Chemically defined mesenchymal stem cell culture medium and its two-dimensional cell culture applications

Abstract

The application discloses a mesenchymal stem cell defined medium, which is composed of a basic medium and an additive component. The additive component is selected from one or more of the following: a binding protein, an adhesion factor, a hormone, a vitamin, an organic acid, an amino acid, a sugar, a growth factor, a trace element-containing substance, a nucleoside, a biological amine, aspirin and a soybean trypsin inhibitor. The medium has a clear chemical composition, does not contain proteolytic components and serum replacement factors, and avoids the instability and pathogenic pollution risks caused by serum or blood replacement components, thereby solving the safety problem of mesenchymal stem cells in clinical application. The medium is added with an anti-aging component of aspirin, which promotes the efficient expansion of mesenchymal stem cells, reduces the cell diameter and maintains the high activity state of the cells.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a chemically defined culture medium for mesenchymal stem cells and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells with the ability to self-renew and differentiate into various cell types, including osteoblasts, chondrocytes, and adipocytes. They can interact with many cell types, modulating the innate and adaptive immune systems through cell-cell contact or the secretion of soluble factors, regulating the tissue microenvironment, and promoting tissue regeneration. They are among the most widely used and experimentally studied adult pluripotent stem cells in clinical treatment. MSCs can be obtained from various tissues, such as bone marrow, adipose tissue, umbilical cord, skin, hair follicles, dental pulp, and peripheral blood. However, the proportion of MSCs in all these tissues is very low; for example, human bone marrow MSCs account for only 0.001%-0.01% of total bone marrow monocytes, and the cell number decreases with donor age. However, clinical applications of MSCs require large numbers of cells. To meet the needs of biological research and clinical applications, researchers are seeking scientific methods for large-scale in vitro expansion of MSCs. The goal is to use effective and safe methods to maintain the key characteristics of MSCs in a short period of time, avoiding cell aging and contamination, while simultaneously achieving in vitro expansion.

[0003] To facilitate the transition of mesenchymal stem cell (MSC) biology from basic research to clinical applications, we need to further develop reagents and equipment for in vitro MSC expansion, with the most crucial aspect being the development and refinement of chemically defined culture media. Compared to MSC media containing serum-replacement factors, chemically defined media offer standardized formulations, mitigating issues such as contamination, cost, batch-to-batch variability, and ethical controversies. Chemically defined media primarily consist of a basal medium and added factors. The basal medium contains glucose, amino acids, vitamins, and inorganic salts essential for maintaining cell growth and metabolism. Added factors include hormones, growth factors, binding proteins, adhesion factors, trace elements, amino acids, nucleotides, lipids, vitamins, sugars, polyamines, organic acids, reducing agents, and buffer systems. Currently available serum-free MSC media are expensive, but their culture results are less than ideal, with lower stem cell proliferation rates and lower expression of stem cell surface markers compared to serum-containing media.

[0004] Aspirin is an acetylsalicylic acid compound obtained by the esterification reaction of salicylic acid (o-hydroxybenzoic acid) and acetic anhydride. It is widely used to treat colds, fever, headaches, toothaches, joint pain, and rheumatism. It can also inhibit platelet aggregation, and is used to prevent and treat ischemic heart disease, angina pectoris, myocardial infarction, and cerebral thrombosis. It is also used in angiogenesis and bypass surgery. In addition, aspirin can treat or delay the progression of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and has a certain effect on the prevention and treatment of colorectal cancer. Multiple studies have confirmed that aspirin inhibits cyclooxygenase COX-2 and cellular reactive oxygen species levels in various tissues and cells, providing a theoretical basis for the clinical application of aspirin to delay human aging. Summary of the Invention

[0005] The purpose of this invention is to provide a novel chemically defined culture medium for mesenchymal stem cells. This culture medium has a clearly defined chemical composition, is free of protein hydrolysates and serum replacement factors, and promotes the efficient expansion of mesenchymal stem cells, thus solving the safety issues of clinical applications of mesenchymal stem cells.

[0006] In a first aspect, the present invention provides a mesenchymal stem cell-defined culture medium, comprising a basal culture medium and additives. The basal culture medium is selected from one or more of IMDM, α-MEM, high-glucose DMEM, DMEM / F12, and 1640, with IMDM being preferred. IMDM provides essential nutrients for cell growth: amino acids, vitamins, inorganic substances, nucleic acid derivatives, etc. The additives are selected from one or more of binding proteins, adhesion factors, hormones, vitamins, organic acids, amino acids, sugars, growth factors, trace elements, nucleosides, biogenic amines, aspirin, and soybean trypsin inhibitors. These additives provide cells with essential nutrients, hormones and various growth factors, binding proteins, and factors that promote cell adhesion and spreading, thus protecting cells from mechanical damage and providing some protection to cells in culture.

[0007] Among them, binding proteins can promote cell growth and also act as chelators for some harmful trace elements. Binding proteins are selected from one or more of transferrin and human serum albumin. Human serum albumin is an important nutrient; the amino acids, energy, or other nitrogen-containing substances produced by its breakdown provide the material basis for cell growth and metabolism. In addition, human serum albumin can provide cells with suitable pH and osmotic pressure, maintain osmotic pressure balance for cell growth, prevent cells from suffering pH fluctuations and mechanical damage, and reversibly bind to hormones, growth factors, vitamins, lipids, and metal ions in the culture medium, acting as a carrier to stabilize and regulate these active substances. Transferrin can bind and transport iron ions, providing the iron element needed for cell metabolism. Simultaneously, transferrin also has antibacterial activity and maintains cell proliferation and differentiation.

[0008] Adhesion factors can promote cell adhesion and growth, significantly increase cell adhesion rate, maintain cell morphology and structure, enhance cell metabolism, promote genetic material synthesis, improve the survival rate of primary cell culture, and greatly shorten cell growth time. Adhesion factor substances are selected from fibroin.

[0009] Hormones play a crucial role in cell growth, metabolism, and signal transduction. For example, insulin plays a vital role in the synthesis and metabolism of mammalian cells, promoting cell proliferation. Hormones are selected from one or more of insulin, parathyroid hormone, estradiol, progesterone, hydrocortisone, and dexamethasone. Insulin regulates key biological processes such as glucose and lipid metabolism, gene expression, protein synthesis, and cell growth, division, and survival. Parathyroid hormone regulates the concentration of calcium ions in extracellular fluid. Steroid hormones can freely enter cells, activate intracellular nuclear receptors, regulate gene transcription, and thus affect the development of the cardiovascular and musculoskeletal systems. Dexamethasone and hydrocortisone participate in the metabolism of carbohydrates and lipids in mesenchymal stem cells and are involved in stem cell immunosuppression.

[0010] Vitamins, used as growth supplements in cell culture media, have antioxidant and anti-aging effects, eliminating peroxides and pathogenic free radicals to protect cells, and are used to increase the growth and vitality of mesenchymal stem cells. Vitamins are selected from one or more of vitamins A and C.

[0011] The organic acid is selected from one or more of taurine and pyruvic acid; taurine has antioxidant properties, cell membrane stabilizing properties, and helps maintain cell calcium levels. 2+Taurine plays a stabilizing role and actively exerts its cell-protective effect. Adding taurine to a culture medium with a defined chemical composition can also increase the yield of proteins of interest. Pyruvate, a weak organic acid, provides an energy source and carbon skeleton for anabolic processes when added to the culture medium. The addition of pyruvate helps maintain cell state when serum concentrations are low, promoting clonal proliferation; pyruvate is also a peroxide scavenger, preventing free radical damage.

[0012] The amino acids mentioned are selected from non-essential amino acids, including L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline, L-serine, and glycine. Adding appropriate concentrations of non-essential amino acids to the cell culture medium can reduce the burden on cells in terms of synthesis and improve the utilization rate of other nutrients.

[0013] The sugar is selected from fructose, which is a structural isomer of glucose. Because its uptake and utilization are more strictly controlled, it can serve as an alternative carbon source for cellular metabolism.

[0014] Growth factors typically bind to specific receptors on target cell membranes, thereby influencing cell mitosis, protein synthesis, and differentiation. They are essential biological supplements for maintaining cell growth, proliferation, division, and differentiation in vitro. For example, transforming growth factor (TGF) can maintain the self-renewal capacity and differentiation potential of stem cells. Growth factors are selected from one or more of the following: platelet-derived factor PDGF-BB, epidermal growth factor EGF, transforming growth factor TGF-β1 and TGF-β3, fibroblast growth factor b-FGF, insulin-like growth factor IGF-I, vascular endothelial growth factor VEGF, connective tissue growth factor CTGF, hepatocyte growth factor HGF, leukemia inhibitory factor LIF, interleukin-like growth factor IL-2, erythropoietin EPO, stem cell growth factor SCF, and tumor necrosis factor TNF-α. The synergistic effect of multiple growth factors stimulates the proliferation and differentiation of mesenchymal stem cells.

[0015] Among them, trace elements such as selenium are the most common. Selenium is an essential trace element for life, participating in the action of antioxidant enzymes (such as glutathione peroxidase-GPx, thioreductase-TrxR, and three deiodinases-D1, D2, and D3), and has strong antioxidant activity, resisting cell damage caused by peroxides and superoxides. The trace element-containing substance is selected from sodium selenite.

[0016] Nucleosides are the building blocks of DNA, participate in the synthesis of cellular genetic material and proteins, and provide energy to maintain cellular metabolism. Nucleoside compounds are selected from one or more of deoxyadenosine, deoxycytidine, and deoxyguanosine.

[0017] Biogenic amines play a crucial role in stimulating cell growth, prolonging the cell cycle, exerting anti-apoptotic effects, and increasing cell proliferation density. Biogenic amines are selected from one or more of ethanolamine and putrescine; amines can synthesize spermine, stabilizing DNA molecules; ethanolamine can promote cell membrane construction.

[0018] Aspirin, in particular, has anti-aging properties and is widely used to improve health and prolong lifespan; it can also enhance cell proliferation.

[0019] Among them, soybean trypsin inhibitors are anti-protease components that neutralize and terminate the digestive action of trypsin during the passage process.

[0020] Preferably, the additives in the chemically defined culture medium for mesenchymal stem cells of the present invention may contain the following components and contents: recombinant human serum albumin 0.5–50 g / L, recombinant human transferrin 0.1–10 mg / L, recombinant human fibronectin FN 0–100 mg / L, insulin 1–100 mg / L, progesterone 0–50 μg / L, estradiol 0–50 μg / L, dexamethasone 1–150 μg / L, hydrocortisone 0.05–5 mg / L, parathyroid hormone 0–10 μg / L, vitamin A 0–1000 μM, vitamin C 25–2000 mg / L, and non-essential amino acids NEAA. 0-500 mg / L, Taurine 0-50 mg / L, Pyruvic acid 0-500 mg / L, Putrescine 0-100 mg / L, Aminoethanol 0.5-50 mg / L, Sodium selenite 2.0-100 μg / L, Soybean trypsin inhibitor 0-100 mg / L, Epidermal growth factor (EGF) 1-500 μg / L, Basic fibroblast growth factor (bFGF) 1-500 μg / L, Platelet-derived growth factor BB (PDGF-BB) 1-500 μg / L, Insulin-like growth factor-I (IGF-I) 1-500 μg / L, Vascular endothelial growth factor (VEGF) 0-500 μg / L, Transforming growth factor β1 (TGF-β1) 1-500 μg / L Transforming growth factor β3 (TGF-β3) 0–500 μg / L, connective tissue growth factor (CTGF) 0–500 μg / L, stem cell factor (SCF) 0–500 μg / L, hepatocyte growth factor (HGF) 0–500 μg / L, interleukin-2 (IL-2) 0–500 μg / L, leukemia inhibitory factor (LIF) 0–500 μg / L, erythropoietin (EPO) 0–100 μg / L, tumor necrosis factor α (TNF-α) 0–100 μg / L, deoxyadenosine 0–500 mg / L, deoxycytidine 0–500 mg / L, deoxyguanosine 0–500 mg / L, fructose 0–500 mg / L, aspirin 0–100 μM. More preferably, the additives contain the following components and contents: recombinant human serum albumin 1.25 g / L, recombinant human transferrin 5.5 mg / L, insulin 10 mg / L, dexamethasone 100 μg / L, hydrocortisone 0.36 mg / L, vitamin C 100 mg / L, aminoethanol 2 mg / L, sodium selenite 10 μg / L, epidermal growth factor (EGF) 20 μg / L, basic fibroblast growth factor (bFGF) 20 μg / L, platelet-derived growth factor BB (PDGF-BB) 20 μg / L, insulin-like growth factor-I (IGF-I) 20 μg / L, and transforming growth factor β1 (TGF-β1) 20 μg / L.

[0021] Preferably, the additives in the mesenchymal stem cell chemical composition-defined culture medium of the present invention may further include: recombinant human serum albumin 0.5–50 g / L, recombinant human transferrin 0.1–10 mg / L, recombinant human fibronectin FN 1–100 mg / L, insulin 1–100 mg / L, progesterone 0.5–50 μg / L, estradiol 0.5–50 μg / L, dexamethasone 1–150 μg / L, hydrocortisone 0.05–5 mg / L, parathyroid hormone 0.1–10 μg / L, vitamin A 10–1000 μM, vitamin C 25–2000 mg / L, and non-essential amino acids NEAA. 1-500 mg / L, Taurine 0.5-50 mg / L, Pyruvic acid 1-500 mg / L, Putrescine 2-100 mg / L, Aminoethanol 0.5-50 mg / L, Sodium selenite 2.0-100 μg / L, Soybean trypsin inhibitor 1-100 mg / L, Epidermal growth factor (EGF) 1-500 μg / L, Basic fibroblast growth factor (bFGF) 1-500 μg / L, Platelet-derived growth factor BB (PDGF-BB) 1-500 μg / L, Insulin-like growth factor-I (IGF-I) 1-500 μg / L, Vascular endothelial growth factor (VEGF) 1-500 μg / L, Transforming growth factor β1 (TGF-β1) 1-500 μg / L Transforming growth factor β3 (TGF-β3) 1–500 μg / L, connective tissue growth factor (CTGF) 1–500 μg / L, stem cell factor (SCF) 1–500 μg / L, hepatocyte growth factor (HGF) 1–500 μg / L, interleukin-2 (IL-2) 1–500 μg / L, leukemia inhibitory factor (LIF) 1–500 μg / L, erythropoietin (EPO) 0.1–100 μg / L, tumor necrosis factor α (TNF-α) 0.1–100 μg / L, deoxyadenosine 1–500 mg / L, deoxycytidine 1–500 mg / L, deoxyguanosine 1–500 mg / L, fructose 0–500 mg / L, aspirin 0–100 μM.More preferably, the additives contain the following components and contents: recombinant human serum albumin 1.25 g / L, recombinant human transferrin 5.5 mg / L, recombinant human fibronectin FN 2 mg / L, insulin 10 mg / L, progesterone 2.7 μg / L, estradiol 1.3 μg / L, dexamethasone 100 μg / L, hydrocortisone 0.36 mg / L, parathyroid hormone 35.74 μg / L, vitamin A 50 μM, vitamin C 100 mg / L, and non-essential amino acids NEAA. 10 mg / L, Taurine 1.25 mg / L, Pyruvic acid 50 mg / L, Putrescine 5.3 mg / L, Aminoethanol 2 mg / L, Sodium selenite 10 μg / L, Soybean trypsin inhibitor 1 mg / L, Epidermal growth factor (EGF) 20 μg / L, Basic fibroblast growth factor (bFGF) 20 μg / L, Platelet-derived growth factor BB (PDGF-BB) 20 μg / L, Insulin-like growth factor-I (IGF-I) 20 μg / L, Vascular endothelial growth factor (VEGF) 20 μg / L, Transforming growth factor β1 (TGF-β) 1) 20 μg / L, Transforming growth factor β3 (TGF-β3) 20 μg / L, Connective tissue growth factor (CTGF) 20 μg / L, Stem cell factor (SCF) 20 μg / L, Hepatocyte growth factor (HGF) 20 μg / L, Interleukin-2 (IL-2) 10 μg / L, Leukemia inhibitory factor (LIF) 20 μg / L, Erythropoietin (EPO) 10 μg / L, Tumor necrosis factor α (TNF-α) 10 μg / L, Deoxyadenosine 10 mg / L, Deoxycytidine 11 mg / L, Deoxyguanosine 10 mg / L.

[0022] Preferably, the additives in the mesenchymal stem cell chemical composition-defined culture medium of the present invention may also include: recombinant human serum albumin 0.5–50 g / L, recombinant human transferrin 0.1–10 mg / L, recombinant human fibronectin FN 1–100 mg / L, insulin 1–100 mg / L, progesterone 0.5–50 μg / L, estradiol 0.5–50 μg / L, dexamethasone 1–150 μg / L, hydrocortisone 0.05–5 mg / L, parathyroid hormone 0.1–10 μg / L, vitamin A 10–1000 μM, vitamin C 25–2000 mg / L, and non-essential amino acids NEAA. 1-500 mg / L, Taurine 0.5-50 mg / L, Pyruvic acid 1-500 mg / L, Putrescine 2-100 mg / L, Aminoethanol 0.5-50 mg / L, Sodium selenite 2.0-100 μg / L, Soybean trypsin inhibitor 1-100 mg / L, Epidermal growth factor (EGF) 1-500 μg / L, Basic fibroblast growth factor (bFGF) 1-500 μg / L, Platelet-derived growth factor BB (PDGF-BB) 1-500 μg / L, Insulin-like growth factor-I (IGF-I) 1-500 μg / L, Vascular endothelial growth factor (VEGF) 1-500 μg / L, Transforming growth factor β1 (TGF-β1) 1-500 μg / L Transforming growth factor β3 (TGF-β3) 1–500 μg / L, connective tissue growth factor (CTGF) 1–500 μg / L, stem cell factor (SCF) 1–500 μg / L, hepatocyte growth factor (HGF) 1–500 μg / L, interleukin-2 (IL-2) 1–500 μg / L, leukemia inhibitory factor (LIF) 1–500 μg / L, erythropoietin (EPO) 0.1–100 μg / L, tumor necrosis factor α (TNF-α) 0.1–100 μg / L, deoxyadenosine 1–500 mg / L, deoxycytidine 1–500 mg / L, deoxyguanosine 1–500 mg / L, fructose 1–500 mg / L, aspirin 1–100 μM.More preferably, the additives contain the following components and contents: recombinant human serum albumin 1.25 g / L, recombinant human transferrin 5.5 mg / L, recombinant human fibronectin FN 2 mg / L, insulin 10 mg / L, progesterone 2.7 μg / L, estradiol 1.3 μg / L, dexamethasone 100 μg / L, hydrocortisone 0.36 mg / L, parathyroid hormone 35.74 μg / L, vitamin A 50 μM, vitamin C 100 mg / L, and non-essential amino acids NEAA. 10 mg / L, Taurine 1.25 mg / L, Pyruvic acid 50 mg / L, Putrescine 5.3 mg / L, Aminoethanol 2 mg / L, Sodium selenite 10 μg / L, Soybean trypsin inhibitor 1 mg / L, Epidermal growth factor (EGF) 20 μg / L, Basic fibroblast growth factor (bFGF) 20 μg / L, Platelet-derived growth factor BB (PDGF-BB) 20 μg / L, Insulin-like growth factor-I (IGF-I) 20 μg / L, Vascular endothelial growth factor (VEGF) 20 μg / L, Transforming growth factor β1 (TGF-β1) 20 μg / L, Transforming growth factor β3 (TGF-β3) 20 μg / L, connective tissue growth factor (CTGF) 20 μg / L, stem cell factor (SCF) 20 μg / L, hepatocyte growth factor (HGF) 20 μg / L, interleukin-2 (IL-2) 10 μg / L, leukemia inhibitory factor (LIF) 20 μg / L, erythropoietin (EPO) 10 μg / L, tumor necrosis factor α (TNF-α) 10 μg / L, deoxyadenosine 10 mg / L, deoxycytidine 11 mg / L, deoxyguanosine 10 mg / L, fructose 20 mg / L, aspirin 10 μM.

[0023] Secondly, the present invention also provides a method for preparing a chemically defined culture medium for mesenchymal stem cells, as follows: The present invention provides a method for preparing a chemically defined culture medium for mesenchymal stem cells: other components except for the basal culture medium are mixed in proportion to obtain additive B, and the obtained additive B is added to the basal culture medium to obtain a chemically defined culture medium for mesenchymal stem cells.

[0024] Thirdly, this invention also provides the application of a chemically defined culture medium for mesenchymal stem cells in two-dimensional experimental culture. The two-dimensional culture experimental steps are as follows: Preheat the water bath to 37°C, and take out an appropriate amount of the prepared defined-component culture medium in advance to rewarm it. Take out the frozen human umbilical cord mesenchymal stem cells and place them in a 37°C water bath for rapid thawing. Immediately aspirate the cell suspension into a 15mL centrifuge tube, add 10mL of the defined-component culture medium restored to room temperature dropwise, and mix gently. Centrifuge, discard the supernatant, add 3-5mL of the defined-component culture medium to resuspend the cells, and accurately count them. Seed at a suitable density (6000-8000 / cm²). 2Recommended 8000 / cm 2 Seed cells into cell culture containers and add an appropriate amount of defined culture medium. Incubate at 37°C, 5% CO2 concentration, and saturated humidity for 3-4 days. When cell confluence reaches 80-85%, passage or harvest is recommended. For cell passage, collect cells using genetically engineered trypsin digestion and seed at a suitable density (6000-8000 / cm²). 2 Recommended 8000 / cm 2 The cells are seeded into a cell culture container, and an appropriate amount of the specified culture medium is added for further culture.

[0025] Beneficial effects of the present invention

[0026] (1) The culture medium described in this invention has a clear chemical composition and is free of protein hydrolysate components and serum replacement factors, thus avoiding the risks of instability and pathogen contamination caused by serum or blood replacement components and solving the safety issues of mesenchymal stem cell clinical application.

[0027] (2) The culture medium of the present invention is enriched with aspirin anti-aging components, which promotes efficient expansion of mesenchymal stem cells, reduces cell diameter, and maintains high cell activity. Attached Figure Description

[0028] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0029] Figure 1 This is a cell morphology diagram of human umbilical cord mesenchymal stem cells cultured in different culture media in Example 1.

[0030] Figure 2 This is a graph showing the cell proliferation fold of three consecutive generations of human umbilical cord mesenchymal stem cells cultured in Example 4.

[0031] Figure 3 This is a series of three generations of cell diameter diagrams of human umbilical cord mesenchymal stem cells cultured in Example 4.

[0032] Figure 4 This is a series of three-generation cell viability graphs of human umbilical cord mesenchymal stem cells cultured in Example 4.

[0033] Figure 5 This is a flow cytometry plot of human umbilical cord mesenchymal stem cells detected in Example 5.

[0034] Figure 6 This is a staining image of aging in human umbilical cord mesenchymal stem cells detected in Example 6. Detailed Implementation

[0035] The following detailed description, in conjunction with embodiments, illustrates the stem cell chemical composition-defined culture medium provided by the present invention and its applications, but these should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1: Effects of different components and contents on two-dimensional culture

[0037] The present invention provides a chemically defined culture medium for mesenchymal stem cells, the composition and concentration of which are as follows. The following components are dissolved and mixed according to their respective characteristics, filtered through a 0.22μm filter membrane for sterilization, and stored at 4 degrees Celsius.

[0038] Element concentration Formula 1 Formula 2 Formula 3 IMDM 1L √ √ √ Recombinant human serum albumin 1.25g / L √ √ √ Recombinant human transferrin 5.5 mg / L √ √ √ insulin 10mg / L √ √ √ Dexamethasone 100μg / L √ √ √ Hydrocortisone 0.36 mg / L √ √ √ Vitamin C 100mg / L √ √ √ Aminoethanol 2mg / L √ √ √ Sodium selenite 10 μg / L √ √ √ EGF 20μg / L √ √ √ b-FGF 20μg / L √ √ √ PDGF-BB 20μg / L √ √ √ IGF-Ⅰ 20μg / L √ √ √ TGF-β1 20μg / L √ √ √ Recombinant human fibronectin FN 2mg / L √ √ Progesterone 2.7 μg / L √ √ estradiol 1.3 μg / L √ √ Parathyroid hormone (PTH) 35.74 ng / L √ √ Vitamin A 50μM √ √ Non-essential amino acids (NEAA) 10mg / L √ √ Taurine 1.25 mg / L √ √ Pyruvic acid 50mg / L √ √ putrescine 5.3 mg / L √ √ Soybean pancreatic enzyme inhibitors 1mg / L √ √ VEGF 20μg / L √ √ TGF-β3 20μg / L √ √ CTGF 20μg / L √ √ SCF 20μg / L √ √ HGF 20μg / l √ √ IL-2 10 μg / l √ √ LIF 20μg / l √ √ EPO 10 μg / l √ √ TNFα 10 μg / l √ √ Deoxyadenosine 10mg / l √ √ Deoxycytidine 11mg / l √ √ Deoxyguanosine 10mg / l √ √ fructose 20mg / L √ aspirin 10uM √

[0039] The culture medium was prepared using the above formula, and umbilical cord mesenchymal stem cells were cultured according to conventional methods. To compare the culture effect of the culture medium of this invention, two commercially available serum-free MSC culture media were used as controls. Serum-free MSC proliferation medium (ME000-N023) was used as Comparative Example 1, and Youkang GMP-grade serum-free mesenchymal stem cell medium (NC0106) was used as Comparative Example 2. Both were co-cultured with the aforementioned medium formulations. Human umbilical cord mesenchymal stem cells (P4) were resuscitated and inoculated (inoculation amount 7.7 × 10⁻⁶). 4 Cells were cultured in six-well plates for 4 days, and the fold increase was measured.

[0040] Table 1

[0041] Experimental group Inoculated cells Cell number Cell proliferation Cell viability % Cell diameter μm Formula 1 77000 590000 7.66 97.52 18.04 Formula 2 77000 1042000 13.53 99.21 16.68 Formula 3 77000 1094000 14.21 98.94 15.86 Comparative Example 1 77000 742000 9.64 100.00 15.60 Comparative Example 2 77000 436000 5.66 97.69 18.11

[0042] Table 1 shows the experimental results. Formula 1 culture medium promoted cell proliferation by 7.66-fold, with cell viability greater than 95% and cell diameter greater than 18 μm. After adding multiple factor combinations, stabilizing factors, and protectants to Formula 1, Formula 2 culture medium increased cell proliferation to 13.53-fold, with cell viability greater than 95% and cell diameter reduced to below 17 μm, showing better culture performance than Formula 1. After adding fructose and aspirin to Formula 2, Formula 3 culture medium increased cell proliferation to 14.21-fold, with cell viability greater than 95% and cell diameter reduced to below 16 μm, showing better culture performance than Formula 2. Neither of the two commercially available culture media showed a cell proliferation increase exceeding 10-fold. The overall results indicate that Formula 3 culture medium of this invention has better culture performance than the two commercially available culture media. The addition of the innovative ingredient aspirin is beneficial in increasing cell proliferation, reducing cell diameter, and maintaining the viability of MSCs. Figure 1 The results showed that the mesenchymal stem cells cultured in formulations 1-3 were spindle-shaped, uniform in size, and normal in morphology.

[0043] Example 2: Effects of replacing different basal culture media on two-dimensional culture

[0044] In this invention, the basal culture medium IMDM in Formula 3 is replaced with α-MEM, high-glucose DMEM, DMEM / F12, and 1640. The remaining components are dissolved and mixed according to their respective characteristics, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4 degrees Celsius. Using the above formula, the culture medium is prepared, and umbilical cord mesenchymal stem cells are cultured according to conventional methods. Human umbilical cord mesenchymal stem cells (P4) are resuscitated and inoculated (inoculation amount 7.7 × 10⁻⁶). 4 Cells were cultured in six-well plates for 4 days, and the fold increase was measured.

[0045] Table 2

[0046]

[0047]

[0048] Table 2 shows the experimental results. The factor combination of the present invention is suitable for culturing in other basal media besides IMDM. The α-MEM and DMEM / F12 basal media groups can promote cell proliferation by 4.7-6.7 times and the cell diameter is less than 18 μm.

[0049] Example 3: Effects of different concentrations of aspirin on two-dimensional culture

[0050] This invention tested the effect of different concentrations of aspirin on the proliferation of mesenchymal stem cells. Aspirin in Formula 3 was prepared at concentration gradients of 1 μM, 10 μM, and 100 μM. The remaining components were dissolved and mixed according to their respective properties, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4 degrees Celsius. Culture medium was prepared using the above formula, and umbilical cord mesenchymal stem cells were cultured according to conventional methods. Human umbilical cord mesenchymal stem cells (P4) were resuscitated and inoculated (inoculation amount 7.7 × 10⁻⁶). 4 Cells were cultured in six-well plates for 4 days, and the fold increase was measured.

[0051] Table 3

[0052] aspirin concentration Inoculated cells Cell number Cell proliferation Cell viability % Cell diameter μm 1μM 77000 431970 5.61 98.11 17.45 10μM 77000 772310 10.03 99.14 16.62 100μM 77000 444290 5.77 98.15 18.10

[0053] Table 3 shows the experimental results, indicating that the optimal concentration of aspirin for promoting mesenchymal stem cell proliferation was 10 μM. Compared to the 1 μM and 100 μM groups, the 10 μM group showed the highest cell proliferation and the smallest cell diameter; therefore, the final composition preferably includes 10 μM aspirin.

[0054] Example 4: Two-dimensional continuous passage of umbilical cord mesenchymal stem cells

[0055] This embodiment tested the two-dimensional continuous passage ability of cells in the serum-free culture medium provided by Formula 3 and the control culture medium. The control group was the serum-free culture medium (NC0106) of Youkang GMP grade mesenchymal stem cells used in Comparative Example 2. The specific experimental method was as follows: human umbilical cord mesenchymal stem cells P4 were resuscitated and seeded (seedling amount 7.7 × 10⁻⁶). 4 Cells were cultured in six-well plates for 3 days, and cell viability, fold increase, and cell diameter were measured using a cell counter. Cells were then continuously transferred to P7.

[0056] Experimental results Figures 2 to 4 The results showed that the cell viability of both Formula 3 and Comparative Formula 2 was high at each passage. However, using Formula 3 could maintain a higher fold increase in MSCs, with all three passages showing an increase of more than 8 times, and also maintain a lower cell diameter, with all three passages showing a cell diameter of less than 17 μm.

[0057] Example 5: Flow cytometry detection of two-dimensional cultured cell phenotypes

[0058] This embodiment compares the phenotypes of P7 generation cells grown in the serum-free culture medium provided in Formula 3 with those in the commercially available serum-free culture medium of Comparative Example 2. The flow cytometry method for identifying surface markers was as follows: when the third generation cells cultured in the defined component culture medium reached 90% confluence, the cells were digested and collected using genetically engineered trypsin, and 1×10⁶ cells were taken. 5 (100 μl) cells were added with 20 μl of CD105, CD90, CD73, CD45, CD34, CD19, CD14 and HLA-DR antibodies respectively, mixed well, incubated in the dark for 30 min, washed twice with PBS, and detected by Backman Clouter FC500 flow cytometer.

[0059] Table 4

[0060] UC-MSC CD105 CD90 CD73 CD45 CD34 CD19 CD14 HLA-DA Formula 3 98.77％ 99.51％ 99.90％ 0.00％ 0.02％ 0.00％ 0.06％ 0.04％ Comparative Example 2 99.59％ 99.71％ 99.54％ 0.19％ 0.40％ 0.42％ 0.35％ 0.44％

[0061] Experimental results are shown in Table 4 and Figure 5 The results showed that MSCs cultured in Formula 3 and Comparative Example 2 both highly expressed mesenchymal stem cell-specific antigens CD73, CD90, and CD105, and expressed low levels of hematopoietic and endothelial cell surface markers CD14, CD19, CD34, and CD45, as well as HLA-DR, a surface marker associated with transplant immune rejection, which is consistent with the 2006 ISCT description of MSC identification.

[0062] Example 6: Detection of Senescence in Two-Dimensional Cultured Cells by β-Galactosidase Staining

[0063] This embodiment assesses the senescence of P7 generation cells cultured in the serum-free medium provided in Formula 3 and the commercially available serum-free medium in Comparative Example 2. The cell senescence detection method is as follows: when the third generation cells cultured in the defined component medium reached 90% confluence, the cells were collected by digestion with genetically engineered trypsin, and 2 × 10⁶ cells were taken. 5 The cells to be tested were seeded in 6-well plates, and 2 mL of fresh culture medium was added at 37°C and CO2. 2 Cells were cultured in an incubator for 24 hours, stained overnight using the β-galactosidase staining method, observed under a microscope, and senescent cells were counted.

[0064] Table 5

[0065]

[0066] Experimental results are shown in Table 5 and Figure 6 The results showed that, compared with the commercially available serum-free culture medium Comparative Example 2, the culture medium provided by Formula 3 of the present invention yielded fewer stained mesenchymal stem cells after three generations of culture, and the number of senescent cells was significantly reduced compared with the control group.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the scope of protection of the present invention and do not depart from the scope defined by the claims of the present invention.

Claims

1. A serum-free mesenchymal stem cell culture medium, characterized in that, The culture medium consists of a basal culture medium and additives, wherein the additives are selected from one or more of the following: binding protein substances, adhesion factor substances, hormone substances, vitamin substances, organic acid substances, amino acid substances, sugar substances, growth factor substances, trace element substances, nucleoside substances, biogenic amine substances, aspirin, and soybean trypsin inhibitors.

2. The culture medium according to claim 1, characterized in that, The basal culture medium is selected from one or more of IMDM, α-MEM, high-glucose DMEM, DMEM / F12, and 1640, with IMDM being preferred.

3. The culture medium according to claim 1 or 2, characterized in that, The binding protein is selected from one or more of transferrin and human serum albumin; the adhesion factor is selected from fibronectin; the hormone is selected from one or more of insulin, parathyroid hormone, estradiol, progesterone, hydrocortisone, and dexamethasone; the vitamin is selected from one or more of vitamin A and vitamin C; the organic acid is selected from one or more of taurine and pyruvate; the amino acid is selected from non-essential amino acids; the sugar is selected from fructose; and the growth factor is selected from platelet-derived factor PDGF-BB, epidermal growth factor EGF, and transforming growth factor TGF. One or more of the following: TGF-β1 and TGF-β3, fibroblast growth factor b-FGF, insulin-like growth factor IGF-I, vascular endothelial growth factor VEGF, connective tissue growth factor CTGF, hepatocyte growth factor HGF, leukemia inhibitory factor LIF, interleukin-like growth factor IL-2, erythropoietin EPO, stem cell growth factor SCF, and tumor necrosis factor TNF-α; the trace element is selected from sodium selenite; the nucleoside compound is selected from one or more of deoxyadenosine, deoxycytidine, and deoxyguanosine; and the biogenic amine is selected from one or more of ethanolamine and putrescine.

4. The culture medium according to claim 1 or 3, characterized in that, The culture medium contains the following components and concentrations: recombinant human serum albumin 0.5–50 g / L, recombinant human transferrin 0.1–10 mg / L, recombinant human fibronectin (FN) 0–100 mg / L, insulin 1–100 mg / L, progesterone 0–50 μg / L, estradiol 0–50 μg / L, dexamethasone 1–150 μg / L, hydrocortisone 0.05–5 mg / L, parathyroid hormone 0–10 μg / L, vitamin A 0–1000 μM, vitamin C 25–2000 mg / L, and non-essential amino acids (NEAA). 0-500 mg / L, Taurine 0-50 mg / L, Pyruvic acid 0-500 mg / L, Putrescine 0-100 mg / L, Aminoethanol 0.5-50 mg / L, Sodium selenite 2.0-100 μg / L, Soybean trypsin inhibitor 0-100 mg / L, Epidermal growth factor (EGF) 1-500 μg / L, Basic fibroblast growth factor (bFGF) 1-500 μg / L, Platelet-derived growth factor BB (PDGF-BB) 1-500 μg / L, Insulin-like growth factor-I (IGF-I) 1-500 μg / L, Vascular endothelial growth factor (VEGF) 0-500 μg / L, Transforming growth factor β1 (TGF-β1) 1-500 μg / L Transforming growth factor β3 (TGF-β3) 0–500 μg / L, connective tissue growth factor (CTGF) 0–500 μg / L, stem cell factor (SCF) 0–500 μg / L, hepatocyte growth factor (HGF) 0–500 μg / L, interleukin-2 (IL-2) 0–500 μg / L, leukemia inhibitory factor (LIF) 0–500 μg / L, erythropoietin (EPO) 0–100 μg / L, tumor necrosis factor α (TNF-α) 0–100 μg / L, deoxyadenosine 0–500 mg / L, deoxycytidine 0–500 mg / L, deoxyguanosine 0–500 mg / L, fructose 0–500 mg / L, aspirin 0–100 μM.

5. The culture medium according to claim 1 or 3, characterized in that, The culture medium contains the following components and concentrations: recombinant human serum albumin 0.5–50 g / L, recombinant human transferrin 0.1–10 mg / L, recombinant human fibronectin (FN) 1–100 mg / L, insulin 1–100 mg / L, progesterone 0.5–50 μg / L, estradiol 0.5–50 μg / L, dexamethasone 1–150 μg / L, hydrocortisone 0.05–5 mg / L, parathyroid hormone 0.1–10 μg / L, vitamin A 10–1000 μM, vitamin C 25–2000 mg / L, and non-essential amino acids (NEAA). 1-500 mg / L, Taurine 0.5-50 mg / L, Pyruvic acid 1-500 mg / L, Putrescine 2-100 mg / L, Aminoethanol 0.5-50 mg / L, Sodium selenite 2.0-100 μg / L, Soybean trypsin inhibitor 1-100 mg / L, Epidermal growth factor (EGF) 1-500 μg / L, Basic fibroblast growth factor (bFGF) 1-500 μg / L, Platelet-derived growth factor BB (PDGF-BB) 1-500 μg / L, Insulin-like growth factor-I (IGF-I) 1-500 μg / L, Vascular endothelial growth factor (VEGF) 1-500 μg / L, Transforming growth factor β1 (TGF-β1) 1-500 μg / L Transforming growth factor β3 (TGF-β3) 1–500 μg / L, connective tissue growth factor (CTGF) 1–500 μg / L, stem cell factor (SCF) 1–500 μg / L, hepatocyte growth factor (HGF) 1–500 μg / L, interleukin-2 (IL-2) 1–500 μg / L, leukemia inhibitory factor (LIF) 1–500 μg / L, erythropoietin (EPO) 0.1–100 μg / L, tumor necrosis factor α (TNF-α) 0.1–100 μg / L, deoxyadenosine 1–500 mg / L, deoxycytidine 1–500 mg / L, deoxyguanosine 1–500 mg / L, fructose 0–500 mg / L, aspirin 0–100 μM.

6. The culture medium according to claim 1 or 3, characterized in that, The culture medium contains the following components and concentrations: recombinant human serum albumin 0.5–50 g / L, recombinant human transferrin 0.1–10 mg / L, recombinant human fibronectin (FN) 1–100 mg / L, insulin 1–100 mg / L, progesterone 0.5–50 μg / L, estradiol 0.5–50 μg / L, dexamethasone 1–150 μg / L, hydrocortisone 0.05–5 mg / L, parathyroid hormone 0.1–10 μg / L, vitamin A 10–1000 μM, vitamin C 25–2000 mg / L, and non-essential amino acids (NEAA). 1-500 mg / L, Taurine 0.5-50 mg / L, Pyruvic acid 1-500 mg / L, Putrescine 2-100 mg / L, Aminoethanol 0.5-50 mg / L, Sodium selenite 2.0-100 μg / L, Soybean trypsin inhibitor 1-100 mg / L, Epidermal growth factor (EGF) 1-500 μg / L, Basic fibroblast growth factor (bFGF) 1-500 μg / L, Platelet-derived growth factor BB (PDGF-BB) 1-500 μg / L, Insulin-like growth factor-I (IGF-I) 1-500 μg / L, Vascular endothelial growth factor (VEGF) 1-500 μg / L, Transforming growth factor β1 (TGF-β1) 1-500 μg / L Transforming growth factor β3 (TGF-β3) 1–500 μg / L, connective tissue growth factor (CTGF) 1–500 μg / L, stem cell factor (SCF) 1–500 μg / L, hepatocyte growth factor (HGF) 1–500 μg / L, interleukin-2 (IL-2) 1–500 μg / L, leukemia inhibitory factor (LIF) 1–500 μg / L, erythropoietin (EPO) 0.1–100 μg / L, tumor necrosis factor α (TNF-α) 0.1–100 μg / L, deoxyadenosine 1–500 mg / L, deoxycytidine 1–500 mg / L, deoxyguanosine 1–500 mg / L, fructose 1–500 mg / L, aspirin 1–100 μM.

7. The culture medium according to claim 4, characterized in that, The culture medium contains the following components and concentrations: recombinant human serum albumin 1.25 g / L, recombinant human transferrin 5.5 mg / L, insulin 10 mg / L, dexamethasone 100 μg / L, hydrocortisone 0.36 mg / L, vitamin C 100 mg / L, aminoethanol 2 mg / L, sodium selenite 10 μg / L, epidermal growth factor (EGF) 20 μg / L, basic fibroblast growth factor (bFGF) 20 μg / L, platelet-derived growth factor BB (PDGF-BB) 20 μg / L, insulin-like growth factor-I (IGF-I) 20 μg / L, and transforming growth factor β1 (TGF-β1) 20 μg / L.

8. The culture medium according to claim 5, characterized in that, The culture medium contained the following components and concentrations: recombinant human serum albumin 1.25 g / L, recombinant human transferrin 5.5 mg / L, recombinant human fibronectin (FN) 2 mg / L, insulin 10 mg / L, progesterone 2.7 μg / L, estradiol 1.3 μg / L, dexamethasone 100 μg / L, hydrocortisone 0.36 mg / L, parathyroid hormone 35.74 μg / L, vitamin A 50 μM, vitamin C 100 mg / L, and non-essential amino acids (NEAA). 10 mg / L, Taurine 1.25 mg / L, Pyruvic acid 50 mg / L, Putrescine 5.3 mg / L, Aminoethanol 2 mg / L, Sodium selenite 10 μg / L, Soybean trypsin inhibitor 1 mg / L, Epidermal growth factor (EGF) 20 μg / L, Basic fibroblast growth factor (bFGF) 20 μg / L, Platelet-derived growth factor BB (PDGF-BB) 20 μg / L, Insulin-like growth factor-I (IGF-I) 20 μg / L, Vascular endothelial growth factor (VEGF) 20 μg / L, Transforming growth factor β1 (TGF-β) 1) 20 μg / L, Transforming growth factor β3 (TGF-β3) 20 μg / L, Connective tissue growth factor (CTGF) 20 μg / L, Stem cell factor (SCF) 20 μg / L, Hepatocyte growth factor (HGF) 20 μg / L, Interleukin-2 (IL-2) 10 μg / L, Leukemia inhibitory factor (LIF) 20 μg / L, Erythropoietin (EPO) 10 μg / L, Tumor necrosis factor α (TNF-α) 10 μg / L, Deoxyadenosine 10 mg / L, Deoxycytidine 11 mg / L, Deoxyguanosine 10 mg / L.

9. The culture medium according to claim 6, characterized in that, The culture medium contained the following components and concentrations: recombinant human serum albumin 1.25 g / L, recombinant human transferrin 5.5 mg / L, recombinant human fibronectin (FN) 2 mg / L, insulin 10 mg / L, progesterone 2.7 μg / L, estradiol 1.3 μg / L, dexamethasone 100 μg / L, hydrocortisone 0.36 mg / L, parathyroid hormone 35.74 μg / L, vitamin A 50 μM, vitamin C 100 mg / L, and non-essential amino acids (NEAA). 10 mg / L, Taurine 1.25 mg / L, Pyruvic acid 50 mg / L, Putrescine 5.3 mg / L, Aminoethanol 2 mg / L, Sodium selenite 10 μg / L, Soybean trypsin inhibitor 1 mg / L, Epidermal growth factor (EGF) 20 μg / L, Basic fibroblast growth factor (bFGF) 20 μg / L, Platelet-derived growth factor BB (PDGF-BB) 20 μg / L, Insulin-like growth factor-I (IGF-I) 20 μg / L, Vascular endothelial growth factor (VEGF) 20 μg / L, Transforming growth factor β1 (TGF-β1) 20 μg / L, Transforming growth factor β3 (TGF-β3) 20 μg / L, connective tissue growth factor (CTGF) 20 μg / L, stem cell factor (SCF) 20 μg / L, hepatocyte growth factor (HGF) 20 μg / L, interleukin-2 (IL-2) 10 μg / L, leukemia inhibitory factor (LIF) 20 μg / L, erythropoietin (EPO) 10 μg / L, tumor necrosis factor α (TNF-α) 10 μg / L, deoxyadenosine 10 mg / L, deoxycytidine 11 mg / L, deoxyguanosine 10 mg / L, fructose 20 mg / L, aspirin 10 μM.

10. A method for preparing the culture medium according to any one of claims 1 to 9, characterized in that, Other components, except for the basal culture medium, are mixed in proportion to prepare an additive, which is then added to the basal culture medium to prepare a mesenchymal stem cell chemically defined culture medium.

11. The use of the culture medium according to any one of claims 1 to 9 in two-dimensional culture of mesenchymal stem cells.

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