A culture medium, a preparation method and a culture method for industrialized preparation of mesenchymal stem cells

Abstract

This invention discloses a culture medium, preparation method, and culture method for the industrial-scale preparation of mesenchymal stem cells, belonging to the field of cell therapy technology. The culture medium comprises a basal medium, cell growth factors, cell proliferation promoters, a vitamin mixture, a nucleoside mixture, and an amino acid mixture; wherein the cell growth factors are selected from one or more of TGF-β, bFGF, VEGF, PDGF, EGF, and IGF. Through the synergistic effect of the composite nutrient system, this invention significantly improves the proliferation rate and cell viability of mesenchymal stem cells, maintains stem cell stemness, trilineage differentiation ability, and secretion levels of key cytokines (VEGF, HGF, TGF-β, IL-10, etc.); it can be stably and continuously passaged up to the 15th generation without abnormal changes in genetic material. This culture medium contains no animal-derived components, has high batch-to-batch stability, and is cost-controllable, making it suitable for GMP-level large-scale industrial preparation of mesenchymal stem cells from various sources such as umbilical cord, bone marrow, adipose tissue, and placenta.

Description

Technical Field

[0001] This invention relates to the field of cell therapy technology, and in particular to a culture medium, preparation method and culture method for the industrial preparation of mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal capacity and multi-lineage differentiation potential. They are widely found in tissues such as bone marrow, umbilical cord, and adipose tissue, and possess various biological functions including immune regulation, tissue repair, and anti-inflammation. They have broad application prospects in regenerative medicine, cell therapy, and drug development. With the industrialization of stem cell technology, higher demands are placed on the large-scale and standardized preparation of MSCs. Culture medium, as a key raw material for in vitro cell expansion and culture, directly determines the cell proliferation efficiency, activity, and biological functions, and is a crucial factor in maintaining cell quantity, batch-to-batch stability, and consistency in the industrialized preparation of MSCs.

[0003] The commonly used culture media for in vitro expansion of mesenchymal stem cells are mainly divided into two categories: basal medium supplemented with fetal bovine serum (FBS) and serum-free medium. The amount of FBS added is usually 8%-15%. FBS has many drawbacks: First, it is limited in source and expensive, which greatly increases the cost of industrial production; second, its composition is complex and uncertain, and the quality varies greatly between different batches, resulting in poor reproducibility of cell culture results and making it difficult to achieve standardized production; third, there is a potential risk of pathogen contamination (such as animal-derived viruses, mycoplasma, etc.) and it may contain foreign proteins, which increases the safety risks of clinical application.

[0004] Conventional serum-free culture media have low proliferation efficiency. Due to the lack of various growth factors and adhesion proteins naturally present in serum, the expansion rate of MSCs is significantly slower than that of traditional serum-containing media, making it difficult to meet the requirements of large-scale, high-yield, and high-activity cell preparation for clinical applications. Secondly, the stem cell retention capacity is insufficient. Long-term in vitro culture can easily lead to problems such as decreased expression of surface markers, weakened trilineage differentiation potential, and accelerated cell senescence, affecting cell quality stability. Thirdly, cell viability and stress resistance are poor. During operations such as digestion and passage, centrifugation and washing, and cryopreservation and thawing, the apoptosis rate increases significantly, and the overall survival and recovery rates are difficult to meet clinical standards. In addition, conventional serum-free culture media generally have many formulation components and high production costs, relying on multiple high-content and high-cost components, which is not conducive to industrial-scale production. At the same time, some additives have large batch-to-batch variations, resulting in significant fluctuations in culture medium performance, making it difficult to meet GMP standardized production requirements. Furthermore, most serum-free culture media have narrow compatibility, showing good culture effects only for MSCs from a single tissue source, with poor compatibility with MSCs from different sources such as umbilical cord, bone marrow, adipose tissue, and placenta, limiting their application scope.

[0005] In summary, existing fetal bovine serum and serum-free culture media cannot simultaneously meet the core technical requirements of efficient expansion, stem cell maintenance, high survival rate, low cost, and high batch-to-batch stability. They cannot meet the actual needs of standardized, regulated, and low-cost preparation of clinical-grade mesenchymal stem cells. Therefore, developing a new and efficient serum-free MSC culture medium has significant practical significance and application value. Summary of the Invention

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is to provide a culture medium for the industrial preparation of mesenchymal stem cells, including a basal culture medium, cell growth factors, cell proliferation promoters, a vitamin mixture, a nucleoside mixture, and an amino acid mixture;

[0007] The cell growth factors include one or more of recombinant human transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF).

[0008] The nucleoside mixture includes two or more of adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine hydrochloride, 2'-deoxyguanosine, and thymidine.

[0009] The vitamin mixture includes two or more of the following: folic acid, vitamin B12, ascorbic acid, riboflavin, thiamine hydrochloride, biotin, and nicotinamide.

[0010] The amino acid mixture includes at least two of the following: L-methionine, L-valine hydrochloride, L-arginine hydrochloride, L-cysteine ​​dihydrochloride, L-histidine hydrochloride, glycine, L-alanine, L-asparagine, and L-aspartic acid.

[0011] The cell proliferation promoters include at least two of L-glutamine, reduced glutathione, ethanolamine, recombinant human insulin, recombinant human transferrin, and platelet lysate;

[0012] The basal culture medium is selected from one of high-glucose DMEM, α-MEM, DMEM / F12, and MEM.

[0013] A method for preparing a culture medium includes the following steps:

[0014] Under sterile, light-protected conditions at 2-8°C, place the basal culture medium in the mixing tank;

[0015] Add the amino acid mixture, vitamin mixture, and nucleoside mixture in sequence, and stir to dissolve.

[0016] Add the cell proliferation promoter and mix well;

[0017] Add cell growth factors in the dark and stir to dissolve.

[0018] Adjust the pH to 7.2-7.4, and the osmotic pressure to 280-320 mOsm / kg;

[0019] After sterile filtration through a 0.22μm filter membrane, it is dispensed, packaged, and refrigerated.

[0020] A method for culturing mesenchymal stem cells in an industrial culture medium, comprising:

[0021] Fifth-generation mesenchymal stem cells were harvested at a concentration of 1×10⁻⁶. 4 -5×10 4 The cells / cm² were inoculated and cultured in the culture medium at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2-3 days, and the cells were passaged or harvested when the confluence reached 80%-90%. The mesenchymal stem cells could be continuously and stably passaged to the 15th generation without any abnormal changes in genetic stability.

[0022] Furthermore, the mesenchymal stem cells are derived from umbilical cord, bone marrow, adipose tissue, or placental tissue.

[0023] Compared with existing technologies, the culture medium and its usage method for the industrial preparation of mesenchymal stem cells provided by this invention have the following significant advantages:

[0024] 1. Significantly promotes cell proliferation and shortens the culture cycle: This invention, through the synergistic effect of a complex nutrient system (a mixture of cell growth factors, proliferation promoters, vitamins, nucleosides, and amino acids), can significantly improve the expansion efficiency of mesenchymal stem cells. Example 5 shows that the number of harvested mesenchymal stem cells from passages 2 to 5 cultured using the medium of this invention increased by 20.0%–25.6% compared to traditional fetal bovine serum-containing medium (control group 1), and by 16.36%–42.57% compared to commercially available serum-free medium (control group 2); simultaneously, the cell population doubling time was shortened to 25.5 ± 3.5 hours, far faster than the control group. This significantly reduces cell expansion time and cost in industrial production, meeting the demand for high cell yields in large-scale preparation.

[0025] 2. Maintaining high cell viability and excellent morphology: The culture medium of this invention effectively reduces cell apoptosis and stress during passage and culture. In Example 5, the cell viability of each passage (P2-P5) in the experimental group was consistently maintained at 94.5%~96.8%, significantly higher than that of the control group (lowest only 87.6%). Furthermore, the cells maintained their typical spindle or fusiform shape for a long period, adhering well to the culture vessel with clear edges, without rounding or suspension death, ensuring the activity and quality stability of each batch of cells, meeting the viability standards for clinical-grade cell preparation.

[0026] 3. Stable Maintenance of Stem Cell Stemness, Phenotypic Characteristics, and Differentiation Potential: The culture medium of this invention effectively inhibits cell senescence and maintains stem cell characteristics during continuous passage. Experiments show that even at the 5th generation, cells still highly express the core stem cell phenotype and retain the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. More importantly, the culture medium of this invention can be stably and continuously passaged up to the 15th generation, and chromosome karyotype analysis and STR gene detection confirm that there are no abnormal changes in the cell genetic material (no translocations, deletions, duplications, or STR site mutations), overcoming the technical bottleneck of limited passage numbers and easy loss of stemness in conventional serum-free culture media.

[0027] 4. Ensuring Key Cytokine Secretion Function: The mesenchymal stem cells cultured in this invention can continuously secrete therapeutically relevant cytokines at high levels. ELISA detection confirmed that the levels of key immunomodulatory and angiogenic factors such as IL-6, VEGF, and TGF-β1 secreted by cells at each passage in the experimental group were significantly higher than those in the control group, and the secretion levels remained stable across passages without decline. This directly ensures that the MSCs prepared on a large scale possess the expected biological activity and therapeutic potential.

[0028] 5. Animal-free, high safety, and good batch-to-batch stability: This culture medium is a chemically defined formula that is completely serum-free and animal-free, avoiding the risk of pathogen (virus, mycoplasma) contamination and foreign protein immunogenicity issues that may be introduced by fetal bovine serum, greatly improving the safety for clinical use. Furthermore, all components are recombinant or chemically synthesized, with minimal batch-to-batch variation and controllable processes, fully meeting the stringent requirements of GMP-level industrial production for raw material consistency.

[0029] 7. Controllable cost and broad applicability: This invention avoids the excessive use of expensive components while ensuring efficient expansion and functional maintenance, resulting in a cost significantly lower than some commercially available serum-free culture media. Furthermore, this culture medium is not only suitable for umbilical cord-derived MSCs but also compatible with mesenchymal stem cells from various tissue sources such as bone marrow, adipose tissue, and placenta, demonstrating excellent broad applicability and providing a unified culture medium solution for the large-scale preparation of MSCs from different sources. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0031] Figure 1 These are cell growth and proliferation curves for each group in this invention;

[0032] Figure 2 This is a graph showing the detection of cytokine secretion in this invention;

[0033] Figure 3 These are karyotype analysis results of three batches of 15th generation cells from this invention.

[0034] Figure 4 These are the STR detection results of three batches of 15th generation cells from this invention;

[0035] Figure 5 This is a map showing the expression of cell surface markers in the experimental group of this invention.

[0036] Figure 6 This is a cell surface marker expression profile of the control group 1 in this invention;

[0037] Figure 7 This is a cell surface marker expression map for the control group 2 of this invention.

[0038] Figure 8 This is a diagram illustrating the cell differentiation induction in each group of the present invention. Detailed Implementation

[0039] This invention proposes a culture medium, preparation method, and culture method for the industrial preparation of mesenchymal stem cells, with the aim of...

[0040] The culture medium, preparation method, and culturing method for the industrial preparation of mesenchymal stem cells proposed in this invention will be described below in specific embodiments:

[0041] Example 1:

[0042] A culture medium for the industrial preparation of mesenchymal stem cells includes a basal culture medium, cell growth factors, cell proliferation promoters, a vitamin mixture, a nucleoside mixture, and an amino acid mixture.

[0043] The cell growth factors include one or more of recombinant human transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF).

[0044] The nucleoside mixture includes two or more of adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine hydrochloride, 2'-deoxyguanosine, and thymidine.

[0045] The vitamin mixture includes two or more of the following: folic acid, vitamin B12, ascorbic acid, riboflavin, thiamine hydrochloride, biotin, and nicotinamide.

[0046] The amino acid mixture includes at least two of the following: L-methionine, L-valine hydrochloride, L-arginine hydrochloride, L-cysteine ​​dihydrochloride, L-histidine hydrochloride, glycine, L-alanine, L-asparagine, and L-aspartic acid.

[0047] The cell proliferation promoters include at least two of L-glutamine, reduced glutathione, ethanolamine, recombinant human insulin, recombinant human transferrin, and platelet lysate;

[0048] The basal culture medium is selected from one of high-glucose DMEM, α-MEM, DMEM / F12, and MEM.

[0049] Example 2:

[0050] A method for preparing a culture medium includes the following steps:

[0051] Under sterile, light-protected conditions at 2-8°C, place the basal culture medium in the mixing tank;

[0052] Add the amino acid mixture, vitamin mixture, and nucleoside mixture in sequence, and stir to dissolve.

[0053] Add the cell proliferation promoter and mix well;

[0054] Add cell growth factors in the dark and stir to dissolve.

[0055] Adjust the pH to 7.2-7.4, and the osmotic pressure to 280-320 mOsm / kg;

[0056] After sterile filtration through a 0.22μm filter membrane, it is dispensed, packaged, and refrigerated.

[0057] Culture medium 1:

[0058] Basic culture medium: MEM 940mL;

[0059] Cell growth factors: bFGF 15ng / mL, IGF 10ng / mL, PDGF 10ng / mL;

[0060] Cell proliferation promoters: L-glutamine 2mM, reduced glutathione 50μM, recombinant human transferrin 10μg / mL;

[0061] Vitamin mixture: riboflavin, thiamine hydrochloride, biotin, nicotinamide;

[0062] Nucleoside mixture: adenosine, cytidine, guanosine, uridine;

[0063] Amino acid mixture: L-arginine hydrochloride, glycine, L-alanine.

[0064] Culture medium 2:

[0065] Basic culture medium: MEM 940mL;

[0066] Cell growth factors: bFGF 10 ng / mL, VEGF 10 ng / mL, PDGF 5 ng / mL;

[0067] Cell proliferation promoters: L-glutamine 2mM, reduced glutathione 50μM, recombinant human transferrin 10μg / mL;

[0068] Vitamin mixture: folic acid, vitamin B12, ascorbic acid, riboflavin, thiamine hydrochloride, biotin, nicotinamide;

[0069] Nucleoside mixture: adenosine, cytidine, 2'-deoxyadenosine, thymidine;

[0070] Amino acid mixture: L-arginine hydrochloride, L-cystine dihydrochloride, glycine.

[0071] Culture medium 3:

[0072] Basic culture medium: DMEM / F12 940mL;

[0073] Cell growth factors: bFGF 10 ng / mL, VEGF 10 ng / mL;

[0074] Cell proliferation promoters: L-glutamine 2mM, reduced glutathione 50μM, 2% platelet lysate;

[0075] Vitamin mixture: riboflavin, thiamine hydrochloride, biotin;

[0076] Nucleoside mixture: adenosine, cytidine, guanosine, uridine;

[0077] Amino acid mixture: L-arginine hydrochloride, L-cystine dihydrochloride, glycine, L-alanine.

[0078] Culture medium 4:

[0079] Basic culture medium: MEM 940mL;

[0080] Cell proliferation promoter: 2% platelet lysate;

[0081] Vitamin mixture: ascorbic acid, riboflavin, thiamine hydrochloride, biotin, nicotinamide;

[0082] Nucleoside mixture: adenosine.

[0083]

[0084] In conclusion, culture medium 1 was the most effective in promoting cell proliferation and maintaining high cell viability, with a cell count of 5.0 × 10⁻⁶. 7 Both the cell count and cell viability (94.0%) were significantly higher than other culture media. In contrast, culture medium 4 only added 2% platelet lysate and a small amount of vitamins and nucleosides, lacking multiple growth factors and a comprehensive mixture of amino acids and nucleosides, resulting in the lowest cell number and viability (2.9 × 10⁻⁶). 7 (83.8%). The effects of media 2 and 3 were intermediate, but neither was as good as media 1. This indicates that the simultaneous addition of multiple cell growth factors (bFGF, IGF, PDGF), a comprehensive mixture of vitamins, nucleosides, and amino acids is crucial for optimizing cell culture conditions. Simplifying or omitting certain key components (such as media 4) significantly weakens cell proliferation and reduces survival rate.

[0085] Example 3:

[0086] A method for culturing mesenchymal stem cells in an industrial culture medium, comprising:

[0087] Fifth-generation mesenchymal stem cells were harvested at a concentration of 1×10⁻⁶.4 -5×10 4 The cells / cm² were inoculated and cultured in the culture medium described above at 37°C, 5% CO2, and saturated humidity. The medium was changed every 2-3 days, and the cells were passaged or harvested when the confluence reached 80%-90%. The mesenchymal stem cells could be continuously and stably passaged to the 15th generation without any abnormal changes in genetic stability.

[0088] Furthermore, the mesenchymal stem cells are derived from umbilical cord, bone marrow, adipose tissue, or placental tissue.

[0089] Example 4:

[0090] A culture medium for the industrial preparation of mesenchymal stem cells, comprising:

[0091] Basic culture medium: DMEM / F12 940mL;

[0092] Cell growth factors: TGF-β 5 ng / mL, bFGF 10 ng / mL, VEGF 10 ng / mL, PDGF 5 ng / mL;

[0093] Cell proliferation promoters: L-glutamine 2mM, reduced glutathione 50μM, recombinant human insulin 10μg / mL, recombinant human transferrin 10μg / mL;

[0094] Vitamin mixture: folic acid, vitamin B12, ascorbic acid, riboflavin, thiamine hydrochloride, biotin, nicotinamide;

[0095] Nucleoside mixtures: adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, thymidine;

[0096] Amino acid mixture: L-arginine hydrochloride, L-cystine dihydrochloride, glycine, L-alanine.

[0097] The culture medium preparation method is as follows:

[0098] Solution preparation environment: Class 10,000 clean area, local Class 100 clean area, temperature 2-8℃, operation in the dark.

[0099] Add the basal culture medium to the mixing tank and turn on the low-speed stirring.

[0100] Add the amino acid mixture, vitamin mixture, and nucleoside mixture in sequence, and stir until completely clear.

[0101] Add the cell proliferation promoter and continue stirring for 10-15 minutes.

[0102] Add cell growth factors under light-protected conditions and stir for 10 minutes, avoiding foaming.

[0103] Adjust the pH to 7.2-7.4 using NaOH / HCl, and maintain an osmotic pressure of 280-320 mOsm / kg.

[0104] Sterilize by filtration through a 0.22μm sterile filter membrane, dispense into sterile culture medium bottles, and store at 2-8℃ protected from light.

[0105] Methods for industrial-scale cultivation and use of 5th generation MSCs:

[0106] Seed cell preparation: Umbilical cord mesenchymal stem cells from 3 healthy donors were selected, cultured to the second generation, and observed under a microscope to be uniform in morphology, spindle-shaped, with a confluence of 85%, and without signs of aging or contamination, and were used as seed cells.

[0107] Seeding and culture: After digesting second-generation mesenchymal stem cells, the cell density was adjusted to resuspended and cultured at 1×10⁻⁶ cells / year. 4 -5×10 4 The cells were seeded at a density of 1 / cm² into an industrial cell factory (20 layers), with the culture medium prepared in Example 4 added to each layer, and the liquid level was 0.6-1cm. The cell factory was then statically cultured in an incubator at 37°C, 5% CO2, and saturated humidity.

[0108] Medium replacement culture: After culturing for 36 hours, replace with fresh medium prepared in Example 4 to remove metabolic waste; thereafter replace with fresh medium every 48 hours and observe cell growth status.

[0109] Subculture: When the cell confluence reaches 85%, trypsin digestion is used for subculture. The subculture ratio is strictly controlled at 1:5. Subculture is carried out to the 3rd, 4th and 5th generations respectively. The medium is changed for each generation according to the above steps to complete the industrial-scale culture.

[0110] Continuous passage to the 15th generation and verification of genetic stability:

[0111] Cells were continuously passaged to the 15th generation using the culture medium from Example 4, with passages every 3-5 days. Chromosomal karyotype analysis and STR gene detection were performed on cells from generations P5, P10, and P15. Results are as follows: Figure 3 , Figure 4 The chromosomes of P5, P10, and P15 generations were all of normal number and structure, with no translocations, deletions, duplications, or other aberrations observed; no mutations, deletions, or abnormal amplifications were found at the STR loci, and the genetic stability remained unchanged.

[0112] Effect verification experiment:

[0113] Three groups were set up. Group 1 was a control group with basal culture medium (DMEM / F12) + 10% FBS. Group 2 was a control group with commercial serum-free MSC culture medium (without the optimized combination of compound nucleosides / vitamins / amino acids). Group 3 was a control group with the culture medium formulated in Example 4. The cell culture method was the same as above, only the culture medium was changed. The umbilical cord mesenchymal stem cells selected in this comparison were derived from the same seed cells from the same donor. The effect comparison was carried out by expanding culture in a 5-layer cell factory. The mesenchymal stem cells of passages 2-5 were tested for the following indicators. Each group was set up with 3 replicates and the average value was taken.

[0114] Cell count detection:

[0115] When the cells reached 85% confluence in each generation of culture, they were digested and collected. The results of the analysis using a cell counter are shown in Table 1 below:

[0116] Table 1:

[0117]

[0118] As shown in Table 1, the number of mesenchymal stem cells cultured in the experimental group (the culture medium of this invention) at each generation was significantly higher than that in the control group, with an increase of more than 20%. This indicates that the culture medium of this invention can significantly promote the proliferation of mesenchymal stem cells and meet the cell quantity requirements of industrial production.

[0119] Cell viability assay:

[0120] Using the AO / PI staining method, cells were digested and collected at 85% confluence in each generation of cells, and cell viability was detected using a cell counter. The results are shown in Table 2 below:

[0121] Table 2:

[0122]

[0123] As shown in Table 2, the cell viability of each generation in the experimental group remained above 94%, which was significantly higher than that in the control group. This indicates that the culture medium of the present invention can effectively maintain the high viability of mesenchymal stem cells, reduce cell apoptosis, and ensure cell quality.

[0124] Cell morphology observation:

[0125] The morphology of cells at each generation was observed using an inverted microscope, such as Figure 6As shown, the experimental group cells all exhibited typical spindle or fusiform shapes, with clear cell edges, homogeneous cytoplasm, good adherence and growth, and tight arrangement, without rounding, suspension, death, or abnormal morphology. In control groups 1 and 2, the morphology of cells in the 2nd and 3rd generations was basically normal, while in the 4th and 5th generations, some cells showed rounding, decreased adhesion, uneven morphology, and a small number of suspended dead cells. This indicates that the culture medium of this invention can effectively maintain the typical morphology of mesenchymal stem cells, avoid morphological abnormalities during passage, and meet the requirements of industrial culture for cell morphological stability.

[0126] Cell growth and proliferation curves and cell population doubling time:

[0127] Fifth-generation cells were selected and seeded into 96-well plates, with three replicates per group. Cell proliferation activity was assessed using the CCK-8 assay after 0, 24, 48, 72, 96, 120, and 144 hours of culture, and proliferation curves were plotted. Figure 1 As shown, the cell proliferation curve of the experimental group showed a stable upward trend. After 96 hours of culture, the absorbance value (OD450) was 1.82±0.05, which was significantly higher than that of control group 1 (1.45±0.04) and control group 2 (1.34±0.13). The cell doubling time of the experimental group was 25.5±3.5 hours, which was shorter than that of control group 1 (28.8±4.1 hours) and control group 2 (30.8±4.6 hours). This indicates that the culture medium of the present invention can significantly improve the proliferation efficiency of mesenchymal stem cells, shorten the culture cycle, and meet the time requirements for large-scale industrial preparation.

[0128] Biological indicator testing:

[0129] (1) Cytokine secretion detection:

[0130] The secretion levels of IL-6, VEGF, and TGF-β1 in the supernatant of cell culture at each passage were detected using ELISA. Figure 2 As shown, the levels of IL-6, VEGF, and TGF-β1 secreted by cells at each generation in the experimental group were significantly higher than those in the control group, and the secretion levels remained stable across generations without a significant decreasing trend. This indicates that the culture medium of the present invention can maintain the cytokine secretion function of mesenchymal stem cells and ensure their biological activity.

[0131] (2) Cell phenotype detection:

[0132] The expression rates of surface markers CD29, CD44, CD90, CD105, CD34, and CD45 in fifth-generation cells were detected by flow cytometry. Figure 7As shown, the positive expression rates of CD29, CD44, CD90, and CD105 in the experimental group cells were all above 98%, while the positive expression rates of CD34 and CD45 were all below 2%, which met the phenotypic criteria of mesenchymal stem cells. In the control group cells, the positive expression rates of CD29, CD44, CD90, and CD105 were above 95%, while the positive expression rates of CD34 and CD45 were below 2%. This indicates that the culture medium of the present invention can effectively maintain the typical phenotype of mesenchymal stem cells and ensure the purity and biological characteristics of the cells.

[0133] (3) Cell differentiation capacity detection:

[0134] Fifth-generation cells were induced to differentiate into osteogenic, adipogenic, and chondrogenic groups, respectively. The differentiation effects were observed after staining. Figure 8 As shown, after osteogenic differentiation, the experimental group cells showed strong positive staining with Alizarin Red, with numerous and large calcium nodules; after adipogenic differentiation, they showed strong positive staining with Oil Red O, with numerous and evenly distributed lipid droplets; after chondrogenic differentiation, they showed strong positive staining with Alcian Blue, with abundant secretion of cartilage matrix. After differentiation, the control group 1 and control group 2 cells showed weaker staining intensity, and fewer calcium nodules, lipid droplets, and cartilage matrix than the experimental group. This indicates that the culture medium of the present invention can effectively maintain the multi-lineage differentiation potential of mesenchymal stem cells and ensure the clinical application value of the cells.

[0135] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A culture medium for the industrial preparation of mesenchymal stem cells, characterized in that, Includes basal culture medium, cell growth factors, cell proliferation promoters, vitamin mixture, nucleoside mixture, and amino acid mixture; The cell growth factors include one or more of recombinant human transforming growth factor-β, basic fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor. The nucleoside mixture includes two or more of adenosine, cytidine, guanosine, uridine, 2'-deoxyadenosine, 2'-deoxycytidine hydrochloride, 2'-deoxyguanosine, and thymidine. The vitamin mixture includes two or more of the following: folic acid, vitamin B12, ascorbic acid, riboflavin, thiamine hydrochloride, biotin, and nicotinamide. The amino acid mixture includes at least two of the following: L-methionine, L-valine hydrochloride, L-arginine hydrochloride, L-cysteine ​​dihydrochloride, L-histidine hydrochloride, glycine, L-alanine, L-asparagine, and L-aspartic acid. The cell proliferation promoters include at least two of L-glutamine, reduced glutathione, ethanolamine, recombinant human insulin, recombinant human transferrin, and platelet lysate; The basal culture medium is selected from one of high-glucose DMEM, α-MEM, DMEM / F12, and MEM.

2. A method for preparing the culture medium as described in claim 1, characterized in that, Includes the following steps: Under sterile, light-protected conditions at 2-8°C, place the basal culture medium in the mixing tank; Add the amino acid mixture, vitamin mixture, and nucleoside mixture in sequence, and stir to dissolve. Add the cell proliferation promoter and mix well; Add cell growth factors in the dark and stir to dissolve. Adjust the pH to 7.2-7.4, and the osmotic pressure to 280-320 mOsm / kg; After sterile filtration through a 0.22μm filter membrane, it is dispensed, packaged, and refrigerated.

3. A method for culturing mesenchymal stem cells in the culture medium as described in claim 1, characterized in that, include: Fifth-generation mesenchymal stem cells were harvested at a concentration of 1×10⁻⁶. 4 -5×10 4 The cells / cm² are inoculated and cultured in the culture medium at 37°C, 5% CO2, and saturated humidity. The medium is changed every 2-3 days. When the cells reach 80%-90% confluence, they are passaged or harvested. The mesenchymal stem cells can be continuously and stably passaged to the 15th generation without any abnormal changes in genetic stability.

4. The method of use according to claim 3, characterized in that, The mesenchymal stem cells are derived from umbilical cord, bone marrow, fat, or placental tissue.

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