A bioactive substance composition, a serum-free culture medium containing the composition, and uses thereof

Abstract

The present invention provides a bioactive substance composition, a serum-free culture medium containing the composition, and uses thereof; the bioactive substance composition is used in a serum-free culture medium and / or composition and its preparation; the serum-free culture medium and / or composition can be used for primary culture and subculture of cells and / or tissues. The cells are selected from any one or more of tendon and / or ligament-derived cells, chondrocytes, meniscus stem cells, mesenchymal stem cells, skeletal stem cells, and muscle stem cells. The tissue is derived from the musculoskeletal system. The bioactive substance composition and / or serum-free culture medium and / or composition can be used to prepare a drug for treating tissue and / or organ damage; the tissue or organ damage is selected from musculoskeletal system tissue or organ damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a serum-free culture medium prepared from bioactive substances and its application. Background Art

[0002] In recent years, advances in cell therapy, tissue engineering, and molecular biology have brought new opportunities for improving tissue repair quality. Researchers have begun exploring the use of tissue engineering and cell therapy to treat and repair tissue defects. Obtaining ideal seed cells is crucial for tissue repair and regeneration. Common cell types used in cell therapy include mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs, or ASCs), tendon and / or ligament-derived cells, and chondrocytes. However, the number of naturally occurring cells in the body suitable for cell therapy is limited and cannot meet the needs of cell therapy. Therefore, stem cells that can be expanded in vitro with high quality and quantity are currently the key to the implementation of stem cell therapy.

[0003] The natural microenvironment for cell growth in the body is extremely complex. With the exception of vascular wall cells and blood-related cells, all other cells in the body reside in serum-free extracellular fluid. This complex natural microenvironment for cell growth is comprised of the complex composition of the extracellular fluid, cell-cell interactions, and cell-extracellular matrix interactions. Cells from different tissues / organs reside in different microenvironments in the body, such as differences in the types and content of extracellular fluid components, and differences in cell-cell and cell-extracellular matrix interactions. These microenvironments are aligned with the functions of the tissues / organs and can effectively promote vital activities such as cell proliferation, phenotypic maintenance, and metabolism in the body, thereby maintaining tissue / organ function. Currently, we know very little about the specific components and interaction mechanisms of this complex and diverse microenvironment in the body. We have only explored the tip of the iceberg and are unable to gain sufficient inspiration to simulate the in vivo microenvironment for cell culture in vitro. At present, the culture environment of stem cells in vitro is mainly based on culture medium containing serum. The commonly used serum can be fetal bovine serum. However, the extracellular fluid microenvironment where cells in the body are located does not contain serum. In addition, due to the complex composition of extracellular fluid, cell-cell interactions, and cell-extracellular matrix interactions, the growth environment of cells in the body is particularly complex, resulting in a huge difference between the in vivo and in vitro environments. The culture environment of stem cells in vitro cannot maintain the proliferation and phenotype of cultured cells. Therefore, how to better simulate the in vivo cell growth environment in vitro is a major unresolved problem in in vitro cell culture.

[0004] Taking the in vitro culture of tendon and / or ligament-derived cells as an example:

[0005] Tendon- and / or ligament-derived cells are a mixed population of cells isolated and extracted from tendon or ligament tissue. They highly express multiple tendon / ligament-specific genes and proteins, including scleraxis (SCX), nestin (NES), tenomodulin (TNMD), thrombospondin-4 (THBS4), collagen type I alpha 1 (COL1, COL1A1), and tenascin-C. These cells include tendon stem / progenitor cells (TSPCs), tendon-derived stem cells (TDSCs), tendon cells (tenocytes), tenoblasts, fibroblasts, ligament stem / progenitor cells, and ligament cells. They are ideal seed cells for tendon injury treatment. Researchers in this field, both domestically and internationally, have isolated tendon stem cells from mouse, human, rat, and rabbit tendons and have thoroughly characterized their function and phenotype, including their multipotency. Studies have shown that these cells not only possess stem cell properties similar to bone marrow mesenchymal stem cells, but also highly express multiple tendon-specific genes and proteins, including scleraxis (SCX), nestin (NES), tenomodulin (TNMD), thrombospondin-4 (THBS4), collagen type I alpha 1 (COL1, COL1A1), and tenascin-C. Therefore, tendon and / or ligament-derived cells, particularly tendon stem cells, are considered suitable seed cells for tendon tissue engineering and cell therapy for tendon injury.

[0006] Due to the small number of cells present in mature tendon and / or ligament tissue, the number of extracted tendon and / or ligament-derived cells is insufficient for use in tendon injury regeneration. Therefore, it is necessary to amplify them in an in vitro culture environment to obtain tendon and / or ligament-derived cells of good quantity and quality. Therefore, in order to meet the clinical treatment objectives of tendon and / or ligament injuries, the in vitro cultured tendon and / or ligament-derived cells must meet certain quality and quantity requirements. Tendon and / or ligament-derived cells have both the common stem cell characteristics of mesenchymal stem cells, adipose stem cells, and other cells, as well as their own unique tendon phenotype. The above-mentioned quality and quantity requirements are mainly evaluated and scored from two major aspects: the common stem cell characteristics and the unique phenotype of tendon / ligament-derived cells, with a total score of 100 points. The specific scoring criteria are as follows (Bi Y et al., Nature medicine, 2007, 13(10): 1219-27. Harvey T, Nature cell biology, 2019, 21(12): 1490-1503. Yin Z et al., Science advances, 2016, 2(11): e1600874. Lee SY, Stem Cells, 2015, 33(10): 2995-3005. Zhang C, Biomaterials, 2018, 172: 66-82.):

[0007] 1. Common characteristics of stem cells (also known as common cell characteristics) (60 points)

[0008] 1. Proliferation rate (30 points): A cell doubling time of 30 hours or less is assigned 30 points. A cell doubling time of 76 hours or less or more than 5 times a week is assigned a passing score of 10 points. A cell doubling time of 76 hours to 100 hours is assigned 5 points. A cell doubling time of more than 100 hours is assigned 0 points.

[0009] 2. Stem Cell Phenotype (20 points): Stem cell phenotype includes stem cell surface markers, clonogenicity, and trilineage differentiation capacity. Stem cell surface markers include positive markers CD105, CD90, and CD44. Expression of these markers must be above 95% to indicate maintenance or improvement of the phenotype. Negative markers CD34 and CD18 must be expressed at or below 1% to indicate maintenance. Clonogenicity refers to the ability of cells to form monoclonal colonies. Clonogenicity that exceeds that of cells cultured with existing serum culture techniques is considered improved, while clonogenicity that is consistent with that of cells cultured with existing serum culture techniques is considered maintained. Trilineage differentiation capacity includes osteogenic, chondrogenic, and adipogenic differentiation capacities. Clonogenicity that exceeds that of cells cultured with existing serum culture techniques is considered improved, while clonogenicity that is consistent with that of cells cultured with existing serum culture techniques is considered maintained. Improvement in all three of these criteria, including stem cell surface markers, clonogenicity, and trilineage differentiation capacity, is worth 20 points. Improvement in one or two criteria while maintenance in the others is worth 15 points. Maintaining all three criteria is worth 10 points, while a decrease in two criteria is worth 5 points. A decrease in all three criteria is worth 0 points. Other scores will be based on the circumstances.

[0010] 3. Safety (10 points): This includes three criteria: normal karyotype, absence of serum residue, and absence of viral mycoplasma contamination. Karyotype analysis indicates that more than 90% of the cell karyotypes are consistent with the normal karyotype of cells of the species, indicating that the cultured cells are normal. A pass score of 10 is awarded if all three criteria are met. A score of 0 is awarded if any of the three criteria are not met. Any cells cultured with serum, whether primary or subcultured, are considered to have residual serum and receive a score of 0. Only cells cultured without serum in both primary and subcultures are considered to be serum-residual free.

[0011] II. Unique phenotypes of cells derived from tendons and / or ligaments (also called cell-specific phenotypes) (40 points)

[0012] 4. Tendon phenotype and tendon differentiation ability (20 points): including tendon gene or protein markers such as SCX, Nestin, TNMD, THBS4, COL1, and collagen formation ability. High expression of three or more tendon gene or protein markers such as SCX, Nestin, TNMD, THBS4, COL1, and the positive rate of tendon markers in cultured cells, such as a Nestin positive rate of more than 90%, is 20 points; high expression of three or more tendon gene or protein markers such as SCX, Nestin, TNMD, THBS4, COL1, and the positive rate of tendon markers in cultured cells, such as a Nestin positive rate of more than 60%, is 15 points; high expression of two tendon genes such as SCX, Nestin, TNMD, THBS4, COL1, and the positive rate of tendon markers in cultured cells, such as a Nestin positive rate of more than 60%, is 15 points. If a gene or protein marker is expressed, and the cultured cells have a tendon marker positivity rate (e.g., Nestin positivity rate of 30% or more), the score is 10. High expression of a tendon marker gene or protein marker, such as SCX, Nestin, TNMD, THBS4, or COL1, and a tendon marker positivity rate (e.g., Nestin positivity rate of 10% or more), the passing score is 5. No or low expression of a tendon marker gene or protein marker, such as SCX, Nestin, TNMD, THBS4, or COL1, and a tendon marker positivity rate (e.g., Nestin positivity rate of less than 10%), the score is 0. Other scores are scored based on the specific situation. "High expression" means that the cultured cells have higher expression of the gene or protein marker compared to cells cultured with existing serum culture techniques; "low expression" means that the cultured cells have lower expression of the gene or protein marker compared to cells cultured with existing serum culture techniques; "no expression" means that the expression of the gene or protein marker in the cultured cells is 0 or extremely low, making it undetectable.

[0013] 5. In vivo tendon and / or ligament repair ability (20 points): If the repaired tissue morphology is close to normal tissue, with dense and neatly arranged collagen, and no non-tendon and / or ligament tissue such as bone, cartilage, or muscle is produced during the repair process, a score of 20 is awarded. If the repaired tissue has a small defect, the collagen formation is relatively neatly arranged, and no non-tendon tissue such as bone or cartilage is produced, a passing score of 10 is awarded. If the repaired tissue has a large defect or non-tendon tissue such as bone, cartilage, or muscle is produced, a score of 0 is awarded. Scoring is based on the specific tissue repair situation.

[0014] Tendon and / or ligament-derived cells cultured in vitro must meet the requirements for clinical treatment of tendon and / or ligament injuries, that is, the five individual items included in the common characteristics of the cells and the cell-specific phenotypes all reach their respective passing lines and the total cell score is greater than or equal to 60 points, which means that the cell indicators are qualified and can meet the requirements of clinical cell therapy. The higher the score, the better the quality and quantity of the cultured cells, and the more in line with the needs of clinical cell therapy; that is, each of the five individual indicators of proliferation rate, stem cell phenotype, safety, tendon phenotype and tendon differentiation ability, and in vivo tendon and / or ligament repair ability must reach their respective passing lines and the total score is greater than or equal to 60 points to be considered qualified for the common characteristics of the cells and the cell-specific phenotypes of the tendon and / or ligament-derived cells cultured in vitro. If any of the five individual indicators does not reach the passing line or the total cell score is less than 60 points, it is considered that the cell indicators are unqualified and not suitable for clinical cell therapy.

[0015] In vitro cell culture is a method that simulates the complex in vivo cell environment, enabling them to survive, grow, reproduce, and maintain their primary structure and function. In vitro cell culture primarily involves two steps: 1) Primary culture: This refers to the initial culture of tissue or cells removed from the body. Strictly speaking, this refers to the inoculation and culture of tissue or cells from the body until the first passage. Cells cultured at this stage are called primary cells (P0). This process involves the transition of cells from the in vivo microenvironment to the in vitro culture environment. To enable cells to adapt to the in vitro environment and proliferate as quickly as possible, the in vitro culture environment for primary cells must closely mimic the complex in vivo microenvironment, requiring more stringent environmental conditions than those for passage culture. 2) Subculture: Primary (P0) cultured cells or cells from other passages that have been cultured in vitro are separated into single cells for subculture. Tendon and / or ligament-derived cells are generally cultured to passage 5 (P5) or higher. Cells of appropriate passages are selected for tendon injury repair or other applications based on their shared stem cell characteristics and the maintenance of their unique phenotype.

[0016] Culture medium is a key factor in in vitro cell expansion. Culture medium refers to a soluble liquid nutrient matrix composed of a combination of different nutrients that supports cell growth and reproduction. Culture medium not only provides nutrition and promotes cell proliferation for cells cultured in vitro, but also serves as an in vitro living environment for cell growth and reproduction, enabling both primary and subculture of cells.

[0017] Because the in vivo microenvironment in which cells live is extremely complex, many components / factors remain to be fully understood. However, animal serum / plasma, such as fetal bovine serum (FBS), is widely available and has a mature preparation process. It contains rich nutrients such as proteins, hormones, and enzymes that can promote cell growth. Therefore, in vitro culture of tendon and / or ligament-derived cells, especially primary culture, currently requires that the in vitro culture environment of primary cells simulate the complex in vivo microenvironment as much as possible in order to enable cells to adapt to the in vitro environment and proliferate as quickly as possible. The environmental requirements are higher than those of subculture, and only culture medium containing serum / plasma can be used to provide cells with an environment relatively similar to that in the organism. However, studies have found that tendon and / or ligament-derived cells cultured in vitro with serum-containing medium cannot simultaneously meet the cell quality and quantity requirements for the clinical treatment of tendon and / or ligament injuries. The total cell score is below 60 points, which means that the cell indicators are unqualified. Specifically, 1) Tendon and / or ligament-derived cells proliferate slowly in serum-containing medium in vitro. As the number of in vitro culture generations increases, replicative senescence occurs, resulting in slower and slower cell proliferation and inability to expand and obtain sufficient cell quantity in a short period of time. 2) As the number of culture generations increases, the tendon phenotype of tendon and / or ligament-derived cells cultured in serum cannot be maintained, which is mainly manifested in the gradual decrease or even complete loss of the expression of tendon genes or proteins such as SCX, DCN, and TNMD ( Figure 1-2 ), high expression of bone genes such as alkaline phosphatase (ALP) and osteocalcin (OCN); 3) Animal experiments have shown that tendons and / or ligaments repaired with serum-cultured tendon and / or ligament-derived cells are composed of a large number of small-diameter collagen fibers, and their function is significantly lower than that of normal tendons ( Figure 3 Furthermore, tendon and / or ligament-derived cells obtained by serum culture are prone to heterotopic ossification when used for tendon and / or ligament repair, that is, bone tissue grows in the position of tendon and / or ligament tissue, resulting in failure of tendon and / or ligament repair ( Figure 4); 4) The complexity and characteristics of serum itself lead to safety risks in tendon and / or ligament-derived cells obtained by culture, including: a. The use of serum carries the risk of contamination with exogenous viruses and pathogenic factors, which can easily lead to contamination of cultured cells with viruses and mycoplasmas; b. The composition of serum is complex and unclear, and it is easy for the recipient to remain in the cell products and cause allergic reactions to the serum, which is not conducive to animal experiments or clinical trials; c. Serum or plasma has batch differences, and the biological activity and factors between different batches of serum are inconsistent, which will lead to poor reproducibility of cell products and experimental results, and thus require a lot of verification work. Therefore, it is very necessary to explore suitable methods to replace existing serum culture technology, reduce or avoid the adverse effects of serum, and amplify tendon and / or ligament-derived cells with a total cell score greater than or equal to 60 points for the clinical treatment of tendon and / or ligament injuries.

[0018] Existing studies have attempted to use single or multiple physical factors to reduce or avoid the adverse effects of serum culture, and to meet the quality and quantity requirements of cells used in the clinical treatment of tendon and / or ligament injuries as much as possible. Physical factors mainly include the surface topology of the culture substrate, hardness and mechanical stimulation. Adding these factors can maintain the tendon phenotype of serum-cultured tendon and / or ligament-derived cells to a certain extent, but their effect on promoting cell proliferation is general, cell proliferation is slow, the amount of cells obtained is small, and it is impossible to ensure a sufficient number of cells at the same time. Moreover, the addition of physical factors still needs to be based on serum culture, which cannot avoid the safety hazards brought by the serum itself, nor can it replace serum. It also increases the complexity of the culture system and increases the difficulty of implementation. Therefore, this culture method cannot simultaneously meet the quality and quantity requirements of cells for the clinical treatment of tendon and / or ligament injuries, and the total score of the cells obtained in culture is lower than the passing line.

[0019] Serum-free culture media refers to liquid nutrient matrices that are free of blood-derived substances such as serum, plasma, platelet-rich plasma (PRP), or blood, and contain a variety of well-defined bioactive substances, inorganic salts, and water. Serum-free culture media are categorized as complete serum-free and partial serum-free. Complete serum-free culture media are serum-free media that can support both serum-free primary and subculture of cells in vitro, providing the nutrients necessary for cell proliferation, phenotypic maintenance, metabolism, and other vital activities. Partial serum-free culture media, on the other hand, cannot be completely free of serum and other blood-derived substances for cell culture and can only support subculture, not primary culture. Because primary culture removes cells from the complex and adaptive microenvironment of the body and transitions them to an in vitro culture environment, they undergo an adaptation process. The closer the in vitro culture environment resembles the complex in vivo microenvironment, the shorter the adaptation period. Therefore, primary culture requires providing cells with a more biomimetic environment than subculture, enabling them to quickly recover and adapt to the in vitro culture environment for expansion.

[0020] The existing serum-free culture media for tendon and / or ligament-derived cells cannot replace the role of serum for primary culture, and can only achieve subculture of tendon and / or ligament-derived cells. Therefore, they are all partially serum-free culture media, and the cells cultured therein cannot meet the requirements of clinical cell therapy. Specifically, the existing technology combines single or multiple growth factors or cytokines with basal culture media such as DMEM or F12 to form a partial serum-free culture medium, which cannot replace serum for primary culture of tendon and / or ligament-derived cells. The cultured cells are still derived from existing serum culture technology, which brings the adverse effects of existing serum culture technology from the source. Subsequent subculture with these partial serum-free culture media can only alleviate to a certain extent but cannot avoid the adverse effects of serum culture. Furthermore, these partially serum-free culture media also have poor results in subculture, with the total score of cultured cells falling below the passing line, and cannot simultaneously meet the requirements for both cell quantity and quality required for the clinical treatment of tendon and / or ligament injuries (Cells Tissues Organs 2013;197:27–36, Chinese Journal of Experimental Surgery. 2014.31(2): 395-398, J. Hand Surg 2005;30:441–447, Biomaterials 2015;69: 99-109). Therefore, the partially serum-free culture media of the existing technology cannot solve the drawbacks of the existing serum culture technology, and completely serum-free culture media for tendon and / or ligament-derived cells have not yet been studied and applied.

[0021] Existing commercial serum-free culture media are developed for cells such as mesenchymal stem cells (MSC), adipose-derived stem cells (ADSC or ASC), pluripotent stem cells (PSC), and neural stem cells (NSC). Common ones include StemProTMMSC SFMXenoFree (Invitrogen, Gibco), MesenCultTM-ACF Plus Medium (STEMCELL Technologies), Mesenchymal Stem Cell Growth Medium DXF (PromoCell), MSC XF (Biological Industries), and StemPro™ NSC SFM (Invitrogen, Gibco). Tendon- and / or ligament-derived cells differ from these stem cells in possessing their own unique characteristics. Specifically, they highly express tendon- and / or ligament-specific genes and proteins, such as scleraxis (SCX), nestin (NES), tenomodulin (TNMD), thrombospondin-4 (THBS4), and collagen type I alpha1 (COL1, COL1A1). Other cells, such as neurons, highly express markers such as PSA-NSAM, p75NTR, Musashi1, ASH1, CD133, and GFAP, which are absent or expressed at low levels in tendon- and / or ligament-derived cells. Consequently, tendon- and / or ligament-derived cells possess specificity that commercial serum-free culture media for other stem cells cannot maintain. Consequently, these commercial serum-free culture media for other stem cells are unsuitable for culturing tendon- and / or ligament-derived cells. Consequently, there is currently no research or application developing a completely serum-free culture medium for tendon- and / or ligament-derived cells that can replace serum for primary and subculture to achieve the cell quantity and quality required for clinical treatment.

[0022] The paper (Cells Tissues Organs 2013;197:27–36) disclosed that 50 ng / mL insulin-like growth factor 1 and 10 ng / mL transforming growth factor β3 can maintain the phenotype of tendon cells in the absence of serum. However, in the experimental methods and materials of this study, the tenocyte isolation and culture method section indicates that the cells used by the institute were primarily cultured in a culture medium containing 20% fetal bovine serum. Therefore, the culture medium of this study is a partially serum-free culture medium, which cannot circumvent the drawbacks of existing serum culture technology, and the safety score of the cultured cells is 0 points. As can be seen from Comparative Example 1, the total score of cells cultured with serum culture technology is less than 60 points, and the paper shows that the proliferation effect of tendon cells under this condition is only 1 / 3 of that of the serum culture group ( Figure 5), collagen formation was only 1 / 2 of that in the serum culture group ( Figure 6 ), the expression of tendon-specific markers such as SCX is also much lower than that in serum culture medium. Therefore, the total score of cells cultured in this way is lower than that of cells cultured with serum culture technology, far below 60 points. Not only can it not replace serum for the in vitro isolation and culture of tendon-derived cells, it also cannot meet the cell requirements for the clinical treatment of tendon injuries.

[0023] A paper (Chinese Journal of Experimental Surgery. 2014. 31(2): 395-398) reported that adding 50 μg / L IGF-1 and 10 μg / L TGF-β3 to serum-free α-MEM culture medium maintained the phenotype of human tenocytes, producing collagen fibers similar to those produced by tenocytes cultured with 10% FBS, while also upregulating the mRNA expression of tenocyte phenotype and differentiation markers. However, this culture method did not promote cell proliferation, with a single cell proliferation score of 0, and the cultured cells could not meet the cell quantity requirements for clinical cell therapy.

[0024] A paper (J. Hand Surg 2005;30:441–447) reported that the addition of platelet-derived growth factor BB (PDGF-BB) and basic fibroblast growth factor (bFGF) to DMEM basal culture medium promoted tenocyte proliferation and collagen formation. The Materials and Methods section, regarding the isolation and culture of tendon fibroblasts, indicates that the cells used were primarily cultured in a medium containing 10% fetal bovine serum. Therefore, the culture medium used in this study was partially serum-free, which did not circumvent the drawbacks of existing serum-based culture techniques and resulted in a safety score of 0. Furthermore, the results of the paper demonstrated that these culture conditions resulted in slow cell proliferation and a low yield, making them insufficient to provide a suitable environment for in vitro cell expansion. Therefore, the overall score of the cells obtained in this paper was below the passing grade, and the results do not indicate that the addition of these two growth factors can replace existing serum-based culture techniques for in vitro culture of tenocytes to obtain the number and quantity of tendon-derived cells required for clinical cell therapy.

[0025] A paper (Biomaterials 2015; 69: 99-109) describes the use of biomimetic microtissue spheroids and specific growth factor supplements to improve tenocyte differentiation in vitro. This culture method, which utilizes a hanging drop technique combined with a low-serum growth medium containing L-ascorbic acid 2-phosphate, insulin, and transforming growth factor (TGF)-1, maintains the tendon phenotype of differentiated tenocytes in vitro. However, the research paper indicates that the culture system still requires low serum levels, which cannot avoid the adverse effects of serum. The safety score was 0, and cell proliferation issues were not addressed, making it impossible to meet the therapeutic and cell quantity requirements for clinical cell therapy.

[0026] Regarding the culture of tendon and / or ligament-derived cells, although the existing technologies have attempted to use single or combined physical factors or biological factors to avoid the drawbacks of current serum-based culture of tendon and / or ligament-derived cells, these technologies still require serum to participate in the primary culture of cells, or even in subculture, and the results are not good. The tendon and / or ligament-derived cells cultured using existing culture technologies cannot simultaneously meet the quality and quantity requirements of cells for clinical treatment of tendon and / or ligament injuries. The total score of cultured cells is less than 60 points, the cell indicators are unqualified, and they are not suitable for clinical cell therapy. The currently commercialized serum-free culture medium is also unable to maintain the specificity of tendon and / or ligament-derived cells and is not suitable for in vitro culture of tendon and / or ligament-derived cells. Therefore, it is an urgent problem to develop a completely serum-free culture medium for tendon and / or ligament-derived cells that is conducive to the efficient proliferation and phenotypic maintenance of tendon and / or ligament-derived cells, avoid the drawbacks of existing tendon and / or ligament-derived cell culture technologies, and meet the requirements of cell quantity and quality required for clinical treatment of tendon and / or ligament injuries. However, the existing technology does not have a corresponding solution. Summary of the Invention

[0027] To address the problems of existing in vitro cell culture, such as slow cell proliferation, low cell numbers, easy phenotypic loss, unstable cell quality, poor safety, and failure of injury repair after transplantation, the present invention provides a composition of a bioactive substance, a serum-free culture medium containing the composition, and uses thereof. Surprisingly, through continuous research, the inventors have developed a completely serum-free culture medium with a well-defined composition that enables completely serum-free primary and subculture of cells. This medium overcomes the prior art's technical bias that primary culture of tendon and / or ligament-derived cells must involve serum, while also meeting the cell quantity and quality requirements for clinical treatment of tendon and / or ligament injuries, achieving unexpected technical results.

[0028] The common cell phenotypes and cell-specific phenotypes of cells cultured in the serum-free medium or composition of the present invention can be maintained or improved, that is, the common cell characteristics (proliferation rate, stem cell phenotype, safety) and cell-specific phenotypes (tendon phenotype and tendon differentiation ability, in vivo tendon and / or ligament repair ability) of cells cultured in the serum-free medium or composition, these five individual items all reach their respective passing lines and the total cell score is greater than or equal to 60 points. Under optimal conditions, the total cell score can reach 100 points, which can simultaneously meet the treatment and quantity requirements of cells required for clinical cell therapy.

[0029] The technical solutions of the present invention are as follows:

[0030] The first object of the present invention is to provide a bioactive substance composition, which contains fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, and selenite.

[0031] Among them, the mass-volume concentration range ratio of each component is:

[0032] Fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 1-50: 1-50: 1-40: 1-11: 10-5000: 10-100000: 10-300000: 1-25000: 1-25: 1-25000: 1-25; preferably, the fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 5-40: 5-40: 2-30: 1-8: 500-4000: 1000-90000: 10 00-200000: 10-15000: 1-15: 2-15000: 1-15; more preferably, fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 10-30: 10-30: 3-20: 2-5: 1000-2000: 10000-80000: 2000-80000: 100-5000: 2-7: 7-10000: 2-7.

[0033] Furthermore, in the bioactive substance composition, the fibroblast growth factor is selected from any one or more of FGF-basic, FGF1, FGF2, FGF4, FGF7, FGF10, FGF18, and fibroblast growth factor synthetic peptides. Preferably, the mass-volume concentration of the fibroblast growth factor in the bioactive substance composition is 1-100 ng / ml, accounting for 0.0000001%-0.00001% by mass; preferably, the mass-volume concentration of the fibroblast growth factor in the bioactive substance composition is 5-70 ng / ml, accounting for 0.0000005%-0.000007% by mass; preferably, the mass-volume concentration of the fibroblast growth factor in the bioactive substance composition is 10-40 ng / ml, accounting for 0.000001%-0.000004% by mass.

[0034] Furthermore, in the bioactive material composition, the platelet-derived growth factor is selected from any one or more of PDGF-AA, PDGF-AB, PDGF-BB, and platelet-derived growth factor synthetic peptides. Preferably, the mass-volume concentration of the platelet-derived factor in the bioactive material composition is 1-100 ng / ml, accounting for 0.0000001%-0.00001% by mass; preferably, the mass-volume concentration of the platelet-derived factor in the bioactive material composition is 5-70 ng / ml, accounting for 0.0000005%-0.000007% by mass; preferably, the mass-volume concentration of the platelet-derived factor in the bioactive material composition is 10-40 ng / ml, accounting for 0.000001%-0.000004% by mass.

[0035] Furthermore, in the bioactive substance composition, the transforming growth factor-β is selected from any one or more of TGF-β1, TGF-β2, TGF-β3, and a transforming growth factor-β synthetic peptide. Preferably, the mass-volume concentration of the transforming growth factor-β in the bioactive substance composition is 0.1-80 ng / ml, accounting for 0.00000001%-0.000008% by mass; preferably, the mass-volume concentration of the transforming growth factor-β in the bioactive substance composition is 2-50 ng / ml, accounting for 0.0000002%-0.000005% by mass; preferably, the mass-volume concentration of the transforming growth factor-β in the bioactive substance composition is 5-25 ng / ml, accounting for 0.0000005%-0.0000025% by mass.

[0036] Furthermore, in the bioactive substance composition, the glucocorticoid is selected from any one or more of dexamethasone or its salt, dexamethasone solvate, hydrocortisone or its salt, hydrocortisone solvate, cortisone acetate, cortisone or its salt, methylprednisolone sodium succinate, prednisone, betamethasone, betamethasone valerate, beclomethasone dipropionate, prednisolone acetate, and prednisolone. Preferably, the molar concentration of the glucocorticoid in the biologically active substance composition is 0.1-90 nM, accounting for 0.0000000039%-0.00000354% by mass; preferably, the molar concentration of the glucocorticoid in the biologically active substance composition is 1-50 nM, accounting for 0.000000039%-0.00000197% by mass; preferably, the molar concentration of the glucocorticoid in the biologically active substance composition is 1-20 nM, accounting for 0.000000039%-0.000000785% by mass.

[0037] Furthermore, in the bioactive substance composition, the heparin or its salt is selected from any one or more of heparin, heparin sodium, and heparin calcium. Preferably, the mass-volume concentration of the heparin or its salt in the bioactive substance composition is 0.1-10 μg / ml, accounting for 0.00001%-0.001% by mass; preferably, the mass-volume concentration of the heparin or its salt in the bioactive substance composition is 0.5-8 μg / ml, accounting for 0.00005%-0.0008% by mass; preferably, the mass-volume concentration of the heparin or its salt in the bioactive substance composition is 1-5 μg / ml, accounting for 0.0001%-0.0005% by mass.

[0038] Furthermore, in the bioactive substance composition, the vitamin C or its derivative is selected from any one or more of vitamin C (i.e., ascorbic acid), ascorbyl glucoside, ethyl ascorbic acid, 3-o-ethyl ascorbic acid, magnesium ascorbic acid phosphate, sodium ascorbic acid phosphate, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, vitamin C tetraisopalmitate, ascorbyl palmitate, ascorbic acid 2-phosphate 6-palmitate, esterified vitamin C, and other solvates of ascorbic acid. Preferably, the mass-volume concentration of the vitamin C or its derivatives in the biologically active substance composition is 0.1-100 μg / ml, accounting for 0.00001%-0.01% by mass; preferably, the mass-volume concentration of the vitamin C or its derivatives in the biologically active substance composition is 1-100 μg / ml, accounting for 0.0001%-0.01% by mass; preferably, the mass-volume concentration of the vitamin C or its derivatives in the biologically active substance composition is 10-80 μg / ml, accounting for 0.001%-0.008% by mass.

[0039] Furthermore, the mass-volume concentration of transferrin in the bioactive substance composition is 0.1-300 μg / ml, accounting for 0.00001%-0.03% by mass; preferably, the mass-volume concentration of transferrin in the bioactive substance composition is 1-200 μg / ml, accounting for 0.0001%-0.02% by mass; preferably, the mass-volume concentration of transferrin in the bioactive substance composition is 1-150 μg / ml, accounting for 0.0001%-0.015% by mass.

[0040] Furthermore, the mass-volume concentration of the insulin in the bioactive substance composition is 0.01-50 μg / ml, accounting for 0.000001%-0.005% by mass; preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 0.1-30 μg / ml, accounting for 0.00001%-0.003% by mass; preferably, the mass-volume concentration of the insulin in the bioactive substance composition is 1-20 μg / ml, accounting for 0.0001%-0.002% by mass.

[0041] Furthermore, the mass-volume concentration of the progesterone in the bioactive substance composition is 0.1-50 ng / ml, accounting for 0.00000001%-0.000005% by mass; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 1-30 ng / ml, accounting for 0.0000001%-0.000003% by mass; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 2-20 ng / ml, accounting for 0.0000002%-0.000002% by mass.

[0042] Furthermore, the putrescine or its salt is selected from any one or more of putrescine and putrescine dihydrochloride. Preferably, the putrescine or its salt has a mass-volume concentration of 0.01-50 μg / ml in the bioactive substance composition, accounting for 0.000001%-0.005% by mass; preferably, the putrescine or its salt has a mass-volume concentration of 0.1-40 μg / ml in the bioactive substance composition, accounting for 0.00001%-0.004% by mass; preferably, the putrescine or its salt has a mass-volume concentration of 1-30 μg / ml in the bioactive substance composition, accounting for 0.0001%-0.003% by mass.

[0043] Furthermore, the selenite is water-soluble; preferably, the selenite is sodium selenite. Preferably, the mass-volume concentration of the selenite in the bioactive substance composition is 0.1-50 ng / ml, accounting for 0.00000001%-0.000005% by mass; preferably, the mass-volume concentration of the selenite in the bioactive substance composition is 1-30 ng / ml, accounting for 0.0000001%-0.000003% by mass; preferably, the mass-volume concentration of the selenite in the bioactive substance composition is 2-20 ng / ml, accounting for 0.0000002%-0.000002% by mass.

[0044] The second object of the present invention is to provide a method for preparing a bioactive substance composition, comprising the steps of mixing fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivative, transferrin, insulin, progesterone, putrescine or its salt, and selenite in the proportions described in the first object, wherein the order of addition of the components is not particular; wherein the mass-volume concentration range ratio of the components is:

[0045] Fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 1-50: 1-50: 1-40: 1-11: 10-5000: 10-100000: 10-300000: 1-25000: 1-25: 1-25000: 1-25;

[0046] Preferably, the fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 5-40: 5-40: 2-30: 1-8: 500-4000: 1000-90000: 1000-200000: 10-15000: 1-15: 2-15000: 1-15;

[0047] More preferably, the ratio of fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite is 10-30: 10-30: 3-20: 2-5: 1000-2000: 10000-80000: 2000-80000: 100-5000: 2-7: 7-10000: 2-7;

[0048] Preferably, the mass-volume concentration of transferrin in the bioactive substance composition is 0.1-300 μg / ml, accounting for 0.00001%-0.03% by mass; preferably, the mass-volume concentration of transferrin in the bioactive substance composition is 1-200 μg / ml, accounting for 0.0001%-0.02% by mass; preferably, the mass-volume concentration of transferrin in the bioactive substance composition is 1-150 μg / ml, accounting for 0.0001%-0.015% by mass;

[0049] Preferably, the mass-volume concentration of insulin in the bioactive substance composition is 0.01-50 μg / ml, accounting for 0.000001%-0.005% by mass; preferably, the mass-volume concentration of insulin in the bioactive substance composition is 0.1-30 μg / ml, accounting for 0.00001%-0.003% by mass; preferably, the mass-volume concentration of insulin in the bioactive substance composition is 1-20 μg / ml, accounting for 0.0001%-0.002% by mass;

[0050] Preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 0.1-50 ng / ml, accounting for 0.00000001%-0.000005% by mass; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 1-30 ng / ml, accounting for 0.0000001%-0.000003% by mass; preferably, the mass-volume concentration of the progesterone in the bioactive substance composition is 2-20 ng / ml, accounting for 0.0000002%-0.000002% by mass;

[0051] Preferably, the mixing temperature is 0-37°C.

[0052] The third object of the present invention is to provide a serum-free culture medium, which comprises a basal culture medium and an additive, wherein the additive comprises a bioactive substance composition in any form as described above or a bioactive substance composition obtained by the method described above. Preferably, the serum-free culture medium is a completely serum-free culture medium; preferably, the culture refers to the primary culture and subculture of cells or tissues; preferably, the culture refers to maintaining or enhancing the proliferation and phenotype of cells or tissues. Preferably, the scores of each single item in the cell-common characteristics and cell-specific phenotypes of cells cultured in the serum-free culture medium reach their respective passing scores and the total cell score reaches 60 points or more.

[0053] Furthermore, the basal culture medium is selected from any one or more of DMEM low glucose medium, DMEM high glucose medium, DMEM / F12 medium, F12 medium, F10 medium, MEM medium, BEM medium, RPMI 1640 medium, Media 199 medium, IMDM medium, mTesR medium, and E8 medium.

[0054] The fourth object of the present invention is to provide a method for preparing a serum-free culture medium, which comprises the step of mixing a basal culture medium and an additive, wherein the additive comprises a bioactive substance composition in any form as described above; preferably, the mixing temperature is 0-37°C.

[0055] The fifth object of the present invention is to provide a composition comprising at least one active ingredient and at least one additive, wherein the active ingredient is selected from at least one of the bioactive material composition in any form as described above, the bioactive material composition obtained by the method as described above, the serum-free culture medium in any form as described above, and the serum-free culture medium obtained by the method as described above. Preferably, the individual scores of the common cell characteristics and cell-specific phenotypes of the cells cultured with the composition reach the passing line and the total score reaches 60 points or more; preferably, the individual scores of the common cell characteristics and cell-specific phenotypes of the cells cultured with the composition reach the passing line and the total score reaches 80 points or more; preferably, the individual scores of the common cell characteristics and cell-specific phenotypes of the cells cultured with the composition reach the passing line and the total score reaches 90 points or more; preferably, the individual scores of the common cell characteristics and cell-specific phenotypes of the cells cultured with the composition reach the passing line and the total score reaches 100 points.

[0056] Furthermore, in the composition, the additive is selected from any one or more of cell culture additives, growth factors, small molecule drugs, hormones, vitamins, adhesion promoting substances, macromolecular proteins, synthetic peptides, amino acids, lipids, enzymes, carbohydrates, pH regulating substances, trace elements and antibiotics;

[0057] Preferably, the cell culture additive comprises any one or more of a B27 cell culture additive, an N2 cell culture additive, a chemically defined lipid concentrate, an ITS, and a fatty acid additive; more preferably, the concentration of the cell culture additive in the composition is 0.1-5X (i.e., 0.1-5 times) based on the volume of the composition; more preferably, the concentration of the cell culture additive in the composition is 0.5-2X (i.e., 0.5-2 times) based on the volume of the composition;

[0058] Preferably, the growth factor is selected from any one or more of vascular endothelial growth factor, vascular endothelial growth factor synthetic peptide, epidermal growth factor, epidermal growth factor synthetic peptide, insulin-like growth factor, insulin-like growth factor synthetic peptide, nerve growth factor, nerve growth factor synthetic peptide, colony stimulating factor, colony stimulating factor synthetic peptide, somatostatin, somatostatin synthetic peptide; more preferably, the mass-volume concentration of the growth factor is 1-100 ng / ml; more preferably, the mass-volume concentration of the growth factor is 1-50 ng / ml; preferably, the mass-volume concentration of the growth factor is 5-40 ng / ml;

[0059] Preferably, the small molecule drug is selected from GSK3 inhibitors; preferably, the GSK3 inhibitor is selected from CHIR99021; preferably, the molar concentration of the small molecule drug is 0.1-10 μM; preferably, the molar concentration of the small molecule drug is 0.1-5 μM;

[0060] Preferably, the amino acid is selected from any one or more of non-essential amino acids, L-glutamic acid, and L-glutamine; more preferably, the molar concentration of the amino acid is 0.01-4 mM;

[0061] Preferably, the carbohydrate is sodium pyruvate; more preferably, the mass-volume concentration of the carbohydrate is 0.01-2 mM;

[0062] Preferably, the pH maintaining agent is selected from any one or more of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and L-phosphoglycerol disodium salt hydrate; more preferably, the molar concentration of the pH maintaining agent is 1-20 mM;

[0063] Preferably, the adhesion promoting substance is selected from any one or more of laminin, fibronectin, vitronectin, collagen, gelatin, and synthetic peptides promoting adhesion; more preferably, the mass-volume concentration range of the laminin is 0.1-100 μg / ml; more preferably, the mass-volume concentration range of the fibronectin is 0.1-200 μg / ml; more preferably, the mass-volume concentration range of the vitronectin is 0.1-100 μg / ml; more preferably, the mass-volume concentration range of the collagen is 0.1-100 mg / ml; more preferably, the mass-volume concentration range of the gelatin is 0.1-100 mg / ml; more preferably, the concentration range of the synthetic peptide promoting adhesion is 0.1 μg-1000 mg / ml;

[0064] Preferably, the antibiotic is selected from any one or more of penicillin, streptomycin, and gentamicin; more preferably, the mass-volume concentration of the antibiotic is in the range of 50 to 100 μg / mL.

[0065] The sixth object of the present invention is to provide a method for preparing a composition, which comprises the step of mixing at least one active component and at least one additive, wherein the active component is selected from at least one of the bioactive substance composition in any form described above, the bioactive substance composition prepared by the method described above, the serum-free culture medium in any form described above, and the serum-free culture medium prepared by the method described above; preferably, the mixing temperature is 0-37°C.

[0066] The seventh object of the present invention is to provide a bioactive substance composition in any form as described above, a bioactive substance composition obtained by the method as described above, a serum-free culture medium in any form as described above, a serum-free culture medium obtained by the method as described above, a composition in any form as described above, or a use of a composition obtained by the method as described above, wherein the use is selected from the culture of cells and / or tissues, or the use in the preparation of a drug for treating tissue and / or organ damage. Preferably, the cells are selected from any one or more of tendon and / or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells; preferably, the tissue is a musculoskeletal tissue; preferably, the musculoskeletal tissue is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, adipose tissue, and muscle tissue; preferably, the tissue and / or organ damage is musculoskeletal tissue and / or organ damage; preferably, the musculoskeletal tissue or organ damage is selected from at least one of tendon and / or ligament damage, cartilage damage, bone damage, muscle damage, skin damage, and vascular damage.

[0067] An eighth object of the present invention is to provide a cell culture method based on the serum-free culture medium and / or composition described in any one of the above items, the culture method comprising the step of contacting cells and / or tissues with the serum-free culture medium and / or composition, the serum-free culture medium is any form of serum-free culture medium as described above, or a serum-free culture medium prepared by the method described above, and the composition is any form of composition as described above, or a composition prepared by the method described above; preferably, the culture method is selected from the group consisting of suspension culture and adherent culture; preferably, the adherent culture method is selected from the group consisting of the method of coating a culture plate with an adhesion-promoting substance and the method of adding an adhesion-promoting substance to the culture medium.

[0068] More preferably, the adhesion-promoting substance culture plate coating method comprises the following steps: 1) treating a culture carrier with an adhesion-promoting substance, preferably, the culture carrier is selected from at least one of a well plate, a culture dish, a culture flask, a microcarrier, a microsphere, a microarray, and a bioactive material; 2) inoculating cells and / or tissues into the culture carrier treated in step 1); 3) adding the serum-free culture medium and / or composition as described above for culturing.

[0069] More preferably, the method of adding an adhesion-promoting substance to a culture medium comprises the following steps: 1) inoculating cells and / or tissues into a culture carrier, preferably, the culture carrier is selected from at least one of a well plate, a culture dish, a culture flask, a microcarrier, a microsphere, a microarray, and a bioactive material; 2) directly adding the adhesion-promoting substance to the serum-free culture medium and / or composition as described above, and then adding the substance to the culture carrier in step 1) for cell culture;

[0070] Preferably, the cells are selected from any one or more of tendon and / or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells; preferably, the tissue is musculoskeletal tissue; preferably, the musculoskeletal tissue is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, adipose tissue, and muscle tissue.

[0071] Furthermore, the adhesion promoting substance is selected from any one or more of laminin, fibronectin, vitronectin, collagen, gelatin, and synthetic peptides of adhesion promoting substances.

[0072] Furthermore, the synthetic peptide promoting adhesion is an artificially synthesized polypeptide, oligopeptide or amino acid sequence that can replace the adhesion promoting substance to promote cell adhesion, including any one or more of laminin synthetic peptide, fibronectin synthetic peptide, vitronectin synthetic peptide, RGD (Arg-Gly-Asp) peptide, and KRSR (Lys-Arg-Ser-Arg) peptide.

[0073] Furthermore, the laminin concentration range is 0.1-100 μg / ml, and / or the fibronectin concentration range is 0.1-200 μg / ml, and / or the vitronectin concentration range is 0.1-100 μg / ml, and / or the collagen concentration range is 0.1-100 mg / ml, and / or the gelatin concentration range is 0.1-100 mg / ml.

[0074] Furthermore, the concentration range of the laminin synthetic peptide is 0.1-100 μg / ml, and / or the concentration range of the fibronectin synthetic peptide is 0.1-200 μg / ml, and / or the vitronectin synthetic peptide is 0.1-100 μg / ml, and / or the concentration range of the RGD (Arg-Gly-Asp) peptide is 50-1000 mg / ml, and / or the concentration of the KRSR (Lys-Arg-Ser-Arg) peptide is 50-1000 mg / ml.

[0075] Preferably, the cell suspension culture method comprises the following steps: 1) inoculating cells into low-adhesion or non-adhesion culture well plates, culture dishes, culture flasks, other culture carriers, or cell dynamic culture bioreactors; 2) adding the serum-free culture medium and / or composition as described above for culture.

[0076] The ninth object of the present invention is to provide a cell and / or tissue, wherein the cell and / or tissue is obtained by culturing using the serum-free medium and / or composition as described above, wherein the serum-free medium is any form of serum-free medium as described above, or a serum-free medium prepared by the method as described above, and the composition is any form of composition as described above, or a composition prepared by the method as described above; preferably, the cell is selected from any one or more of tendon and / or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells; preferably, the tissue is a musculoskeletal tissue; preferably, the musculoskeletal tissue is selected from tendon tissue, Ligament tissue, meniscus tissue, cartilage tissue, adipose tissue, muscle tissue; preferably, the single score of each single item in the common cell characteristics and cell-specific phenotypes of the cells reaches the passing line and the total score reaches 60 points or more; preferably, the score of each single item in the common cell characteristics and cell-specific phenotypes of the cells reaches the respective passing line and the total score of the cells reaches 80 points or more; preferably, the score of each single item in the common cell characteristics and cell-specific phenotypes of the cells reaches the respective passing line and the total score of the cells reaches 90 points or more; preferably, the score of each single item in the common cell characteristics and cell-specific phenotypes of the cells reaches the respective passing line and the total score of the cells reaches 100 points.

[0077] The following further describes certain technical terms involved in the present invention. These descriptions are merely examples of how the present invention is implemented and do not constitute any limitation to the present invention.

[0078] Bioactive substance composition

[0079] The bioactive substance composition comprises fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivative, transferrin, insulin, progesterone, putrescine or its salt, and selenite.

[0080] The fibroblast growth factor (FGF), also known as heparin-binding growth factor, mainly includes two categories: acidic and alkaline. It refers to a class of active proteins or polypeptide substances that can promote cell growth, including but not limited to FGF-basic, FGF1, FGF2, FGF4, FGF7, FGF10, FGF18, fibroblast growth factor synthetic peptides, etc.

[0081] Platelet-derived growth factor (PDGF) is a low-molecular-weight mitogen, a peptide regulatory factor that stimulates the growth of cells in connective tissue and other tissues. The platelet-derived growth factor family includes platelet growth factor (PDGF) and vascular endothelial growth factor (VEGF). Each growth factor receptor is a tyrosine kinase (RTK) receptor. Members of the platelet-derived growth factor family include PDGFA, PDGFB, PDGFC, PDGFD, placental growth factor (PGF), vascular endothelial growth factor (VEGF), VEGF41, VEGFB, VEGFC, FIGF (VEGFD), homo- or heterodimers of two polypeptide chains linked by disulfide bonds, including PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD, as well as synthetic platelet-derived growth factor peptides.

[0082] The transforming growth factor-β (TGF-β) belongs to the transforming growth factor superfamily of multifunctional cytokines, which has the function of regulating cell growth and differentiation and maintaining cell phenotype, including but not limited to TGF-β1, TGF-β2, TGF-β3, transforming growth factor-β synthetic peptides, etc.

[0083] The glucocorticoids are hormones that have important regulatory effects on cell growth, differentiation, metabolism, anti-inflammation, and immunity. Common glucocorticoids include glucocorticoids, glucocorticoid derivative salts, and glucocorticoid solvates, including but not limited to dexamethasone or its salts, dexamethasone solvates, hydrocortisone or its salts, hydrocortisone solvates, cortisone acetate, cortisone or its salts, methylprednisolone sodium succinate, prednisone, betamethasone, betamethasone valerate, beclomethasone dipropionate, prednisolone acetate, and prednisolone.

[0084] Heparin or its salts refers to heparin or its salts. Heparin, named after its discovery in the liver, is a polysaccharide sulfate composed of alternating components of glucosamine, L-iduronide, N-acetylglucosamine, and D-glucuronic acid. It has an average molecular weight of 15 kDa and is strongly acidic. It is a natural anticoagulant in animals. It can promote cell proliferation during cell culture. Heparin or its salts include, but are not limited to, heparin, heparin sodium, heparin calcium, and heparin.

[0085] Vitamin C or its derivatives refer to vitamin C, its salts, and its solvates. Vitamin C (ascorbic acid), also known as ascorbic acid, has antioxidant properties, can inhibit cell aging, and promote cell growth and phenotype maintenance. Vitamin C or its derivatives include, but are not limited to, vitamin C, ascorbyl glucoside, ethyl ascorbic acid, 3-o-ethyl ascorbic acid, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, ascorbyl tetraisopalmitate, ascorbyl palmitate, ascorbyl 2-phosphate 6-palmitate, esterified vitamin C, and other solvates of ascorbic acid.

[0086] additive

[0087] The additives include but are not limited to growth factors, small molecule drugs, hormones, vitamins, cell culture additives, adhesion promoting substances, macromolecular proteins, synthetic peptides, amino acids, lipids, enzymes, carbohydrates, pH regulating substances, trace elements, antibiotics, etc.

[0088] Growth factors are a class of polypeptide substances that regulate cell growth and other cellular functions by binding to specific, high-affinity cell membrane receptors. They play a crucial regulatory role in human immunity, hematopoiesis, tumorigenesis, inflammation and infection, wound healing, angiogenesis, cell differentiation, apoptosis, morphogenesis, and embryogenesis. Growth factors are widely present in various tissues of the body, including mature and embryonic tissues, regulating the proliferation and differentiation of various cells through autocrine and / or paracrine pathways. Many cells cultured in vitro can also release growth factors. In the present invention, any trace organic substance that is essential for microbial life activities and cannot be synthesized by the microorganisms themselves can be referred to as a growth factor. There are many types of growth factors, including fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), insulin, epidermal growth factor (EGF), insulin-like growth factor (IGF), nerve growth factor (NGF), colony-stimulating factor (CSF), and somatostatin (SRIH) described in the above-mentioned bioactive substance composition. In the present application, the fibroblast growth factor, platelet-derived growth factor, and transforming growth factor described in the above-mentioned bioactive substance composition are essential for the activity of the bioactive substance composition or serum-free culture medium or composition. They work together with other components in the bioactive substance composition to achieve cell and / or tissue culture and tissue repair. The function of adding other growth factors mentioned above is mainly to further enhance the activity of the composition, which is not an essential condition.

[0089] Synthetic peptides refer to: 1) amino acid sequences derived from effective fragments or combinations of fragments of a protein or polypeptide; or 2) peptides or oligopeptides obtained through chemical synthesis that share the same amino acid sequence as a growth factor or adhesion factor; or 3) peptide mimetics of a protein or polypeptide. These peptide mimetics can be obtained by screening peptide libraries using receptors for known proteins or polypeptides. Their amino acid sequences differ from those of their corresponding cytokines or adhesion factors, but they possess cytokine or adhesion factor activity and possess the advantage of a smaller molecular weight. For effective protein or polypeptide fragments, the corresponding amino acid sequences can be identified based on published literature, followed by synthesis through primer design or by outsourcing synthesis to a biopharmaceutical company. For unreported fragments, optimized peptides can be identified through enzymatic protein hydrolysis and peptide mapping analysis, followed by synthesis through primer design or outsourcing synthesis to a biopharmaceutical company. Similarly, peptide mimetics of a protein or polypeptide can be synthesized by outsourcing synthesis to a biopharmaceutical company based on the amino acid sequences of known protein or polypeptide mimetics. Synthetic peptides include, but are not limited to, synthetic peptides for growth factors and synthetic peptides for adhesion promoters. Synthetic growth factor peptides include, but are not limited to, one or more of fibroblast growth factor synthetic peptides, platelet-derived growth factor synthetic peptides, transforming growth factor synthetic peptides, epidermal growth factor synthetic peptides, insulin-like growth factor synthetic peptides, vascular endothelial growth factor synthetic peptides, nerve growth factor synthetic peptides, and colony-stimulating factor synthetic peptides. Synthetic peptides for promoting adhesion include, but are not limited to, any one or more of laminin synthetic peptides, fibronectin synthetic peptides, vitronectin synthetic peptides, RGD (Arg-Gly-Asp) peptides, KRSR (Lys-Arg-Ser-Arg) peptides, and synthetic peptides of other extracellular matrix components.

[0090] The small molecule drugs refer to a class of chemical small molecule drugs that can easily penetrate the membrane and enter cells through diffusion or carrier proteins to act on the intracellular pathway, including but not limited to one or more of heparin or its salts, putrescine, selenious acid or its salts, CHIR99021, SB431542, etc.

[0091] The hormones include but are not limited to one or more of glucocorticoids, progesterone, insulin, progesterone, cortisol, corticosterone, triiodothyronine, thyroxine (T3), etc.

[0092] The vitamins include but are not limited to one or more of vitamin A, vitamin B, vitamin C or its derivatives, vitamin D, vitamin E, vitamin K, vitamin H, vitamin P, vitamin PP, vitamin M, vitamin T, vitamin U, water-soluble vitamins, biotin, choline chloride, D-calcium pantothenate, folic acid, inositol, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, coenzyme Q10, vitamin B12, putrescine dihydrochloride, tocopheryl acetate, tocopherol, L-carnitine hydrochloride, acetyl-L-carnitine, etc.

[0093] The cell culture additive refers to a mixture of nutrients that can provide nutrition to cells, promote cell proliferation and maintain phenotype, including any one or more of B27 cell culture additives (including but not limited to Gibco's B-27™ Supplement (50X), the original concentration is 50 times, i.e. 50X), N2 cell culture additives (including but not limited to Gibco's N-2 additive (100X), the original concentration is 100 times, i.e. 100X), chemically defined lipid concentrates (including but not limited to chemically defined lipid concentrates produced by Gibco), ITS (i.e., Insulin-Transferrin-Selenium mixed solution, including but not limited to ITS produced by Gibco and Sigma), and fatty acid additives (including but not limited to fatty acid additives produced by Gibco).

[0094] The adhesion-promoting substance refers to a type of substance that can promote cell adhesion and growth, including but not limited to any one or more of laminin, fibronectin, vitronectin, collagen, gelatin, and other extracellular matrix components.

[0095] The macromolecular proteins described include, but are not limited to, albumin, albumin-related proteins, and other non-growth factor proteins that promote cell growth. Examples of albumin-related proteins include, but are not limited to, retinol binding protein, α-2-glycoprotein, transthyretin, hemoglobin α, and keratin precursors. Albumin can replace serum in cell culture, providing physiological and mechanical protection and acting as a carrier, promoting the growth and survival of mammalian cells. In this application, albumin is an optional ingredient; the serum-free culture medium of the present invention can maintain cell proliferation and phenotype even without the addition of albumin.

[0096] The amino acids include, but are not limited to, any one or more of MEM non-essential amino acid solution (NEAA), glutathione, L-glutamine, L-carnitine, adenine, guanine, uracil, thymine, cytosine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine. MEM non-essential amino acid solution, which includes seven non-essential amino acids: L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, and glycine, effectively improves cell culture medium composition, reduces the side effects of cell culture's own production of non-essential amino acids, and promotes cell proliferation and metabolism. It is a commonly used additive in cell culture.

[0097] The lipids include but are not limited to any one or more of linoleic acid, oleic acid, linolenic acid, and cholesterol.

[0098] Enzymes are a class of reducing agents that scavenge free radicals, have potent antioxidant activity, and can protect cells from the cytotoxicity caused by glucose and glucose oxidase. They are primarily protein-based and include, but are not limited to, any one or more of superoxide dismutase or its analogs, and catalase or its analogs.

[0099] The carbohydrates refer to a class of substances that provide the main energy source for cell growth, some of which are components for synthesizing proteins and nucleic acids, including but not limited to any one or more of galactose, glucose, ribose, deoxyribose, chondroitin sulfate, sodium pyruvate, and acetic acid.

[0100] The pH regulating substance refers to a class of substances or mixtures that maintain the pH stability of the cell culture environment and maintain the osmotic pressure balance of the cells, including but not limited to any one or more of ethanolamine and its salts, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, L-glycerophosphate disodium salt hydrate, phenol red, sodium dihydrogen phosphate, trisodium phosphate, sodium pyrophosphate, potassium pyrophosphate, sodium bicarbonate, potassium hydroxide, ammonium hydroxide, triethanolamine, and citric acid.

[0101] The trace elements are mainly involved in cell composition and metabolism, including but not limited to one or more of iron, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, silver, tin, aluminum, barium, boron, rubidium, etc.

[0102] Movement system

[0103] The musculoskeletal system, in a broad sense, consists of the central nervous system, peripheral nerves, and neuromuscular junctions; skeletal muscles; and the cardiopulmonary and metabolic support systems. The musculoskeletal system, in a narrow sense, is composed of three organs: bones, bone connections, and skeletal muscles. Bones are linked together in various ways to form the skeleton. They form the basic shape of the human body and provide attachment for muscles. Under neural control, muscles contract, pulling on the bones to which they are attached, using the movable bone connections as a pivot to produce lever motion.

[0104] Mesenchymal stem cells

[0105] Mesenchymal stem cells, also known as multipotent stromal cells (MSCs), are a type of multipotent stem cell that belongs to the mesoderm. They are primarily found in connective tissue and organ mesenchyme. They can be found in tissues such as bone marrow, umbilical cord, fat, mucosa, bone, muscle, dental pulp, lung, liver, and pancreas, as well as amniotic fluid, amniotic membrane, and placenta. They possess the ability to self-renew and, under appropriate conditions, can differentiate into various tissues, including fat, bone, and cartilage. These include, but are not limited to, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose tissue-derived stem cells, mucosal mesenchymal stem cells, dental pulp mesenchymal stem cells, amniotic fluid mesenchymal stem cells, amniotic membrane mesenchymal stem cells, and placental mesenchymal stem cells.

[0106] Tendon and / or ligament-derived cells

[0107] Tendon and / or ligament-derived cells are a type of mixed cell isolated and extracted from tendon or ligament tissue. They highly express multiple tendon / ligament tissue-specific genes and proteins, including scleraxis (SCX), nestin (NES), tenomodulin (TNMD), thrombospondin-4 (THBS4), collagen type I alpha 1 (COL1, COL1A1), and tenascin-C. They include tendon stem / progenitor cells (TSPCs, tendon derived stem cells, TDSCs, tendon stem cells, TSCs), tenocytes, tenoblasts, fibroblasts, ligament stem / progenitor cells, and ligament cells, and are the most ideal seed cells for tendon injury treatment.

[0108] Common cell characteristics

[0109] Common cell characteristics refer to cell survival, proliferation, and safety. For stem cells, the common cell characteristics described in this application refer to the survival, proliferation, stem cell phenotype, and safety of the stem cells, also known as common stem cell characteristics.

[0110] Cell-specific phenotype

[0111] In this application, a cell-specific phenotype refers to a unique phenotype of a cell that is different from the common characteristics of stem cells. Taking tendon stem cells as an example, the cell-specific phenotype is the tendon phenotype and tendon differentiation ability, and the ability to repair tendons and / or ligaments in vivo.

[0112] nourish

[0113] Cultivation refers to a method of simulating the in vivo environment (sterility, suitable temperature, pH, and specific nutritional conditions) in vitro to enable cells to survive, grow, proliferate, and maintain their primary structure and function (i.e., phenotype). Cultivation, as used in this invention, refers to primary and subculture processes designed to maintain or enhance the proliferation and phenotype of cells or tissues.

[0114] Bioactive Materials

[0115] Bioactive materials refer to a class of natural or synthetic materials that are non-toxic or low-toxic to cells and / or tissues, have good biocompatibility, and can support cell and / or tissue culture.

[0116] Mass-volume concentration

[0117] Mass-volume concentration refers to the mass-volume concentration per unit volume (1m 3 The concentration is expressed as the mass of solute in a solution (1 L, 1 ml, etc.), expressed using symbols such as g / m³, mg / L, mg / ml, μg / ml, or ng / ml. Mass-volume concentration = mass of solute divided by the volume of the solution. For example, if 1 ml of serum-free medium contains 10 ng of FGF2, the concentration of FGF2 is 10 ng / ml.

[0118] Molar concentration

[0119] Molar concentration generally refers to the amount of substance, which is defined as the amount of solute B in a solution divided by the volume of the mixture and is represented by the symbol c(B). In the formula, c(B) = n(B) / V, where Cb represents the molar concentration of the solute, n(B) represents the amount of solute B, and V represents the volume of the solution. Unless otherwise specified, the solvent is assumed to be water. The SI unit for amount of substance is mol·m⁻³, and the commonly used unit is mol·L⁻¹, abbreviated as M. mM, μM, and nM are commonly used in this patent. The formula for converting mass to molar concentration is: mass (mg) = molar concentration (mM) × volume (mL) × molecular weight (g / mol).

[0120] Mass-volume concentration to molar concentration conversion

[0121] The present invention relates to converting molar concentration to mass-volume concentration. The specific conversion formula is mass-volume concentration (mg / ml) = molar concentration (mM) × molecular weight (g / mol). For example, the molecular weight of dexamethasone is 392.46, and the mass-volume concentration of 10nM dexamethasone is 3.9246ng / ml.

[0122] Various concentration calculation methods involved in the present invention

[0123] The mass-volume concentration ratio range of each component involved in the present invention is calculated by first converting the concentration of each component into mass-volume concentration. The mass ratio in the present invention refers to mass percentage, and the solution is regarded as water for calculation. For example, if the mass-volume concentration of fibroblast growth factor is 100ng / ml, then its weight ratio in 1ml solution (1ml water is 1g) is calculated as: (100ng / 1g)*100%, that is (100ng / 10 9 ng)*100%= 0.00001%. For example, if the molar concentration of dexamethasone is 10nM, first convert it to mass-volume concentration of 3.9246ng / ml, then the weight ratio in 1ml solution is calculated as: (3.9246ng / 1g)*100%, that is, (3.9246ng / 10 9 ng)*100% = 0.00000039246%.

[0124] Beneficial effects of the present invention:

[0125] 1) The present invention provides, for the first time, a highly active bioactive substance composition. The components of the bioactive substance composition are unique and indispensable, and the unique ratios make it highly active. The completely serum-free culture medium or composition prepared therefrom has clear ingredients and can achieve completely serum-free primary culture and subculture of cells. In particular, it overcomes the technical bias of the prior art that primary culture of tendon and / or ligament-derived cells must involve serum. The cultured cells can simultaneously meet the requirements for cell quantity and quality required for the clinical treatment of tendon and / or ligament injuries, achieving unexpected technical effects. At the same time, the bioactive substance composition can also be used to prepare drugs for treating tissue and / or organ injuries in vivo.

[0126] 2) This invention provides, for the first time, a completely serum-free culture medium or composition with defined ingredients that promotes cell proliferation and phenotypic maintenance. This serum-free culture medium or composition is free of serum, platelet-rich plasma, or other blood-derived substances, and supports both primary and subculture of cells. The added ingredients effectively replace serum components during the cell culture process through various mechanisms, resulting in excellent cell growth and significantly superior cell morphology, density, viability, and function to those achieved in serum-containing culture medium. Cells cultured in this serum-free culture medium or composition meet both the quality and quantity requirements for clinical treatment of tissue damage, achieving a total score of 60 or higher, and under optimal conditions, a full score of 100.

[0127] 3) The serum-free culture medium or composition provided by the present invention can be used for the in vitro culture of tendon and / or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells.

[0128] 4) The serum-free culture medium or composition of the present invention is particularly suitable for culturing cells derived from tendons and / or ligaments, and in particular, provides for the first time a completely serum-free culture medium with a defined composition for cells derived from tendons and / or ligaments. Taking cells derived from tendons and / or ligaments as an example, specifically: for the first time, completely serum-free in vitro culture of cells derived from tendons and / or ligaments is achieved, including serum-free primary culture and subculture, achieving the following effects: a) Rapid proliferation rate and short cell doubling time, capable of expanding cells 5-fold or more in one week, and optimally, reaching 100,000-fold expansion in four weeks of subculture, allowing sufficient cell numbers to be obtained in a relatively short period of time ( Figure 10-11 , Table 1); b). High safety, normal cell karyotype, no serum residue, no virus mycoplasma contamination ( Figure 13-14 c). Maintained stem cell surface markers, colony formation ability and tri-lineage differentiation ability and other stem cell phenotypes ( Figure 15-17 , Table 2, Table 3); d). Cell-specific phenotypes were improved, such as the tendon phenotype and tendon differentiation ability of tendon and / or ligament-derived cells were significantly improved ( Figure 18-20, Table 4); 5. Enhanced cell tissue repair ability ( Figure 21-25 Each individual score of the cultured cells reached the passing line and the total score reached above 60 points (Table 5), meeting the quality and quantity requirements for clinical cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 The GPF fluorescence image and Scx+ positive rate statistical results of Scx-GFP tendon stem cells cultured using existing culture technology show that the existing technology causes the loss of the SCX phenotype, a tendon-specific marker of tendon-derived cells (Biomaterials 2018;172:66-82);

[0130] Figure 2 The results of the DCN content test of tendon-derived cells cultured using existing culture technology indicate that the existing culture technology leads to the loss of the DCN phenotype, a tendon-specific marker, in tendon-derived cells (Tissue Eng 2006; 12(7): 1843-9);

[0131] Figure 3 Transmission electron micrograph of the collagen cross-section of tendon repaired by tendon-derived cells cultured using existing culture techniques and normal tendon tissue. The results show that the tissue repaired by cells cultured using existing culture techniques is composed of a large number of small collagens, which are much smaller than the diameter of normal tendon collagen.

[0132] Figure 4 CT images of tendons repaired with tendon-derived cells cultured using existing culture techniques show that tendons regenerated with cells cultured using existing culture techniques have ectopic calcification and repair failure (STEM CELLS 2016;34:1083–1096);

[0133] Figure 5 A line graph shows the number of proliferating cells in tendon cell culture using insulin-like growth factor-1 and transforming growth factor-β3 instead of FBS. The number of proliferating cells in the growth factor group was only 1 / 3 of that in the serum control group (Cells Tissues Organs 2013;197:27–36).

[0134] Figure 6 A bar graph shows the collagen content of tenocytes cultured using insulin-like growth factor-1 and transforming growth factor-β3 instead of FBS. The optimal combination of growth factors produced only half the amount of collagen as the serum control group (Cells Tissues Organs 2013;197:27–36).

[0135] Figure 7This is a growth morphology diagram of primary tendon stem cells (P0) cultured in serum-free medium under an inverted microscope (4x magnification) in Example 1 of the present invention;

[0136] Figure 8 The growth morphology of tendon stem cells from generations P1 to P6 of the serum-free culture experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 under an inverted microscope (4x magnification) is shown;

[0137] Figure 9 This is a graph showing the growth morphology of tendon stem cells at generation P3 in the serum-free culture experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 under an inverted microscope (20x magnification);

[0138] Figure 10 This is a line graph showing the multiplication of cell proliferation folds per generation of tendon stem cells P1-P6 in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1;

[0139] Figure 11 This is a bar graph of the doubling time of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0140] Figure 12 This is a statistical diagram of the diameters of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0141] Figure 13 This is a diagram showing the karyotype analysis of tendon stem cells in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 of the present invention.

[0142] Figure 14 The mycoplasma detection results of serum-free culture medium, serum and mycoplasma positive control in Example 1 of the present invention are shown in FIG.

[0143] Figure 15 The results of ALP staining experiments of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 were used to test their osteogenic ability.

[0144] Figure 16 The results of Alcian blue staining of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 were used to test their chondrogenic ability.

[0145] Figure 17 The results of Oil Red O staining of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 were used to test their adipogenic ability.

[0146] Figure 18 This is a bar graph showing the relative expression of tendon-related genes SCX, Nestin, and TNMD in the tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0147] Figure 19 This is a diagram showing the results of picrosirius red staining of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 after 14 days of tendon lineage induction.

[0148] Figure 20 This is a graph showing the results of collagen formation detected by transmission electron microscopy after tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1 were induced to roll into cell sheets after 14 days of tendon induction.

[0149] Figure 21 The relative quantitative results of the expression of scx, nestin, col1a1, and tnmd tendon system genes in the ectopic formation of tendon tissue by tendon stem cells in nude mice in the serum-free experimental group of Example 1 and the serum control group of Comparative Example 1 are shown.

[0150] Figure 22 These are the results of HE staining and Masson staining of ectopically formed tendons in nude mice by tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0151] Figure 23 This is an immunofluorescence image of the expression of scx and col1 tendon-related proteins in the ectopically formed tendon tissue of nude mice by tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0152] Figure 24 These are the HE staining and Masson staining results of the in situ patellar tendon repair samples of rats using tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0153] Figure 25 This is an immunofluorescence image of the expression of nestin, a tendon-related protein, in the in situ patellar tendon repair samples of tendon stem cells in the serum-free experimental group of Example 1 of the present invention and the serum control group of Comparative Example 1.

[0154] Figure 26 This is a growth morphology diagram of human ligament stem cells in the serum-free culture experimental group of Example 2 of the present invention and the serum control group of Comparative Example 1 under an inverted microscope (20 times magnification).

[0155] Figure 27 This is a growth morphology diagram of the tendon stem cells in the serum-free culture experimental group of Example 3 of the present invention and the serum control group of Comparative Example 1 under an inverted microscope (20 times).

[0156] Figure 28 This is a picture of the growth morphology of human adipose-derived stem cells in the serum-free culture experimental group of Example 4 of the present invention and the serum control group of Comparative Example 1 under an inverted microscope (20 times).

[0157] Figure 29 These are growth morphologies of tendon stem cells cultured under an inverted microscope (20x magnification) in the serum-free culture experimental group of Example 5 of the present invention, the serum control group of Comparative Example 1, the commercial Biological Industries MSC serum-free culture medium (BI SFM) of Comparative Example 2, and the commercial Gibco MSC serum-free culture medium (ST SFM) of Comparative Example 3.

[0158] Figure 30 This is a bar graph showing the relative expression of tendon stem cell-related genes in the serum-free medium (SFM) group of Example 5 of the present invention, the serum medium (SCM) group of Comparative Example 1, and the commercial Biological Industries MSC serum-free medium (BI SFM) group of Comparative Example 2.

[0159] Figure 31 This is a three-dimensional growth morphology diagram of the tendon stem cells in the serum-free culture experimental group of Example 6 of the present invention and the serum control group of Comparative Example 1 under an inverted fluorescence microscope (10 times).

[0160] Figure 32 This is a three-dimensional growth morphology diagram of the tendon stem cells in the serum-free culture experimental group of Example 7 of the present invention and the serum control group of Comparative Example 1 under an inverted fluorescence microscope (10 times).

[0161] Figure 33 1 and 2 show the growth morphology of human mesenchymal stem cells cultured in the serum-free culture experimental group of Example 8 and Comparative Example 4 (SFM-Ctrl4) under an inverted microscope (4 times magnification).

[0162] Figure 34 This is a bar graph showing the cell proliferation ratio of human mesenchymal stem cells cultured for 3 days in the serum-free culture experimental group of Example 8 of the present invention and in Comparative Example 4 (SFM-Ctrl4).

[0163] Figure 35 This is a bar graph of the doubling time of human mesenchymal stem cells cultured in the serum-free culture experimental group of Example 8 of the present invention and in Comparative Example 4 (SFM-Ctrl4).

[0164] Figure 36 This is a growth morphology diagram of three-dimensional culture of human chondrocytes in the serum-free culture experimental group of Example 9 of the present invention under an inverted fluorescence microscope (4 times).

[0165] Figure 37 This is a cell growth morphology diagram of human skeletal stem cells in the serum-free culture experimental group of Example 10 of the present invention and the serum control group of Comparative Example 1 under an inverted fluorescence microscope (20 times).

[0166] Figure 38 This is a growth morphology diagram of tendon stem cells in the control group culture medium containing only B27 cell culture additives in Comparative Example 5 of the present invention under an inverted microscope (20 times).

[0167] Figure 39 This is a growth morphology diagram of tendon stem cells in serum-free culture medium of Comparative Example 6 of the present invention under an inverted microscope (4 times).

[0168] Figure 40 This is a growth morphology diagram of tendon stem cells in serum-free culture medium of Comparative Example 7 of the present invention under an inverted microscope (4 times).

[0169] Figure 41 This is a growth morphology diagram of tendon stem cells in serum-free culture medium of Comparative Example 8 of the present invention under an inverted microscope (4 times).

[0170] Table 1 shows the cell viability in each embodiment and comparative example.

[0171] Table 2 shows the expression of cell surface markers in various examples and comparative examples.

[0172] Table 3 shows the statistics of cell clone formation ability in various examples and comparative examples.

[0173] Table 4 shows the percentage of Nestin+ cells cultured in various examples and comparative examples.

[0174] Table 5 shows the scores of cells cultured in various embodiments and comparative examples. DETAILED DESCRIPTION

[0175] The present invention is described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit this discovery in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the present invention. These all fall within the scope of protection of the present invention.

[0176] Example 1

[0177] In this example, two culture media were prepared: a serum-free culture medium and a serum control culture medium. The cultured cells were tendon stem cells (hTSPCs) extracted from normal human tendon tissue. The following are detailed experimental and testing procedures:

[0178] Culture medium preparation

[0179] Serum-free medium (SFM)

[0180] The serum-free medium comprises a basal medium and supplemental components; the basal medium is selected from DMEM / F12 medium, and per 500 mL of DMEM / F12 medium, 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added. The supplemental components are added such that the concentration ratio of the components in the serum-free medium is: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite: epidermal growth factor: CHIR99021 = 10:10:5:2:1000:25000:2000:500:2:500:2:10:251. The medium also contains 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 1 mM sodium pyruvate, and 1X B27 cell culture supplement.

[0181] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0182] FGF2 20ng / ml

[0183] PDGF-BB 20ng / ml

[0184] TGF-β3 10ng / ml

[0185] Dexamethasone 10 nM

[0186] Heparin sodium 2 μg / ml

[0187] Vitamin C 50 μg / ml

[0188] Transferrin 4 μg / ml

[0189] Insulin 1 μg / ml

[0190] Progesterone 4ng / ml

[0191] Putrescine 1 μg / ml

[0192] Sodium selenite 4ng / ml

[0193] Epidermal growth factor 20ng / ml

[0194] CHIR99021 1μM

[0195] Non-essential amino acids 0.1 mM

[0196] L-glutamate 2mM

[0197] Sodium pyruvate 1 mM

[0198] B27 Cell Culture Supplement 1X

[0199] Comparative Example 1

[0200] Serum control medium (SCM)

[0201] The culture medium of the serum control group was selected from DMEM low-glucose culture medium, and 55 mL of fetal bovine serum, 5 mmol of HEPES, 10,000 U of penicillin and 10,000 U of streptomycin were added to every 500 mL of DMEM low-glucose culture medium.

[0202] Example 1 Biological Activity Experiment

[0203] Cultured cell treatment

[0204] Primary cell isolation and culture

[0205] Remove the tendon and place it in a culture dish containing 10% polysaccharide (PS) in PBS, 5% PS, 2% PS, and 1% PS in PBS for 1 minute each to sterilize. Add the tendon tissue to a 0.2% collagenase solution, mince until a paste forms, and add the digestion solution to 10ml. Submerge the tendon in the digestion medium as evenly as possible and incubate the medium in a 37°C incubator. Air-dry the tendon every hour until fully digested. Centrifuge the digested cell suspension at 1200 rpm for 5 minutes, discard the supernatant, and add it to a fibronectin-coated 10cm culture dish. Add 10ml of SFM and incubate at 37°C with 5% CO2 in a cell culture incubator. Change the medium every 3-5 days. Once the cells have attached to the wall and are 80-90% confluent, digest and passage the cells for subsequent experiments. Cryopreserve any excess cells.

[0206] Passaging cell culture

[0207] Tendon stem cells from generations P1 to P6 were collected at a rate of approximately 9 x 10^3 / cm 2 The cells were seeded at a density of 100 μg / ml into well plates, culture dishes or culture flasks coated with 80 μg / ml fibronectin, and cultured with serum-free medium and serum control medium, respectively. The cells were cultured in a cell culture incubator at 37°C and 5% CO2, with the medium changed every 3 days until they were basically full. Experiments such as cell photography, cell counting, gene expression detection, trilineage differentiation, and immunofluorescence were performed.

[0208] Experimental results analysis

[0209] Cell morphological observation

[0210] During the above-mentioned cell culture treatment process, the growth and morphological changes of the tendon stem cells in the serum-free culture experimental group and the serum culture control group were observed with an inverted phase contrast microscope, and recorded by microscopic photography.

[0211] like Figure 7 As shown, under a 4X microscope, the tendon stem cells cultured in serum-free primary culture grew into monoclonal growth, with vigorous cell growth, uniform size, translucent and abundant cytoplasm, and good adhesion to the wall, indicating that the serum-free medium supports the primary culture of cells and the culture effect is very good.

[0212] like Figure 8 and 9 As shown, Figure 8 The growth of cells from P1 to P6 when they are basically full grown is shown under a 4X microscope. The results show that the tendon stem cells cultured in SFM (serum-free medium) grow vigorously, and the cell proliferation is significantly better than that in the SCM (serum medium) group. Figure 9 As shown, the cell morphology when the cells are basically full is displayed under a 20X microscope. The results show that compared with the SCM (serum culture medium) group, the tendon stem cells cultured in SFM (serum-free culture medium) have more uniform cell morphology and size, bright and abundant cytoplasm, good adhesion, and more cells.

[0213] Cell counting and cell proliferation analysis

[0214] Tendon stem cells from passages P1-P6 were seeded at a density of approximately 9 x 10^3 / cm2 in 10 cm culture dishes coated with fibronectin. Cells were cultured in serum-free medium or serum-controlled medium until nearly confluent. The medium was then discarded, the cells were washed once with 1X PBS, trypsinized, centrifuged at 1200 rpm for 5 minutes, the supernatant discarded, and the pellet resuspended in 1 ml of culture medium and mixed thoroughly. Cells were counted using the trypan blue counting method. The cell suspension was mixed with 0.4% trypan blue solution in a ratio of 9:1 (final trypan blue concentration 0.04%).

[0215] Cell count automatic technique: Pipette 20 μl of cell suspension and count automatically using a cell count counter.

[0216] Manual counting method with hemocytometer: Pipette cell suspension into hemocytometer and count under microscope. The counting method is: (total number of cells in four grids / 4)×10 4 × dilution factor = number of cells in cell suspension / mL.

[0217] If the cell membrane is intact and the cell is not stained with trypan blue, it is a normal cell; if the cell membrane is incomplete or ruptured, the trypan blue dye enters the cell and the cell turns blue, which is a necrotic cell.

[0218] Cell viability was calculated as follows: viable cell rate (%) = total number of viable cells / (total number of viable cells + total number of dead cells) × 100%.

[0219] The number of days of cell culture, the number of harvested cells and the cell diameter at harvest were recorded, and relevant graphs were drawn.

[0220] like Figure 10 As shown, from P1 to P6 (four weeks), the cells in the SFM (serum-free medium) group proliferated 1.8X10^5 times, while those in the SCM (serum medium) group only proliferated 40 times. Therefore, the cell proliferation multiples in the SFM (serum-free medium) group were 4500 times that of the SCM group. The proliferation rate of tendon stem cells in the SFM (serum-free medium) group was significantly higher than that in the SCM (serum medium) group.

[0221] As shown in Table 1, the cell viability of tendon stem cells in the SFM (serum-free medium) group was significantly higher than that in the SCM (serum medium) group.

[0222] Cell doubling time analysis

[0223] Tendon stem cells from generations P1 to P6 were collected at a rate of approximately 9 x 10^3 / cm 2 The cells were seeded at a density of 10 cm in a fibronectin-coated culture dish. The cells were cultured with serum-free medium and serum control medium respectively until they were basically confluent. The number of days of cell culture was recorded, and the cell doubling time was calculated according to the formula DT=t*[lg2 / (lgNt-lgNo)] (t is the culture time, No is the number of cells recorded for the first time, and Nt is the number of cells after t time), and the relevant graphs were drawn.

[0224] like Figure 11 As shown, the doubling time of tendon stem cells in the experimental group (SFM) was less than 30 hours, significantly lower than that in the SCM (serum-free medium) control group. The doubling time in the SCM group was 4.2 times that of the SFM group. The shorter the doubling time, the faster the cell proliferation. In other words, the proliferation rate of tendon stem cells in the experimental group (SFM) was significantly faster than that in the SCM (serum-free medium) control group. This indicates that the serum-free medium can effectively replace the role of serum and is significantly better at promoting cell proliferation than serum.

[0225] Cell size comparison

[0226] Tendon stem cells were collected at a rate of approximately 9 x 10^3 / cm 2 The cells were planted at a density of 10 cm in a coated culture dish and cultured in serum-free and serum-containing conditions until they were basically full. The culture medium was discarded, the cells were washed once with 1XPBS, digested with trypsin, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, the pellet was resuspended in 1 ml of culture medium, mixed, 20 μl of cell suspension was aspirated, and the cells were counted using a cell counter. The cell diameter at the time of harvest was recorded and a bar graph was drawn.

[0227] like Figure 12As shown in the figure, the diameter of the harvested tendon stem cells in the SFM (serum-free medium) experimental group was significantly smaller than that in the SCM (serum-containing medium) control group. This indicates that the cells cultured in SFM (serum-free medium) are more consistent with stem cell characteristics than those cultured in the SCM (serum-containing medium) control group.

[0228] Karyotype analysis

[0229] Tendon stem cells were collected at a rate of approximately 9 x 10^3 / cm 2 The cells were planted at a density of 10 cm in a coated culture dish and cultured in serum-free and serum-containing conditions until they were basically full. The cells were then sent to a genetic diagnosis company for karyotype analysis.

[0230] like Figure 13 As shown in the figure, the results showed that tendon stem cells in both the SFM (serum-free medium) experimental group and the SCM (serum medium) control group were normal (the proportion of cells with normal karyotype was greater than 90%). This indicates that the cells cultured in this serum-free medium are normal cells with no karyotype variation.

[0231] Mycoplasma testing

[0232] The supernatant and FBS of cells cultured for 3 days in the SFM group, as well as a mycoplasma positive control, were collected and tested for mycoplasma using a one-step rapid mycoplasma detection kit. The main principle is that if the cell culture is contaminated with mycoplasma, the conserved sequences of mycoplasma DNA will be amplified rapidly and in large quantities, causing the reaction solution to change from blue-purple to sky blue. The results are visible to the naked eye without the need for electrophoresis.

[0233] like Figure 14 As shown, the results showed that the detection reagent of the SFM group was the original blue-purple, the mycoplasma positive control was sky blue, and the detection color of the FBS group was slightly sky blue. This result shows that the serum-free culture medium of the invention is free of mycoplasma contamination and the cultured cells are safe, while this batch of FBS has slight mycoplasma contamination, which also shows that adding FBS to culture cells has a great risk of mycoplasma contamination, and the serum-free culture medium of the invention can avoid this risk.

[0234] Flow cytometry analysis to detect the expression of stem cell surface markers

[0235] P3 or P5 tendon stem cells were placed at a rate of approximately 9 x 10^3 / cm 2The cells were seeded at a density of 10 cm in a coated 10 cm culture dish and cultured in serum-free and serum-containing conditions until they were basically full. The culture medium was discarded, the cells were washed once with 1XPBS, trypsinized, centrifuged for 5 minutes, the supernatant was discarded, the pellet was resuspended in blocking solution and blocked for 30 minutes. The stem cell surface CD markers were stained separately, and CD146, CD105, CD90, CD44, CD34, CD18 and other flow cytometry antibodies were stained for 30 minutes. The cells were then added with 1XPBS, mixed, centrifuged and washed twice. The cells were resuspended in 500 ul PBS and mixed in a flow cytometer to analyze the expression of CD markers.

[0236] As shown in Table 2, CD105, CD90, and CD44 are positive markers for tendon stem cells, while CD34 and CD18 are negative markers for tendon stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. These results also indicate that cells cultured in the SFM (serum-free medium) experimental group more closely resemble tendon stem cell characteristics than those in the SCM (serum-free medium) control group. This suggests that cells cultured in the SFM (serum-free medium) medium exhibit more stem cell characteristics than cells cultured in the SCM (serum-free medium) control group.

[0237] Clonogenic ability assay

[0238] P3 or P5 tendon stem cells were seeded in 6 cm culture dishes at a density of 100 cells / dish in triplicate and cultured in serum-free and serum-containing conditions for 10-12 days, stained with 1% crystal violet, and colonies with a diameter >2 mm were counted.

[0239] As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in serum-free medium are more consistent with stem cell characteristics than those cultured in serum-containing medium.

[0240] Three-lineage differentiation capacity assay

[0241] Take the serum-free medium from P3 to P5 and the serum control medium and culture the cells to the well plate for osteoinduction, chondrogenic induction and adipogenic induction respectively. The specific methods are as follows:

[0242] Method and identification of directional induction of osteoblast differentiation: tendon stem cells were collected and cultured, and the digested cells were divided into 1×10 4 cells / cm 2Cells were seeded in 24-well plates. After most cells adhered to the plate as observed under a microscope, the supernatant was removed and the culture medium was replaced with high-glucose DMEM supplemented with 10% FBS. The osteogenic induction system was added and the medium was completely replaced every three days for 14 days. The ossification-inducing ability of the cells was qualitatively assessed using an alkaline phosphatase (ALP) kit and quantitatively assessed by Alizarin Red staining (ARS). Cell nuclei were labeled with DAPI; Alizarin Red was then eluted with 5% SDS-HCl solution. OD values were obtained using a microplate reader. The difference in the ratio of OD value to cell number represents the quantitative osteogenic ability of the cells.

[0243] Method and identification of directional induction of chondrocyte differentiation: Method and identification of directional induction of chondrocyte differentiation: TSPCs cells were collected and cultured, and the digested cells were divided into 2×10 5 Drop the cells at a concentration of TSPCs / 10ul in the center of a 12-well plate, place it in an incubator, and add chondrogenic induction solution after the cells adhere to the wall. Replace the solution every 2-3 days, and fix it after 2 weeks for Aclian blue staining.

[0244] Method and identification of directional induction of adipocyte differentiation: TSPCs were collected and cultured, and the digested cells were counted at 1×10 4 cells / cm 2 Cells were seeded in 24-well plates. After most cells adhered, the medium was switched to high-glucose DMEM supplemented with 10% FBS and an adipogenic induction system was added. After 2 weeks, intracellular lipid droplet formation was observed under a microscope and stained with Oil Red O. The red color was eluted with isopropanol, and the cells were read on a microplate reader to obtain the adipogenic capacity (X ± SD).

[0245] like Figure 15-17 As shown in the results, the SFM (serum-free medium) experimental group had significantly better osteogenic and chondrogenic differentiation abilities than the SCM (serum medium) control group, while the adipogenic ability of the SFM (serum-free medium) and SCM (serum medium) groups was comparable. This indicates that the serum-free medium has stronger trilineage differentiation abilities than the serum-containing control.

[0246] qPCR detection of gene expression

[0247] Tendon stem cells were seeded in coated 12-well plates and cultured in serum-free medium and serum control medium respectively until the 5th day. The medium was discarded and the cells were washed once with 1XPBS. 500ul RNA cell lysis solution was added to each well of the 12-well plate containing cultured cells. The cellular RNA was extracted using an RNA extraction kit and reverse transcribed into cDNA. The samples were then loaded onto the machine for qPCR detection of the relative expression of tendon-related genes in the cells. The results were analyzed and a bar graph of the relative expression of the gene was drawn with the group as the horizontal axis and the relative expression of the gene as the vertical axis.

[0248] like Figure 18 As shown in the figure, compared with the SFM (serum-free medium) control group, the tendon-related genes SCX, nestin, and TNMD were significantly overexpressed in the serum-free experimental group. Therefore, it is shown that SFM (serum-free medium) is more conducive to the maintenance of the tendon phenotype of tendon stem cells than SCM (serum medium).

[0249] In vitro tendon differentiation ability assay

[0250] Cells cultured in serum-free medium and serum control group medium for P3-P5 generations were passaged into 12-well plates at a rate of 4×10^4 cells / well, with three replicates per group. When the cells were basically confluent in serum-free medium and serum control group medium, the tendon induction medium was replaced every 2-3 days. After one to two weeks of induction, the cells were stained with picrosirius red, rolled into cell sheets, and photographed under a transmission electron microscope to evaluate collagen formation.

[0251] like Figure 19 As shown by picrosirius red staining, the amount of collagen formation in the SFM (serum-free medium) experimental group was significantly increased compared with the SCM (serum medium) control group, indicating that under tendon induction conditions, tendon stem cells cultured in this serum-free medium have stronger tendon differentiation ability than tendon stem cells in serum medium.

[0252] like Figure 20 As shown in the figure, transmission electron microscopy results showed that compared with the SCM (serum culture medium) control group, the amount of collagen formation in the SFM (serum-free culture medium) experimental group was significantly increased, and the diameter of the collagen formed was significantly larger than that in the control group, indicating that under tendon induction conditions, tendon stem cells cultured in this serum-free culture medium have stronger tendon differentiation ability than tendon stem cells in serum culture medium.

[0253] Flow cytometry analysis to detect Nestin expression

[0254] P3 or P5 tendon stem cells were placed at a rate of approximately 9 x 10^3 / cm 2 The cells were planted at a density of 10 cm in a coated culture dish and cultured in serum-free and serum-containing conditions until they were basically full. The culture medium was discarded, the cells were washed once with 1XPBS, digested with trypsin, centrifuged for 5 minutes, the supernatant was discarded, the pellet was resuspended in blocking solution and blocked for 30 minutes. The membrane was broken and the stem cells were stained for Nestin in separate tubes. After 30 minutes, 1XPBS was added and mixed, centrifuged and washed twice, 500ul of PBS was added and resuspended, and the expression of Nestin markers was analyzed.

[0255] As shown in Table 4, the results showed that the expression of Nestin-positive markers in the SFM (serum-free medium) experimental group was greater than 30%, while the expression of Nestin-positive markers in the SCM control group of comparative example 1 was less than 5%, indicating that the tendon phenotype of cells cultured in the SFM (serum-free medium) experimental group was significantly improved compared with that in the SCM (serum medium) control group.

[0256] In vivo tendon formation ability assay

[0257] Take the serum-free medium of P5 generation and the serum control medium to culture cells at a temperature of about 9X10^3 / cm 2 The cells were seeded at a density of 10 cm in a coated culture dish and cultured in serum-free medium and serum control medium until they were basically full. Then, they were cultured in tendon induction medium with the medium changed every 2-3 days. After two weeks of induction, they were rolled into cell sheets and implanted subcutaneously on the back of nude mice. Samples were collected two weeks later and the tendon formation of the implanted cells was evaluated by HE staining, Masson staining, immunofluorescence staining, qPCR and other experiments.

[0258] like Figure 21 As shown in the figure, QPCR results showed that compared with the SFM (serum-free medium) control group, the expression of tendon-related genes such as SCX, Nestin, TNMD, and COL1A1 in the serum-free experimental group was significantly increased, indicating that the tendon stem cells cultured in this serum-free medium have stronger tendon differentiation ability and more mature tendon tissue formed in vivo.

[0259] like Figure 22 As shown in the figure, histological results showed that compared with the SCM (serum culture medium) control group, the collagen of the tendon tissue formed in the SFM (serum-free culture medium) experimental group was more neatly arranged and denser, indicating that the tendon stem cells cultured in this serum-free culture medium have a stronger ability to form tendons in vivo.

[0260] like Figure 23 As shown in the figure, the immunofluorescence results showed that compared with the SFM (serum-free medium) control group, the expression of tendon-related proteins such as SCX and COL1A1 was significantly increased in the serum-free experimental group. Therefore, it was shown that the serum-free medium was more conducive to the differentiation of tendon stem cells into the tendon system and the formation of tendon tissue than the serum medium.

[0261] In vivo assessment of tendon repair capacity in situ

[0262] Take the serum-free medium of P5 generation and the serum control medium to culture cells at a temperature of about 9X10^3 / cm 2The cells were seeded at a density of 10 cm in a coated culture dish, and the medium was changed every 2-3 days. When they were basically confluent in serum-free culture medium and serum control culture medium, respectively, the cells were digested into single cells, mixed with fibrin gel to form a colloid, and then implanted into the local defect site of the rat patellar tendon. Samples were collected after four or eight weeks, and the tendon formation of the implanted cells was evaluated by HE staining, Masson staining, immunofluorescence staining and other experiments.

[0263] like Figure 24 As shown in the figure, histological results showed that compared with the serum control group (SCM group), the collagen of the repaired tendon tissue in the SFM (serum-free medium) experimental group was more neatly arranged and denser, indicating that the tendon stem cells cultured in this serum-free medium have a stronger in situ tendon repair ability in vivo.

[0264] like Figure 25 As shown in the figure, the immunofluorescence results showed that compared with the serum control group (SCM group), the expression of tendon-related protein Nestin in the serum-free experimental group was significantly increased. Therefore, it was shown that the serum-free medium was more conducive to the differentiation of tendon stem cells into the tendon system and the formation of tendon tissue than the serum medium.

[0265] Example 1 Cell score calculation

[0266] From the results of "Cell Doubling Time Analysis" in Example 1, it can be seen that the doubling time of the cells cultured in the serum-free medium in Example 1 is less than 30 hours, so the cell proliferation rate is scored as 30 points.

[0267] The results of "Flow Cytometry Detection of Stem Cell Surface Marker Expression" in Example 1 show that the cells cultured in serum-free medium in Example 1 expressed greater than 95% positive markers and less than 1% negative markers, and were more consistent with tendon stem cell characteristics than the SCM (serum medium) control group, indicating that the cells cultured in serum-free medium in Example 1 had increased stem cell surface marker expression. The results of "Clonogenic Ability Assay" in Example 1 show that the SFM (serum-free medium) experimental group had significantly better colonogenic ability (25 cells / well) than the SCM (serum medium) control group (12 cells / well). The results of "Tri-lineage Differentiation Ability Assay" in Example 1 show that the SFM (serum-free medium) experimental group had significantly better osteogenic and chondrogenic differentiation abilities than the SCM (serum medium) control group, while the adipogenic ability of the SFM (serum-free medium) and SCM (serum medium) groups was comparable. This suggests that cells cultured in serum-free medium have stronger tri-lineage differentiation abilities than those in the serum-supplemented control group. Based on the above results, it can be seen that, compared with cells cultured in conventional serum medium in the prior art, the cells obtained by culture in serum-free medium in Example 1 have improved stem cell surface marker expression, clone formation ability and tri-lineage differentiation ability, so the stem cell phenotype score is 20 points.

[0268] As can be seen from the results of "Karyotype Analysis" in Example 1, the karyotype of cells cultured in the serum-free medium of Example 1 is normal; as can be seen from the results of "Mycoplasma Detection" in Example 1, the serum-free medium of Example 1 is free of mycoplasma contamination, and the cultured cells are safe, while this batch of FBS has slight mycoplasma contamination; combined with the fact that the serum-free medium of the present invention is a completely serum-free medium, it can achieve primary and subculture of cells without the involvement of serum throughout the process, so there is no serum residue. In summary, the cell karyotype obtained by culturing in the serum-free medium of Example 1 is normal, there is no serum residue, and there is no mycoplasma contamination, so the safety score is 10 points.

[0269] The results of "qPCR gene expression" in Example 1 indicate that, compared with the SCM (serum culture medium) control group, the cells cultured in the serum-free medium of Example 1 showed significantly higher expression of the tendon-related genes SCX, Nestin, and TNMD in the serum-free experimental group. The results of "Flow cytometry analysis of Nestin expression" in Example 1 indicate that the Nestin positivity rate in the cells cultured in the serum-free medium of Example 1 reached 94%, and the results of "in vitro tendon differentiation ability assay" also indicate that the collagen formation ability of the cells cultured in the serum-free medium of Example 1 was significantly enhanced compared to the serum control group. In summary, the tendon phenotype and tendon differentiation ability obtained from the serum-free medium culture of Example 1 were significantly improved compared to the serum control group, with high expression of the three tendon-related genes SCX, Nestin, and TNMD, and a Nestin positivity rate greater than 90%. Therefore, the tendon phenotype and tendon differentiation ability were scored as 20 points.

[0270] The results of the "In Vivo Tendon Formation Ability Assay" and "In Vivo In Situ Tendon Repair Ability Assessment" in Example 1 indicate that, compared with the serum control group (SCM group), the tendon tissue repaired by cells cultured in serum-free medium in Example 1 had more uniformly arranged and denser collagen, lacking non-tendon tissues such as bone, cartilage, and muscle, and more closely resembling normal tissue morphology. Therefore, the in vivo tendon and / or ligament repair ability score was 20 points.

[0271] In summary, the total score of the cells obtained by serum-free culture in Example 1 was 100 points, which met the cell quantity and quality requirements for clinical cell therapy of tendon and / or ligament injury.

[0272] Example 2

[0273] In this example, two culture media were prepared: a serum-free culture medium and a serum control culture medium. The cultured cells were ligament stem cells isolated and cultured from human ligament tissue. The following are detailed experimental and testing steps:

[0274] Culture medium preparation

[0275] Serum-free medium (SFM)

[0276] The serum-free culture medium comprises a basal culture medium and additives; the basal culture medium is selected from F10 culture medium, and 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added to every 500 mL of F10 culture medium, and the additives are added so that the concentration range ratio of each component in the serum-free culture medium is: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 15:10:6:2:1500:25,000:75,000:3,000:4:7:4.

[0277] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0278] FGF-basic 30ng / ml

[0279] PDGF-AA 10ng / ml

[0280] PDGF-BB 10ng / ml

[0281] TGF-β1 3ng / ml

[0282] TGF-β3 3ng / ml

[0283] Dexamethasone 10 nM

[0284] Heparin sodium 3 μg / ml

[0285] Vitamin C 50 μg / ml

[0286] Transferrin 150 μg / ml

[0287] Insulin 6 μg / ml

[0288] Progesterone 8ng / ml

[0289] Putrescine 14 μg / ml

[0290] Sodium selenite 8ng / ml

[0291] Example 2 Biological Activity Experiment

[0292] Cultured cell treatment

[0293] P3-P6 human ligament stem cells were cultured at a rate of approximately 9 x 10^3 / cm 2 The cells were seeded at a density of 100 μg / ml laminin, 200 μg / ml fibronectin and 100 ug / ml vitronectin coated well plates or culture dishes, and cultured with serum-free medium and serum control medium, respectively. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 3 days until the cells were basically full, and the cell growth was observed by taking pictures.

[0294] Experimental results analysis

[0295] Cell morphological observation

[0296] The method is the same as in Example 1. Figure 26 As shown, the cell morphology when the cells are basically full is displayed under a 20X microscope. The results show that compared with the SCM (serum culture medium) group, the human ligament stem cells cultured in SFM (serum-free culture medium) have more uniform morphology and size, bright and abundant cytoplasm, good adhesion to the wall, and larger number.

[0297] Flow cytometry analysis to detect the expression of stem cell surface markers

[0298] The method was the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive markers for human ligament stem cells, while CD34 and CD18 are negative markers for human ligament stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. This group also showed a higher degree of conformity with human ligament stem cell characteristics than the SCM (serum-free medium) control group. This suggests that cells cultured in the serum-free medium (SFM) compared to the SCM (serum-free medium) control group exhibited a higher degree of conformity with stem cell characteristics.

[0299] Clonogenic ability assay

[0300] The method was the same as in Example 1. As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in the serum-free medium are more consistent with stem cell characteristics than those cultured in the serum-containing control group.

[0301] Example 3

[0302] In this example, two culture media were prepared: a serum-free culture medium and a serum control culture medium. The cultured cells were tendon stem cells (hTSPCs) extracted from normal human tendon tissue. The following are detailed experimental and testing procedures:

[0303] Culture medium preparation

[0304] Serum-free medium (SFM)

[0305] The serum-free culture medium comprises a basal culture medium and additives; the basal culture medium is selected from DMEM / F12 culture medium, and 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added to each 500 mL of DMEM / F12 culture medium, and the additives are added so that the concentrations of the additives in the serum-free culture medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 50: 50: 40: 11: 1,000: 100,000: 300,000: 25,000: 25: 25,000: 25; the culture medium also contains 0.1 mM non-essential amino acids, 0.1 mM L-glutamic acid, 0.1 mM sodium pyruvate, and 0.1X B27 cell culture additive.

[0306] Furthermore, the concentrations of the components of the serum-free medium are as follows:

[0307] FGF2 20ng / ml

[0308] FGF4 20ng / ml

[0309] FGF18 10ng / ml

[0310] PDGF-AA 25ng / ml

[0311] PDGF-AB 25ng / ml

[0312] TGF-β1 20ng / ml

[0313] TGF-β2 20ng / ml

[0314] Dexamethasone 15 nM

[0315] Hydrocortisone 14nM

[0316] Heparin 1 μg / ml

[0317] Vitamin C 50 μg / ml

[0318] Vitamin C sodium phosphate 50μg / ml

[0319] Transferrin 300 μg / ml

[0320] Insulin 25 μg / ml

[0321] Progesterone 25ng / ml

[0322] Putrescine 15 μg / ml

[0323] Putrescine dihydrochloride 10 μg / ml

[0324] Sodium selenite 25ng / ml

[0325] Non-essential amino acids 0.1 mM

[0326] L-glutamate 0.1 mM

[0327] Sodium pyruvate 0.1 mM

[0328] B27 Cell Culture Supplement 0.1X

[0329] Example 3 Biological Activity Experiment

[0330] Cultured cell treatment

[0331] P3-P6 tendon stem cells were cultured at a rate of approximately 9 x 10^3 / cm 2 The cells were seeded at a density of 100 μg / ml into 40 mg / ml gelatin-coated well plates, culture dishes or culture flasks, and cultured with serum-free medium and serum control medium, respectively. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days until the cells were basically full, and the cell growth was observed by taking pictures.

[0332] Experimental results analysis

[0333] Cell morphological observation

[0334] The method is the same as in Example 1. Figure 27As shown, the cell morphology when the cells are basically full is displayed under a 20X microscope. The results show that compared with the SFM (serum-free medium) group and the SCM (serum medium) group, the tendon stem cells cultured in SFM (serum-free medium) grow vigorously, and the cell proliferation is significantly better than that in the SCM (serum medium) group. The cell morphology and size are more uniform, the cytoplasm is translucent and rich, and the adhesion is good.

[0335] Flow cytometry analysis to detect the expression of stem cell surface markers

[0336] The method was the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive markers for tendon stem cells, while CD34 and CD18 are negative markers for tendon stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. This group also showed a higher degree of tendon stem cell characteristics than the SCM (serum-free medium) control group. This suggests that cells cultured in the serum-free medium (SFM) are more consistent with stem cell characteristics than cells cultured in the SCM (serum-free medium) control group.

[0337] Clonogenic ability assay

[0338] The method was the same as in Example 1. As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in the serum-free medium are more consistent with stem cell characteristics than those cultured in the serum-containing control group.

[0339] Example 4

[0340] In this example, two culture media were prepared: a serum-free culture medium and a serum control culture medium. The cultured cells were adipose-derived stem cells (ADSCs) extracted from human fat. The following are detailed experimental and testing steps:

[0341] Culture medium preparation

[0342] Serum-free medium (SFM)

[0343] The serum-free culture medium comprises a basal culture medium and additives; the basal culture medium is selected from F12 culture medium, and 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added to each 500 mL of F12 culture medium, and the additives are added so that the concentrations of the additives in the serum-free culture medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 1: 1: 1: 1: 10: 10: 10: 1: 1: 1: 1; the culture medium also contains 1 mM non-essential amino acids, 4 mM L-glutamic acid, 2 mM sodium pyruvate, 2X B27 cell culture supplement, 1 μg / ml vitronectin, 1 μg / ml fibronectin, and 1 μg / ml laminin.

[0344] bFGF 10 ng / ml

[0345] PDGF-AA 10ng / ml

[0346] TGF-β2 10ng / ml

[0347] Dexamethasone 25 nM

[0348] Heparin sodium 0.1 μg / ml

[0349] Vitamin C 0.1 μg / ml

[0350] Transferrin 0.1 μg / ml

[0351] Insulin 0.01 μg / ml

[0352] Progesterone 10 ng / ml

[0353] Putrescine 0.01 μg / ml

[0354] Sodium selenite 10ng / ml

[0355] Nonessential amino acids 1mM

[0356] L-glutamate 4mM

[0357] Sodium pyruvate 2 mM

[0358] B27 Cell Culture Supplement 2X

[0359] Vitronectin 1 μg / ml

[0360] Fibronectin 1 μg / ml

[0361] Laminin 1 μg / ml

[0362] Example 4 Biological Activity Experiment

[0363] Cultured cell treatment

[0364] P3-P6 adipose-derived stem cells were cultured at a rate of approximately 5 x 10^3 / cm 2 The cells were seeded at a density of 100 μg / mL in well plates, culture dishes or culture flasks, and cultured with serum-free medium and serum control medium, respectively. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 3 days until the cells were basically full, and the cell growth was observed by taking pictures.

[0365] Experimental results analysis

[0366] Cell morphological observation

[0367] The method is the same as in Example 1. Figure 28 , The cell morphology when the cells are basically full is shown under a 20X microscope. The adipose-derived stem cells cultured in SFM (serum-free medium) grow vigorously, and the cell proliferation is significantly better than that in the SCM (serum medium) group. In addition, the adipose-derived stem cells cultured in SFM (serum-free medium) have more uniform cell morphology and size, rich and transparent cytoplasm, and good adhesion to the wall.

[0368] Flow cytometry analysis to detect the expression of stem cell surface markers

[0369] The method was the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive markers for adipose-derived stem cells, while CD34 and CD18 are negative markers for adipose-derived stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. This group also showed a better fit for adipose-derived stem cell characteristics than the SCM (serum-free medium) control group. This suggests that cells cultured in SFM (serum-free medium) are more consistent with stem cell characteristics than cells cultured in the SCM (serum-free medium) control group.

[0370] Clonogenic ability assay

[0371] The method was the same as in Example 1. As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in the serum-free medium are more consistent with stem cell characteristics than those cultured in the serum-containing control group.

[0372] Example 5

[0373] In this example, four culture media were prepared: serum-free culture medium, serum control culture medium, commercial Biological Industries MSC serum-free culture medium, and commercial Gibco MSC serum-free culture medium. The cultured cells were tendon stem cells (hTSPCs) extracted from normal human tendon tissue. The following are detailed experimental and testing procedures:

[0374] Culture medium preparation

[0375] Serum-free medium (SFM)

[0376] The serum-free medium includes a basal medium and supplementary components; the basal medium is selected from DMEM / F12 medium, and 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added to each 500 mL of DMEM / F12 medium, and the supplementary components are added so that the concentrations of the supplementary components in the serum-free medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite: epidermal growth factor: CHIR99021 = 30:30:5:2:2000:80,000:50,000:100:5:100:5:10:1004. The medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, and 1X B27 cell culture supplement.

[0377] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0378] FGF2 30ng / ml

[0379] PDGF-BB 30ng / ml

[0380] TGF-β3 5ng / ml

[0381] Dexamethasone 5 nM

[0382] Heparin sodium 2 μg / ml

[0383] Vitamin C 80 μg / ml

[0384] Transferrin 50 μg / ml

[0385] Insulin 0.1 μg / ml

[0386] Progesterone 5ng / ml

[0387] Putrescine 0.1 μg / ml

[0388] Sodium selenite 5ng / ml

[0389] Epidermal growth factor 10ng / ml

[0390] CHIR99021 2μM

[0391] Non-essential amino acids 0.1 mM

[0392] L-Glutamic acid 1mM

[0393] Sodium pyruvate 0.5 mM

[0394] B27 Cell Culture Supplement 1X

[0395] Comparative Example 2

[0396] Commercialized Biological Industries MSC serum-free medium (BI SFM):

[0397] MSC NutriStem®XF Basal Medium (Culture Medium): MSC NutriStem®XF Supplement (Additive): Blue Chain Antibody = 500ml: 3ml: 5ml.

[0398] Comparative Example 3

[0399] Commercial Gibco MSC serum-free culture (ST SFM, or StemPro SFM):

[0400] StemPro ® MSC SFM Supplement CTS ™: StemPro ® MSC SFM Basal Medium CTS ™: L-glutamine or GlutaMAX ™ -I CTS ™ = 15ml: 84ml: 1ml, with the final concentration of L-glutamine or GlutaMAX ™ -I CTS ™ being 2mM.

[0401] Example 5 Biological Activity Experiment

[0402] Cultured cell treatment

[0403] P3-P6 tendon stem cells were cultured at a rate of approximately 9 x 10^3 / cm 2The cells were seeded at a density of 100 μg / ml in well plates, culture dishes or culture flasks coated with laminin. The cells were cultured with serum-free medium, serum control medium, commercial MSC serum-free medium from Biological Industries, and commercial MSC serum-free medium from Gibco, respectively. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days. Cell photography and qPCR experiments were performed to observe cell growth and tendon gene expression.

[0404] Experimental results analysis

[0405] Cell morphological observation

[0406] The method is the same as in Example 1. Figure 29 As shown, the cell morphology when the cells are basically full is displayed under a 20X microscope. The results show that the tendon stem cells cultured in SFM (serum-free medium group) grow vigorously, the cells in BI SFM and SCM groups proliferate slowly, the cells in STSFM group grow poorly, basically do not proliferate, and produce a lot of cell secretion impurities. Therefore, the cell proliferation in the SFM group is significantly better than that in the SCM (serum medium group), BI SFM group and ST SFM group, indicating that this serum-free medium is more suitable for tendon stem cell proliferation than the two commercial serum-free culture media and serum control group culture medium, and the commercial Gibco MSC serum-free culture (STSFM) is completely unsuitable for tendon stem cell proliferation in vitro.

[0407] QPCR detection of tendon gene expression

[0408] The method is the same as in Example 1. Figure 30 As shown, compared with the serum-free culture experimental group (SFM), serum culture control group (SCM), and commercial Biological Industries MSC serum-free culture medium (BI SFM), tendon-related genes such as SCX, nestin, THBS4, and TNMD were significantly highly expressed in the SFM (serum-free culture medium) group, while their expression in the BI SFM commercial culture medium was lower and no different from the serum control group. Therefore, this indicates that the serum-free culture medium is more conducive to the maintenance of the tendon phenotype of tendon stem cells than the serum control group culture medium and the commercial BI MSC serum-free culture medium.

[0409] Flow cytometry analysis to detect the expression of stem cell surface markers

[0410] The method was the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive markers for tendon stem cells, while CD34 and CD18 are negative markers for tendon stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. This group also showed a higher degree of tendon stem cell characteristics than the SCM (serum-free medium) control group. This suggests that cells cultured in the serum-free medium (SFM) are more consistent with stem cell characteristics than cells cultured in the SCM (serum-free medium) control group.

[0411] Clonogenic ability assay

[0412] The method was the same as in Example 1. As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in the serum-free medium are more consistent with stem cell characteristics than those cultured in the serum-containing control group.

[0413] Example 6

[0414] In this example, two culture media were prepared: a serum-free culture medium and a serum control culture medium. The cultured cells were tendon stem cells extracted from normal tendon tissue of Scx-GFP mice (Scx-GFP mTSPCs). The following are detailed experimental and detection steps:

[0415] Culture medium preparation

[0416] Serum-free medium (SFM)

[0417] The serum-free medium comprises a basal medium and supplemental components; the basal medium is selected from DMEM / F12 medium, and per 500 mL of DMEM / F12 medium, 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added. The supplemental components are added such that the concentrations of the supplemental components in the serum-free medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 20:20:16:7:2000:4000:2000:10000:10:10000:10. The medium also contains 0.01 mM non-essential amino acids, 0.01 mM L-glutamic acid, 0.01 mM sodium pyruvate, and 0.2X B27 cell culture supplement.

[0418] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0419] FGF-basic 100ng / ml

[0420] PDGF-AA 100ng / ml

[0421] TGF-β1 80ng / ml

[0422] Dexamethasone 90 nM

[0423] Heparin sodium 10 μg / ml

[0424] Vitamin C 20 μg / ml

[0425] Transferrin 10 μg / ml

[0426] Insulin 50 μg / ml

[0427] Progesterone 50ng / ml

[0428] Putrescine 50 μg / ml

[0429] Sodium selenite 50ng / ml

[0430] Non-essential amino acids 0.01 mM

[0431] L-glutamic acid 0.01 mM

[0432] Sodium pyruvate 0.01 mM

[0433] B27 Cell Culture Supplement 0.2X

[0434] Example 6 Biological Activity Experiment

[0435] Cultured cell treatment

[0436] P3-P6 tendon stem cells were cultured at a rate of approximately 5 x 10^4 / cm 2 The cells were seeded at a density of 100 μg / mL in a low-adhesion 6-well plate and cultured with serum-free medium and serum control medium, 2 ml per well, 3 replicates per group, and cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and experiments such as cell photography were performed to observe cell growth and tendon gene expression.

[0437] Experimental results analysis

[0438] Cell morphological observation

[0439] The method is the same as in Example 1. Figure 31As shown, the cell morphology when the cells were basically full was displayed under a 10X microscope. The results showed that the three-dimensional cell spheres formed by Scx-GFP mTSPCs cultured in SFM (serum-free medium group) were larger than those in the SCM control group, indicating that the cell proliferation in the SFM group was significantly better than that in the SCM group. At the same time, the GFP fluorescence intensity of the three-dimensional cell spheres formed by Scx-GFP mTSPCs cultured in SFM (serum-free medium group) was stronger, indicating that the serum-free medium was more suitable for maintaining the SCX phenotype of tendon stem cells than the serum control group medium. At the same time, this result shows that our culture medium also supports three-dimensional culture of cells.

[0440] Flow cytometry analysis to detect the expression of stem cell surface markers

[0441] The method was the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive markers for tendon stem cells, while CD34 and CD18 are negative markers for tendon stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. This group also showed a higher degree of tendon stem cell characteristics than the SCM (serum-free medium) control group. This suggests that cells cultured in the serum-free medium (SFM) are more consistent with stem cell characteristics than cells cultured in the SCM (serum-free medium) control group.

[0442] Clonogenic ability assay

[0443] The method was the same as in Example 1. As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in the serum-free medium are more consistent with stem cell characteristics than those cultured in the serum-containing control group.

[0444] Example 7

[0445] In this example, two culture media were prepared: a serum-free culture medium and a serum control culture medium. The cultured cells were tendon stem cells extracted from normal tendon tissue of Scx-GFP mice (Scx-GFP mTSPCs). The following are detailed experimental and detection steps:

[0446] Culture medium preparation

[0447] Serum-free medium (SFM)

[0448] The serum-free culture medium includes a basal culture medium and additives; the basal culture medium is selected from DMEM / F12 culture medium, and 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added to each 500 mL of DMEM / F12 culture medium, and the additives are added so that the concentrations of the additives in the serum-free culture medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite: epidermal growth factor: CHIR99021=50:50:5:2:5000:100000:5000:5000:5:5000:5:1500:2510. The culture medium also contains 0.01 mM non-essential amino acids, 0.01 mM L-glutamic acid, 0.01 mM sodium pyruvate, and 2X B27 cell culture supplement.

[0449] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0450] FGF2 1ng / ml

[0451] PDGF-BB 1ng / ml

[0452] TGF-β2 0.1ng / ml

[0453] Dexamethasone 0.1 nM

[0454] Heparin calcium 0.1 μg / ml

[0455] Vitamin C 2 μg / ml

[0456] Transferrin 0.1 μg / ml

[0457] Insulin 0.1 μg / ml

[0458] Progesterone 0.1ng / ml

[0459] Putrescine dihydrochloride 0.1 μg / ml

[0460] Sodium selenite 0.1ng / ml

[0461] Epidermal growth factor 30ng / ml

[0462] CHIR99021 0.1μM

[0463] Non-essential amino acids 0.01 mM

[0464] L-glutamic acid 0.01 mM

[0465] Sodium pyruvate 0.01 mM

[0466] B27 Cell Culture Supplement 2X

[0467] Example 7 Biological Activity Experiment

[0468] Cultured cell treatment

[0469] P3-P6 mouse tendon stem cells were cultured at a rate of approximately 1×10^5 / cm 2 The cells were seeded at a density of 100 μg / mL in a low-adhesion 6-well plate and cultured with serum-free medium and serum control medium, 2 ml per well, 3 replicates per group, and cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and experiments such as cell photography were performed to observe cell growth and tendon gene expression.

[0470] Experimental results analysis

[0471] Cell morphological observation

[0472] The method is the same as in Example 1. Figure 32 As shown, the 10X microscope displays the cell morphology when the cells are nearly confluent. The results show that the Scx-GFP mTSPCs cultured in SFM (serum-free medium group) formed more three-dimensional cell spheres than the SCM control group, indicating that the cell proliferation in the SFM group was significantly better than that in the SCM group. Furthermore, the GFP fluorescence intensity of the three-dimensional cell spheres formed by the Scx-GFP mTSPCs cultured in SFM (serum-free medium group) was stronger, indicating that this serum-free medium is more suitable for maintaining the SCX phenotype of tendon stem cells than the serum control medium. This result also shows that our culture medium also supports three-dimensional cell culture.

[0473] Flow cytometry analysis to detect the expression of stem cell surface markers

[0474] The method was the same as in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive markers for tendon stem cells, while CD34 and CD18 are negative markers for tendon stem cells. The results show that the SFM (serum-free medium) experimental group had positive marker expression greater than 95%, while negative marker expression was less than 1%. This group also showed a higher degree of tendon stem cell characteristics than the SCM (serum-free medium) control group. This suggests that cells cultured in the serum-free medium (SFM) are more consistent with stem cell characteristics than cells cultured in the SCM (serum-free medium) control group.

[0475] Clonogenic ability assay

[0476] The method was the same as in Example 1. As shown in Table 3, the results show that the SFM (serum-free medium) experimental group had significantly better colony formation ability than the SCM (serum-containing medium) control group. This indicates that cells cultured in the serum-free medium are more consistent with stem cell characteristics than those cultured in the serum-containing control group.

[0477] Example 8

[0478] In this example, two culture media were prepared: a serum-free culture medium and a serum-free culture medium for stem cells described in Chinese Patent (CN111206017A). The cultured cells were human mesenchymal stem cells. The following are detailed experimental and testing procedures:

[0479] Culture medium preparation

[0480] The serum-free medium comprises a basal medium and supplemental components; the basal medium is selected from DMEM low-glucose medium, and 1 mmol of HEPES is added to every 500 mL of DMEM low-glucose medium. Supplemental components are also added such that the concentrations of the supplemental components in the serum-free medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite: epidermal growth factor: CHIR99021 = 40:15:5:2:3000:20000:50000:100:5:100:5:20:1004. The medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, 1X B27 cell culture supplement, 3 μg / ml vitronectin synthetic peptide, and 3 μg / ml fibronectin synthetic peptide.

[0481] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0482] FGF-basic 40ng / ml

[0483] PDGF-BB 15ng / ml

[0484] TGF-β3 5ng / ml

[0485] Dexamethasone 5 nM

[0486] Heparin sodium 3 μg / ml

[0487] Vitamin C 20 μg / ml

[0488] Transferrin 50 μg / ml

[0489] Insulin 0.1 μg / ml

[0490] Progesterone 5ng / ml

[0491] Putrescine 0.1 μg / ml

[0492] Sodium selenite 5ng / ml

[0493] Epidermal growth factor 20ng / ml

[0494] CHIR99021 2μM

[0495] Non-essential amino acids 0.1 mM

[0496] L-Glutamic acid 1mM

[0497] Sodium pyruvate 0.5 mM

[0498] B27 Cell Culture Supplement 1X

[0499] Vitronectin synthetic peptide 3 μg / ml

[0500] Fibronectin synthetic peptide 3 μg / ml

[0501] Penicillin (10000U) 5mmoL

[0502] Streptomycin (10000U) 5mmoL

[0503] Comparative Example 4

[0504] Stem cell serum-free culture medium described in Chinese patent (CN111206017A):

[0505] The following components were added to each 500 mL of DMEM low glucose basal medium (GIBCO) to a concentration of:

[0506] Recombinant human serum albumin 20 mg / mL

[0507] Recombinant human PDGF-AA 20ng / mL

[0508] Recombinant human PDGF-BB 20ng / mL

[0509] Recombinant human bFGF 5ng / mL

[0510] Recombinant human TGF-β1 5ng / mL

[0511] Recombinant human EGF 20ng / mL

[0512] Recombinant IGF 20 ng / mL

[0513] Recombinant human fibronectin 5 μg / mL

[0514] Heparin 5 μg / mL

[0515] Lipid concentrate 0.1% (v / v)

[0516] Recombinant human insulin 2 μg / mL

[0517] Transferrin 1 μg / mL

[0518] Sodium selenite 1 ng / mL

[0519] Galactose 20 mM

[0520] L-glutamine 292mg / L

[0521] Putrescine 50 μM

[0522] Progesterone 20nM

[0523] Hydrocortisone 100nM

[0524] Vitamin C 200 μM

[0525] Vitamin A 50 μM

[0526] Sodium bicarbonate 20.5 mM

[0527] Penicillin (10000U) 5mmoL

[0528] Streptomycin (10000U) 5mmoL

[0529] Example 8 Biological Activity Experiment

[0530] Cultured cell treatment

[0531] Example 8 Human mesenchymal stem cells from generations P3 to P6 were cultured at a concentration of approximately 5 × 10^3 / cm 2 The cells were inoculated at a density of 100 nm in a well plate or culture dish, cultured with serum-free culture medium of Example 8 and Comparative Example 4, respectively, and cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and experiments such as cell photography were performed to observe cell growth and tendon gene expression.

[0532] Experimental results analysis

[0533] Cell morphological observation

[0534] The method is the same as in Example 1. Figure 33As shown, the cell morphology of cells grown for 3 days was displayed under a 4X microscope. The results showed that under the same conditions and in the same field of view, the number of human mesenchymal stem cells cultured in SFM (serum-free medium group) was significantly greater than that in the serum-free control group of Comparative Example 4, and the cell morphology and size in the SFM group were more uniform, the cells were smaller, the cytoplasm was more transparent and abundant, and the adhesion was better, indicating that the cell proliferation in the SFM group was significantly better than that in the serum-free control group of Comparative Example 4.

[0535] Cell counting and cell proliferation analysis

[0536] The method is the same as in Example 1. Figure 34 As shown, the number of cells obtained in the serum-free control group of Comparative Example 4 is 1, and the cell proliferation multiple of the SFM (serum-free medium) group of Example 8 is 5.17 times that of Comparative Example 4. This result shows that the proliferation rate of human mesenchymal stem cells in the SFM (serum-free medium) group is significantly higher than that of the serum-free control group of Comparative Example 4.

[0537] Cell doubling time analysis

[0538] The method is the same as in Example 1. Figure 35 As shown, the doubling time of human mesenchymal stem cells in the serum-free medium (SFM) of Example 8 was significantly lower than that in the serum-free control group (SFM-Ctrl4) of Comparative Example 4. The doubling time of the SFM-Ctrl4 group was 1.52 times that of the SFM group. The shorter the doubling time, the faster the cell proliferation. That is, the proliferation rate of human mesenchymal stem cells in the serum-free medium (SFM) of the experimental group was significantly faster than that in the serum-free control group (SFM-Ctrl4) of Comparative Example 4. This demonstrates that the serum-free medium composed of the bioactive substances of the present invention is significantly superior to the stem cell serum-free medium described in Chinese Patent No. 202010104684.5 in promoting cell proliferation.

[0539] Flow cytometry analysis to detect the expression of stem cell surface markers

[0540] The method is the same as that in Example 1. As shown in Table 2, CD105, CD90, and CD44 are positive expression markers of human mesenchymal stem cells, and CD34 and CD18 are negative expression markers of human mesenchymal stem cells. The results show that the expression of positive markers in both SFM groups is greater than 95%, the expression of negative markers of human mesenchymal stem cells in SFM (serum-free medium) of Example 8 is less than 1%, and the expression of negative markers of human mesenchymal stem cells in SFM-Ctrl4 (serum-free control group of Comparative Example 4) is greater than 1%, indicating that the serum-free medium composed of the bioactive substance of the present invention has slightly better stem cell characteristics than the stem cell serum-free medium described in Chinese Patent (202010104684.5).

[0541] Clonogenic ability assay

[0542] The method was the same as in Example 1. As shown in Table 3, the results show that the clone-forming ability of the SFM (serum-free medium) experimental group in Example 8 was significantly superior to that of the SFM-Ctrl4 (serum-free control group in Comparative Example 4). This demonstrates that the serum-free medium composed of the bioactive substance of the present invention has superior stem cell characteristics for culturing cells to the serum-free medium described in Chinese Patent No. 202010104684.5.

[0543] In summary, the Chinese patent (CN111206017A) discloses a serum-free culture medium for stem cells and its application. Based on the experimental data provided by the invention patent and the comparative experiments conducted by the present invention, it was found that the proliferation rate of stem cells cultured in the patent was significantly slower than that of the serum-free culture medium prepared by the bioactive substance composition of the present invention ( Figures 32-34 Furthermore, the patent does not provide sufficient evidence to prove that the culture medium disclosed in the patent can be used for primary culture, does not prove the safety of the cultured cells, and does not contain any in vivo animal experiments to prove that the cultured cells can be used for tissue engineering and injury repair. It cannot prove that the cultured cells can be used for clinical cell therapy.

[0544] Example 9

[0545] In this example, a serum-free culture medium was prepared and the cells cultured were human chondrocytes. The following are the detailed experimental and detection steps:

[0546] Culture medium preparation

[0547] The serum-free medium comprises a basal medium and supplemental components; the basal medium is selected from MEM medium, and 1 mmol of HEPES is added to every 500 mL of MEM medium. The supplemental components are also added such that the concentrations of the supplemental components in the serum-free medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite: epidermal growth factor: CHIR99021 = 40:40:3:2:5000:10000:1000:10:1:2:1:20:1004. The medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamic acid, 0.5 mM sodium pyruvate, and 1X B27 cell culture supplement.

[0548] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0549] FGF2 synthetic peptide 40ng / ml

[0550] PDGF-BB synthetic peptide 40ng / ml

[0551] TGF-β3 synthetic peptide 3ng / ml

[0552] Dexamethasone 5 nM

[0553] Heparin sodium 5 μg / ml

[0554] Vitamin C 10 μg / ml

[0555] Transferrin 1 μg / ml

[0556] Insulin 0.01 μg / ml

[0557] Progesterone 1ng / ml

[0558] Putrescine 2ng / ml

[0559] Sodium selenite 1ng / ml

[0560] Epidermal growth factor synthetic peptide 20ng / ml

[0561] CHIR99021 2μM

[0562] Non-essential amino acids 0.1 mM

[0563] L-Glutamic acid 1mM

[0564] Sodium pyruvate 0.5 mM

[0565] B27 Cell Culture Supplement 1X

[0566] Penicillin (10000U) 5mmoL

[0567] Streptomycin (10000U) 5mmoL

[0568] Example 9 Biological Activity Experiment

[0569] Cultured cell treatment

[0570] P3-P6 human chondrocytes were cultured at a rate of approximately 1×10^4 / cm 2 The cells were inoculated at a density of 100 μg / cm2 in a common 10 cm culture dish and cultured in a serum-free culture medium in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and cell photography and other experiments were performed to observe the cell growth.

[0571] Experimental results analysis

[0572] Cell morphological observation

[0573] The method is the same as in Example 1. Figure 36As shown, under a 4X microscope, human chondrocytes cultured in SFM (serum-free medium group) were able to form large three-dimensional cell spheroids, and the spheroid diameter showed an increasing trend, indicating that this serum-free medium is suitable for chondrocyte culture. This result also shows that our culture medium also supports three-dimensional cell culture.

[0574] Cell counting and cell proliferation analysis

[0575] The method was the same as that in Example 1. The cell counting results showed that the number of harvested cells was 4.29×10^6 and the total number of initial cell inoculation was 5.5×10^5. Therefore, the cells proliferated 7.8 times. This result shows that the serum-free culture medium composed of the bioactive substance of the present invention is suitable for the in vitro expansion and culture of chondrocytes.

[0576] Example 10

[0577] In this example, two groups of culture media were prepared, namely a serum-free culture medium and a serum control culture medium. The cells cultured were human skeletal stem cells. The following are detailed experimental and detection steps:

[0578] Culture medium preparation

[0579] The serum-free medium includes a basal medium and additives; the basal medium is selected from BEM medium, and 1 mmol of HEPES is added to every 500 mL of BEM medium, and the additives are added so that the concentrations of the additives in the serum-free medium are: fibroblast growth factor synthetic peptide: platelet-derived growth factor synthetic peptide: transforming growth factor-β synthetic peptide: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite: epidermal growth factor synthetic peptide: CHIR99021 = 30:30:20:5:2000:80000:80000:5000:7:10000:7:20:1004. The culture medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamine, 0.5 mM sodium pyruvate, 1X B27 cell culture supplement, 0.1 μg / ml vitronectin synthetic peptide, 0.1 μg / ml fibronectin synthetic peptide, and 0.1 μg / ml laminin synthetic peptide.

[0580] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0581] FGF-basic 20ng / ml

[0582] FGF1 synthetic peptide 10ng / ml

[0583] PDGF-AA 20ng / ml

[0584] PDGF-AB synthetic peptide 10ng / ml

[0585] TGF-β3 10ng / ml

[0586] TGF-β1 synthetic peptide 10ng / ml

[0587] Dexamethasone 12.5 nM

[0588] Heparin sodium 2 μg / ml

[0589] Vitamin C 80 μg / ml

[0590] Transferrin 80 μg / ml

[0591] Insulin 5 μg / ml

[0592] Progesterone 7ng / ml

[0593] Putrescine 10 μg / ml

[0594] Sodium selenite 7ng / ml

[0595] Epidermal growth factor 20ng / ml

[0596] CHIR99021 2μM

[0597] Non-essential amino acids 0.1 mM

[0598] L-glutamine 1mM

[0599] Sodium pyruvate 0.5 mM

[0600] B27 Cell Culture Supplement 1X

[0601] Vitronectin synthetic peptide 0.1 μg / ml

[0602] Fibronectin synthetic peptide 0.1 μg / ml

[0603] Laminin synthetic peptide 0.1 μg / ml

[0604] Penicillin (10000U) 5mmoL

[0605] Streptomycin (10000U) 5mmoL

[0606] Example 10 Biological Activity Experiment

[0607] Cultured cell treatment

[0608] P3-P6 generation human skeletal stem cells were cultured at a rate of approximately 1X10^4 / cm 2The cells were seeded at a density of 500 mg / ml RGD (Arg-Gly-Asp) peptide and 200 mg / ml KRSR (Lys-Arg-Ser-Arg) peptide in a 10 cm culture dish, and cultured in serum-free medium in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 2-3 days, and cell photography and other experiments were performed to observe cell growth.

[0609] Experimental results analysis

[0610] Cell morphological observation

[0611] The method is the same as in Example 1. Figure 37 As shown, observation under a 20X microscope showed that human skeletal stem cells cultured in SFM (serum-free medium) grew vigorously, with significantly better cell proliferation than those in the SCM (serum-free medium) group. Furthermore, human skeletal stem cells cultured in SFM (serum-free medium) had more uniform cell morphology and size, with translucent and abundant cytoplasm and good adhesion to the wall. These results indicate that the composition composed of the bioactive substances of the present invention is suitable for the in vitro expansion and culture of skeletal stem cells.

[0612] Clonogenic ability assay

[0613] The method was the same as in Example 1. As shown in Table 3, the results show that the clone-forming ability of the serum-free medium (SFM) experimental group in Example 10 was significantly superior to that of the serum-free serum control group. This indicates that the stem cell characteristics of skeletal stem cells cultured in the serum-free medium composed of the bioactive substances of the present invention are superior to those of skeletal stem cells cultured in the serum control group.

[0614] Comparative Example 5

[0615] Medium containing only B27 Cell Culture Supplement (B27)

[0616] The culture medium containing only B27 cell culture additives is selected from DMEM / F12 medium, and 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added per 500 mL of DMEM / F12 medium. The supplemental components are then added to a concentration of:

[0617] Non-essential amino acids 0.1 mM

[0618] L-glutamate 2mM

[0619] Sodium pyruvate 1 mM

[0620] B27 Cell Culture Supplement 1X

[0621] Comparative Example 5 Biological Activity Experiment

[0622] Cultured cell treatment

[0623] P3-P6 tendon stem cells were cultured at a rate of approximately 9 x 10^3 / cm 2 The cells were seeded at a density of 100 μg / ml in a 12-well plate coated with 20 mg / ml type I collagen. The cells were cultured with serum-free medium, B27 control group medium, and serum control group medium, respectively, with 1 ml per well and 3 replicates per group. The plates were cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 3 days. The culture was continued for 5 days, and cell photography was performed to observe cell growth.

[0624] Experimental results analysis

[0625] Cell morphological observation

[0626] The method is the same as in Example 1. Figure 38 As shown, the growth of cells after 5 days of culture was displayed under a 20X microscope. The results showed that the cells in the B27 group basically did not proliferate, indicating that the cell culture additives alone could not effectively proliferate the cells.

[0627] Comparative Example 6

[0628] In this comparative example, a serum-free culture medium was prepared according to a paper published in Chinese Journal of Experimental Surgery, 2014, 31(2): 395-398. The cells cultured were human tendon stem cells (hTSPCs) extracted from normal human tendon tissue. The following are the detailed experimental and testing procedures:

[0629] Culture medium preparation

[0630] Comparative serum-free culture medium

[0631] The serum-free culture medium includes a basal culture medium and additional components; the basal culture medium is selected from α-MEM culture medium, and 25 μg IGF-1 and 5 TGF-β3 are added to each 500 mL of α-MEM culture medium, that is, 50 μg / L IGF-1 and 10 μg / L TGF-β3.

[0632] Comparative Example 6 Biological Activity Experiment

[0633] Cultured cell treatment

[0634] The primary culture of the cultured cells was carried out in serum medium, and after passage, the cells were cultured in the comparative medium. Other methods were the same as those in Example 1.

[0635] Experimental results analysis

[0636] Cell morphological observation

[0637] The method is the same as in Example 1. Figure 39As shown, the growth of cells after 5 days of culture was shown under a 4X microscope. The results showed that the tendon stem cells cultured in the serum-free medium of the excessive concentration control example proliferated slowly. This result shows that the culture medium in this paper cannot maintain cell proliferation. At the same time, because the cultured cells are derived from serum medium, there is serum residue, and the safety score is 0. Therefore, this comparative example shows that the cells obtained by primary culture in serum medium and subsequent subculture in serum-free culture cannot meet the cell quantity and quality requirements for clinical treatment.

[0638] Comparative Example 7

[0639] In this comparative example, a serum-free culture medium was prepared with a concentration far exceeding the existing range of bioactive substances, and the cultured cells were tendon stem cells (hTSPCs) extracted from normal human tendon tissue. The following are the detailed experimental and testing steps:

[0640] Culture medium preparation

[0641] Comparative serum-free culture medium

[0642] The serum-free medium comprises a basal medium and supplemental components; the basal medium is selected from DMEM / F12 medium, and per 500 mL of DMEM / F12 medium, 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added. The supplemental components are added such that the concentrations of the supplemental components in the serum-free medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 203:120:100:120:13,000:200,000:400,000:100,000:100:100,000:100. The medium also contains 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 1 mM sodium pyruvate, and 6X B27 cell culture supplement.

[0643] FGF-basic 203ng / ml

[0644] PDGF-AA 120ng / ml

[0645] TGF-β1 100ng / ml

[0646] Dexamethasone 300 nM

[0647] Heparin sodium 13 μg / ml

[0648] Vitamin C 200 μg / ml

[0649] Transferrin 400 μg / ml

[0650] Insulin 100 μg / ml

[0651] Progesterone 100ng / ml

[0652] Putrescine 100 μg / ml

[0653] Sodium selenite 100ng / ml

[0654] Non-essential amino acids 0.1 mM

[0655] L-glutamate 2mM

[0656] Sodium pyruvate 1 mM

[0657] B27 Cell Culture Supplement 6X

[0658] Comparative Example 6 Biological Activity Experiment

[0659] Cultured cell treatment

[0660] P3-P6 tendon stem cells were cultured at a rate of approximately 9 x 10^3 / cm 2 The cells were seeded at a density of 100 μg / ml in a 12-well plate coated with 20 mg / ml type I collagen. The cells were cultured with serum-free medium, B27 control group medium, and serum control group medium, respectively, with 1 ml per well and 3 replicates per group. The plates were cultured in a cell culture incubator at 37°C and 5% CO2. The medium was changed every 3 days. The culture was continued for 5 days, and cell photography was performed to observe cell growth.

[0661] Experimental results analysis

[0662] Cell morphological observation

[0663] The method is the same as Example 40. Figure 39 As shown, the growth of cells after 5 days of culture was displayed under a 4X microscope. The results showed that the tendon stem cells cultured in the serum-free culture medium with excessive concentration did not proliferate at all, and even died. This result shows that the concentration range of each component of the serum-free culture medium developed by the present invention is unique.

[0664] Comparative Example 8

[0665] The culture medium prepared in this comparative example did not contain fibroblast growth factor, and other conditions were the same as those in Example 2.

[0666] Experimental results analysis

[0667] Cell morphological observation

[0668] The method is the same as in Example 1. Figure 41As shown, the growth of cells after 5 days of culture was shown under a 4X microscope. The results showed that the tendon stem cells cultured in the serum-free culture medium of the comparative example basically did not proliferate. This result shows that the components of the bioactive composition and serum-free culture medium developed by the present invention are necessary for them to function, indicating the uniqueness of each component of the bioactive substance composition of the present invention.

[0669] Comparative Example 9

[0670] The culture medium prepared in this comparative example did not contain transforming growth factor-β, but was added with 5 ng / ml of epidermal growth factor. Other conditions were the same as those in Example 2.

[0671] Experimental results analysis

[0672] The cell culture effect of this comparative example shows that cell proliferation slowed down and the ability to maintain the phenotype decreased significantly, indicating that each component of the bioactive composition developed by the present invention is necessary for its function and cannot be replaced by other components, which illustrates the uniqueness of each component of the bioactive substance composition of the present invention.

[0673] Example 11

[0674] In this example, a serum-free culture medium was prepared to culture tendon stem cells (Scx-GFP mTSPCs) extracted from normal tendon tissue of Scx-GFP mice. The following are the detailed experimental and detection steps:

[0675] Culture medium preparation

[0676] The serum-free medium comprises a basal medium and supplemental components; the basal medium is selected from DMEM / F12 medium, and per 500 mL of DMEM / F12 medium, 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added. The supplemental components are added such that the concentrations of the supplemental components in the serum-free medium are: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 5:5:2:1:4,000:90,000:200,000:15,000:15:15,000:15. The medium also contains 0.1 mM non-essential amino acids, 1 mM L-glutamine, 0.5 mM sodium pyruvate, and 1X B27 cell culture supplement.

[0677] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0678] FGF-basic 5ng / ml

[0679] PDGF-AA 5ng / ml

[0680] TGF-β3 2ng / ml

[0681] Dexamethasone 2.5 nM

[0682] Heparin sodium 4 μg / ml

[0683] Vitamin C 90 μg / ml

[0684] Transferrin 200 μg / ml

[0685] Insulin 15 μg / ml

[0686] Progesterone 15ng / ml

[0687] Putrescine 15 μg / ml

[0688] Sodium selenite 15ng / ml

[0689] Non-essential amino acids 0.1 mM

[0690] L-glutamine 1mM

[0691] Sodium pyruvate 0.5 mM

[0692] B27 Cell Culture Supplement 1X

[0693] Penicillin (10000U) 5mmoL

[0694] Streptomycin (10000U) 5mmoL

[0695] Example 11 Biological Activity Experiment

[0696] The cultured cells were treated as in Example 6.

[0697] Experimental results analysis

[0698] The cell culture effect of this example is similar to that of Example 6, indicating that the culture medium of the present invention supports the culture of mouse tendon stem cells and also supports cell suspension culture.

[0699] Example 12

[0700] In this example, a serum-free culture medium was prepared, and the cells cultured were ligament stem cells isolated and cultured from human ligament tissue. The following are detailed experimental and testing steps:

[0701] Culture medium preparation

[0702] Serum-free medium (SFM)

[0703] The serum-free medium comprises a basal medium and supplemental components. The basal medium is selected from F10 medium, and per 500 mL of F10 medium, 5 mmol of HEPES, 10,000 U of penicillin, and 10,000 U of streptomycin are added. The supplemental components are added such that the concentration ratio of the components in the serum-free medium is: fibroblast growth factor: platelet-derived growth factor: transforming growth factor-β: glucocorticoid: heparin or its salt: vitamin C or its derivative: transferrin: insulin: progesterone: putrescine or its salt: selenite = 26:15:30:8:500:1000:75000:3000:4:7:4. The medium also contains 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 1 mM sodium pyruvate, 1X B27 cell culture supplement, and 2 μg / ml vitronectin.

[0704] Furthermore, the concentrations of the components in the serum-free medium are as follows:

[0705] FGF-basic 26ng / ml

[0706] PDGF-BB 15ng / ml

[0707] TGF-β2 30ng / ml

[0708] Dexamethasone 20 nM

[0709] Heparin sodium 0.5 μg / ml

[0710] Vitamin C 1 μg / ml

[0711] Transferrin 75 μg / ml

[0712] Insulin 3 μg / ml

[0713] Progesterone 4ng / ml

[0714] Putrescine 7 μg / ml

[0715] Sodium selenite 4ng / ml

[0716] Non-essential amino acids 0.1 mM

[0717] L-glutamate 2mM

[0718] Sodium pyruvate 1 mM

[0719] B27 Cell Culture Supplement 1X

[0720] Vitronectin 2ug / ml

[0721] Penicillin (10000U) 5mmoL

[0722] Streptomycin (10000U) 5mmoL

[0723] Example 12 Biological Activity Experiment

[0724] Cultured cells were treated as in Example 2

[0725] Experimental results analysis

[0726] The cell culture effect of this example is similar to that of Example 2, indicating that the culture medium of the present invention supports the culture of human ligament stem cells.

[0727] Table 1 Cell viability in different examples and comparative examples

[0728]

[0729] Table 2 Flow cytometry results show the expression of CD markers on the cell surface in different examples and comparative examples

[0730]

[0731] Note: \ indicates that cells did not proliferate in this culture medium and the cell quantity was too small to be detected.

[0732] Table 3 CFU test results show the tendon clone-forming ability of P3 generation cells in different embodiments and comparative examples.

[0733]

[0734] Table 4 Percentage of Nestin+ cells cultured in each embodiment and comparative example

[0735]

[0736] Table 5 Scores of cells cultured in various examples and comparative examples

[0737]

[0738] Comparison of each embodiment with comparative example 1 shows that the serum-free culture medium and / or composition described in the present application is a completely serum-free culture medium, which can completely replace the serum-containing culture medium, realize the primary culture and subculture of cells, and realize the rapid proliferation of cells in vitro and the maintenance or improvement of phenotype.

[0739] Comparison between Example 1 and Comparative Example 1 shows that the cells obtained by culturing with the serum-free culture medium and / or composition described in the present application proliferate rapidly and have a significantly improved phenotype. These cells with excellent proliferation and phenotype cultured in vitro are still highly functional when transplanted into the body and can repair tissue damage. The tissue damage repair effect is significantly better than that of the cells obtained by culturing with the serum-containing culture medium control group in Comparative Example 1. This shows that the cells obtained by culturing with the serum-free culture medium and / or composition described in the present application are highly functional and can quickly participate in the regeneration of the damaged area and repair tissue damage when implanted in the body. The tissue or organ damage is selected from the group consisting of musculoskeletal system tissue or organ damage; preferably, the musculoskeletal system tissue or organ damage is selected from at least one of tendon and / or ligament damage, cartilage damage, bone damage, muscle damage, skin damage, and vascular damage.

[0740] The cells cultured in Examples 6, 7, and 11 were of animal origin, while the cells cultured in the other Examples were of human origin, demonstrating that the serum-free culture media and / or compositions described herein are capable of culturing cells of either human or animal origin in vitro. Furthermore, Examples 6 and 7 employed three-dimensional suspension culture, while the other Examples employed adherent culture, demonstrating that the serum-free culture media and / or compositions described herein are capable of culturing cells in both suspension and adherent conditions.

[0741] The comparison results of Example 5 with Comparative Examples 2 and 3 (common commercial serum-free culture media for MSC) show that the proliferation ability, stem cell phenotype and tendon phenotype of cells cultured with the serum-free culture medium and / or composition described in the present application are significantly better than those of Comparative Examples 2 and 3, indicating that the serum-free culture medium and / or composition described in the present application is more suitable for the in vitro culture and phenotypic maintenance / improvement of cells.

[0742] The experimental results of Comparative Example 7 demonstrate that the bioactive material and serum-free medium developed by the present invention have unique working concentration ranges for each component, and that they are ineffective beyond these concentration ranges. The experimental results of Comparative Examples 8 and 9 demonstrate that each component of the bioactive composition and serum-free medium developed by the present invention is essential for their function and cannot be replaced. This demonstrates the unique composition and concentration of each component of the bioactive material composition developed by the present invention.

[0743] In summary, the present application is verified by adjusting the basal culture medium, bioactive substance composition, additives and their contents of the serum-free culture medium used in the in vitro expansion process of stem cells, and / or the bioactive substance composition, additives and their contents of the composition, using different cell cultures to obtain a serum-free culture medium and / or composition that can improve the proliferation ability and phenotype of cells in vitro. The cells are selected from any one or more of tendon and / or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells; preferably, the single score of each single item in the cell common characteristics and cell-specific phenotype of the cells cultured in the serum-free culture medium and / or composition reaches the respective passing line and the total score reaches 60 points or more; preferably, the single score of each single item in the cell common characteristics and cell-specific phenotype of the cells cultured in the serum-free culture medium reaches the respective passing line and the total score reaches 80 points or more; preferably, the single score of each single item in the cell common characteristics and cell-specific phenotype of the cells cultured in the serum-free culture medium reaches the respective passing line and the total score reaches 90 points or more.

[0744] As can be seen from the various embodiments and comparative examples, the serum-free culture medium and / or composition described herein can achieve in vitro expansion and phenotypic maintenance of cells such as tendon and / or ligament-derived cells, mesenchymal stem cells, meniscus stem cells, chondrocytes, skeletal stem cells, and muscle stem cells, and these cells are the main cell members of the musculoskeletal system and play an important role in the formation and function of musculoskeletal tissue. For example, tendon is composed of two major components: cells derived from tendon and collagen matrix. Cells derived from tendon are the only cell members of tendon tissue and are the main players in the development, homeostasis maintenance, and injury repair of tendon tissue. Moreover, the collagen matrix in tendon is also formed by secretion of tendon-derived cells. The serum-free culture medium and / or composition described herein achieves in vitro proliferation and phenotypic maintenance of tendon stem cells, and therefore, can also achieve in vitro culture and functional maintenance of tendon tissue. Thus, the serum-free culture medium and / or composition described herein can be used for in vitro culture and functional maintenance of musculoskeletal system-derived tissue. Preferably, the musculoskeletal system tissue is selected from tendon tissue, ligament tissue, meniscus tissue, cartilage tissue, adipose tissue, and muscle tissue.

[0745] At the same time, it can be seen from the various embodiments that serum-free culture media and compositions can promote the in vitro expansion of cells and the maintenance or improvement of phenotypes, and the core components of these two substances are bioactive material compositions, which also shows that the bioactive material compositions have super strong activity and can be used to prepare cell culture reagents. Bioactive material compositions, serum-free culture media, and compositions use various components to simulate the complex microenvironment of cell growth in vivo to achieve in vitro cell culture. During tissue damage, the microenvironment of the tissue damage site is damaged, resulting in slow tissue regeneration / repair. Injecting and / or applying the bioactive material composition and / or serum-free culture media and / or composition described in this application to the damage site can quickly reshape the microenvironment of the damage site in the body, accelerating damage repair and tissue regeneration. Therefore, the bioactive material composition and / or serum-free culture media and / or composition described in this application have uses in the preparation of drugs for the treatment of tissue and / or organ damage. The tissue or organ damage is selected from musculoskeletal system tissue or organ damage; preferably, the musculoskeletal system tissue or organ damage is selected from at least one of tendon and / or ligament damage, cartilage damage, bone damage, muscle damage, skin damage, and vascular damage.

Claims

1. A bioactive substance composition for serum-free in vitro culture of tendon stem cells or ligament stem cells, characterized in that: The bioactive substance composition is composed of the following substances: fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivative, transferrin, insulin, progesterone, putrescine or its salt, and selenite, wherein the concentration range of each component is: Fibroblast growth factor 1-100 ng / ml, platelet-derived growth factor 1-100 ng / ml, transforming growth factor-β 0.1-80 ng / ml, glucocorticoid 0.1-90 nM, heparin or its salt 100-10,000 ng / ml, vitamin C or its derivative 1,000-100,000 ng / ml, transferrin 100-300,000 ng / ml, insulin 100-50,000 ng / ml, progesterone 0.1-50 ng / ml, putrescine or its salt 100-50,000 ng / ml, selenite 0.1-50 ng / ml; The fibroblast growth factor is selected from any one or more of FGF-basic, FGF4, and FGF18; The platelet-derived growth factor is selected from any one or more of PDGF-AA, PDGF-AB, and PDGF-BB; The transforming growth factor-β is selected from any one or more of TGF-β1, TGF-β2, and TGF-β3; The glucocorticoid is selected from any one or more of dexamethasone or its salt, dexamethasone solvate, hydrocortisone or its salt, and hydrocortisone solvate.

2. The bioactive substance composition according to claim 1, characterized in that: The concentration ranges of the fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, and selenite are respectively: 20-50 ng / ml, 15-30 ng / ml, 5-40 ng / ml, 5-29 nM, 500-3000 ng / ml, 1000-100000 ng / ml, 4000-300000ng / ml, 100-25000 ng / ml, 4-25 ng / ml, 100-25000 ng / ml, and 4-25 ng / ml.

3. The bioactive substance composition according to claim 1, characterized in that: The concentration ranges of the fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, and selenite are respectively: 20-30 ng / ml, 20-30 ng / ml, 5-12 ng / ml, 5-10 nM, 2000-3000 ng / ml, 50000-80000 ng / ml, 4000-150000 ng / ml, 100-6000 ng / ml, 4-8 ng / ml, 100-14000 ng / ml, and 4-8 ng / ml.

4. The bioactive substance composition according to claim 1, wherein: The heparin or its salt is selected from any one or more of heparin, heparin sodium, and heparin calcium.

5. The bioactive substance composition according to claim 1, characterized in that: The vitamin C or its derivative is selected from any one or more of vitamin C, ascorbyl glucoside, ethyl ascorbic acid, 3-o-ethyl ascorbic acid, ascorbic acid magnesium phosphate, ascorbic acid sodium phosphate, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, ascorbic acid tetraisopalmitate, ascorbyl palmitate, ascorbic acid-2-phosphate-6-palmitate, esterified vitamin C, and other solvates of ascorbic acid.

6. The bioactive substance composition according to claim 1, characterized in that: The putrescine or its salt is selected from any one or more of putrescine and putrescine dihydrochloride.

7. The bioactive substance composition according to claim 1, characterized in that: The selenite is water-soluble selenite.

8. The bioactive substance composition according to claim 1, characterized in that: The selenite is sodium selenite.

9. A method for preparing the bioactive substance composition according to claim 1, characterized in that: The preparation of the bioactive substance composition comprises the steps of mixing fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, and selenite, and the order of adding the components is not particular.

10. The preparation method according to claim 9, characterized in that: The mass-volume concentration ranges of the fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, and selenite are: 20-50 ng / ml, 15-30 ng / ml, 5-40 ng / ml, 5-29 nM, 500-3000 ng / ml, 1000-100000 ng / ml, 4000-300000 ng / ml, 100-25000 ng / ml, 4-25 ng / ml, 100-25000 ng / ml, and 4-25 ng / ml, respectively.

11. The preparation method according to claim 9, characterized in that: The mass-volume concentration ranges of the fibroblast growth factor, platelet-derived growth factor, transforming growth factor-β, glucocorticoid, heparin or its salt, vitamin C or its derivatives, transferrin, insulin, progesterone, putrescine or its salt, and selenite are: 20-30 ng / ml, 20-30 ng / ml, 5-12 ng / ml, 5-10 nM, 2000-3000 ng / ml, 50000-80000 ng / ml, 4000-150000 ng / ml, 100-6000 ng / ml, 4-8 ng / ml, 100-14000 ng / ml, and 4-8 ng / ml, respectively.

12. The preparation method according to claim 9, characterized in that: The mixing temperature is 0-37°C.

13. A serum-free culture medium for tendon stem cells or ligament stem cells, characterized in that: The serum-free culture medium comprises a basal culture medium and an additive, wherein the additive is the bioactive substance composition according to any one of claims 1 to 8 or the bioactive substance composition prepared by the preparation method according to any one of claims 9 to 12.

14. The serum-free culture medium according to claim 13, wherein: The serum-free culture medium is a complete serum-free culture medium.

15. The serum-free culture medium according to claim 13, wherein: The serum-free culture medium cultured primary or passaged tendon stem cells or ligament stem cells.

16. The serum-free culture medium according to claim 13, wherein: The serum-free culture medium is used to maintain the proliferation and phenotype of tendon stem cells or ligament stem cells, or to enhance the proliferation and phenotype of tendon stem cells or ligament stem cells.

17. The serum-free culture medium according to claim 13, wherein: The scores of each single item in the common characteristics of tendon stem cells or ligament stem cells cultured in the serum-free medium and the cell-specific phenotypes of tendon stem cells or ligament stem cells reach their respective passing lines and the total cell score reaches more than 60 points.

18. The serum-free culture medium according to claim 13, wherein: The basal culture medium is selected from any one or more of DMEM low glucose medium, DMEM high glucose medium, DMEM / F12 medium, F12 medium, F10 medium, MEM medium, BEM medium, RPMI 1640 medium, Media 199 medium, IMDM medium, mTesR medium, and E8 medium.

19. A method for preparing the serum-free culture medium according to claim 13, characterized in that: The preparation method comprises the step of mixing a basal culture medium and an additive, wherein the additive is the bioactive substance composition according to any one of claims 1 to 8.

20. The method for preparing a serum-free culture medium according to claim 19, wherein: The mixing temperature is 0-37°C.

21. A composition for culturing tendon stem cells or ligament stem cells, characterized in that: The composition comprises an active component and at least one additive, wherein the active component is selected from at least one of the bioactive substance composition according to any one of claims 1 to 8, the bioactive substance composition obtained by the preparation method according to any one of claims 9 to 12, the serum-free culture medium according to any one of claims 13 to 18, and the serum-free culture medium obtained by the preparation method according to claim 19 or 20.

22. The composition according to claim 21, characterized in that: The scores of each single item in the common characteristics of tendon stem cells or ligament stem cells cultured by the composition and the unique phenotypes of tendon stem cells or ligament stem cells reach the respective passing lines and the total cell score reaches more than 60 points.

23. The composition according to claim 21, characterized in that: The scores of each single item in the common characteristics of tendon stem cells or ligament stem cells cultured by the composition and the unique phenotypes of tendon stem cells or ligament stem cells reach the respective passing lines and the total cell score reaches more than 80 points.

24. The composition according to claim 21, characterized in that: The scores of each single item in the common characteristics of tendon stem cells or ligament stem cells cultured by the composition and the unique phenotypes of tendon stem cells or ligament stem cells reach the respective passing lines and the total cell score reaches more than 90 points.

25. The composition according to claim 21, characterized in that: The scores of each single item in the common characteristics of tendon stem cells or ligament stem cells cultured by the composition and the unique phenotypes of tendon stem cells or ligament stem cells reach their respective passing lines and the total cell score reaches 100 points.

26. The composition according to claim 21, characterized in that: The additive is selected from any one or more of the following components: B27 cell culture additive, N2 cell culture additive, chemically defined lipid concentrate, amino acids, 4-hydroxyethylpiperazineethanesulfonic acid, sodium pyruvate, growth factors, GSK3 inhibitors, laminin, fibronectin, vitronectin, collagen, gelatin, and antibiotics.

27. The composition according to claim 26, characterized in that: Calculated based on the total volume of the composition, the final concentration of any one of the B27 cell culture supplement, the N2 cell culture supplement, and the chemically defined lipid concentrate is 0.1-5×.

28. The composition according to claim 26, characterized in that: The final concentration of any one of the B27 cell culture supplement, the N2 cell culture supplement, and the chemically defined lipid concentrate is 0.5-2× based on the total volume of the composition.

29. The composition according to claim 26, characterized in that: The growth factor is selected from any one or more of vascular endothelial growth factor, vascular endothelial growth factor synthetic peptide, epidermal growth factor, epidermal growth factor synthetic peptide, insulin-like growth factor, insulin-like growth factor synthetic peptide, nerve growth factor, nerve growth factor synthetic peptide, colony stimulating factor, colony stimulating factor synthetic peptide, growth hormone release inhibitory factor, and growth hormone release inhibitory factor synthetic peptide.

30. The composition according to claim 26, wherein: The mass-volume concentration of the growth factor is 1-100 ng / ml.

31. The composition according to claim 26, wherein: The mass-volume concentration of the growth factor is 1-50 ng / ml.

32. The composition according to claim 26, wherein: The mass-volume concentration of the growth factor is 5-40 ng / ml.

33. The composition according to claim 26, wherein: The GSK3 inhibitor is selected from CHIR99021.

34. The composition according to claim 26, wherein: The molar concentration of the GSK3 inhibitor is 0.1-10 μM.

35. The composition according to claim 26, characterized in that: The molar concentration of the GSK3 inhibitor is 0.1-5 μM.

36. The composition according to claim 26, wherein: The amino acid is selected from any one or more of L-glutamic acid and L-glutamine.

37. The composition according to claim 26, characterized in that: The molar concentration of the amino acid is 0.01-4 mM.

38. The composition according to claim 26, wherein: The mass-volume concentration of the sodium pyruvate is 0.01-2 mM.

39. The composition according to claim 26, characterized in that: The molar concentration of the 4-hydroxyethylpiperazineethanesulfonic acid is 1-20 mM.

40. The composition according to claim 26, characterized in that: The laminin mass-volume concentration range is 0.1-100 μg / ml.

41. The composition according to claim 26, wherein: The mass-volume concentration of the fibronectin is in the range of 0.1-200 μg / ml.

42. The composition according to claim 26, wherein: The vitronectin mass-volume concentration range is 0.1-100 μg / ml.

43. The composition according to claim 26, characterized in that: The collagen mass-volume concentration range is 0.1-100 mg / ml.

44. The composition according to claim 26, characterized in that: The gelatin mass-volume concentration is 0.1-100 mg / ml.

45. The composition according to claim 26, characterized in that: The antibiotic is selected from any one or more of penicillin, streptomycin and gentamicin.

46. The composition according to claim 26, characterized in that: The mass-volume concentration range of the antibiotic is 50-100 μg / mL.

47. A method for preparing the composition of claim 21, characterized in that: The preparation method comprises the step of mixing an active component and at least one additive, wherein the active component is selected from at least one of the bioactive substance composition described in any one of claims 1-8, the bioactive substance composition obtained by the preparation method described in any one of claims 9-12, the serum-free culture medium described in any one of claims 13-18, and the serum-free culture medium obtained by the preparation method described in claim 19 or 20; and the additive is selected from one or more of the additives described in claim 26.

48. The method for preparing the composition according to claim 47, wherein: The mixing temperature is 0-37°C.

49. Use of the bioactive substance composition according to any one of claims 1 to 8, the bioactive substance composition obtained by the preparation method according to any one of claims 9 to 12, the serum-free culture medium according to any one of claims 13 to 18, the serum-free culture medium obtained by the preparation method according to claim 19 or 20, the composition according to any one of claims 21 to 46, or the composition obtained by the preparation method according to claims 47 to 48, characterized in that: The use is selected from the culture of tendon stem cells or ligament stem cells.

50. A method for culturing tendon stem cells or ligament stem cells, characterized in that: The culture method comprises the step of contacting the cells with a serum-free medium and / or a composition, wherein the serum-free medium is the serum-free medium described in any one of claims 13-18, or the serum-free medium prepared by the preparation method described in claim 19 or 20, and the composition is the composition described in any one of claims 21-46, or the composition prepared by the preparation method described in claims 47-48.

51. The method for culturing tendon stem cells or ligament stem cells according to claim 50, wherein: The culture method is selected from the group consisting of suspension culture and adherent culture.

52. The method for culturing tendon stem cells or ligament stem cells according to claim 51, wherein: The adherent culture method is selected from the group consisting of a method of coating a culture plate with an adhesion-promoting substance and a method of adding an adhesion-promoting substance to a culture medium.

53. The method for culturing tendon stem cells or ligament stem cells according to claim 52, wherein: The adhesion-promoting substance culture plate coating method comprises the following steps: 1) treating a culture carrier with an adhesion-promoting substance; 2) inoculating cells and / or tissues into the culture carrier treated in step 1); and 3) adding the serum-free culture medium and / or composition for culture.

54. The method for culturing tendon stem cells or ligament stem cells according to claim 53, wherein: In step 1), the culture carrier is selected from at least one of a well plate, a culture dish, a culture bottle, a microarray, and a bioactive material.

55. The method for culturing tendon stem cells or ligament stem cells according to claim 52, wherein: The method of adding the adhesion promoting substance to the culture medium comprises the following steps: 1) inoculating cells into a culture carrier; 2) directly adding the adhesion promoting substance to the serum-free culture medium and / or composition, and then adding it to the culture carrier in step 1) for cell culture.

56. The method for culturing tendon stem cells or ligament stem cells according to claim 55, wherein: The culture carrier is selected from at least one of a well plate, a culture dish, a culture bottle, a microarray, and a bioactive material.

57. The method for culturing tendon stem cells or ligament stem cells according to claim 55, wherein: The adhesion promoting substance is selected from any one or more of laminin, fibronectin, vitronectin, collagen, gelatin, and synthetic peptides of adhesion promoting substances.

58. The method for culturing tendon stem cells or ligament stem cells according to claim 57, wherein: The synthetic peptides promoting adhesion are selected from any one or more of laminin synthetic peptides, fibronectin synthetic peptides, vitronectin synthetic peptides, Arg-Gly-Asp peptides, and Lys-Arg-Ser-Arg peptides.

59. The method for culturing tendon stem cells or ligament stem cells according to claim 57, wherein: The laminin concentration ranges from 0.1 to 100 μg / ml, and / or the fibronectin concentration ranges from 0.1 to 200 μg / ml, and / or the vitronectin concentration ranges from 0.1 to 100 μg / ml, and / or the collagen concentration ranges from 0.1 to 100 mg / ml, and / or the gelatin concentration ranges from 0.1 to 100 mg / ml.

60. The method for culturing tendon stem cells or ligament stem cells according to claim 58, wherein: The concentration of the laminin synthetic peptide is in the range of 0.1-100 μg / ml, and / or the concentration of the fibronectin synthetic peptide is in the range of 0.1-200 μg / ml, and / or the vitronectin synthetic peptide is in the range of 0.1-100 μg / ml, and / or the concentration of the Arg-Gly-Asp peptide is in the range of 50-1000 mg / ml, and / or the concentration of the Lys-Arg-Ser-Arg peptide is in the range of 50-1000 mg / ml.

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