Mesenchymal stem cell serum-free medium with limited chemical components as well as preparation method and application of mesenchymal stem cell serum-free medium

Abstract

The invention relates to a serum-free culture medium with limited chemical components for mesenchymal stem cells as well as a preparation method and application of the serum-free culture medium. The culture medium comprises a basic culture medium, growth factors and a carrier protein stabilizer, the growth factors comprise a proliferation promoting factor, an activating agent, a signal factor and nutrients; the activating agent comprises Wnt7a protein and LIF protein. According to the invention, Wnt7a, LIF, Noggin, Activin A and TGF-beta3 are combined together according to a specific concentration and proportion (especially Wnt7a and LIF) for the first time, and cooperate with other factors (FGF-2 and IGF-1) to construct a brand new signal network capable of simulating an in-vivo dry microenvironment.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and cell engineering technology, and in particular to a chemically defined serum-free culture medium for mesenchymal stem cells, its preparation method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) have shown great promise in regenerative medicine, immunotherapy, and tissue engineering due to their multipotent differentiation potential, potent immunomodulatory functions, and paracrine effects. However, the clinical application of MSCs faces a major bottleneck: difficulties in in vitro expansion. Traditional MSC culture typically relies on culture media containing fetal bovine serum (FBS).

[0003] Traditional culture media have the following problems: Their composition is unclear, containing hundreds or even thousands of unknown proteins, hormones, and growth factors, resulting in large batch-to-batch variations and poor experimental reproducibility and cell quality stability. They also pose high safety risks, potentially carrying pathogens such as viruses and mycoplasma, introducing immunogenicity, and presenting potential biosafety risks, severely hindering the standardization and approval of cell therapy products. Furthermore, FBS (Follicular Unit Extract) has functional limitations; it cannot optimally support the long-term expansion of MSCs, and cells typically show signs of aging such as slowed proliferation, flattened morphology, spontaneous differentiation, and loss of stemness markers after 5-10 passages, significantly reducing their therapeutic efficacy.

[0004] To address these issues, serum-free culture media have been developed. However, some media still contain human-derived components, such as human platelet lysate (hPL). While superior to FBS, these media still present risks related to pathogens, batch-to-batch variability, and uncertain composition, and are not truly "chemically limited." Growth factor combinations are often limited, with most formulations containing only a few growth factors such as FGF-2. This fails to mimic the complex microenvironment of the in vivo stem cell nest and cannot effectively maintain the primitive stemness and long-term proliferative capacity of MSCs. Simultaneous maintenance of proliferation and function is impossible; some formulations may promote rapid cell proliferation, but at the cost of early loss of cell function (such as decreased immunosuppressive capacity). Furthermore, the lack of regulation of key signaling pathways means that the media fails to systematically and synergistically regulate signaling pathways crucial for stem cell fate, such as Wnt, LIF, and TGF-β / BMP.

[0005] Therefore, there is an urgent need in this field to develop a serum-free culture medium with completely defined components, free of any animal / human impurities, capable of synergistically activating multiple stemness maintenance pathways, thereby supporting the long-term stable expansion of MSCs and with complete functionality. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a chemically defined serum-free culture medium for mesenchymal stem cells, its preparation method, and its application. This invention is the first to combine Wnt7a, LIF, Noggin, Activin A, and TGF-β3 at specific concentrations and ratios (especially Wnt7a and LIF), and synergize them with other factors (FGF-2, IGF-1) to construct a novel signaling network that can simulate the in vivo stem microenvironment.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a serum-free culture medium for mesenchymal stem cells, the culture medium comprising a basal culture medium, growth factors and a carrier protein stabilizer; the growth factors comprising proliferative factors, activators, signaling factors and nutrients; the activators comprising Wnt7a protein and LIF protein.

[0009] In this invention, the combination of Wnt7a and LIF is creatively introduced, and it is found that when their mass ratio is between 5:1 and 2:1, a significant synergistic effect can be produced, effectively activating stemness-related signaling pathways (such as STAT3), while avoiding differentiation caused by overactivation of the Wnt pathway, which is crucial for maintaining the undifferentiated state of MSCs.

[0010] Preferably, the mass ratio of the Wnt7a protein to the LIF protein is (2-5):1. The (2-5) can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, or 5.

[0011] Preferably, the signaling factor includes any one or a combination of at least two of Noggin protein, Activin A, or TGF-β3.

[0012] In this invention, specific signaling factors are added, with Noggin (a BMP signaling inhibitor) and Activin A / TGF-β3 (a TGF-β signaling activator) forming a delicate regulatory "switch". This combination mimics the signal balance of the in vivo niche, effectively inhibiting the spontaneous differentiation of MSCs (especially osteogenic differentiation) and promoting the maintenance of their immunomodulatory phenotype.

[0013] Preferably, the proliferative factors include FGF-2 and / or IGF-1.

[0014] Preferably, the nutrients include recombinant human insulin and / or recombinant human transferrin.

[0015] Preferably, the culture medium contains a proliferation-promoting factor concentration of 15-45 μg / mL, an activator concentration of 4-15 ng / mL, a signaling factor concentration of 6.5-22 ng / mL, and a nutrient concentration of 2-15 ng / mL. The 15-45 μg / mL concentration can be, for example, 15 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, or 45 μg / mL. The 4-15 ng / mL concentration can be, for example, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, or 15 ng / mL. The 6.5-22 ng / mL can be, for example, 6.5 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL or 22 ng / mL, etc. The 2-15 ng / mL can be, for example, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 14 ng / mL or 15 ng / mL, etc.

[0016] Preferably, the proliferative factors include FGF-2 and IGF-1.

[0017] Preferably, the concentration of FGF-2 in the culture medium is 5-15 ng / mL, and the concentration of IGF-1 is 10-30 ng / mL. The 5-15 ng / mL concentration can be, for example, 5 ng / mL, 7 ng / mL, 9 ng / mL, 11 ng / mL, 13 ng / mL, or 15 ng / mL. The 10-30 ng / mL concentration can be, for example, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, or 30 ng / mL.

[0018] Preferably, the signaling factors include Noggin protein, Activin A, and TGF-β3.

[0019] Preferably, the concentration of Noggin protein in the culture medium is 5-15 ng / mL, the concentration of Activin A is 0.5-2 ng / mL, and the concentration of TGF-β3 is 1-5 ng / mL. The 5-15 ng / mL concentration can be, for example, 5 ng / mL, 7 ng / mL, 9 ng / mL, 11 ng / mL, 13 ng / mL, or 15 ng / mL. The 0.5-2 ng / mL concentration can be, for example, 0.5 ng / mL, 0.7 ng / mL, 0.9 ng / mL, 1.1 ng / mL, 1.3 ng / mL, 1.5 ng / mL, 1.7 ng / mL, 1.9 ng / mL, or 2 ng / mL. The 1-5 ng / mL concentration can be, for example, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, or 5 ng / mL.

[0020] Preferably, the nutrients include recombinant human insulin and recombinant human transferrin.

[0021] Preferably, the concentration of recombinant human insulin in the culture medium is 1-10 μg / mL and the concentration of recombinant human transferrin is 1-5 μg / mL. The 1-10 μg / mL concentration can be, for example, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL. Similarly, the 1-5 μg / mL concentration can be, for example, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, or 5 μg / mL.

[0022] Preferably, the carrier protein stabilizer comprises recombinant human serum albumin and / or polyethylene glycol-modified liposomes.

[0023] In this invention, adding recombinant human serum albumin and polyethylene glycol-modified liposomes to the culture medium can provide fatty acids and cholesterol, and stabilize various growth factors in the solution, preventing them from adsorbing onto the container wall and degrading.

[0024] Preferably, the carrier protein stabilizer comprises recombinant human serum albumin and polyethylene glycol-modified liposomes.

[0025] Preferably, the concentration of recombinant human serum albumin in the culture medium is 0.5-5 mg / mL. The 0.5-5 mg / mL concentration can be, for example, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL.

[0026] Preferably, the volume concentration of polyethylene glycol-modified liposomes in the culture medium is 0.005-0.02%. The 0.005-0.02% can be, for example, 0.005%, 0.007%, 0.009%, 0.011%, 0.013%, 0.015%, 0.017%, 0.019%, or 0.02%.

[0027] Preferably, the basal culture medium includes α-MEM culture medium and additives.

[0028] Preferably, the additives include trace elements, metabolic regulators, and antioxidants.

[0029] Preferably, the trace elements include any one or at least a combination of sodium selenate, zinc sulfate, or copper sulfate.

[0030] Preferably, the metabolic regulator includes α-ketoglutarate and / or nicotinamide.

[0031] Preferably, the antioxidant comprises L-ascorbic acid-2-phosphate magnesium and / or reduced glutathione.

[0032] Preferably, the culture medium contains trace element concentrations of 5.6-11.5 μM, metabolic regulator concentrations of 4-13 mM, and antioxidant concentrations of 1.05-2.1 mM. The 5.6-11.5 μM concentration can be, for example, 5.6 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, or 11.5 μM.

[0033] Preferably, the trace elements include sodium selenate, zinc sulfate, and copper sulfate.

[0034] Preferably, the concentration of sodium selenate in the culture medium is 0.1-0.5 μM, the concentration of zinc sulfate is 5-10 μM, and the concentration of copper sulfate is 0.5-1 μM. The 0.1-0.5 μM can be, for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, or 0.5 μM. The 0.5-1 μM can be, for example, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM. The 5-10 μM can be, for example, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

[0035] In this invention, a specific combination of trace elements is added. At a specific concentration, they act as cofactors for multiple key enzymes, working synergistically to regulate cellular antioxidant stress capacity (selenium, zinc), DNA synthesis (zinc), and cellular energy metabolism (copper).

[0036] Preferably, the metabolic regulator includes α-ketoglutarate and nicotinamide.

[0037] In this invention, α-ketoglutarate and nicotinamide are added. α-ketoglutarate is an intermediate product of the tricarboxylic acid cycle, which can promote energy metabolism and affect the epigenetic state of cells; nicotinamide is a precursor of NAD+, which helps maintain cellular energy and redox homeostasis. The combination of these two substances reprograms the metabolic pattern of MSCs, making them more adapted to rapid proliferation.

[0038] Preferably, the concentration of α-ketoglutarate in the culture medium is 1-5 mM and the concentration of nicotinamide is 3-8 mM. The 1-5 mM can be, for example, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM. The 3-8 mM can be, for example, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM.

[0039] Preferably, the antioxidant includes L-ascorbic acid-2-phosphate magnesium and reduced glutathione.

[0040] In this invention, the addition of antioxidants provides strong and stable intracellular and extracellular antioxidant protection, effectively neutralizing reactive oxygen species (ROS) accumulated in in vitro culture, which is key to delaying cell aging.

[0041] Preferably, the concentration of L-ascorbic acid-2-phosphate magnesium in the culture medium is 50-100 μg / mL and the concentration of reduced glutathione is 1-2 mM. The 50-100 μg / mL concentration can be, for example, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL. The 1-2 mM concentration can be, for example, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, or 2 mM.

[0042] Preferably, the basal culture medium further includes a buffer solution.

[0043] Preferably, the buffer solution comprises HEPES and sodium bicarbonate.

[0044] Preferably, the HEPES concentration in the culture medium is 10-25 mM (the 10-25 mM can be, for example, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM or 25 mM, etc.) and the sodium bicarbonate concentration is 10-25 mM (the 10-25 mM can be, for example, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM or 25 mM, etc.).

[0045] In a second aspect, the present invention provides a method for preparing the serum-free culture medium for mesenchymal stem cells described in the first aspect, the method comprising filtering and mixing a basal culture medium, growth factors and a carrier protein stabilizer.

[0046] Thirdly, the present invention provides a method for culturing mesenchymal stem cells, the method comprising seeding mesenchymal stem cells into the serum-free culture medium for mesenchymal stem cells described in the first aspect for culturing.

[0047] Compared with the prior art, the present invention has at least the following beneficial effects:

[0048] 1. This invention is the first to combine Wnt7a, LIF, Noggin, Activin A and TGF-β3 in specific concentrations and ratios (especially Wnt7a:LIF) and synergize them with other factors (FGF-2, IGF-1) to construct a novel signaling network that can simulate the in vivo dry microenvironment.

[0049] 2. This invention is not a simple additive process, but rather a synergistic regulation of multiple key signaling pathways (Wnt, JAK-STAT, TGF-β / BMP) based on a deep understanding of the biological characteristics of MSCs. This "multi-target" strategy is not obvious and has achieved unexpected technical results: stable expansion of MSCs for more than 15 generations with intact function has been achieved under completely serum-free, platelet lysate-free, and chemically-constrained conditions. It creatively resolves the contradiction between "maintaining proliferation" and "maintaining stemness / function." Comparative examples demonstrate that the absence of any key factor (such as Noggin or alteration of the Wnt7a:LIF ratio) significantly reduces the effectiveness.

[0050] 3. All components of this invention are chemically synthesized or recombinantly expressed, free of platelet lysates, human and animal origins, completely eliminating pathogen contamination and immunogenicity, and meeting the high standards of raw material regulation for cell therapy products. It supports efficient amplification of MSCs from multiple sources with extremely high batch-to-batch consistency, laying the foundation for the industrial and standardized production of MSCs. It is suitable for both traditional two-dimensional culture and microcarrier-based three-dimensional bioreactor culture, facilitating large-scale amplification and possessing extremely high industrial transformation value. Attached Figure Description

[0051] Figure 1 A comparison chart of amplification folds over 72 hours.

[0052] Figure 2 Comparison of cell morphology under different treatments.

[0053] Figure 3 This is an image showing the identification of surface markers in the 5th generation MSC.

[0054] Figure 4 This is an image showing the identification of surface markers in the 15th generation MSC.

[0055] Figure 5 The images show the results of the adipogenic differentiation function identification. In the image, A is a bar chart and B is a cell comparison chart.

[0056] Figure 6 The results of osteogenic differentiation function identification are shown in Figure A, which is a bar chart, and Figure B is a cell comparison chart.

[0057] Figure 7 The results of the chondrogenic differentiation function identification are shown in Figure A, which is a bar chart, and Figure B is a slice of cell clusters.

[0058] Figure 8 This is a diagram showing the cell morphology of adipose-derived mesenchymal stem cells.

[0059] Figure 9 This is a diagram showing the cell morphology of bone marrow mesenchymal stem cells.

[0060] Figure 10 This is a diagram of the cell morphology of human amniotic mesenchymal stem cells.

[0061] Figure 11 This is a comparison chart showing the effects of Wnt7a and LIF at different scales.

[0062] Figure 12 This is a comparison chart showing the impact of adding Wnt7a and LIF on the effects.

[0063] Figure 13 This is a comparison chart of the effects of different signal factors. Detailed Implementation

[0064] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0065] The reagents used in the following examples were sourced from the following sources:

[0066] Recombinant human insulin: Brand: McLean, Product No.: R917753-500 mg.

[0067] Recombinant human transferrin: Brand: Sigma, Product No.: T3705-5G.

[0068] Recombinant human serum albumin: Brand: Heyuan Biotechnology, Product No.: HYC001C01.

[0069] α-MEM medium: Brand: Thermo, Product No.: 12000063.

[0070] Wnt7a: Brand: abcam, Item No.: ab171487.

[0071] LIF: Brand: MCE, Item No.: HY-P73278.

[0072] Example 1

[0073] This embodiment describes the preparation of a chemically defined serum-free culture medium for mesenchymal stem cells.

[0074] (1) Preparation of growth factors and carrier protein stabilizers

[0075] Under a sterile laminar flow hood, take a sterile container and add 80 mL of water for injection. Weigh or measure each component according to 100 times the final concentration: recombinant human FGF-2, IGF-1, Wnt7a, LIF, Noggin, Activin A, TGF-β3, recombinant human insulin, recombinant human transferrin, recombinant human serum albumin, and PEGylated liposomes. Add the above components one by one to the water, stir slowly to dissolve completely, and bring the volume to 100 mL. Filter the solution sterilely using a 0.22 μm PES filter membrane.

[0076] (2) Preparation of basal culture medium

[0077] Take 1 L of α-MEM dry powder culture medium and dissolve it in ultrapure water according to its instructions. Add sodium selenate, zinc sulfate, copper sulfate, α-ketoglutarate, nicotinamide, L-ascorbic acid-2-phosphate magnesium, reduced glutathione, and HEPES. After stirring and dissolving thoroughly, add sodium bicarbonate to adjust the pH to 7.4. Add ultrapure water to the final volume of 1 L and filter sterilely using a 0.22 μm filter membrane.

[0078] Take 495 mL of basal culture medium, add 5 mL of 100-fold growth factor and carrier protein stabilizer, and gently shake to prepare the culture medium. In this example, three groups of serum-free mesenchymal stem cell culture media were prepared, with specific formulations shown in Table 1. The cell culture results are shown in Table 2. It can be seen that cells cultured using the above formulation showed good expansion performance, and the expansion effect was relatively stable within the specified range.

[0079] Table 1

[0080]

[0081] Table 2

[0082]

[0083] Example 2

[0084] This embodiment describes the isolation and primary culture of human umbilical cord-derived MSCs.

[0085] In this embodiment, fresh human umbilical cords were collected with the informed consent of the mother. The tissue block adhesion method was used: Wharton's jelly tissue from the umbilical cord was cut into 1 mm³ pieces and evenly attached to the bottom of a T25 culture flask, with 3 mL of the complete culture medium prepared in Example 1 added. The flask was incubated statically at 37°C in a 5% CO2 incubator. After 6 days, spindle-shaped cells were observed to emerge from the edge of the tissue block. The medium was completely changed every 3 days. When the cell confluence reached 85%, the flask was passaged.

[0086] Example 3

[0087] This embodiment describes the long-term subculturing of MSCs and the effect of the culture medium.

[0088] The primary MSCs (P0 generation) obtained in Example 2 were digested with trypsin-EDTA at a concentration of 1 × 10⁻⁶. 4 Cells were seeded at a density of cells / cm² in new culture flasks. The culture media used were Group 1 from Example 1, commercial serum-free medium, and DMEM medium containing 10% FBS. The medium was changed every 3 days, and the cell number was counted and the fold increase was calculated per passage. Specific results are shown below. Figure 1 As shown, compared to commercial serum-free culture medium and DMEM medium containing 10% FBS, the cell proliferation rate in the culture medium group of this invention was the fastest, expanding 12-fold after 72 hours of culture. Cell observation was also performed, as shown... Figure 2 As shown, the cells in the culture medium of this invention can be stably passaged to the 15th generation, while the proliferation of commercial serum-free culture medium slows down significantly after the 8th generation, and DMEM culture medium containing 10% FBS shows obvious senescence morphology in the 8th generation.

[0089] Example 4

[0090] This embodiment performs MSC surface marker identification.

[0091] MSCs cultured to passage 5 and passage 15 using the method in Group 1 of Example 3 were analyzed for surface markers by flow cytometry. Specific results are as follows: Figure 3 and Figure 4 As shown, cells from passages 5 and 15 highly expressed CD73, CD90, and CD105 (positive rates >98%), but did not express hematopoietic cell markers CD34, CD45, and HLA-DR (positive rates <2%). This indicates that the culture medium of this invention can maintain the phenotype of MSCs stably and for a long period of time.

[0092] Example 5

[0093] This embodiment describes the identification of MSC multi-directional differentiation potential.

[0094] In this embodiment, MSCs were cultured to the 8th generation using the method in Group 1 of Example 3, and osteogenic, adipogenic, and chondrogenic differentiation were induced respectively. Osteogenic induction: After 21 days of culture in osteogenic induction medium (containing dexamethasone, sodium β-glycerophosphate, and ascorbic acid), obvious red calcium nodules were observed after Alizarin Red staining. Adipogenic induction: After 21 days of culture in adipogenic induction medium (containing IBMX, dexamethasone, insulin, and indomethacin), red lipid droplets were observed filling the cells after Oil Red O staining. Chondrogenic induction: Using the microcluster culture method, after 21 days of culture in chondrogenic induction medium (containing TGF-β3, ITS, ascorbic acid, etc.), a large amount of blue proteoglycan deposition was observed after Alcian blue staining.

[0095] Specific results from Figure 5 , Figure 6 and Figure 7 As shown, this demonstrates that MSCs amplified using the medium of this invention still retain their complete multi-directional differentiation ability.

[0096] Adipogenic differentiation function, such as Figure 5 As shown in Figures A and B, after adipogenic differentiation, the cell morphology changed from elongated spindle shape to oval or round shape. Transparent lipid droplets were visible in the cytoplasm of some cells. After Oil Red O staining, a large number of round lipid droplets of varying sizes were visible, while the control group cells remained fibroblast-like and failed to stain. Gene expression showed that the expression of FABP4 and PPAR-γ in the induced group was significantly higher than that in the control group.

[0097] Osteogenic differentiation function, such as Figure 6 As shown in Figures A and B, after osteogenic differentiation, the cell morphology changes to polygonal or irregular shapes. Alizarin Red S staining reveals red calcified matrix deposits between cells, while the control group cells remain fibroblast-like and fail to stain. Gene expression shows that the expression of OCN and ALP in the induced group is significantly higher than that in the control group.

[0098] Chondrogenic differentiation function, such as Figure 7 As shown in Figures A and B, after the cells differentiate into chondrocytes, the cell clusters form milky white spheres with a smooth, non-porous surface and a certain degree of toughness. They stain blue with Alcian blue. Gene expression shows that the expression of COL2A1 and ACAN in the induced group is significantly higher than that in the control group.

[0099] Example 6

[0100] This embodiment verifies the culture of MSCs from different tissue sources.

[0101] In this embodiment, MSCs were isolated from human adipose tissue (liposuction waste), human bone marrow (donor), and human amnion (postpartum placenta), and cultured using the method described in Group 1 of Example 3. Figure 8 , Figure 9 and Figure 10As shown, MSCs from all sources adhered and proliferated well, exhibiting uniform morphology and a typical spindle shape, demonstrating the wide applicability of the culture medium of this invention.

[0102] Example 7

[0103] This embodiment explores the effect of activators on efficacy.

[0104] (1) Verification of the effects of different ratios of Wnt7a and LIF

[0105] Based on Group 1 of Example 3, with other components remaining unchanged, three different Wnt7a:LIF mass ratio culture media were prepared: Group A: 5:1, Group B: 2:1, and Group C: 1:1, for culturing human umbilical cord MSCs up to the 10th generation. Results are as follows... Figure 11 As shown, the culture media in groups A and B were most effective in maintaining cell proliferation rate. In group C, cell proliferation slowed down in the later stages. This demonstrates that Wnt7a and LIF have the best synergistic effect within a specific ratio range of 5:1 to 2:1.

[0106] (2) Verification of the effect of adding Wnt7a and LIF

[0107] Based on Group 1 of Example 3, with other components remaining unchanged, three different groups were prepared. Group A contained Wnt7a and LIF; Group B did not contain LIF, but the weight proportion of LIF was allocated to Wnt7a according to the proportion in Group 1; Group C did not contain Wnt7a, but the weight proportion of Wnt7a was allocated to LIF according to the proportion in Group 1. Specific results are as follows: Figure 12 As shown, the absence of either Wnt7a or LIF significantly reduces the proliferation effect, indicating that Wnt7a and LIF have a significant synergistic effect.

[0108] Example 8

[0109] This embodiment explores the influence of signal factors on the effect.

[0110] Based on Group 1 of Example 3, with other components remaining unchanged, three different groups were prepared. Group A contained Noggin, Activin A, and TGF-β3; Group B did not contain Noggin and distributed Noggin to Activin A and TGF-β3 according to the ratio in Group 1; Group C did not contain Activin A and distributed Activin A to Noggin and TGF-β3 according to the ratio in Group 1; Group D did not contain TGF-β3 and distributed TGF-β3 to Activin A and TGF-β3 according to the ratio in Group 1. Specific results are as follows... Figure 13As shown, Noggin, Activin A, and TGF-β3 have a significant synergistic effect, and this combination can promote cell proliferation.

[0111] Example 9

[0112] Application of this embodiment in a microcarrier three-dimensional culture system

[0113] Place the 3D StarPore® Max microcarriers in a 125 mL spin bottle and equilibrate with PBS. Inoculate with fifth-generation umbilical cord MSCs and culture using the complete culture medium prepared based on group 1 in Example 3. Maintain the spin speed at 80 rpm and change 50% of the medium every 2 days.

[0114] The specific results are shown in Table 3. The cells adhered well to the microcarriers and proliferated rapidly, reaching high density after 4 days. Fluorescent staining revealed that the cells formed dense multilayers on the microcarrier surface. After digestion and collection, cell counts showed a viability >95%, and the phenotype remained unchanged. This demonstrates that the culture medium of this invention is fully suitable for large-scale three-dimensional bioreactor culture.

[0115] Table 3

[0116]

[0117] Example 10

[0118] This embodiment detects cellular immune regulation function.

[0119] MSCs cultured to passage 10 using the method in Group 1 of Example 3 were co-cultured with human peripheral blood mononuclear cells (PBMCs) activated by phytohemagglutinin (PHA). The immunosuppressive capacity of MSCs was assessed by detecting PBMC proliferation (CCK-8 assay).

[0120] Compared with the control group without MSCs, the MSCs cultured in the medium of this invention can significantly inhibit the proliferation of PBMCs, with an inhibition rate of over 70%, and the effect is better than that of MSCs cultured in FBS medium at the same time, indicating that their immunomodulatory function is better maintained.

[0121] Example 11

[0122] This embodiment involves a culture medium stability test.

[0123] Three batches of the complete culture medium prepared at different times according to this invention were stored at 4°C for 0, 1, 2, and 4 weeks, and then used to culture the same batch of P3 generation umbilical cord MSCs. The viable cell density and doubling time were measured on day 3 after cell seeding.

[0124] The three batches of culture medium showed no significant difference in cell growth performance during the 4-week storage period (p>0.05), demonstrating that the culture medium of the present invention has good batch-to-batch consistency and short-term storage stability.

[0125] In summary, this invention is the first to combine Wnt7a, LIF, Noggin, Activin A and TGF-β3 in specific concentrations and ratios (especially Wnt7a and LIF), and in synergy with other factors (FGF-2, IGF-1), to construct a novel signaling network that can simulate the in vivo dry microenvironment.

[0126] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A serum-free culture medium for mesenchymal stem cells, characterized in that, The culture medium includes a basal culture medium, growth factors, and carrier protein stabilizers; The growth factors include proliferation-promoting factors, activators, signaling factors, and nutrients; The activators include Wnt7a protein and LIF protein.

2. The serum-free culture medium for mesenchymal stem cells according to claim 1, characterized in that, The mass ratio of Wnt7a protein to LIF protein is (2-5):1; Preferably, the signaling factor includes any one or a combination of at least two of Noggin protein, Activin A, or TGF-β3; Preferably, the proliferative factors include FGF-2 and / or IGF-1; Preferably, the nutrients include recombinant human insulin and / or recombinant human transferrin.

3. The serum-free culture medium for mesenchymal stem cells according to claim 1 or 2, characterized in that, The culture medium contains a proliferation-promoting factor concentration of 15-45 μg / mL, an activator concentration of 4-15 ng / mL, a signaling factor concentration of 6.5-22 ng / mL, and a nutrient concentration of 2-15 ng / mL.

4. The serum-free culture medium for mesenchymal stem cells according to any one of claims 1-3, characterized in that, The proliferative factors include FGF-2 and IGF-1; Preferably, the concentration of FGF-2 in the culture medium is 5-15 ng / mL, and the concentration of IGF-1 is 10-30 ng / mL; Preferably, the signaling factors include Noggin protein, Activin A, and TGF-β3; Preferably, the concentration of Noggin protein in the culture medium is 5-15 ng / mL, the concentration of Activin A is 0.5-2 ng / mL, and the concentration of TGF-β3 is 1-5 ng / mL; Preferably, the nutrients include recombinant human insulin and recombinant human transferrin; Preferably, the concentration of recombinant human insulin in the culture medium is 1-10 μg / mL and the concentration of recombinant human transferrin is 1-5 μg / mL.

5. The serum-free culture medium for mesenchymal stem cells according to any one of claims 1-4, characterized in that, The carrier protein stabilizer includes recombinant human serum albumin and / or polyethylene glycol-modified liposomes; Preferably, the carrier protein stabilizer comprises recombinant human serum albumin and PEGylated liposomes; Preferably, the concentration of recombinant human serum albumin in the culture medium is 0.5-5 mg / mL; Preferably, the volume concentration of polyethylene glycol-modified liposomes in the culture medium is 0.005-0.02%.

6. The serum-free culture medium for mesenchymal stem cells according to any one of claims 1-5, characterized in that, The basal culture medium includes α-MEM medium and additives; Preferably, the additives include trace elements, metabolic regulators, and antioxidants; Preferably, the trace elements include any one or a combination of at least two of sodium selenate, zinc sulfate, or copper sulfate; Preferably, the metabolic regulator comprises α-ketoglutarate and / or nicotinamide; Preferably, the antioxidant comprises L-ascorbic acid-2-phosphate magnesium and / or reduced glutathione.

7. The serum-free culture medium for mesenchymal stem cells according to any one of claims 1-6, characterized in that, The culture medium contains trace element concentrations of 5.6-11.5 μM, metabolic regulator concentrations of 4-13 mM, and antioxidant concentrations of 1.05-2.1 mM. Preferably, the trace elements include sodium selenate, zinc sulfate, and copper sulfate; Preferably, the concentration of sodium selenate in the culture medium is 0.1-0.5 μM, the concentration of zinc sulfate is 5-10 μM, and the concentration of copper sulfate is 0.5-1 μM. Preferably, the metabolic regulator includes α-ketoglutarate and nicotinamide; Preferably, the concentration of α-ketoglutarate in the culture medium is 1-5 mM and the concentration of nicotinamide is 3-8 mM; Preferably, the antioxidant comprises L-ascorbic acid-2-phosphate magnesium and reduced glutathione; Preferably, the concentration of L-ascorbic acid-2-phosphate magnesium in the culture medium is 50-100 μg / mL and the concentration of reduced glutathione is 1-2 mM.

8. The serum-free culture medium for mesenchymal stem cells according to any one of claims 1-7, characterized in that, The basic culture medium also includes a buffer solution; Preferably, the buffer solution comprises HEPES and sodium bicarbonate; Preferably, the concentration of HEPES in the culture medium is 10-25 mM and the concentration of sodium bicarbonate is 10-25 mM.

9. A method for preparing serum-free culture medium for mesenchymal stem cells according to any one of claims 1-8, characterized in that, The preparation method includes filtering the basal culture medium, growth factors and carrier protein stabilizer, and then mixing them to obtain the final product.

10. A method for culturing mesenchymal stem cells, characterized in that, The method includes seeding mesenchymal stem cells into the serum-free culture medium for mesenchymal stem cells according to any one of claims 1-8 for culture.

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