. 5Cells were cultured at 37°C in a 5% CO2 incubator with 2 mL of culture medium until confluent. The culture medium 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 the mixture was vortexed and allowed to stand for 10 min. The cells were then centrifuged at 12000×g for 15 min at 4°C. The supernatant was transferred to a new 1.5 mL RNase-free EP tube, and an equal volume of isopropanol was added. The tube was inverted and mixed thoroughly, allowed to stand at room temperature for 5 min, and then centrifuged at 12000×g for 15 min at 4°C. The supernatant was discarded, and 0.5 mL of 75% ethanol was added to wash the precipitate. The tube was then centrifuged at 12000×g for 5 min at 4°C. After discarding the supernatant, the tube was air-dried, and 10 μL of DEPC water was added. The RNA was dissolved at 4°C, allowed to stand for 30 min, and then centrifuged by vortexing. 1 μL of the solution was taken to measure the concentration. The sample was processed using the Toyobo reverse transcription kit according to the instructions. The sample was incubated at 65°C for 5 min and then quickly placed on ice. 10⁻⁶ cells were counted. The total sample volume for 1000 ngRNA was added in a 10 μL system. 2 μL of reverse transcription reagent was added to each group, and double-distilled water was added to make up to 10 μL. Reverse transcription was performed using a thermal cycler to obtain cDNA, as shown in Table 6. qPCR experiments were performed using the Takara TB Green dye quantitative kit, as shown in Table 7. The expression levels of NES, SCX, and THBS4 genes were detected. A 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 sequences, and 5 μL of 2×TB Green Mix. Primer sequences are shown in Table 8. Results analysis: 2-ΔΔCt was used as the relative expression level of the gene. The relative expression level of tendon-derived mesenchymal stem cells cultured in fetal bovine serum-containing medium combination 3 was used as the unit "1". The calculation results are shown in Table 9.

[0108] Table 6. Reverse Transcription Procedure

[0109]

[0110] Table 7. qPCR Procedure

[0111]

[0112] Table 8. qPCR primer sequences

[0113]

[0114] Table 9. Relative expression levels of SCX, NES, and THBS4 genes.

[0115]

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

[0117] Furthermore, the expression level of NES protein in tendon-derived mesenchymal stem cells cultured using the above-mentioned serum-free culture medium combination 1 (the present invention) and fetal bovine serum-containing culture medium combination 3 via primary culture method one was detected. The specific method is as follows: eight-chamber culture was used, and 1 × 10⁶ cells were seeded in each culture well. 4 Cells were cultured at 37 °C for 48 h using 200 μL of culture medium per well. After culture, the cells were washed three times with 200 μL PBS, fixed with 200 μL of 4% paraformaldehyde at room temperature for 20-30 min, and washed three times with PBS. The cells were then permeabilized with 200 μL of permeabilizing agent for 10 min, and washed three times with PBS. 100 μL of blocking buffer was added, and the cells were blocked at room temperature for 30 min. The blocking buffer was removed, 50 μL of primary antibody was added, and the cells were incubated overnight at 4 °C in a humidified chamber. The cells were then warmed to room temperature for 30 min, washed three times with PBS, and incubated with 100 μL of secondary antibody at room temperature in the dark for 1 h, followed by washing three times with PBS. The cells were then treated with 200 μL of DAPI-containing mounting medium, stored in a humidified chamber at 4 °C, and photographed using a confocal fluorescence microscope. The results are as follows: Figure 6As shown, tendon-derived mesenchymal stem cells cultured in serum-free culture medium combination 1 express more NES protein and have stronger pluripotency than those cultured in culture medium combination 3 containing fetal bovine serum. This indicates that the mesenchymal stem cells can differentiate into multiple cell types, such as osteoblasts, chondrocytes, and adipocytes, under suitable conditions.

[0118] 3.2 Verification that tendon-derived mesenchymal stem cells cultured using primary culture method 1 in serum-free culture medium combination 1 possess tendon differentiation capacity

[0119] Furthermore, to verify whether tendon-derived mesenchymal stem cells cultured in the above-mentioned serum-free culture medium combination 1 (the present invention) using primary culture method one can differentiate into tendon stem cells, the specific method is as follows: the suspension of tendon-derived mesenchymal stem cells primary cultured in serum-free culture medium combination 1 is mixed with 5×10 4 Cells were seeded per well in 12-well plates, with 1 mL of complete culture medium added to each well. Cells were cultured until nearly confluent. In the control group, the culture medium was discarded, and staining was performed with photographs. In the experimental group, the culture medium was discarded and replaced with tendon induction medium, with medium changes every three days. After approximately two weeks of culture, the culture medium was aspirated, the cells were washed once with PBS, fixed with 70% ethanol for 30 min, rinsed three times with double-distilled water, stained with 500 μL of Sirius red staining solution for approximately 15 min, and quickly rinsed twice with double-distilled water. Macroscopic images were scanned and photographed under a microscope at 10× field of view (3 fields per group) to observe the staining. Cells were then stored in double-distilled water. Staining results are shown below. Figure 7 As shown, the mesenchymal stem cells cultured in serum-free culture medium combination 1 show 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 tendon-derived mesenchymal stem cells cultured using primary culture method 1 in serum-free culture medium combination 1 possess trilineage differentiation capability

[0121] As verified in section 3.2 above, the tendon-derived mesenchymal stem cells cultured by primary culture method 1 of the present invention can differentiate into tendon stem cells. However, the gene detection results in section 3.1 prove that the tendon-derived mesenchymal stem cells also have pluripotency. The following experiment will verify whether the tendon-derived mesenchymal stem cells also have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes. The osteoblastic, chondrocyte, and adipocyte differentiation abilities of the mesenchymal stem cells will be detected according to the following experimental methods.

[0122] 1) The method for detecting osteogenic differentiation capacity is as follows: Tendon-derived mesenchymal stem cell suspension cultured in serum-free culture medium combination 1 using primary culture method 1 is inoculated with 1×10⁻⁶... 4Cells were seeded per well in 24-well plates, with 0.5 mL of osteogenic induction medium added to each well. After 12 h of culture, cells adhered to the plate. In the control group, the medium was discarded, staining was performed, and photographs were taken. In the experimental group, the medium was discarded and replaced with osteogenic induction medium, with the medium changed every three days. For ALP staining, after one week of induction culture, discard the culture medium, fix with 500 μL / well of 4% paraformaldehyde for 20 min, wash three times with PBS for 5 min each time, add 500 μL of ALP staining working solution to each well, ensuring the working solution fully covers the sample, incubate at room temperature, and observe every 10 minutes. If the experimental group has developed color and is significantly different from the control group, stop staining, wash twice with distilled water to terminate the staining reaction, aspirate the distilled water, and quickly scan the macroscopic image using a scanner. For ARS staining, stop culturing cells after obvious calcium precipitation, which requires approximately 2 weeks of induction culture, discard the culture medium, fix with 500 μL / well of 4% paraformaldehyde for 10 min, wash three times with double-distilled water, add 500 μL of 2% pH=4.2 ARS solution to each well, incubate at room temperature for at least 30 min, rinse three times with distilled water, aspirate the distilled water, quickly scan the macroscopic image using a scanner, and add 500 μL of ARS solution to each well. PBS buffer was used, and the staining was observed by taking pictures under a microscope at 10× field of view, with 3 fields of view per group. For DAPI fluorescent staining, 300 μL of DAPI staining solution (1:8000) was added to each well and stained for 20 minutes. Then, the well was washed three times with double-distilled water for 5 minutes each time. 500 μL of double-distilled water was added to each well, and the staining was observed by taking pictures under a microscope at 10× field of view, with 3 fields of view per group.

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

[0124] ARS staining results are as follows Figure 9 As shown, tendon-derived mesenchymal stem cells cultured in serum-free culture medium combination 1 using primary culture method 1 showed deeper ARS staining after induction, indicating a higher calcium salt content. This suggests that the induced mesenchymal stem cells have begun to differentiate into osteogenic cells, and the cells are actively engaged in mineralization activities.

[0125] The results of DAPI fluorescence staining are as follows: Figure 10 As shown, mesenchymal stem cells cultured in serum-free culture medium combination 1 using primary culture method 1 exhibit higher DAPI fluorescence intensity after induction, indicating that the mesenchymal stem cells proliferate actively.

[0126] 2) The method for detecting chondrogenic differentiation capacity is as follows: Tendon-derived mesenchymal stem cell suspension cultured in serum-free culture medium combination 1 using primary culture method 1 is inoculated at 2.5 × 10⁻⁶ mg / L. 5 Seeds were placed in 12-well plates at a dose of 10 μL per well. The plates were kept moist and cultured for 3-4 hours until the cells adhered. The negative control group was stained directly. The experimental group was treated with 1 mL of chondrogenic medium added to each well. The medium was changed every 3 days. On day 14, the induction medium was discarded, and the cells were stained and photographed using Alcian Blue or Safranin O. For Alcian blue staining, after culturing for 14 days, discard the culture medium, wash three times with PBS buffer for 5 min each time, fix with 1 mL of 4% paraformaldehyde at room temperature for 30 min, wash three times with PBS, rinse with 1 mL of 0.1 N HCl solution for 5 min, stain with 1 mL of Alcian blue overnight, wash three times with 0.1 N HCl for 5 min each time, remove the background, scan the gross image, and take pictures under a microscope at 10× field of view. Observe the staining status in 3 fields of view for each group. For Safranin O staining, after culturing for 14 days, discard the culture medium, wash three times with PBS buffer for 5 min each time, fix with 1 mL of 4% paraformaldehyde at room temperature for 30 min, wash three times with PBS, stain with 1 mL of Safranin O staining solution for 3 min, decolorize with 95% ethanol, and take pictures under a microscope.

[0127] Alcian blue staining results as follows Figure 11 As shown, the tendon-derived mesenchymal stem cells cultured in serum-free culture medium combination 1 using primary culture method 1 showed deeper Alcian blue staining after induction, indicating that the intracellular or extracellular matrix of the induced mesenchymal stem cells contained higher levels of acidic mucopolysaccharides, glycosaminoglycans, and other components, suggesting that the mesenchymal stem cells had differentiated into chondrocytes.

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

[0129] 3) The method for detecting adipogenic differentiation capacity is as follows: Tendon-derived mesenchymal stem cell suspension cultured in serum-free culture medium combination 1 using primary culture method 1 is inoculated at 5×10⁻⁶. 4Cells were seeded per well in 24-well plates, with 0.5 mL of adipogenic induction medium added to each well. After 12 h of culture, cells adhered. In the control group, the medium was discarded, and staining was performed with photographs. In the experimental group, the medium was discarded and replaced with adipogenic induction medium, with medium changes every three days. Culture was continued for approximately 2 weeks until obvious lipid droplets appeared. If no obvious lipid droplets were observed, culture could continue for 3 weeks before staining and photographing. The Oil Red O staining working solution was prepared as follows: Oil Red O stock solution was mixed with double-distilled water at a ratio of 3:2, filtered through filter paper, and incubated at room temperature for 10 min. The diluted solution was used within several hours. The Oil Red O staining procedure was as follows: In the experimental group, the medium was aspirated from the cells, and the cells were washed once with PBS. 500 μL of 4% paraformaldehyde was used for fixation for 30 min, followed by two washes with PBS. 500 μL of Oil Red O staining working solution was added, and staining was performed for approximately 30 min, with real-time observation. Cells were then rapidly destained with 75% ethanol, and 500 μL of PBS was added. Microscope photographs were taken at 20× field of view, with 3 fields of view per group, to observe the staining results.

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

[0131] 3.4 Detection of tendon-derived mesenchymal stem cells cultured in serum-free medium combination 2 (primary) and P2 (secondary) cultures to assess their tendon lineage differentiation and trilineage differentiation capabilities.

[0132] 1) The tendon differentiation capacity of primary and P2 generation cultured tendon-derived mesenchymal stem cells in serum-free culture medium combination 2 was detected, using the method described in 3.2 of this example. Sirius red staining results showed that the primary cultured tendon-derived mesenchymal stem cells stained lighter than those in the P2 generation, indicating that the primary cultured tendon-derived mesenchymal stem cells did not express a large amount of collagen fibers after induction, resulting in poor differentiation into tendon stem cells. However, the Sirius red staining of the P2 generation cultured cells after induction was not as deep as that of the primary cultured cells in serum-free culture medium combination 1 in 3.2.

[0133] 2) The osteogenic differentiation capacity of primary and P2 generation cultured tendon-derived mesenchymal stem cells in serum-free culture medium combination 2 was detected, using the method described in Example 3.3. ALP, ARS, and DAPI fluorescence staining results showed that the primary cultured tendon-derived mesenchymal stem cells stained lighter than those in the P2 generation, indicating that the primary cultured tendon-derived mesenchymal stem cells had a poorer differentiation into osteoblasts after induction, with lower calcium salt content and less active cell proliferation after induction. Simultaneously, 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 in serum-free culture medium combination 1 in Example 3.3.

[0134] 3) The chondrogenic differentiation capacity of primary and P2 generation cultured tendon-derived mesenchymal stem cells in serum-free culture medium combination 2 was detected, using the method described in Example 3.3. Alcian blue and Safranin O staining results showed that the primary cultured tendon-derived mesenchymal stem cells stained lighter than those in the P2 generation, indicating that the primary cultured tendon-derived mesenchymal stem cells had lower levels of acidic mucopolysaccharides, glycosaminoglycans, and proteoglycans in their intracellular or extracellular matrix after induction, suggesting a poorer ability for differentiation into chondrocytes. Furthermore, 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 in serum-free culture medium combination 1 in Example 3.3.

[0135] 4) The adipogenic differentiation capacity of primary and P2 generation cultured tendon-derived mesenchymal stem cells in serum-free culture medium combination 2 was detected, using the method described in Example 3.3. Oil Red O staining results showed that the primary cultured tendon-derived mesenchymal stem cells stained lighter than those in the P2 generation, indicating that the primary cultured tendon-derived mesenchymal stem cells synthesized and accumulated only a small amount of lipids after induction, suggesting a poor ability to differentiate into adipocytes. Furthermore, the Oil Red O staining of the P2 generation cultured cells after induction was also lighter than that of the primary cultured cells in serum-free culture medium combination 1 in Example 3.3.

[0136] The above experimental results demonstrate that primary cultured tendon-derived mesenchymal stem cells (MDSCs) using culture medium combination 2 provided in Example 1 of CN113692282A directly result in poor cell differentiation effects for tendon lineage differentiation and trilineage differentiation. Passaging is necessary to improve the differentiation effect of tendon-derived MDSCs. However, the culture medium combination 1 provided by this invention, using only primary cultured tendon-derived MDSCs, can achieve and exceed the tendon lineage and trilineage differentiation effects achievable only after induction of P2 generation tendon-derived MDSCs cultured in culture medium combination 2. Furthermore, the tendon-derived MDSCs successfully differentiated into tendon stem cells, osteoblasts, chondrocytes, and adipocytes after induction with different induction media.

[0137] Example 2: Effect of B-27 serum-free additive on serum-free cultured mesenchymal stem cells

[0138] This embodiment investigates the effect of adding B-27 serum-free additive to serum-free culture medium combination 1 of Example 1 on the proliferation and differentiation capacity of primary cultured tendon-derived mesenchymal stem cells, and compares it with the serum-free culture medium in CN113692282A previously applied for by the team of this invention.

[0139] The following five groups of culture media are set up as shown in Table 10 below.

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

[0141]

[0142] The above-mentioned culture medium combinations 1-2 and 5 were used to culture tendon-derived mesenchymal stem cells according to the primary culture method in Example 1. The proliferation and differentiation abilities of the primary cultured tendon-derived mesenchymal stem cells were detected, and the detection method was the same as in Example 1. Culture medium combinations 3-4 were first cultured according to the primary culture method in Example 1, and then passaged to the P2 generation. The proliferation and differentiation abilities of the P2 generation tendon-derived mesenchymal stem cells were detected, and the detection method was the same as in Example 1.

[0143] Figure 14 The growth morphology of primary cultured tendon-derived mesenchymal stem cells in the culture medium of Combination 1 (the present invention) at harvest time after 14 days is recorded. Figure 15 The growth morphology of primary cultured tendon-derived mesenchymal stem cells in combination 2 culture medium was compared with that of the two. The stem cells cultured in combination 1 (the present invention) were evenly distributed and had good growth morphology, while the addition of 1×B-27 serum-free additive in combination 2 culture medium caused local over-aggregation or sparseness of the cultured stem cells, and the cell growth morphology was poor. Figure 16This study aimed to determine the growth morphology of tendon-derived mesenchymal stem cells cultured in combination 3 culture medium until harvest at passage P2. Figure 17 The growth morphology of tendon-derived mesenchymal stem cells cultured in combination 4 medium was compared to that of stem cells harvested at P2 generation. There was no significant difference in the growth morphology of stem cells cultured in the two media. However, the lack of 1×B-27 serum additive in combination 4 medium resulted in sparser stem cells compared to those cultured in combination 3 medium. Figure 18 To serve as a control, we studied the growth morphology of primary tendon-derived mesenchymal stem cells cultured in combination 5 medium containing fetal bovine serum until harvest at 14 days.

[0144] The results of the proliferation and cell viability detection of tendon-derived mesenchymal stem cells are shown in Table 11 below. The results of the immunophenotype detection are shown in Table 12 below. The 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 in tendon-derived mesenchymal stem cells cultured in fetal bovine serum culture medium combination 5 are represented by "1".

[0145] Table 11. Effects of serum-free culture medium containing B-27 serum-free additive on stem cell proliferation.

[0146]

[0147] Table 12. Immunophenotyping results of stem cell cultured in serum-free culture medium containing B-27 serum-free additive.

[0148]

[0149] Table 13. Results of Detection of Relative Gene Expression Levels in Stem Cells Cultured in Serum-Free Culture Medium with or without B-27 Serum-Free Additive

[0150]

[0151] Based on the results in Table 11, there was a significant difference in the number of P2 generation tendon-derived mesenchymal stem cells cultured in serum-free medium between combinations 3 and 4. Combination 4, without the addition of 1×B-27 serum-free additive, resulted in a significant decrease in the number of cultured stem cells. Figures 16-17 This indicates that the serum-free culture medium provided in Example 1 of CN113692282A, previously applied for by our team, must contain 1×B-27 serum-free additive to improve the proliferation of tendon-derived mesenchymal stem cells during passage; and comparing the number of tendon-derived mesenchymal stem cells cultured in serum-free culture media of combinations 1 and 2, the number of stem cells cultured in combination 1 (this invention) is higher than that in combination 2. Figures 14-15This indicates that adding 1×B-27 serum-free additive to Combination 1 actually inhibited the primary proliferation of tendon-derived mesenchymal stem cells. It is speculated that various growth factors and hormones in the 1×B-27 serum-free additive overlap with certain components in the culture medium of Combination 1, leading to excessively high concentrations of certain components, nutrient imbalance, and metabolic disorders, thereby inhibiting normal cell growth. The number of cells cultured in the serum-free culture medium of Combination 1 (this invention) was significantly higher than that of Combinations 3 and 4, indicating that the serum-free culture medium provided by this invention, compared to the serum-free culture medium provided by our team in Example 1 of CN113692282A, not only eliminates the need for the addition of 1×B-27 serum-free additive, but also allows for a high number of tendon-derived mesenchymal stem cells (5.7 × 10⁻⁶) with only primary culture. 7 This exceeds the proliferation effect achieved by culturing to the P2 generation in the previous culture medium (1.9 × 10⁻⁶). 7 B-27 serum-free additive is expensive, and the serum-free culture medium provided by this invention can reduce the cost of culturing tendon-derived mesenchymal stem cells.

[0152] Analysis of the results in Table 12 shows that the tendon-derived mesenchymal stem cells cultured in serum-free culture medium combinations 1-4 all met the immunophenotypic requirements of mesenchymal stem cells and complied with safety indicators. Furthermore, the surface antigen detection results of mesenchymal stem cells showed that the expression levels of CD73, CD105, and CD90 were all ≥95%, while the expression levels of CD45, CD34, CD14, CD19, and HLA-DR were all ≤2%. This indicates that the cells cultured in these four groups conform to the mesenchymal stem cell phenotype and have the potential to differentiate into tendon stem cells.

[0153] According to the results in Table 13, the relative expression levels of SCX, NES, and THBS4 genes in P2 generation tendon-derived mesenchymal stem cells cultured in serum-free medium of combination 4 were significantly lower than those in combination 3. This indicates that the addition of 1×B-27 serum-free additive to the serum-free medium provided in Example 1 of CN113692282A, previously filed by our team, can improve the differentiation ability of tendon-derived mesenchymal stem cells. However, the relative expression levels of SCX, NES, and THBS4 genes in primary cultured tendon-derived mesenchymal stem cells in serum-free medium of combination 1 (the present invention) were not significantly different from those in combination 2. This indicates that the addition of 1×B-27 serum-free additive to the serum-free medium provided by the present invention only affects the expansion efficiency of primary cultured stem cells and 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 serum-free medium of Combination 1 (the present invention) are comparable to those in P2 generation tendon-derived mesenchymal stem cells cultured in serum-free medium of Combination 3. This indicates that the serum-free medium provided by the present invention does not require the addition of 1×B-27 serum-free additive to achieve the relative expression levels of the three genes in primary cultured cells that are equivalent to those in P2 generation cells cultured in serum-free medium provided in Example 1 of CN113692282A, further improving the tendon differentiation ability and pluripotency of primary cultured tendon-derived mesenchymal stem cells.

[0154] Example 3: Effects of different basal culture media on the proliferation and differentiation capacity of mesenchymal stem cells

[0155] 1. Effects of serum-free culture media containing different basal media on the proliferation capacity of mesenchymal stem cells.

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

[0157] Group 1: Zhongke Ruiji mesenchymal stem cell serum-free culture medium Pro (Zhongke Ruiji RGM1051);

[0158] Group 2: Serum-free culture medium for mesenchymal stem cells from Zhongke Ruiji (Zhongke Ruiji RGM1071).

[0159] Group 3: Huakan 3D FloTrix mesenchymal stem cell serum-free culture medium (Huakan Bio RMZ112-PYJ);

[0160] Group 4: Youkang mesenchymal stem cell serum-free culture medium (Youkang Bio NC0106);

[0161] Group 5: Culture medium containing fetal bovine serum.

[0162] The effects of the above five culture media on the proliferation capacity of tendon-derived mesenchymal stem cells were investigated. The primary culture method for mesenchymal stem cells was the same as that in Example 1, and the cell count detection method was the same as that in Example 1. After harvesting on day 14 of culture, the cell count and cell viability of each culture medium were detected, and the results are shown in Table 14 below.

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

[0164]

[0165] Analysis of the data in Table 14 shows that the serum-free culture media containing different basal media (groups 1-3) all exhibited stronger proliferative capacity for tendon-derived mesenchymal stem cells compared to the fetal bovine serum-containing culture medium (group 5). The proliferative effect of culture medium group 4 was not as good as that of culture medium group 5. Comparing the cell counts after 14 days of culture in media group 1-3, group 1 (Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Culture Medium Pro) showed the strongest cell proliferation effect and is the preferred choice. The basic components for maintaining cell growth in the four commercial mesenchymal stem cell culture media mentioned above should be similar. However, the unknown components added to Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Culture Medium Pro significantly improved its primary culture effect compared to the other three commercial media. It is speculated that human serum albumin, human platelet lysate, or other substances may have been added to the serum-free culture medium to enhance cell proliferation.

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

[0167] Furthermore, the tendon-derived mesenchymal stem cells cultured in the five culture media in Table 14 above were subjected to flow cytometry analysis by a testing company to detect the surface markers of the cultured mesenchymal stem cells. The test results are shown in Table 15 below, and the reference ranges for the test indicators are shown in Table 5 of Example 1.

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

[0169]

[0170] Analysis of the data in Table 15 showed that the tendon-derived mesenchymal stem cells cultured in serum-free medium in groups 1-4 all met the immunophenotypic requirements for mesenchymal stem cells and complied with safety criteria. CD73 was highly expressed on the surface of mesenchymal stem cells, while CD105 and CD90 were highly expressed on both mesenchymal and tendon stem cells. CD14, CD19, and HLA-DR were lowly expressed or absent on the surface of mesenchymal stem cells, and CD45 and CD34 were lowly expressed or absent on the surface of tendon stem cells. Detection of surface antigens in the tendon-derived mesenchymal stem cells cultured in serum-free medium in groups 1-4 showed that the expression levels of CD73, CD105, and CD90 were ≥95%, while the expression levels of CD45, CD34, CD14, CD19, and HLA-DR were ≤2%. This indicates that the tendon-derived cells cultured in the mediums of groups 1-4 conformed to the mesenchymal stem cell phenotype and have a high potential to differentiate into tendon stem cells.

[0171] Furthermore, the expression of the tendon-derived mesenchymal stem cells cultured in these 5 groups of culture media was detected by the expression of the tendon gene SCX and the NES and THBS4 genes. The detection method is the same as in Example 1. The relative expression levels of the three genes are shown in Table 16 below.

[0172] Table 16. Effects of serum-free culture media containing different basal media on the relative gene expression levels of mesenchymal stem cells.

[0173]

[0174] According to the data analysis in Table 16, the relative expression levels of SCX, NES, and THBS4 genes in tendon-derived mesenchymal stem cells cultured in serum-free medium in groups 1-4 with different basal media were all higher than those in group 5 with fetal bovine serum, indicating that tendon-derived mesenchymal stem cells cultured in media in groups 1-4 all possess tendon differentiation ability and pluripotency. Among them, the relative expression levels of these three genes in stem cells cultured in group 1 were significantly higher than those in the other three media, indicating that stem cells cultured in group 1 had stronger tendon differentiation ability and pluripotency. Therefore, the preferred basal medium is Zhongke Ruiji mesenchymal stem cell serum-free medium Pro.

[0175] Example 4: Screening of bioactive substances

[0176] 1. Effects of serum-free culture media containing different bioactive substances on mesenchymal stem cell culture

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

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

[0179]

[0180]

[0181] The above 18 groups of primary culture medium for tendon-derived mesenchymal stem cells were used to detect cell proliferation. The culture method was the same as that in Example 1, and the detection method was the same as that in Example 1. The detection results are shown in Table 18 below.

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

[0183]

[0184] Based on the results in Table 18, the effects of serum-free culture medium groups 1-7 on the primary culture of tendon-derived mesenchymal stem cells were compared. In serum-free culture medium groups 2-7, one bioactive substance was replaced in serum-free culture medium group 1. The results showed that the number of stem cells and cell viability of stem cells cultured in serum-free culture medium groups 2-6 were not significantly different from those in serum-free culture medium group 1, but the number of stem cells cultured in serum-free culture medium group 1 was the highest.

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

[0186] Comparing the effects of serum-free culture medium group 1 and serum-free culture medium groups 13-17 on the primary culture of tendon-derived mesenchymal stem cells, serum-free culture medium groups 13-17 were lacking one of the bioactive substances in serum-free culture medium group 1. The results showed that the number of stem cells cultured in serum-free culture medium groups 13-17 was significantly lower than that in serum-free culture medium 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 culture of mesenchymal stem cells.

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

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

[0189]

[0190] Analysis of the data in Table 19 shows that the tendon-derived mesenchymal stem cells cultured in serum-free medium in groups 1-17 all met the immunophenotypic requirements for mesenchymal stem cells and complied with safety indicators. Surface antigen detection results of the mesenchymal stem cells cultured in serum-free medium in groups 1-17 showed that the expression levels of CD73, CD105, and CD90 were all ≥95%, while the expression levels of CD45, CD34, CD14, CD19, and HLA-DR were all ≤2%. This indicates that the cells cultured in serum-free medium in groups 1-17 conformed to the mesenchymal stem cell phenotype and possessed good potential for differentiation into tendon stem cells.

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

[0192] Table 20. Results of relative gene expression levels in mesenchymal stem cells cultured in serum-free medium containing different bioactive substances.

[0193]

[0194] Analysis of the data in Table 20 compared the relative expression levels of SCX, THBS4, and NES genes in primary mesenchymal stem cells cultured in serum-free medium in groups 1 and 13-17 showed that the relative expression levels of SCX, THBS4, and NES genes in primary mesenchymal stem cells cultured in serum-free medium in groups 13-17 were all lower than those in group 1, with the most significant decrease in the relative expression level of SCX gene. This indicates that the absence of any one of the five bioactive substances in the serum-free medium in group 1 leads to a decrease in the ability of cultured mesenchymal stem cells to differentiate into tendon stem cells and a decrease in pluripotency. Comparison of groups 1 and 2-7, and groups 1 and 8-12 in serum-free medium... The relative expression levels of SCX, THBS4, and NES genes in primary cultured tendon-derived mesenchymal stem cells in serum culture medium were as follows: Group 1 showed the highest relative expression levels of the three genes, indicating that the stem cells have stronger tendon differentiation ability and pluripotency, making them the preferred choice. The lower relative expression level of 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 culture medium, it may lead to receptor competition and signaling pathway antagonism between the two transforming growth factors, resulting in a decrease in the relative expression level of SCX gene.

[0195] 2. Effects of serum-free culture media containing different concentrations of bioactive substances on mesenchymal stem cell culture

[0196] Furthermore, serum-free culture medium group 1 of the preferred scheme 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 capacity of tendon-derived mesenchymal stem cells.

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

[0198] Table 21. Serum-free culture media containing different concentrations of bioactive substances

[0199]

[0200] 2.1 Effect of bioactive substance concentration on the proliferative capacity of primary cultured tendon-derived mesenchymal stem cells

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

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

[0203]

[0204] Based on the data analysis in Table 22, primary culture of tendon-derived mesenchymal stem cells was performed in groups 1-21 using serum-free culture media containing different concentrations of bioactive substances, and the cell count in each group reached 1×10⁻⁶. 7 The results showed high cell viability, all above 90%, indicating that tendon-derived mesenchymal stem cells cultured in serum-free medium containing different concentrations of bioactive substances in groups 1-21 all exhibited good proliferative capacity.

[0205] Furthermore, by replacing the aforementioned vitamin C with L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, heparin with heparin sodium, PDGF-BB with PDGF-AA or a combination of PDGF-BB and PDGF-AA, FGF-basic with FGF-7 or a combination of FGF-basic and FGF-7, and TGF-β3 with TGF-β1 or TGF-β2, serum-free culture media of different concentrations were prepared. The cultured tendon-derived mesenchymal stem cells all exhibited good proliferative capacity, with primary culture cell numbers reaching 1×10⁻⁶. 7And maintain high cell viability. Therefore, the final concentration of vitamin C or its derivatives added is 0.1-100 μg / ml, the final concentration of heparin or its salts added is 0.1-10 μg / ml, the final concentration of transforming growth factor-β added is 0.1-80 ng / ml, the final concentration of fibroblast growth factor added is 1-100 ng / ml, and the final concentration of platelet-derived growth factor added is 1-100 ng / ml.

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

[0207] The immunophenotypes of the 22 groups of primary cultured tendon-derived mesenchymal stem cells in serum-free medium in Table 21 were detected. The detection method was the same as in Example 1. The immunophenotype detection results are shown in Table 23 below.

[0208] Table 23. Immunophenotyping results of mesenchymal stem cells cultured in serum-free culture medium containing different concentrations of bioactive substances.

[0209]

[0210] Analysis of the data in Table 23 shows that the tendon-derived mesenchymal stem cells cultured in serum-free medium in groups 1-21 all met the immunophenotypic requirements for mesenchymal stem cells and complied with safety indicators. Surface antigen detection results of the mesenchymal stem cells cultured in serum-free medium in groups 1-21 showed that the expression levels of CD73, CD105, and CD90 were all ≥95%, while the expression levels of CD45, CD34, CD14, CD19, and HLA-DR were all ≤2%. This indicates that the cells cultured in serum-free medium in groups 1-21 conformed to the mesenchymal stem cell phenotype and possessed good potential for differentiation into tendon stem cells.

[0211] Furthermore, the relative expression levels of SCX, NES, and THBS4 genes in 22 groups of primary cultured tendon-derived mesenchymal stem cells in serum-free medium were detected. The detection method was the same as in Example 1. The results of the relative expression level detection of the three genes are shown in Table 24 below.

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

[0213]

[0214] Analysis of the results in Table 24, comparing the results of groups 1 and 2-5, groups 1 and 6-9, groups 1 and 10-13, groups 1 and 14-17, and groups 1 and 18-21, revealed high relative expression levels of SCX, THBS4, and NES genes. Specifically, the SCX and NES gene expression levels were highest in the culture medium of group 1. These results indicate that the addition of vitamin C or its derivatives at final concentrations ranging from 0.1-100 μg / ml, heparin or its salts at final concentrations ranging from 0.1-10 μg / ml, transforming growth factor-β at final concentrations ranging from 0.1-80 ng / ml, fibroblast growth factor at final concentrations ranging from 1-100 ng / ml, and platelet-derived growth factor at final concentrations ranging from 1-100 ng / ml can significantly increase the relative expression levels of these three genes in tendon-derived mesenchymal stem cells, and also enhance the stem cells' tendon differentiation capacity and pluripotency.

[0215] Furthermore, by replacing the above-mentioned vitamin C with L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, heparin with heparin sodium, PDGF-BB with PDGF-AA or a combination of PDGF-BB and PDGF-AA, FGF-basic with FGF-7 or a combination of FGF-basic and FGF-7, and TGF-β3 with TGF-β1 or TGF-β2, serum-free culture media of different concentrations were prepared. The cultured tendon-derived mesenchymal stem cells all exhibited good tendon differentiation ability and pluripotency.

[0216] Example 5: Effects of serum-free culture medium on the proliferation and differentiation of primary cultured umbilical cord-derived mesenchymal stem cells.

[0217] Example 1 demonstrates that the serum-free culture medium combination 1 provided by this invention is particularly suitable for the primary culture of tendon-derived mesenchymal stem cells. It not only improves the proliferation of primary cultured tendon-derived mesenchymal stem cells but also enhances their tendon differentiation capacity and trilineage differentiation capacity. This example investigates whether the serum-free culture medium combination 1 can affect the primary culture proliferation and differentiation capacity of mesenchymal stem cells from other sources. The specific methods are as follows:

[0218] Umbilical cord cells were cultured according to the primary culture method 1 of Example 1 using serum-free culture medium combination 1 of Example 1, with the fetal bovine serum-containing culture medium combination 3 of Example 1 serving as a control. At cell harvest, the proliferation, immunophenotype, and relative expression levels of the three genes SCX, THBS4, and NES were measured, using the same methods as in Example 1. The growth morphology of umbilical cord-derived mesenchymal stem cells cultured in serum-free culture medium combination 1 of Example 1 on day four of culture is shown below. Figure 19As shown, the cultured umbilical cord-derived mesenchymal stem cells maintain good adherence, have abundant cytoplasm, exhibit a spindle-shaped cell morphology, and are densely packed. The growth morphology of umbilical cord-derived mesenchymal stem cells cultured on day six in a medium containing fetal bovine serum is as follows. Figure 20 As shown, although the cultured umbilical cord-derived mesenchymal stem cells maintained good adherence and exhibited a spindle-shaped cell morphology, the cell number was sparse. The cell number and viability at harvest are shown in Table 25, the immunophenotype results are shown in Table 26, and the relative expression levels of the three genes SCX, THBS4, and NES are shown in Table 27.

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

[0220]

[0221] Based on the data analysis in Table 25, primary culture of umbilical cord-derived mesenchymal stem cells using the serum-free culture medium provided by this invention can also achieve a quantity of 1×10⁻⁶. 7 Furthermore, the cell viability was high, but compared with the number of tendon-derived mesenchymal stem cells cultured in Example 1, it indicates that the serum-free culture medium is more effective for the proliferation of tendon-derived mesenchymal stem cells.

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

[0223]

[0224] According to the data analysis in Table 26, the primary umbilical cord-derived mesenchymal stem cells cultured using the serum-free culture medium provided by this invention all met the immunophenotypic requirements of mesenchymal stem cells and complied with the safety indicators.

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

[0226]

[0227] Analysis of the data in Table 27 shows that the relative expression level of NES in primary cultured umbilical cord-derived mesenchymal stem cells (MSCs) using the serum-free medium provided by this invention is significantly higher than that of SCX and THBS4, indicating that the MSCs possess high self-renewal capacity and multi-lineage differentiation potential. However, comparing the relative expression level of SCX in primary cultured tendon-derived MSCs using the serum-free medium in Example 1, the relative expression level of SCX in the cultured umbilical cord-derived MSCs is significantly lower than that in the tendon-derived MSCs, indicating that the cultured umbilical cord-derived MSCs have poor tendon differentiation ability. This suggests that the serum-free medium provided by this invention is more suitable for the primary culture of tendon-derived MSCs and can significantly improve their tendon differentiation ability.

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

[0229] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A serum-free culture medium, characterized in that, The medium includes bioactive substances and a basal culture medium; the bioactive substances are vitamin C, heparin, TGF-β3, FGF-basic, and PDGF-BB; the serum-free culture medium does not contain B-27 serum-free additive; the mass ratio of the bioactive substances to the basal culture medium is (0.302-190.2):100000000; the final concentration of vitamin C is 100 μg / ml, the final concentration of heparin is 10 μg / ml, the final concentration of TGF-β3 is 80 ng / ml, the final concentration of FGF-basic is 100 ng / ml, and the final concentration of PDGF-BB is 100 ng / ml; the basal culture medium is Zhongke Ruiji Mesenchymal Stem Cell Serum-Free Culture Medium Pro, the model of which is Pro Zhongke Ruiji RGM1051, with its accompanying additives added.

2. The application of the serum-free culture medium as described in claim 1 in the primary culture of tendon-derived mesenchymal stem cells, characterized in that, The application aims to improve the proliferation capacity of primary cultured tendon-derived mesenchymal stem cells and the expression levels of SCX, THBS4, and NES.

Images