Mesenchymal stem cell culture medium, preparation method and application thereof

Abstract

This invention provides a mesenchymal stem cell culture medium, its preparation method, and its application. The mesenchymal stem cell culture medium of this invention includes a basal culture medium and functional additives. The functional additives include: recombinant human albumin, recombinant transferrin, recombinant bone morphogenetic protein; recombinant insulin or insulin-like growth factor, recombinant fibroblast growth factor, recombinant epidermal growth factor, recombinant platelet-derived growth factor; thyroid hormones, ethanolamine, carnitine, glucocorticoid regulators, retinoic acid compounds; heparin sodium, signal-regulating lipids, cholesterol, DL-α-tocopherol acetate, linolenic acid, oleic acid, stearic acid; prolactin F68, and Tween 80. The mesenchymal stem cell culture medium of this invention does not contain immunogenic proteins or blood products derived from animals or humans. All components can be supplied with GMP-compliant raw materials and the process can be controlled, ensuring the controllability, safety, and reproducibility of the culture process.

Description

Technical Field

[0001] This invention relates to the fields of cell biology, biomedicine, or tissue engineering, and more specifically, to a mesenchymal stem cell culture medium, its preparation method, and its application. Background Technology

[0002] Human mesenchymal stem cells (MSCs) are adult cells derived from tissues such as bone marrow, adipose tissue, and umbilical cord. They can proliferate in vitro and differentiate into various specialized cell types, and have been widely used in clinical trials for immune or inflammatory diseases, such as knee osteoarthritis and graft-versus-host disease. However, the widely used MSC culture systems in laboratories and clinics currently involve adding a certain amount of serum or blood products from animals or humans, such as fetal bovine serum (FBS), to the basal medium. While these systems can support MSC culture to some extent, animal or human blood products have complex origins, difficult-to-define components, and contain large amounts of xenogeneic or allogeneic immunogenic components such as proteins, enzymes, and growth factors. These components can have uncontrollable effects on the function, efficacy, and phenotype of MSCs. Furthermore, traceability and quantifiability cannot fully meet the needs of clinical-grade cell preparation production, thus limiting the clinical translation and application of MSCs. Furthermore, serum products are produced from animals, and significant variations exist between different origins and batches, making it difficult to guarantee the consistency of culture systems. This negatively impacts the reproducibility and reliability of research, hindering the standardization of cell preparation production and the development of in-depth mechanistic studies. In addition, animal- or human-derived serum may carry viruses, mycoplasma, or other potential pathogens, posing immunogenicity and safety risks, which complicates quality control of cell products and regulatory compliance for clinical applications. Compared to animal serum, human-derived alternatives, such as human serum or platelet lysate, are scarce, cannot be mass-produced, and have limited sources. The use of human-derived products also raises ethical concerns.

[0003] Although commercially available serum-free MSC media have achieved the goals of being serum-free, animal-free, and having defined chemical components, they still rely on exogenous matrix proteins or coatings to achieve cell adhesion. At the same time, the formulations often contain complex mixtures, such as B27, non-essential amino acids, and lipid additives with defined chemical components. This not only increases costs and weakens batch-to-batch consistency, but also lacks the ability to flexibly and precisely control the culture platform, making it difficult to meet the requirements of formulation production and basic scientific research for culture medium stability, reproducibility, and mechanism elucidation. Summary of the Invention

[0004] The present invention aims to provide a mesenchymal stem cell culture medium with a clearly defined chemical composition and free from immunogenic proteins or blood products derived from animals or humans, so as to ensure the controllability, safety and reproducibility of the cell culture process, while meeting the requirements for large-scale expansion of mesenchymal stem cells and preparation of formulations.

[0005] To address the above problems, a first aspect of the present invention provides a mesenchymal stem cell culture medium, comprising a basal culture medium and functional additives;

[0006] The functional additives include proteins, growth factors, small molecule compounds, fatty acids and lipids, polysaccharides, and adjuvants, wherein:

[0007] The proteins include recombinant human albumin, recombinant transferrin, and recombinant bone morphogenetic protein;

[0008] The growth factors include recombinant insulin or insulin-like growth factor, recombinant fibroblast growth factor, recombinant epidermal growth factor, and recombinant platelet-derived growth factor.

[0009] The small molecule compounds include thyroid hormones, ethanolamine, carnitine, glucocorticoid regulators, and retinoic acid compounds;

[0010] The fatty acids and lipids include signal-regulating lipids, cholesterol, DL-α-tocopheryl acetate, linolenic acid, oleic acid, and stearic acid;

[0011] The polysaccharide includes sodium heparin;

[0012] The adjuvants include Pronik F68 and Tween 80.

[0013] A second aspect of the present invention provides a method for preparing a mesenchymal stem cell culture medium, for preparing the mesenchymal stem cell culture medium as described in the first aspect, comprising the following steps:

[0014] Provide basic culture medium;

[0015] Proteins and growth factors were added to the basal culture medium and mixed thoroughly to prepare the first culture medium;

[0016] Small molecule compounds and polysaccharides were added to the first culture medium and mixed evenly to obtain the second culture medium.

[0017] Fatty acids, lipids, and additives are added to the second culture medium and mixed thoroughly to obtain the mesenchymal stem cell culture medium.

[0018] A third aspect of the present invention provides an application of a mesenchymal stem cell culture medium, wherein the mesenchymal stem cell culture medium as described in the first aspect or the mesenchymal stem cell culture medium prepared by the preparation method described in the second aspect is used for in vitro culture of mesenchymal stem cells.

[0019] The mesenchymal stem cell culture medium, its preparation method, and its application described in this invention, by adding functional additives to a basal culture medium, can form a mesenchymal stem cell culture medium with a clearly defined chemical composition. This mesenchymal stem cell culture medium does not contain animal or human blood products. Compared with related technologies that rely on animal or human blood products for mesenchymal stem cell culture, the mesenchymal stem cell culture medium provided in this invention has the following advantages:

[0020] (1) It can significantly improve the controllability of components and batch consistency. The mesenchymal stem cell culture medium provided in this embodiment has clear chemical composition and clear components. It does not contain complex biological extracts, which can effectively avoid component uncertainty and batch differences. It is conducive to establishing a standardized and reproducible cell culture and production process and technology. Moreover, the mesenchymal stem cell culture medium has clear components, strong traceability, and is suitable for large-scale preparation and automated production processes, which is conducive to process scale-up and compliance with GMP standards.

[0021] (2) It offers better safety and traceability, containing no animal or human blood products (such as fetal bovine serum, human serum, etc.), nor any non-recombinant enzymes, albumins, globulins, growth factors, or other biologically derived components with potential immunogenicity or batch instability. All components can use GMP-grade raw materials, reducing the risk of potential pathogen contamination and infectious diseases, while also lowering immunogenicity and better meeting the requirements for raw materials in cell therapy products;

[0022] (3) The mesenchymal stem cell culture medium provided in this embodiment can form and maintain MSC spherical aggregates under carrier-free conditions, and maintain the integrity and proliferation of spherical aggregates, which can support the process scale-up and GMP conversion; it can support the adherent culture of mesenchymal stem cells without coating or the addition of exogenous extracellular matrix proteins or adhesion molecules.

[0023] Furthermore, compared with mesenchymal stem cell culture media with clearly defined chemical compositions in related technologies, the mesenchymal stem cell culture medium provided in this invention does not contain complex compositions such as non-essential amino acids, lipid concentrates with clearly defined chemical compositions, or B27, significantly reducing the compositional complexity of the mesenchymal stem cell culture medium and improving its controllability and consistency. In this embodiment, the mesenchymal stem cell culture medium, by adding signal-regulating lipids, retinoic acid compounds, and FGF2, and using a low concentration of recombinant human albumin, enables the mesenchymal stem cell culture medium to regulate cell adhesion-related signaling pathways, cytoskeletal protein homeostasis, and extracellular matrix autosecretion. This achieves cell adhesion and two-dimensional adherent growth of mesenchymal stem cells on conventional tissue culture surfaces without relying on exogenous matrix proteins or coating materials. It enables cell adhesion without the addition of exogenous matrix proteins or coating materials, reducing process complexity and the safety and quality control risks associated with introducing additional exogenous proteins, thus further improving the safety of the mesenchymal stem cell culture medium. Attached Figure Description

[0024] Figure 1 Microscopic images of UC-MSCs cultured in mesenchymal stem cell culture medium in Example 2 for 72 hours;

[0025] Figure 2 The results of PDL obtained after culturing UC-MSCs in the mesenchymal stem cell culture medium of Example 2 and the conventional FBS culture system for 96 hours;

[0026] Figure 3 The bar chart shows the PDL obtained after culturing human MSCs for 96 hours in three control media formed by adding 10% FBS to three different basal media (DMEM / F-12, MEM Alpha and RPMI1640) and the three complete media with clearly defined chemical composition of the present invention.

[0027] Figure 4 The image shows the cell doubling level results obtained after 96 hours of cell culture using basal culture medium, traditional FBS culture system, and mesenchymal stem cell culture medium of the present invention for human umbilical cord-derived MSCs (UC-MSCs), adipose tissue-derived MSCs (AD-MSCs), colon-derived MSCs (Colon MSCs), liver-derived MSCs (Liver MSCs), and T-MSCs obtained by extraembryonic lineage induction of pluripotent stem cells.

[0028] Figure 5The graph shows the results of flow cytometry analysis of the expression levels of positive and negative biomarkers of UC-MSCs after three generations of culture in the traditional FBS culture system and the mesenchymal stem cell culture medium of the present invention.

[0029] Figure 6 The images show the cell morphology micrographs of mesenchymal stem cells cultured directly in tissue culture plates without any surface coating treatment for three days, along with the corresponding cell counting results and cell proliferation statistics.

[0030] Figure 7 The images show the results of microscopic imaging of UC-MSCs cultured at different initial seeding densities for 24 hours and 120 hours in a 10% FBS system and the mesenchymal stem cell culture medium of the present invention, along with the results of statistical microscopic imaging of spherical cell aggregates. Detailed Implementation

[0031] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0032] Furthermore, the terms "comprising," "including," "containing," and "having" are non-restrictive and can refer to the addition of other steps and components that do not affect the results. Unless otherwise specified, all materials, equipment, and reagents are commercially available.

[0033] Furthermore, although the present invention describes each step in the preparation process in the form of S110, S120, S130, etc., this description is only for ease of understanding. The forms such as S110, S120, S130 do not indicate a limitation on the order of the steps.

[0034] A first aspect of this application provides a mesenchymal stem cell culture medium, including a basal culture medium and functional additives;

[0035] Functional additives include proteins, growth factors, small molecule compounds, fatty acids and lipids, polysaccharides, and adjuvants, among which:

[0036] The proteins include recombinant human albumin, recombinant transferrin (or Optiferrin), and recombinant bone morphogenetic protein. In this embodiment, proteins support the adhesion and survival of MSCs. Specifically, recombinant human albumin can stabilize cell membrane structure, act as an essential carrier to assist in the transport and utilization of fat-soluble vitamins and fatty acids, and protect cells from metabolic waste and toxic substances (such as impurities in the CO2 atmosphere) inevitably introduced from the culture environment during culture. At higher concentrations, recombinant human albumin can non-specifically block various adhesion molecules and intercellular junction ligands on the surface of cell spherical aggregates, inhibiting cell-to-container adhesion and cell-to-cell adhesion and aggregation, thereby better maintaining the structural integrity of cell spherical aggregates in suspension. Recombinant transferrin is an iron ion transporter that can support cell metabolism and DNA synthesis by providing iron, while reducing cell damage caused by free radicals, which is essential for maintaining rapidly proliferating cells such as stem cells. Recombinant bone morphogenetic protein is an indispensable component for maintaining the stem cell properties of mesenchymal stem cells, cell expansion, and ensuring normal cell adhesion.

[0037] Growth factors include recombinant insulin or insulin-like growth factor, recombinant fibroblast growth factor 2 (FGF2), recombinant epidermal growth factor (EGF), and recombinant platelet-derived growth factor (PDGF). In this embodiment, growth factors are used to regulate the proliferation, stemness maintenance, or differentiation direction of MSCs. Specifically, recombinant insulin or insulin-like growth factor is an important component for maintaining normal cellular energy metabolism, while promoting protein synthesis through the mTOR signaling pathway and inhibiting apoptosis through the PI3K / AKT signaling pathway. Recombinant fibroblast growth factor, recombinant epidermal growth factor, and recombinant platelet-derived growth factor are indispensable components for maintaining the stem cell properties of mesenchymal stem cells, as well as cell expansion and ensuring normal cell adhesion.

[0038] The small molecule compounds include thyroid hormone, ethanolamine (EtA), carnitine, glucocorticoid regulators, and retinoic acid compounds. In this embodiment, the small molecule compounds are used to enhance the expansion efficiency of MSCs and inhibit their spontaneous differentiation. Specifically, thyroid hormone, ethanolamine, carnitine, glucocorticoid regulators, and retinoic acid compounds can effectively support key metabolic pathways of mesenchymal stem cells, maintain fatty acid balance, and support cell membrane structure, making them necessary additives for maintaining the expansion of mesenchymal stem cells in vitro; furthermore, retinoic acid compounds can synergistically promote cell adhesion and migration with FGF2.

[0039] Fatty acids and lipids include signal-regulating lipids, cholesterol, DL-α-tocopherol, linolenic acid, oleic acid, and stearic acid. In this embodiment, fatty acids and lipids are used to improve cell membrane structure and function. Specifically, cholesterol, DL-α-tocopherol, linolenic acid, oleic acid, and stearic acid can effectively support key metabolic pathways of mesenchymal stem cells, maintain fatty acid balance, and support cell membrane structure, making them necessary additives for maintaining mesenchymal stem cell proliferation in vitro. Lipids with signal-regulating activity are core components for promoting mesenchymal stem cell proliferation under serum-free and low-lipid conditions. They can mediate the activation of key pathways such as PI3K / AKT and ERK, promote proliferation, and regulate cell cycle progression and apoptosis balance.

[0040] The polysaccharide includes heparin sodium. In this embodiment, heparin sodium is an indispensable component for maintaining the stem cell properties of mesenchymal stem cells, as well as for cell proliferation and ensuring normal cell adhesion.

[0041] The adjuvants include Pluronic F68 (or Poloxamer 188 or Kolliphor 188) and Tween 80 (or Polysorbate 80). In this embodiment, Pluronic F68 and Tween 80 are crucial surfactants, shear protectants, and stabilizers, essential for the stability of the cell culture medium itself and the viability of cells in three-dimensional culture.

[0042] In this embodiment, recombinant fibroblast growth factor, recombinant epidermal growth factor, recombinant platelet-derived growth factor, recombinant bone morphogenetic protein, and sodium heparin are all indispensable components for maintaining the stem cell properties of mesenchymal stem cells, cell expansion, and ensuring normal cell adhesion. The lack of one or more of these components will lead to a decrease in the activity or homeostasis of signaling pathways closely related to the morphology and properties of mesenchymal cells during in vitro culture of mesenchymal stem cells, resulting in a decrease in cell morphology, differentiation potential, migration and adhesion ability, and proliferation ability.

[0043] The mesenchymal stem cell culture medium provided in this embodiment, by adding functional additives to the basal culture medium, can form a mesenchymal stem cell culture medium with a clearly defined chemical composition. This mesenchymal stem cell culture medium does not contain animal-derived or human-derived blood products. Compared with related technologies that rely on animal-derived or human-derived blood products, the mesenchymal stem cell culture medium provided in this embodiment has the following advantages:

[0044] (1) It can significantly improve the controllability of components and batch consistency. The mesenchymal stem cell culture medium provided in this embodiment has clear chemical composition and clear components. It does not contain complex biological extracts, which can effectively avoid component uncertainty and batch differences. It is conducive to establishing a standardized and reproducible cell culture and production process and technology. Moreover, the mesenchymal stem cell culture medium has clear components, strong traceability, and is suitable for large-scale preparation and automated production processes, which is conducive to process scale-up and compliance with GMP standards.

[0045] (2) Better safety and traceability: The components of the mesenchymal stem cell culture medium provided in this embodiment do not contain animal or human blood products (such as fetal bovine serum, human serum, etc.), nor do they contain non-recombinant enzymes, albumin, globulin, growth factors, or other biologically derived components with potential immunogenicity or batch instability from animals or humans. All components can use GMP-grade raw materials, reducing the risk of potential pathogen contamination and infectious diseases, while also reducing immunogenicity, which is more in line with the requirements for raw materials of cell therapy products;

[0046] (3) The mesenchymal stem cell culture medium provided in this embodiment can form and maintain MSC spherical aggregates under carrier-free conditions, and maintain the integrity and proliferation of spherical aggregates, which can support the process scale-up and GMP conversion; it can support the adherent culture of mesenchymal stem cells without coating or the addition of exogenous extracellular matrix proteins or adhesion molecules.

[0047] Furthermore, compared with mesenchymal stem cell culture media with clearly defined chemical compositions in related technologies, the mesenchymal stem cell culture medium provided in this embodiment does not contain complex compositions such as non-essential amino acids, lipid concentrates with clearly defined chemical compositions, or B27, significantly reducing the compositional complexity of the mesenchymal stem cell culture medium and improving its controllability and consistency. Moreover, by adding signal-regulating lipids, retinoic acid compounds, and FGF2, and using a low concentration of recombinant human albumin, the mesenchymal stem cell culture medium provided in this embodiment can regulate cell adhesion-related signaling pathways, cytoskeletal protein homeostasis, and extracellular matrix autosecretion. This enables mesenchymal stem cells to adhere to and grow in two dimensions on conventional tissue culture surfaces without relying on exogenous matrix proteins or coating materials. It achieves cell adhesion without adding exogenous matrix proteins or coating materials, reducing process complexity and the safety and quality control risks associated with introducing additional exogenous proteins, thus further improving the safety of the mesenchymal stem cell culture medium.

[0048] Based on the above embodiments, as an optional implementation, the contents of the functional additives in the mesenchymal stem cell culture medium are as follows:

[0049] The concentration of recombinant human albumin was 0.02% w / v to 0.5% w / v;

[0050] The concentration of recombinant transferrin was 2 µg / mL to 20 µg / mL;

[0051] The concentration of recombinant insulin or insulin-like growth factor was 2 µg / mL to 20 µg / mL;

[0052] The concentration of recombinant fibroblast growth factor was 10 ng / mL to 50 ng / mL;

[0053] The concentration of recombinant epidermal growth factor was 10 ng / mL to 50 ng / mL;

[0054] The concentration of recombinant platelet-derived growth factor was 10 ng / mL to 50 ng / mL;

[0055] The concentration of recombinant bone morphogenetic protein was 5 ng / mL to 20 ng / mL;

[0056] The concentration of heparin sodium is 5 µg / mL to 10 µg / mL;

[0057] The concentration of thyroid hormones ranges from 1 ng / mL to 50 ng / mL;

[0058] The concentration of ethanolamine ranges from 1 µmol / L to 40 µmol / L;

[0059] The concentration of carnitine ranges from 1 µmol / L to 100 µmol / L;

[0060] The concentration of glucocorticoid regulators ranges from 10 nmol / L to 300 nmol / L;

[0061] The concentration of retinoids ranged from 10 nmol / L to 300 nmol / L;

[0062] The concentrations of lipids with signal-regulating activity ranged from 10 nmol / L to 10 µmol / L;

[0063] Cholesterol concentrations ranged from 2.5 µg / mL to 15 µg / mL;

[0064] The concentration of DL-α-tocopherol acetate is from 1 µg / mL to 10 µg / mL;

[0065] The concentration of linolenic acid ranges from 30 ng / mL to 3000 ng / mL;

[0066] The concentration of oleic acid is from 30 ng / mL to 300 ng / mL;

[0067] The concentration of stearic acid is from 30 ng / mL to 300 ng / mL;

[0068] The concentration of Pronic F68 ranges from 0.01 mg / mL to 1 mg / mL;

[0069] The concentration of Tween 80 ranges from 10 µg / mL to 100 µg / mL.

[0070] In this embodiment, the content of functional additives is controlled within the above-mentioned range, which can effectively avoid precipitation or impact on cell membrane structure integrity and homeostasis caused by excessively high concentrations of lipids (such as cholesterol and fatty acids), ensuring the stable dispersion and bioavailability of fatty acids and lipids; at the same time, it avoids the risk of apoptosis or stem cell inhibition caused by excessively high concentrations of small molecules such as glucocorticoid regulators; the concentrations of growth factors and protein components are optimized to maintain their proliferative activity while preventing receptor saturation or signal desensitization due to excessive concentrations, significantly reducing the ineffective loss of expensive recombinant proteins; controlling the content of functional additives within the above-mentioned range balances functional synergy, safety and economy, reduces the cost of large-scale production, and improves the industrialization feasibility of stem cell culture medium.

[0071] Based on the above embodiments, as an optional implementation, the lipids with signal-regulating activity are lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), platelet-activating factor (PAF), or ceramides. These lipids with signal-regulating activity have a good proliferative effect on mesenchymal stem cells. As a preferred embodiment, the concentration of the lipids with signal-regulating activity is from 100 nmol / L to 10 µmol / L. Of course, those skilled in the art can select other suitable lipids with signal-regulating activity according to the actual situation.

[0072] In this embodiment, the thyroid hormone is triiodothyronine (T3) or thyroxine (T4). As a preferred embodiment, T3 is used because it is a directly bioactive thyroid hormone that can directly bind to intracellular nuclear receptors, promoting cell maintenance in vitro and exhibiting higher conversion efficiency. T4, on the other hand, is a precursor to T3. The bioactivity of T4 depends on the deiodination activation by 5′ deiodinase, but the expression level of 5′ deiodinase is limited in mesenchymal stem cells, thus limiting the conversion efficiency of T4. Therefore, in this embodiment, T3 is preferably used as the thyroid hormone.

[0073] Based on the above embodiments, as an optional implementation, glucocorticoid regulators include hydrocortisone, dexamethasone, prednisolone, or betamethasone. Of course, those skilled in the art can select other suitable glucocorticoid regulators according to the actual situation.

[0074] Based on the above embodiments, as an optional implementation, the retinoic acid compound includes all-trans retinoic acid, 9-cis retinoic acid, or 13-cis retinoic acid. As a preferred embodiment, the retinoic acid compound is all-trans retinoic acid. Of course, those skilled in the art can select other suitable retinoic acid compounds according to the actual situation.

[0075] Based on the above embodiments, as an optional implementation, the mesenchymal stem cell culture medium further includes a signal regulating factor. This signal regulating factor is a non-animal-derived component capable of inhibiting the TGFβ signaling pathway. The signal regulating factor includes SB431542 or A83-01. Of course, those skilled in the art can select other suitable signal regulating factors according to actual conditions. By adding a signal regulating factor to the mesenchymal stem cell culture medium, the TGFβ signaling pathway can be inhibited, maintaining the morphology and integrity of cell aggregates under three-dimensional culture conditions, reducing shear force damage caused by adhesion between aggregates, thereby regulating the in vitro state of MSCs, maintaining undifferentiated characteristics, or improving expansion performance. As an optional implementation, if the signal regulating factor is SB431542, the concentration of SB431542 is 3 µmol / L to 50 µmol / L; if the signal regulating factor is A83-01, the concentration of A83-01 is 0.5 µmol / L to 10 µmol / L.

[0076] Based on the above embodiments, as an optional implementation, the basal culture medium includes DMEM medium, DMEM-HG medium, DMEM / F-12 medium, RPMI-1640 medium, Opti-MEM medium, or MEM alpha medium, or equivalent variants of the above-mentioned media within the acceptable range under normal experimental conditions. The above-mentioned basal culture media do not contain any animal or human-derived components and provide inorganic salts, amino acids, vitamins, carbon sources, buffer systems, and trace elements to provide the nutrition and environment required for MSC growth. It should be noted that the above-mentioned basal culture media can be commercially available products or basal culture media with clearly defined chemical compositions prepared using publicly available formulations.

[0077] Based on the above embodiments, as an optional implementation, the pH value of the mesenchymal stem cell culture medium is 7.1 to 7.4. In specific operation, an aqueous solution of hydrochloric acid or sodium bicarbonate can be used to adjust the pH value of the mesenchymal stem cell culture medium to between 7.1 and 7.4. Therefore, this pH value provides a suitable "microenvironment" for mesenchymal stem cells, which is beneficial to their growth.

[0078] The second aspect of this application provides a method for preparing a mesenchymal stem cell culture medium, used to prepare the mesenchymal stem cell culture medium as described in the first aspect, comprising the following steps:

[0079] Step S110: Provide the basic culture medium;

[0080] Step S120: Add protein and growth factors to the basal culture medium, mix well, and prepare the first culture medium; specifically, add recombinant human albumin, recombinant transferrin, recombinant insulin or insulin-like growth factor, recombinant fibroblast growth factor, recombinant epidermal growth factor, recombinant platelet-derived growth factor and recombinant bone morphogenetic protein to the basal culture medium, mix well, and prepare the first culture medium.

[0081] Step S130: Add small molecule compounds and polysaccharides to the first culture medium, mix well, and prepare the second culture medium; specifically, add heparin sodium, thyroid hormone, ethanolamine, carnitine, glucocorticoid regulators and retinoic acid compounds to the first culture medium, mix well, and prepare the second culture medium.

[0082] Step S140: Add fatty acids, lipids, and adjuvants to the second culture medium, mix well, and obtain mesenchymal stem cell culture medium; specifically, add signal-regulating lipids, cholesterol, DL-α-tocopherol acetate, linolenic acid, oleic acid, stearic acid, Pronicotinic F68, and Tween 80 to the second culture medium, mix well, and obtain the mesenchymal stem cell culture medium.

[0083] The method for preparing mesenchymal stem cell culture medium provided in this embodiment effectively avoids mutual interference between protein and lipid components and their solvents during the mixing process by adding proteins, growth factors, small molecule compounds, fatty acids and lipids to the basal culture medium in batches. This significantly improves the structural stability and biological activity of recombinant proteins (such as growth factors and albumin). At the same time, it promotes the dispersion and dissolution of hydrophobic lipids (such as cholesterol, fatty acids and tocopherol esters) in the aqueous phase, thereby improving their bioavailability. The gentle mixing in each step avoids the loss of activity caused by shearing or precipitation, ensuring that the functional added components synergistically exert the best culture efficiency.

[0084] Based on the above embodiments, as an optional implementation, before adding the protein and growth factor to the basal culture medium, the method further includes: preparing the protein and growth factor into a high-concentration stock solution; specifically, dissolving and diluting the protein and growth factor in sterile water for injection, phosphate buffer, or basal culture medium to prepare a high-concentration stock solution, and then adding it to the basal culture medium in proportion to achieve the final concentration. As a preferred implementation, to improve the stability of the above-mentioned protein and growth factor and reduce their denaturation due to adsorption, denaturation, and aggregation during preparation and storage, one or more stabilizers may be added to the above-mentioned high-concentration stock solution. The stabilizers include, but are not limited to: recombinant human albumin (0.01% to 1% (w / v)), Pronico F68 (0.001% to 0.1%), trehalose (1% to 10% (w / v)), and polyethylene glycol (molecular weight 200 to 6000, 0.1% to 5%).

[0085] Based on the above embodiments, as an optional implementation, before adding the small molecule compound to the first culture medium, the method further includes: preparing a solution of the small molecule compound; specifically, dissolving the small molecule compound in sterile water for injection or phosphate buffer solution, and, if necessary, adding dimethyl sulfoxide to ensure the full dissolution and bioavailability of the small molecule compound.

[0086] Based on the above embodiments, as an optional implementation, before adding fatty acids and lipids to the second culture medium, the method further includes: preparing a fatty acid and lipid solution; specifically, dissolving the fatty acids and lipids in sterile water for injection or phosphate buffer solution, and using ethanol or dimethyl sulfoxide as a co-solvent to ensure sufficient dissolution of the fatty acids and lipids. As a preferred implementation, to improve the dispersibility and stability of fatty acids and lipids in an aqueous system, Pronique F68 or Tween 80 may be further added to the fatty acid and lipid solution as a carrier, wherein the concentration of Pronique F68 is preferably 0.02% to 0.1%, and the concentration of Tween 80 is preferably 0.02% to 0.1%.

[0087] In this embodiment, each step is mixed using a gentle mixing method to reduce the degradation or deactivation of the functional additives.

[0088] Based on the above embodiments, as an optional implementation, after the mesenchymal stem cell culture medium is prepared, if aseptic treatment is required, it is filtered and sterilized using a 0.22μm PES filter in a Class A laminar flow hood, then dispensed and sealed, and stored in the dark at 2℃ to 8℃, or frozen at -20℃, to avoid repeated freeze-thaw cycles.

[0089] A third aspect of this application provides an application of a mesenchymal stem cell culture medium, such as the mesenchymal stem cell culture medium described in the first aspect or the mesenchymal stem cell culture medium prepared by the preparation method described in the second aspect, for the in vitro culture of mesenchymal stem cells. Specifically, the in vitro culture of mesenchymal stem cells provided in this embodiment includes: expansion of MSCs, maintenance of MSCs, functional regulation of MSCs (including regulation of immune regulation, secretory function, or multilineage differentiation potential), directed differentiation of MSCs into specific cell lineages, preparation of MSC-derived derivatives, and preparation of MSC cell preparations or exosomes.

[0090] The mesenchymal stem cell culture medium provided in this embodiment has a wide range of applications. It can be used not only for the maintenance, expansion, functional regulation, and differentiation of MSCs, but also for the preparation of cell preparations or exosome production. Furthermore, the mesenchymal stem cells provided in this embodiment can support the in vitro maintenance and culture of mesenchymal stem cells from various tissue sources, and have broad applicability.

[0091] To provide a more detailed description of the present invention, specific embodiments will be described below. Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are all commercially available. For example: recombinant human albumin was purchased from Yuanpei S479JJ; recombinant transferrin was purchased from Yuanpei S482T0; recombinant insulin was purchased from Yuanpei S454T0; recombinant insulin-like growth factor was purchased from PeproTech 100-11; recombinant fibroblast growth factor was purchased from PeproTech GMP100-18B; recombinant epidermal growth factor was purchased from PeproTech GMP100-15; recombinant platelet-derived growth factor was purchased from Novoprotein GMP-C199; recombinant bone morphogenetic protein was purchased from R&D Systems #314E-GMP; sodium heparin was purchased from Sigma H3393; triiodothyronine was purchased from Sigma T6397; thyroxine was purchased from Sigma T2376; and ethanolamine was purchased from Sigma. 398136; L-carnitine was purchased from Sigma C0287; all-trans retinoic acid was purchased from Sigma R2625; 9-cis retinoic acid was purchased from MCE HY-15128; 13-cis retinoic acid was purchased from Sigma R3255; hydrocortisone was purchased from Sigma H4001; dexamethasone was purchased from Sigma D4902; prednisone was purchased from MCE HY-B0214; prednisolone was purchased from MCE HY-17463; betamethasone was purchased from MCE HY-13570; lysophosphatidylcholine was purchased from ENZOBML-LP100; cholesterol was purchased from Yuanpei S497T7; DL-α-tocopherol acetate was purchased from Sigma 29992; linolenic acid was purchased from Sigma L2376; oleic acid was purchased from Sigma O1383; stearic acid was purchased from Sigma S4751; Pronico F68 was purchased from Sigma K4894; Tween 80 was purchased from Sigma P4780; SB431542 was purchased from SelleckChem S1067; A83-01 was purchased from MCE HY-10432. Furthermore, regarding the mesenchymal stem cell culture medium provided in this invention, those skilled in the art should understand that, without departing from the spirit and scope of this invention, all pharmaceutically acceptable substitutions of the compounds with salts, esters, isomers, derivatives, etc., should be included within the scope of protection of this invention.

[0092] Example 1

[0093] This embodiment provides a basal culture medium free of immunogenic proteins or blood products derived from animals or humans. The basal culture medium uses DMEM / F-12 solution or dry powder as the basal culture medium, and the pH value of the basal culture medium is 7.1 to 7.4.

[0094] The basal culture medium was prepared using the following method:

[0095] (1) Add the selected basic culture medium powder to sterile water for injection of cell culture grade according to the instructions to prepare a liquid base solution, or directly use a liquid base solution with the same composition that has been dissolved.

[0096] (2) After heating the liquid base solution to 35°C to 37°C, add a small amount of sterile injectable aqueous solution of hydrochloric acid or sodium bicarbonate of cell culture grade to adjust the pH to 7.1 to 7.4 to obtain the basic culture medium; if additional buffering is required during the preparation process, 10 mmol / L to 25 mmol / L of HEPES can be added (which can be prepared by 1 mol / L of HEPES stock solution, which can be purchased from Yuanpei B110JV);

[0097] (3) If the basic culture medium needs to be sterilized, it should be filtered and sterilized using a 0.22μm PES filter in a Class A laminar flow hood, then dispensed and sealed, and stored in the dark at 2℃~8℃, or frozen at -20℃, avoiding repeated freeze-thaw cycles.

[0098] (4) Before use, the clarity, pH and osmotic pressure of the basic culture medium should be tested; microscopic examination should be performed to detect no microbial contamination, and the medium can be used after passing the test.

[0099] Example 2

[0100] This embodiment provides a mesenchymal stem cell culture medium with a clearly defined chemical composition. This culture medium includes a basal culture medium and functional additives that promote the in vitro maintenance and expansion of MSCs. The basal culture medium is the one prepared in Example 1, and the functional additives include:

[0101] Recombinant human albumin, at a concentration of 0.05% w / v;

[0102] Recombinant transferrin at a concentration of 10 µg / mL;

[0103] Recombinant insulin at a concentration of 10 µg / mL;

[0104] Recombinant fibroblast growth factor at a concentration of 20 ng / mL;

[0105] Recombinant epidermal growth factor at a concentration of 20 ng / mL;

[0106] Recombinant platelet-derived growth factor at a concentration of 10 ng / mL;

[0107] Recombinant bone morphogenetic protein at a concentration of 5 ng / mL;

[0108] Sodium heparin, at a concentration of 5 µg / mL;

[0109] Triiodothyronine, at a concentration of 2 ng / mL;

[0110] Ethanolamine, with a concentration of 16.37 µmol / L;

[0111] L-carnitine, with a concentration of 12.4 µmol / L;

[0112] Hydrocortisone, with a concentration of 100 nmol / L;

[0113] All-trans retinoic acid, with a concentration of 100 nmol / L;

[0114] Lysophosphatidic acid, with a concentration of 1 µmol / L;

[0115] Cholesterol, at a concentration of 7.5 µg / mL;

[0116] DL-α-tocopherol acetate, with a concentration of 1.7 µg / mL;

[0117] Linolenic acid, at a concentration of 1 µg / mL;

[0118] Oleic acid, at a concentration of 100 ng / mL;

[0119] Stearic acid, at a concentration of 100 ng / mL;

[0120] Pronico F68 (Kolliphor 188), with a concentration of 0.9 mg / mL;

[0121] Tween 80, with a concentration of 22 µg / mL.

[0122] The basal culture medium was prepared using the following method:

[0123] (1) Add protein and growth factor to the basal culture medium and mix evenly to prepare the first culture medium; specifically, add recombinant human albumin, recombinant transferrin, recombinant insulin or insulin-like growth factor, recombinant fibroblast growth factor, recombinant epidermal growth factor, recombinant platelet-derived growth factor and recombinant bone morphogenetic protein to the basal culture medium and mix gently evenly to prepare the first culture medium; wherein, before adding protein and growth factor to the basal culture medium, dissolve and dilute protein and growth factor in sterile water for injection, phosphate buffer or basal culture medium respectively to prepare high-concentration stock solutions, and then add them to the basal culture medium in proportion to achieve the final target concentration; and in order to improve stability, one or more stabilizers may be added to the high-concentration stock solutions.

[0124] (2) Add small molecule compounds and polysaccharides to the first culture medium, mix them evenly, and prepare the second culture medium; specifically, add heparin sodium, thyroid hormone, ethanolamine, carnitine, glucocorticoid regulator and retinoic acid compound to the first culture medium, mix them evenly, and prepare the second culture medium; wherein, before adding small molecule compounds to the first culture medium, dissolve the small molecule compounds in sterile water for injection or phosphate buffer solution, and dimethyl sulfoxide may be added for dissolution;

[0125] (3) Add fatty acids, lipids and adjuvants to the second culture medium and mix evenly to obtain mesenchymal stem cell culture medium; specifically, add signal-regulating lipids, cholesterol, DL-α-tocopherol acetate, linolenic acid, oleic acid, stearic acid, Pronic F68 and Tween 80 to the second culture medium and mix gently evenly to obtain mesenchymal stem cell culture medium; wherein, before adding fatty acids and lipids to the second culture medium, dissolve fatty acids and lipids in sterile water for injection or phosphate buffer solution, and ethanol or dimethyl sulfoxide can be used as a cosolvent, and add Pronic F68 (0.02% to 0.1%) as a carrier.

[0126] If the mesenchymal stem cell culture medium requires aseptic treatment, it should be filtered and sterilized using a 0.22μm PES filter in a Class A laminar flow hood, then dispensed, sealed, and stored in the dark at 2℃~8℃.

[0127] Example 3

[0128] The ability of the mesenchymal stem cell culture medium with clearly defined chemical composition prepared in this embodiment to support the in vitro proliferation of MSCs was verified using the following method:

[0129] Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were processed at a density of 8,000 cells / cm². 2The cells were seeded at a density specified in the chemical composition of the mesenchymal stem cell culture medium prepared in Example 2 to form the complete culture medium group of the present invention.

[0130] UC-MSCs were cultured at a density of 8,000 cells / cm². 2 The cells were inoculated at a density in a conventional serum culture system (i.e., a control medium made by adding 10% (v / v) fetal bovine serum (FBS) to commercially available DMEM / F-12 basal medium) to form an FBS control group.

[0131] The complete culture medium group and the FBS control group of this invention were cultured at 37°C, 5% CO2, and saturated humidity. All culture medium was replaced every 48 hours. After 96 hours, cells were digested and collected using TrypLE™ Select enzyme digest solution supplemented with 25 U / mL Benzonase nuclease. Cells were counted by flow cytometry and the Population Doubling Level (PDL) was calculated.

[0132] Figure 1 Microscopic images of UC-MSCs cultured in the mesenchymal stem cell culture medium of Example 2 for 72 hours. Figure 1 It can be seen that, Figure 1 No morphological abnormalities or enlargement of UC-MSCs were observed, indicating that UC-MSCs can be maintained normally in the mesenchymal stem cell culture medium described in Example 2 of this invention.

[0133] Figure 2 shows the proliferative density (PDL) results of UC-MSCs after culturing for 96 hours in the mesenchymal stem cell culture medium of Example 2 and the conventional FBS culture system. The PDL results were used to assess the proliferation level of UC-MSCs in the two culture systems. Figure 2 It can be seen that the mesenchymal stem cell culture medium in Example 2 of the present invention is comparable to the traditional FBS culture system in terms of the proliferation capacity of UC-MSCs. No significant difference was observed in the cell doubling level between the two groups of cells within 72 hours of culture, suggesting that the mesenchymal stem cell culture medium of the present invention can achieve cell expansion effect comparable to the traditional serum system and can effectively support the in vitro proliferation of mesenchymal stem cells.

[0134] Example 4

[0135] The universality of the functional additives in this embodiment in different basal culture media was verified using the following methods:

[0136] Three commercially available liquid base solutions, DMEM / F-12, MEM Alpha, and RPMI1640, were selected as the base culture media that do not contain immunogenic proteins or blood products derived from animals or humans. The functional additives from Example 2 were added to the above three base culture media according to the formula and concentration to form three different complete culture media with clearly defined chemical compositions.

[0137] UC-MSCs were cultured at a density of 8,000 cells / cm². 2 Cells were seeded at a density in three different complete culture media. The three different complete culture media were cultured at 37°C, 5% CO2, and saturated humidity. All culture media were replaced every 48 hours. After 96 hours, cells were digested and collected using TrypLE™ Select enzyme digest solution supplemented with 25 U / mL Benzonase nuclease. Cells were counted by flow cytometry and the Population Doubling Level (PDL) was calculated.

[0138] Figure 3 shows the PDL (Proteinization Product Length) of three control media (DMEM / F-12, MEM Alpha, and RPMI 1640) with 10% FBS added, compared to the three chemically defined complete media of the present invention, after culturing human MSCs for 96 hours. This PDL chart measures the suitability of the functional additives in the present invention in different basal media. Figure 3 As can be seen, under three different basal media, the cell proliferation levels obtained by the chemically defined complete culture medium of this invention were consistent with those of FBS, with no significant differences. This result indicates that the chemically defined culture medium formulation of this invention can effectively support the expansion of MSCs in different basal media, demonstrating good applicability and broad versatility.

[0139] Example 5

[0140] The applicability of the mesenchymal stem cell culture medium with clearly defined chemical composition prepared in this embodiment to MSCs from various sources was verified using the following methods:

[0141] Mesenchymal stem cells from different sources, including human umbilical cord-derived mesenchymal stem cells (UC-MSCs), adipose tissue-derived mesenchymal stem cells (AD-MSCs), colon-derived mesenchymal stem cells (Colon MSCs), liver-derived mesenchymal stem cells (Liver MSCs), and T-MSCs derived from pluripotent stem cells through differentiation from trophoblastic ectoderm or extraembryonic lineages, were divided into groups at a density of 8,000 cells / cm². 2The density was inoculated into the basal culture medium prepared in Example 1 to form the basal culture medium group;

[0142] Mesenchymal stem cells from different sources, including human umbilical cord-derived mesenchymal stem cells (UC-MSCs), adipose tissue-derived mesenchymal stem cells (AD-MSCs), colon-derived mesenchymal stem cells (Colon MSCs), liver-derived mesenchymal stem cells (Liver MSCs), and T-MSCs derived from pluripotent stem cells through differentiation from trophoblastic ectoderm or extraembryonic lineages, were divided into groups at a density of 8,000 cells / cm². 2 The cells were seeded at a density specified in the chemical composition of the mesenchymal stem cell culture medium prepared in Example 2 to form the complete culture medium group of the present invention.

[0143] Mesenchymal stem cells from different sources, including human umbilical cord-derived mesenchymal stem cells (UC-MSCs), adipose tissue-derived mesenchymal stem cells (AD-MSCs), colon-derived mesenchymal stem cells (Colon MSCs), liver-derived mesenchymal stem cells (Liver MSCs), and T-MSCs derived from pluripotent stem cells through differentiation from trophoblastic ectoderm or extraembryonic lineages, were divided into groups at a density of 8,000 cells / cm². 2 The cells were inoculated at a density in a conventional serum culture system (i.e., a control medium made by adding 10% (v / v) fetal bovine serum (FBS) to commercially available DMEM / F-12 basal medium) to form an FBS control group.

[0144] The basal culture medium group, the complete culture medium group of this invention, and the FBS control group were cultured at 37°C, 5% CO2, and saturated humidity. All culture media were replaced every 48 hours. After 96 hours, cells were digested and collected using TrypLE™ Select enzyme digest solution supplemented with 25 U / mL Benzonase nuclease. Cells were counted by flow cytometry and the Population Doubling Level (PDL) was calculated.

[0145] Figure 4 shows the cell doubling level results obtained after 96 hours of cell culture using basal culture medium, traditional FBS culture system, and the mesenchymal stem cell culture medium of the present invention for human umbilical cord-derived MSCs (UC-MSCs), adipose tissue-derived MSCs (AD-MSCs), colon-derived MSCs (Colon MSCs), liver-derived MSCs (Liver MSCs), and T-MSCs obtained by extraembryonic lineage induction of pluripotent stem cells.

[0146] Depend on Figure 4As can be seen, under the basal culture medium conditions described in Example 1 alone, several different MSCs could not proliferate effectively; cell growth was stagnant, and the PDL remained almost 0. This indicates that the basal culture medium itself is insufficient to support the in vitro expansion of mesenchymal stem cells. In contrast, the traditional serum culture system with 10% fetal bovine serum added and the well-defined mesenchymal stem cell culture medium constructed in this invention achieved significant cell multiplication, with the PDL of MSCs from different sources reaching approximately 3 after 96 hours of culture. This further confirms that the mesenchymal stem cell culture medium provided by this invention has consistent expansion efficiency with the traditional serum culture system, indicating that the mesenchymal stem cell culture medium of this invention can be used to support the proliferation of mesenchymal stem cells from different sources and has broad applicability.

[0147] Example 6

[0148] The ability of the mesenchymal stem cell culture medium with clearly defined chemical composition prepared in this embodiment to maintain the cell properties of MSCs in vitro was verified by the following method:

[0149] Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were processed at a density of 8,000 cells / cm². 2 Cells were seeded at the density specified in Example 2 on the chemically defined mesenchymal stem cell culture medium and cultured at 37°C, 5% CO2, and saturated humidity. The entire culture medium was replaced every 48 hours until cell confluence reached approximately 80%. Cells were then digested and collected using TrypLE™ Select digestion solution supplemented with 25 U / mL Benzonase, and re-seeded and passaged at the same initial seeding density. After passage to the third generation in the corresponding culture medium, cells were digested and collected again using TrypLE™ Select digestion solution supplemented with 25 U / mL Benzonase, and the expression of cell surface markers (including CD90, CD105, and CD73) was analyzed using the BD Stemflow™ Human MSC Analysis Kit.

[0150] Figure 5 shows the results of flow cytometry analysis of the expression levels of positive and negative biomarkers in UC-MSCs after three passages in both the traditional FBS culture system and the mesenchymal stem cell culture medium of this invention. Positive biomarkers included CD105 (PerCP5.5), CD90 (FITC), and CD73 (APC). Negative biomarkers were detected using a negative biomarker cocktail, including CD34 (PE), CD11b (PE), CD19 (PE), CD45 (PE), and HLA-DR (PE). The black curve represents the signal of the target biomarker expression after antigen-antibody staining, and the gray curve represents the isotype control. Figure 5 It can be seen that MSCs cultured in both the traditional FBS culture system and the mesenchymal stem cell culture medium of the present invention exhibit typical surface antigen expression characteristics. Among them, the positive markers CD105, CD90, and CD73 all showed high expression levels with obvious positive peaks. Meanwhile, the negative markers, which were not expressed, did not show significant peak shifts in either culture medium, indicating that the cell characteristics did not shift, nor did they differentiate into hematopoietic or immune lineages. These results demonstrate that the chemically defined mesenchymal stem cell culture medium of the present invention can maintain the phenotypic stability of mesenchymal stem cells in vitro, comparable to a serum system.

[0151] Example 7

[0152] To verify whether the mesenchymal stem cell culture medium with clearly defined chemical composition prepared in this embodiment can support the proliferation of mesenchymal stem cells without the need for extracellular matrix proteins and coating, the specific method is as follows:

[0153] Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) were processed at a density of 8,000 cells / cm². 2 Cells were seeded at a density in the chemically defined mesenchymal stem cell culture medium prepared in Example 2 and cultured at 37°C with 5% CO2. No coating treatment, such as with collagen, fibronectin, or matrix gel, was applied to the culture surface during the culture process. All culture medium was replaced every 48 hours. After three consecutive days of culture, the cells were observed under a microscope, and cell morphology images were acquired. Simultaneously, cell counts were performed after three days of culture, and the cell proliferation rate was calculated.

[0154] Figure 6 shows microscopic images of mesenchymal stem cells directly cultured in tissue culture plates without any surface coating treatment for three days, along with corresponding cell counting and fold proliferation statistics. Figure 6As can be seen, under uncoated conditions, mesenchymal stem cells (MSCs) can effectively adhere to the culture surface, exhibiting a typical fibroblast-like morphology with uniform cell morphology and good condition. Furthermore, even without coating, MSCs can still achieve significant expansion, with the final cell number and expansion fold comparable to traditional FBS-containing culture media. These results demonstrate that the MSC culture medium of this invention can support the adhesion, growth, and proliferation of MSCs without relying on exogenous matrix coating, indicating synergistic effects among the components of the MSC culture medium, endowing the medium with the ability to directly support the adhesion and proliferation of MSCs, a type of stromal cell that secretes its own extracellular matrix proteins. This invention simplifies the culture process, reduces culture costs, and minimizes batch variations and potential risks associated with exogenous coating materials, making it suitable for research and industrial applications of MSC culture.

[0155] Example 8

[0156] The ability of the mesenchymal stem cell culture medium with clearly defined chemical composition prepared in this embodiment to induce mesenchymal stem cells to form cell spheroids and maintain 3D formation was verified. The specific method is as follows:

[0157] SB431542 was added to the mesenchymal stem cell culture medium with a clearly defined chemical composition in Example 2 at a concentration of 20 µmol / L, and the concentration of recombinant human albumin was increased to 0.5% (w / v) to obtain an optimized mesenchymal stem cell culture medium. Using the optimized mesenchymal stem cell culture medium, UC-MSCs were cultured in low-adsorption U-shaped bottom 96-well plates. Specifically, UC-MSCs were seeded into the optimized mesenchymal stem cell culture medium at densities of 5,000, 10,000, and 20,000 cells per well to form the complete culture medium group of this invention.

[0158] UC-MSCs were seeded at densities of 5,000, 10,000, and 20,000 cells per well in a conventional serum culture system (i.e., a control medium consisting of commercially available DMEM / F-12 basal medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 0.2% human recombinant albumin) to form the FBS control group.

[0159] The complete culture medium group and the FBS control group of the present invention were cultured at 37°C, 5% CO2, and saturated humidity. After inoculation, the medium was centrifuged at 300g for 2 minutes in a horizontal rotor centrifuge, and then centrifuged at 100g for 1 minute. After static culture for 24 hours, the medium was transferred to a horizontal shaker at 37 rpm for culture. Half of the culture medium was replaced every 48 hours.

[0160] After 24 hours of static culture and subsequent cell spheroidization, and after 120 hours of maintenance, the cell spheroids were imaged using a microscopic imaging system and their diameter was analyzed using ImageJ.

[0161] Figure 7 The images show the results of microscopic imaging of UC-MSCs cultured at different initial seeding densities for 24 hours and 120 hours in a 10% FBS system and the mesenchymal stem cell culture medium of the present invention, along with the results of statistical microscopic imaging of spherical cell aggregates. Figure 7 The left side shows a microscopic image of spherical aggregates of cells, and the right side shows a quantitative analysis of the change in the diameter of the aggregates over time. Figure 7 As can be seen from the microscopic imaging results, UC-MSCs cultured in both the 10% FBS system and the mesenchymal stem cell culture medium of the present invention, at different initial seeding densities (5,000, 10,000, and 20,000 cells per 96-well plate), formed typical spherical aggregates in a low-adsorption U-shaped bottom environment 24 hours after seeding. In both different culture systems, an increase in the diameter of the cell spherical aggregates was observed with increasing cell number, indicating that the initial density significantly affects the initial value of the cell spherical size. In the mesenchymal stem cell culture medium of the present invention, the diameter of the cell aggregates was larger, suggesting that the mesenchymal stem cell culture medium of the present invention can more effectively complete cell aggregation and maintain initial cell proliferation.

[0162] After 120 hours of continued culture, the diameter of all cell aggregates in the 10% FBS system showed a significant decrease, with an average diameter reduction ranging from 27.31% to 40.77%. This suggests that the cell aggregates in the 10% FBS system contracted and disintegrated, indicating that the 10% FBS system cannot efficiently maintain the morphology and stability of three-dimensional cell spheroids. Conversely, in the mesenchymal stem cell culture medium of the present invention, cell aggregates with different initial seeding densities were able to maintain their spherical structure and size, with highly consistent diameters between 24 and 120 hours, and no obvious contraction or disintegration was observed. In particular, in the mesenchymal stem cell culture medium of the present invention, spherical aggregates formed by seeding 5,000 cells per 96 wells showed an average diameter increase of 7.13% after 96 hours of continued culture, indicating that the mesenchymal stem cell culture medium of the present invention can not only maintain the stability of the three-dimensional cell spheroid structure, but also support further cell proliferation in the form of spherical aggregates.

[0163] The above results demonstrate that inhibition of the TGFβ signaling pathway and increased albumin concentration in the culture medium enable more successful three-dimensional cell culture with almost no observed adhesion of spherical aggregates to low-adhesion surfaces. Furthermore, compared to culture systems containing FBS, the mesenchymal stem cell culture medium of this invention is not only suitable for adherent culture and expansion of MSCs, but also better maintains cells under three-dimensional culture conditions, effectively preserving the structure and size of MSC spheroids, exhibiting superior three-dimensional stability and cell aggregation support compared to traditional serum systems.

Claims

1. A mesenchymal stem cell culture medium, characterized in that, Includes basal culture medium and functional additives; The functional additives include proteins, growth factors, small molecule compounds, fatty acids and lipids, polysaccharides, and adjuvants, wherein: The proteins include recombinant human albumin, recombinant transferrin, and recombinant bone morphogenetic protein; The growth factors include recombinant insulin or insulin-like growth factor, recombinant fibroblast growth factor, recombinant epidermal growth factor, and recombinant platelet-derived growth factor. The small molecule compounds include thyroid hormones, ethanolamine, carnitine, glucocorticoid regulators, and retinoic acid compounds; The fatty acids and lipids include signal-regulating lipids, cholesterol, DL-α-tocopheryl acetate, linolenic acid, oleic acid, and stearic acid; The polysaccharide includes sodium heparin; The adjuvants include Pronik F68 and Tween 80.

2. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The concentration of the recombinant human albumin is from 0.02% w / v to 0.5% w / v; The concentration of the recombinant transferrin is from 2 µg / mL to 20 µg / mL; The concentration of the recombinant insulin or insulin-like growth factor is from 2 µg / mL to 20 µg / mL; The concentration of the recombinant fibroblast growth factor is from 10 ng / mL to 50 ng / mL; The concentration of the recombinant epidermal growth factor is from 10 ng / mL to 50 ng / mL; The concentration of the recombinant platelet-derived growth factor is from 10 ng / mL to 50 ng / mL; The concentration of the recombinant bone morphogenetic protein is from 5 ng / mL to 20 ng / mL; The concentration of the heparin sodium is from 5 µg / mL to 10 µg / mL; The concentration of the thyroid hormone is from 1 ng / mL to 50 ng / mL; The concentration of the ethanolamine is from 1 µmol / L to 40 µmol / L; The concentration of carnitine is from 1 µmol / L to 100 µmol / L; The concentration of the glucocorticoid regulator is from 10 nmol / L to 300 nmol / L; The concentration of the retinoic acid compound is from 10 nmol / L to 300 nmol / L; The concentration of the lipids with signal-regulating activity is from 10 nmol / L to 10 µmol / L; The cholesterol concentration is from 2.5 µg / mL to 15 µg / mL; The concentration of DL-α-tocopherol acetate is from 1 µg / mL to 10 µg / mL; The concentration of the linolenic acid is from 30 ng / mL to 3000 ng / mL; The concentration of oleic acid is from 30 ng / mL to 300 ng / mL; The concentration of stearic acid is from 30 ng / mL to 300 ng / mL; The concentration of the pronicotinic F68 is from 0.01 mg / mL to 1 mg / mL; The concentration of Tween 80 is from 10 µg / mL to 100 µg / mL.

3. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The lipids with signal-regulating activity are lysophosphatidic acid, sphingosine-1-phosphate, platelet-activating factor, or ceramide.

4. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The thyroid hormone in question is triiodothyronine.

5. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The functional additives also include signal modulation factors, including SB431542 or A83-01; If the signal modulation factor is SB431542, then the concentration of SB431542 is from 3 µmol / L to 50 µmol / L; If the signal regulation factor is A83-01, then the concentration of A83-01 is 0.5 µmol / L to 10 µmol / L.

6. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The basal culture medium includes DMEM medium, DMEM-HG medium, DMEM / F-12 medium, RPMI-1640 medium, Opti-MEM medium, or MEMalpha medium.

7. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The pH value of the mesenchymal stem cell culture medium is 7.1 to 7.

4.

8. A method for preparing a mesenchymal stem cell culture medium, characterized in that, The preparation of the mesenchymal stem cell culture medium as described in any one of claims 1 to 7 comprises the following steps: Provide basic culture medium; Proteins and growth factors were added to the basal culture medium and mixed thoroughly to prepare the first culture medium; Small molecule compounds and polysaccharides were added to the first culture medium and mixed evenly to obtain the second culture medium. Fatty acids, lipids, and additives are added to the second culture medium and mixed thoroughly to obtain the mesenchymal stem cell culture medium.

9. An application of a mesenchymal stem cell culture medium, characterized in that, The mesenchymal stem cell culture medium as described in any one of claims 1 to 7, or the mesenchymal stem cell culture medium prepared by the preparation method as described in claim 8, is used for in vitro culture of mesenchymal stem cells.

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