Stem cell clone culture method and serum-free culture medium

Abstract

The invention discloses a stem cell clone culture method and a serum-free culture medium. According to the method, the heterogeneity of gene phenotypes in a cell population is reduced from the source, the problem of quality instability caused by proportion change of cells of different phenotypes in a traditional culture method is avoided, the safety and controllability of the culture process are greatly improved, the risks of exogenous pollution and adverse reaction are reduced, and the method is more suitable for clinical application. The stem cells can be cultured and subcultured to 40 generations or above, large-scale amplification is achieved, and supply of a large number of cells needed by clinical treatment is ensured. The obtained cell population is more uniform, and the characteristics and functions of the cell population are more stable, so that the cell treatment effect and safety are remarkably improved, and the uncertainty and unpredictability of the treatment effect are favorably reduced. The invention provides a solution which is simpler, more convenient, more reliable and high in cost benefit, and a new prospect is opened up for clinical application of stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for culturing stem cell clones and a serum-free medium. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells with multi-directional differentiation potential and immunomodulatory functions, and are widely used in regenerative medicine and tissue engineering. However, in the prior art, there is a lack of standardization in the isolation and culture methods of umbilical cord MSCs, especially in the suspension sphere culture of MSCs and the long-term adherent culture in chemically defined media, and there is no mature technical solution.

[0003] The culture methods of stem cells mainly include the following common techniques and strategies: First, adherent culture is one of the most common methods, especially suitable for mesenchymal stem cells (MSCs). In this method, after the stem cells are isolated from the tissue sample, they adhere and grow in a specific culture dish or flask, and start to proliferate to form a monolayer of cells after adhesion. Second, suspension culture is suitable for stem cell types that do not require adherent growth, such as hematopoietic stem cells. In suspension culture, the cells grow suspended in the culture medium, and sometimes a shaking flask or a bioreactor is used to maintain the suspended state. The three-dimensional culture technique is another method, including using scaffold materials, colloids or microcarriers, etc., to allow the stem cells to grow in a three-dimensional structure. This method can better simulate the in vivo environment and is beneficial to the proliferation and differentiation of stem cells. In addition, microcarrier culture is a method of attaching cells to tiny particles (microcarriers) and culturing them in a stirred bioreactor, which can increase the culture surface area and thus improve the cell yield. Finally, hypoxic culture helps to maintain the undifferentiated state of stem cells and improve their proliferation ability by culturing stem cells under hypoxic conditions to mimic the microenvironment of in vivo stem cells, such as the environment of hematopoietic stem cells in the bone marrow. Medium optimization is also crucial. A specially designed medium containing specific growth factors and nutrients is used to promote the proliferation of stem cells and maintain their undifferentiated state.

[0004] However, the above methods do not screen and distinguish the initially adherent cells, but culture all cells that can survive in a specific environment without discrimination. This leads to a significant problem, that is, there are cells with different gene phenotypes in the initial cell population. With the expansion and passage of cells, this heterogeneity of gene phenotypes will lead to changes in the proportions of cells with different gene phenotypes, and the resulting cell population will have significant differences in characteristics and functions. This quality instability not only affects the repeatability of basic research, but is more likely to lead to uncertainty and unpredictability of treatment effects in clinical applications, thus limiting the wide application of stem cell therapy.

[0005] There are currently several culture techniques for improving the homogeneity of cell populations: Limited dilution method: This method achieves monoclonal culture by diluting the stem cell population until there is only one cell in each culture dish. In theory, this method can obtain a homogeneous cell population. However, in actual operation, this method is complex, time-consuming, has a low success rate, and is difficult to be widely applied. Fluorescence-activated cell sorting (FACS): FACS technology sorts cells based on specific surface markers on the cells through fluorescence labeling and flow cytometry. This method can precisely select a cell population expressing specific markers, thereby improving homogeneity. However, FACS equipment is expensive and the operation is complex. In addition, the high-speed sorting process may cause certain damage to the cells. Magnetic bead sorting: This method uses magnetic labeling and magnetic field separation technology to separate cells with specific surface markers from a mixed cell population. Compared with FACS, magnetic bead sorting has a lower cost and is easier to operate, but the purity and selectivity are usually lower than those of FACS, and it is difficult to completely eliminate the heterogeneity of gene phenotypes.

[0006] Among them, FACS and magnetic bead sorting improve the homogeneity of cell populations to a certain extent, but they all have certain limitations, such as complex operation, expensive equipment, and possible damage to cells. In addition, these methods still cannot fundamentally avoid the cumulative effect of gene phenotype heterogeneity in long-term culture, resulting in the inability to fully guarantee the stability of cell quality. In the process of isolating and culturing mesenchymal stem cells by the limited dilution method, it often leads to low survival rate, poor state, and low final yield, unable to provide a sufficient number of cells, and it is even more difficult to achieve cell proliferation beyond 15 passages. Summary of the Invention

[0007] Object of the Invention: The technical problem to be solved by the present invention is to provide a serum-free medium that can achieve continuous subculture and amplification of cells for more than 40 passages.

[0008] Another technical problem to be solved by the present invention is to provide the application of the serum-free medium in stem cell clone culture.

[0009] The last technical problem to be solved by the present invention is to provide a stem cell clone culture method that can achieve continuous subculture of cells for more than 40 passages and has a homogeneous cell gene phenotype.

[0010] Technical solution: To solve the above technical problems, the present invention provides a serum-free medium. The serum-free medium includes α-MEM medium. Based on the α-MEM medium, it further includes 15-25 mg / L insulin, 10-20 μg / L selenium, 55-75 mg / L L-ascorbic acid, 5-25 μg / L basic fibroblast growth factor (b-FGF), 5-25 μg / L epidermal growth factor (EGF), 1-5 μg / L transforming growth factor β1 (TGF-β1), 200-500 μg / L hydrocortisone, 10-30 μg / L insulin-like growth factor (IGF), 15-30 mg / L heparin, 450-500 mg / L NaHCO3 and 250-400 mg / L transporter protein, and 15-30 nM R-spondin 1, 0.5-1 mM N-acetylcysteine (NAC), which is named CDM1 medium.

[0011] Among them, the serum-free medium further includes B27, N2, methylcellulose, which is named CDM2 medium. Preferably, the CDM2 medium contains 1×B27, 1×N2, 2-4% (w / v) methylcellulose; preferably, the methylcellulose is 3%.

[0012] The present invention also includes a medium combination, which includes the above-mentioned serum-free medium and a conventional mesenchymal stem cell medium.

[0013] Among them, the components of the conventional mesenchymal stem cell medium are: including α-MEM medium. Based on the α-MEM medium, it further includes 15-25 mg / L insulin, 10-20 μg / L selenium, 55-75 mg / L L-ascorbic acid, 5-25 μg / L b-FGF, 5-25 μg / L EGF, 1-5 μg / L TGF-β1, 200-500 μg / L hydrocortisone, 10-30 μg / L IGF, 15-30 mg / L heparin, 450-500 mg / L NaHCO3 and 250-400 mg / L transporter protein.

[0014] The present invention also includes the application of the above-mentioned serum-free medium or the medium combination in the clonal culture of stem cells.

[0015] The present invention also includes a method for clonal culture of stem cells, which includes the following steps: (1) Early culture of mesenchymal stem cells: The above-mentioned serum-free medium is evenly dispersed to separate and amplify mesenchymal stem cells from tissues or tunica albuginea cells, and adherent cells of P0-P4 generations are obtained after adherent culture. (2)Preparation of cell spheres from single cells: Any generation of cells among the obtained P0 - P4 generation cells is digested and dissociated into a single cell suspension, placed in a low - attachment culture well plate, ensuring one cell per well, and cultured in the serum - free medium. The medium is changed every three days until cell spheres grow. After the cell spheres reach a diameter of 50 - 400 microns, they are digested and dissociated into a single cell suspension; (3)Expansion of single - cell - derived cells: The single cell suspension obtained in step (2) is inoculated into a conventional cell culture plate at a density of 50,000 cells per milliliter and cultured with a conventional mesenchymal stem cell medium. When the cells reach 85 - 90% confluence, continuous passage and expansion are carried out.

[0016] Among them, the mesenchymal stem cells in step (1) include umbilical cord blood mesenchymal stem cells, umbilical cord mesenchymal stem cells, or adipose mesenchymal stem cells.

[0017] Among them, the separation and expansion steps of the umbilical cord blood mesenchymal stem cells in step (1) are: The buffy coat cells are obtained from umbilical cord blood by methods such as density gradient centrifugation, and then inoculated on an LVF culture plate in the serum - free medium for adherent culture.

[0018] Preferably, the serum - free medium in step (1) is CDM1 medium.

[0019] Among them, the separation and expansion steps of the umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells in step (1) are: The umbilical cord, adipose tissue obtained by liposuction, or subcutaneous adipose tissue collected under sterile conditions is cut into pieces, diluted with PBS, centrifuged for 10 min, the supernatant is removed, and the remaining tissue pieces are dispersed and diluted with fresh PBS and washed again under the same conditions. The tissue pieces deposited at the bottom of the centrifuge tube can be seen. The collected tissue pieces are evenly dispersed with the serum - free medium and inoculated on a coated cell culture plate. The medium is changed every 3 days until the cells crawl out and grow to 85 - 90% confluence.

[0020] Among them, the low - attachment culture well plate in step (2). Preferably, the serum - free medium in step (2) is CDM2 medium.

[0021] Among them, the conventional mesenchymal stem cell medium in step (3) is named CDM3 medium, and its components include: α-MEM medium. Based on the α-MEM medium, it also includes 15 - 25 mg / L insulin, 10 - 20 μg / L selenium, 55 - 75 mg / L L-ascorbic acid, 5 - 25 μg / L b-FGF, 5 - 25 μg / L EGF, 1 - 5 μg / L TGF-β, 200 - 500 μg / L hydrocortisone, 10 - 30 μg / L IGF, 15 - 30 mg / L heparin, 450 - 500 mg / L NaHCO3, and 250 - 400 mg / L transporter protein.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: In view of the problem of heterogeneous gene phenotypes in stem cell culture, the present invention proposes an innovative single-cell screening and culture method, which has significant innovations and advantages in many aspects. First of all, the core of the present invention lies in the application of single-cell screening technology, which ensures that only homogeneous stem cells are amplified, reducing the heterogeneity of gene phenotypes within the cell population from the source and avoiding the quality instability problem caused by changes in the proportion of different phenotype cells in traditional culture methods. Secondly, the present invention uses chemically defined media and digestive enzymes, greatly improving the safety and controllability of the culture process, reducing the risk of exogenous contamination and adverse reactions, and making it more suitable for clinical applications. In addition, the present invention can subculture stem cells for more than 40 generations, achieving large-scale amplification and ensuring a large supply of cells required for clinical treatment. Since the obtained cell population is more homogeneous, its characteristics and functions are more stable, which significantly improves the effectiveness and safety of cell therapy, helping to reduce the uncertainty and unpredictability of treatment effects. Finally, compared with existing methods such as limited dilution method, fluorescence-activated cell sorting (FACS), and magnetic bead sorting, the present invention overcomes the problems of complex operation, expensive equipment, and potential cell damage of these technologies, providing a more simple, reliable, and cost-effective solution. Therefore, the present invention has made an important breakthrough in stem cell culture technology, not only improving the reproducibility of research but also opening up new prospects for the clinical application of stem cells. Description of the Drawings

[0023] Figure 1 It is a graph showing the results of the number of passages and cell proliferation ability of different types of stem cells cultured with different medium combinations; Figure 2 It is a comparison of the cell passage ability under the culture conditions of the single-cell culture method described in the present invention and the traditional culture method: the ordinate represents the maximum number of cell passages, and the abscissa is different culture systems; Figure 3Comparison of the homogeneity of cell gene phenotypes under the culture methods described in the present invention and traditional culture methods. The vertical axis represents the Pearson correlation coefficient (ranging from 0 to 1, where 1 indicates complete consistency in gene expression) at different passages (5th, 15th, 40th), and the horizontal axis represents the types of stem cells treated with different culture systems. Detailed implementation manners

[0024] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0025] The reagent sources of the present invention are shown in Table 1.

[0026] Table 1 Reagent sources of the present invention

[0027] The "homogeneity of cell gene phenotypes" described in the present invention refers to the consistency of gene expression patterns in cells at multiple passages, and the evaluation index is the Pearson correlation coefficient of the whole transcriptome RNA expression profile. The formula is:

[0028] where r represents the gene expression similarity between a certain passage of cells and P0 cells at the whole transcriptome level (i.e., the "homogeneity of cell gene phenotypes"), x i represents the expression level of P0 cells at the i-th gene (such as TPM or FPKM value), y i represents the expression level of the comparison passage (such as the 5th, 15th, 40th) at the i-th same gene, represents the average value of all gene expression values of P0 cells, represents the average value of all gene expression values of the comparison passage.

[0029] Example 1 Preparation of CDM1 medium The present invention proposes to use CDM1 medium and a culture plate coated with a protein matrix in cooperation therewith. Within the scope of protection proposed by the present invention, the specific concentrations of each component of the medium are adjusted to prepare CDM1 medium: α-MEM medium and additives. Based on the α-MEM medium (by volume), the addition amounts of the additives are 25 mg / L insulin, 20 μg / L selenium, 55 mg / L L-ascorbic acid, 25 μg / L b-FGF, 25 μg / L EGF, 5 μg / L TGF-β, 200 μg / L hydrocortisone, 10 μg / L IGF, 30 mg / L heparin, 500 mg / L NaHCO3, and 250 mg / L Transferrin (transfer protein), 30 nM R-spondin 1, and 0.8 mM NAC.

[0030] This embodiment also configured media with combinations of different concentrations of the following components: CDM1.A medium: α-MEM medium and added components. Calculated based on the volume of α-MEM medium, the added amounts of the added components are 15 mg / L insulin, 10 μg / L selenium, 75 mg / L L-ascorbic acid, 5 μg / L b-FGF, 5 μg / L EGF, 1 μg / L TGF-β, 500 μg / L hydrocortisone, 30 μg / L IGF, 15 mg / L heparin, 450 mg / L NaHCO3, and 400 mg / L Transferrin, 15 nM R-spondin 1, 0.5 mM NAC.

[0031] CDM1.B medium: α-MEM medium and added components. Calculated based on the volume of α-MEM medium, the added amounts of the added components are 20 mg / L insulin, 15 μg / L selenium, 65 mg / L L-ascorbic acid, 15 μg / L b-FGF, 15 μg / L EGF, 3 μg / L TGF-β, 300 μg / L hydrocortisone, 20 μg / L IGF, 20 mg / L heparin, 480 mg / L NaHCO3, and 300 mg / L Transferrin, 20 nM R-spondin 1, 1 mM NAC.

[0032] CDM1.C medium: α-MEM medium and added components. Calculated based on the volume of α-MEM medium, the added amounts of the added components are 10 mg / L insulin, 30 μg / L selenium, 40 mg / L L-ascorbic acid, 30 μg / L b-FGF, 2 μg / L EGF, 8 μg / L TGF-β, 800 μg / L hydrocortisone, 50 μg / L IGF, 50 mg / L heparin, 600 mg / L NaHCO3, and 600 mg / L Transferrin, 10 nM R-spondin 1, 0.2 mM NAC.

[0033] CDM1.D medium: α-MEM medium and added components. Calculated based on the volume of α-MEM medium, the added amounts of the added components are 30 mg / L insulin, 5 μg / L selenium, 80 mg / L L-ascorbic acid, 22 μg / L b-FGF, 30 μg / L EGF, 0.5 μg / L TGF-β, 150 μg / L hydrocortisone, 5 μg / L IGF, 10 mg / L heparin, 300 mg / L NaHCO3, and 300 mg / L Transferrin, 50 nM R-spondin 1, 2 mM NAC.

[0034] Example 2 Preparation of CDM2 Medium The CDM2 medium was prepared as follows: α-MEM medium and additives. Based on the α-MEM medium (by volume), the addition amounts of the additives were 25 mg / L insulin, 20 μg / L selenium, 55 mg / L L-ascorbic acid, 25 μg / L b-FGF, 25 μg / L EGF, 5 μg / L TGF-β, 200 μg / L hydrocortisone, 10 μg / L IGF, 30 mg / L heparin, 500 mg / L NaHCO3, and 250 mg / L Transferrin, 30 nM R-spondin 1, 1×B27 (i.e., diluted at a volume ratio of 1:50), 1×N2 (i.e., diluted at a volume ratio of 1:100), and 3% (w / v) methylcellulose.

[0035] In this example, media with different concentration combinations of the following components were also prepared simultaneously: CDM2.A: α-MEM medium and additives. Based on the α-MEM medium (by volume), the addition amounts of the additives were 15 mg / L insulin, 10 μg / L selenium, 75 mg / L L-ascorbic acid, 5 μg / L b-FGF, 5 μg / L EGF, 1 μg / L TGF-β, 500 μg / L hydrocortisone, 30 μg / L IGF, 15 mg / L heparin, 450 mg / L NaHCO3, and 400 mg / L Transferrin, 15 nM R-spondin 1, 2×B27 (i.e., diluted at a volume ratio of 1:25), 2×N2 (i.e., diluted at a volume ratio of 1:50), and 2% (w / v) methylcellulose.

[0036] CDM2.B: α-MEM medium and additives. Based on the α-MEM medium (by volume), the addition amounts of the additives were 20 mg / L insulin, 15 μg / L selenium, 65 mg / L L-ascorbic acid, 15 μg / L b-FGF, 15 μg / L EGF, 3 μg / L TGF-β, 300 μg / L hydrocortisone, 20 μg / L IGF, 20 mg / L heparin, 480 mg / L NaHCO3, and 300 mg / L Transferrin, 20 nM R-spondin 1, 0.5×B27 (i.e., diluted at a volume ratio of 1:100), 0.5×N2 (i.e., diluted at a volume ratio of 1:200), and 4% methylcellulose.

[0037] CDM2.C: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 10 mg / L insulin, 30 μg / L selenium, 40 mg / L L-ascorbic acid, 30 μg / L b-FGF, 2 μg / L EGF, 8 μg / L TGF-β, 800 μg / L hydrocortisone, 50 μg / L IGF, 50 mg / L heparin, 600 mg / L NaHCO3, and 600 mg / L Transferrin, 10 nM R-spondin 1, 1×B27, 1×N2, and 1% methylcellulose.

[0038] CDM2.D: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 30 mg / L insulin, 5 μg / L selenium, 80 mg / L L-ascorbic acid, 22 μg / L b-FGF, 30 μg / L EGF, 0.5 μg / L TGF-β, 150 μg / L hydrocortisone, 5 μg / L IGF, 10 mg / L heparin, 300 mg / L NaHCO3, and 300 mg / L Transferrin, 50 nM R-spondin 1, 1×B27, 1×N2, and 6% methylcellulose.

[0039] Example 3 Preparation of CDM3 Medium Prepare CDM3: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 15 mg / L insulin, 10 μg / L selenium, 75 mg / L L-ascorbic acid, 5 μg / L b-FGF, 5 μg / L EGF, 1 μg / L TGF-β, 500 μg / L hydrocortisone, 30 μg / L IGF, 15 mg / L heparin, 450 mg / L NaHCO3, and 400 mg / L Transferrin.

[0040] The present invention also simultaneously prepares media with combinations of different concentrations of each component: CDM3.A: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 15 mg / L insulin, 10 μg / L selenium, 75 mg / L L-ascorbic acid, 25 μg / L b-FGF, 5 μg / L EGF, 1 μg / L TGF-β, 500 μg / L hydrocortisone, 30 μg / L IGF, 15 mg / L heparin, 450 mg / L NaHCO3, and 300 mg / L Transferrin.

[0041] CDM3.B: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 25 mg / L insulin, 20 μg / L selenium, 55 mg / L L-ascorbic acid, 15 μg / L b-FGF, 25 μg / L EGF, 5 μg / L TGF-β, 200 μg / L hydrocortisone, 10 μg / L IGF, 30 mg / L heparin, 500 mg / L NaHCO3, and 250 mg / L Transferrin.

[0042] CDM3.C: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 10 mg / L insulin, 30 μg / L selenium, 40 mg / L L-ascorbic acid, 30 μg / L b-FGF, 2 μg / L EGF, 8 μg / L TGF-β, 800 μg / L hydrocortisone, 50 μg / L IGF, 50 mg / L heparin, 600 mg / L NaHCO3, and 600 mg / L Transferrin.

[0043] CDM3.D: α-MEM medium and additives. Calculated based on the volume of α-MEM medium, the addition amounts of additives are 30 mg / L insulin, 5 μg / L selenium, 80 mg / L L-ascorbic acid, 22 μg / L b-FGF, 30 μg / L EGF, 0.5 μg / L TGF-β, 150 μg / L hydrocortisone, 5 μg / L IGF, 10 mg / L heparin, 300 mg / L NaHCO3, and 300 mg / L Transferrin.

[0044] Example 4 Single-cell culture of umbilical cord blood mesenchymal stem cells Under sterile conditions, 100 mL of umbilical cord blood from normal or premature fetuses was obtained and anticoagulated with heparin. The isolation and purification process of umbilical cord blood mesenchymal stem cells was carried out within 24 hours after delivery. The following preferred operation is the CB group for cell culture.

[0045] (1)Isolation and amplification of umbilical cord blood mesenchymal stem cells: 50 mL of umbilical cord blood collected under sterile conditions was mixed and diluted with α-MEM medium (50 mL) at a volume ratio of 1:1 to obtain 100 mL, which was then mixed with methylcellulose at a concentration of 5 g / L at a volume ratio of 4:1, and allowed to stand for 30 minutes to sediment red blood cells. The supernatant was aspirated, and after centrifugation, a single-cell suspension was prepared with PBS (Gibco, Cat.10010023), which was layered onto lymphocyte separation medium Ficoll-Hypaque (GE Healthcar, 17-1440-02) with a relative density of 1.077, and centrifuged at 2500 rpm for 20 minutes. The interface layer was taken, and a single-cell suspension was prepared with PBS and centrifuged and washed. Since the specific gravity of Ficoll-Hypaque is 1.077 g / ml, which is heavier than monocytes but lighter than red blood cells, monocytes can be separated from the remaining red blood cells. Relatively pure monocytes can be collected by collecting the interface layer. The obtained monocytes were diluted with PBS, centrifuged at 2000 rpm for 10 minutes, the supernatant was removed, and new PBS was added to disperse and dilute. Washing was performed again under the same conditions, and the cells deposited at the bottom of the centrifuge tube were visible.

[0046] Subsequently, 10 mL of the CDM1 medium prepared in Example 1 was used to uniformly disperse the collected cells. After the cells were dispersed, they were inoculated onto the coated cell culture plate at a density of 10 6 / ml, and the medium was changed after 7 days. The culture plate was washed to remove non-adherent cells. 90% confluent cells were digested with trypsin and inoculated onto the culture plate (Corning 96-well culture plate standard transparent plate (3596), the same below), and cultured with the same CDM1 medium as in the primary culture, and the medium was changed every 3 days. Until confluence, and the next passage was carried out until the 2nd passage.

[0047] (2)After digestion of the P2-generation cells, they were dispersed into a cell suspension with CDM2 medium and inoculated onto a low-attachment culture plate at a density of 10 5 / ml, and it was ensured that there was one cell per well. The medium was changed by half every three days with CDM2 medium until cell spheres grew to a diameter of 200 μm; (3)1 mL of 0.25% trypsin was used to digest the cell spheres one by one, and they were uniformly dispersed into a single-cell suspension with CDM3 medium, and inoculated onto the culture plate (Corning 3596) at a density of 50,000 cells per milliliter, and cultured with CDM3 medium. The medium was changed every three days, and passage was carried out after 85-90% confluence, and it could be passed 42 generations.

[0048] The present invention also simultaneously tested the culture effects of media with different concentration combinations of each component on culturing umbilical cord blood mesenchymal stem cells.

[0049] Parallel experimental group CB-A: During the experiment, CDM1.A, CDM2.A, and CDM3.A were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0050] Parallel experimental group CB-B: During the experiment, CDM1.B, CDM2.B, and CDM3.B were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0051] Parallel experimental group CB-C: During the experiment, CDM1.C, CDM2.C, and CDM3.C were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0052] Parallel experimental group CB-D: During the experiment, CDM1.D, CDM2.D, and CDM3.D were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0053] The results are as Figure 1 . From Figure 1 it can be seen that the highest passage number of CB group cells reached 42 generations. The CB-A and CB-B experimental groups were the next, but both reached more than 35 generations. While the CB-C and CB-D experimental groups had lower passage numbers, less than 10 generations, indicating that compared with the CB-C and CB-D experimental groups, CB, CB-A, and CB-B could significantly maintain cell viability, with high passage numbers and cell proliferation ability.

[0054] Example 5 Single-cell amplification of umbilical cord mesenchymal stem cells Under aseptic conditions, umbilical cords of normal or premature fetuses were obtained. The separation and purification process of umbilical cord mesenchymal stem cells was carried out within 24 hours after delivery. The following preferred operation is the UC group for cell culture.

[0055] (1) Isolation and amplification of umbilical cord mesenchymal stem cells: The umbilical cord collected under aseptic conditions was cut into 0.5 mm 3 fragments, diluted with PBS, centrifuged at 2000 rpm for 10 min, the supernatant was removed, and new PBS was added to disperse and dilute. Wash again under the same conditions, and the tissue fragments deposited at the bottom of the centrifuge tube can be seen. Subsequently, 10 mL of the CDM1 medium prepared in Example 1 was used to uniformly disperse the collected tissue fragments, inoculated on a coated cell culture plate, and the medium was changed every 3 days until cells crawled out and grew to 85-90% confluence. The 85-90% confluent cells were digested with 1 mL of 0.25% trypsin and inoculated on a general culture plate (Corning 3596), and cultured with the same CDM1 as in the original culture, and the medium was changed every 3 days. Until confluence, and the next passage was carried out until the 2nd passage.

[0056] (2)After digestion of P2 cells, they were dispersed into a cell suspension with CDM2 medium, inoculated into a low-attachment culture plate, and ensured that there was one cell per well. The medium was changed by half every three days until cell spheres grew to a diameter of 200 μm. (3)The cell spheres were digested one by one with trypsin and dispersed into a single-cell suspension evenly with CDM3. They were inoculated into a culture plate at a density of 50,000 cells per milliliter and cultured with CDM3. The medium was changed every three days. After 85 - 90% confluence, they were passaged and could be passaged 35 times.

[0057] To fully illustrate the protection scope of the present invention, the present invention also simultaneously tested the culture effects of media with different concentration combinations of each component on culturing umbilical cord blood mesenchymal stem cells.

[0058] Parallel experimental group UC - A: During the experiment, CDM1.A, CDM2.A, and CDM3.A were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0059] Parallel experimental group UC - B: During the experiment, CDM1.B, CDM2.B, and CDM3.B were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0060] Parallel experimental group UC - C: During the experiment, CDM1.C, CDM2.C, and CDM3.C were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0061] Parallel experimental group UC - D: During the experiment, CDM1.D, CDM2.D, and CDM3.D were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0062] The results are as Figure 1 . From Figure 1 it can be seen that the highest passage number of cells in the UC group reached 35 generations. The UC - A and UC - B experimental groups were the next, but both reached more than 30 generations. While the UC - C and UC - D experimental groups had lower passage numbers, only about 5 generations, indicating that compared with the UC - C and UC - D experimental groups, UC, UC - A, and UC - B could significantly maintain cell viability and had high passage numbers and cell proliferation ability.

[0063] Example 6 Single - cell amplification of adipose - derived mesenchymal stem cells Adipose tissue after liposuction was obtained under sterile conditions. The isolation and purification process of adipose - derived mesenchymal stem cells was carried out within 24 hours after liposuction. The following preferred operations are regarded as the AD experimental group.

[0064] (1)Isolation and amplification of adipose - derived mesenchymal stem cells: The adipose tissue collected under sterile conditions was minced into 0.5 mm 3The fragments were diluted with PBS, centrifuged at 2000 rpm for 10 min, the supernatant was removed, and new PBS was added to disperse and dilute them. They were washed again under the same conditions, and the tissue fragments deposited at the bottom of the centrifuge tube could be seen. Subsequently, 10 mL of the CDM1 medium prepared in Example 3 was used to uniformly disperse the collected tissue fragments, which were inoculated into the coated cell culture plate. The medium was changed every 3 days until cells crawled out and grew to 85 - 90% confluence. The 85 - 90% confluent cells were digested with 1 mL of trypsin and inoculated into a general culture plate or an LVF culture plate, and cultured with the same CDM1 as in the original culture, and the medium was changed once every 3 days. Until confluence, and the next passage was carried out until the 2nd passage.

[0065] (2)After digestion of the P2 - generation cells, they were dispersed into a cell suspension with CDM2 culture medium and inoculated into a low - absorption culture plate, ensuring one cell per well. The medium was changed by half every three days until cell spheres grew to a diameter of 200 μm; (3)The cell spheres were digested one by one with trypsin and dispersed into a single - cell suspension with CDM3 uniformly. They were inoculated into a general culture plate at a density of 50,000 cells per milliliter and cultured with CDM3. The medium was changed every three days, and passage was carried out after 85 - 90% confluence, and up to 32 passages could be achieved.

[0066] The present invention also simultaneously tested the culture effects of media with different - concentration combinations of each component on culturing umbilical cord blood mesenchymal stem cells.

[0067] Parallel experimental group AD - A: During the experiment, CDM1.A, CDM2.A, and CDM3.A were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0068] Parallel experimental group AD - B: During the experiment, CDM1.B, CDM2.B, and CDM3.B were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0069] Parallel experimental group AD - C: During the experiment, CDM1.C, CDM2.C, and CDM3.C were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0070] Parallel experimental group AD - D: During the experiment, CDM1.D, CDM2.D, and CDM3.D were used to replace CDM1, CDM2, and CDM3 in the above operations respectively.

[0071] The results are as Figure 1 。From Figure 1It can be seen that the highest passage number of the cells in the AD group reached 32 passages. The AD-A and AD-B experimental groups followed, but both reached more than 25 passages. The AD-C and AD-D experimental groups had lower passage numbers, less than about 10 passages, indicating that compared with the AD-C and AD-D experimental groups, AD, AD-A, and AD-B can significantly maintain cell viability and have high passage numbers and cell proliferation ability.

[0072] Comparative Example 1 Isolation and culture of umbilical cord blood mesenchymal stem cells Under aseptic conditions, 100 mL of umbilical cord blood from normal or premature fetuses was obtained and anticoagulated with heparin. The isolation and purification process of umbilical cord blood mesenchymal stem cells was carried out within 24 hours after delivery.

[0073] Isolation and amplification of umbilical cord blood mesenchymal stem cells: The umbilical cord blood (containing cells) collected under aseptic conditions was mixed and diluted in α-MEM medium at a volume ratio of 1:1, and mixed with 5 g / L methylcellulose at a ratio of 4:1, and allowed to stand for 30 minutes to sediment red blood cells. The supernatant was aspirated, and after centrifugation, a single-cell suspension was prepared with PBS and overlaid on lymphocyte separation medium Ficoll-Hypaque (Sigma, USA) with a relative density of 1.077. Centrifuged at 2500 rpm for 20 min, the interface layer was taken, and a single-cell suspension was prepared with PBS and centrifuged and washed.

[0074] The subsequent steps were cultured and amplified according to the culture method in Example 4 of the Chinese patent application with the publication number CN113774019A.

[0075] Comparative Example 2 Isolation and culture of umbilical cord mesenchymal stem cells Under aseptic conditions, umbilical cords from normal or premature fetuses were obtained. The isolation and purification process of umbilical cord mesenchymal stem cells was carried out within 24 hours after delivery.

[0076] Isolation and amplification of umbilical cord mesenchymal stem cells: The umbilical cord collected under aseptic conditions was cut into 0.5 mm 3 fragments, diluted with PBS, centrifuged at 2000 rpm for 10 min, the supernatant was removed, and new PBS was added to disperse and dilute. Washed again under the same conditions, and the tissue fragments deposited at the bottom of the centrifuge tube could be seen.

[0077] Subsequently, the collected tissue fragments were evenly dispersed using a commercial conventional mesenchymal stem cell medium (Gibco™ MSC Growth MediumKit; Gibco, A1082901), inoculated on a coated cell culture plate, and the commercial conventional mesenchymal stem cell medium was replaced every 3 days until the cells crawled out. After growing to 85-90% confluence, they were passaged.

[0078] Comparative Example 3 Isolation and culture of adipose mesenchymal stem cells Obtain adipose tissue after liposuction under aseptic conditions. The isolation and purification process of adipose mesenchymal stem cells is carried out within 24 hours after liposuction.

[0079] Isolate and expand adipose mesenchymal stem cells: Cut the umbilical cord collected under aseptic conditions into 0.5 mm 3 fragments, dilute with PBS, centrifuge at 2000 rpm for 10 min, discard the supernatant, and add new PBS to disperse and dilute. Wash again under the same conditions, and the tissue fragments deposited at the bottom of the centrifuge tube can be seen.

[0080] Subsequently, use a commercial conventional mesenchymal stem cell medium to evenly disperse the collected tissue fragments, inoculate them on a coated cell culture plate, and change the commercial conventional mesenchymal stem cell medium every 3 days until the cells crawl out. Passage when they grow to 85 - 90% confluence.

[0081] Comparative Example 4 Isolation and Culture of Umbilical Cord Blood Mesenchymal Stem Cells Obtain umbilical cord blood. After isolating PBMC from umbilical cord blood tissue, culture it adherently with a general medium, change the medium every 3 - 5 days until the cells adhere, and culture and passage with the general medium, passage every 3 - 5 days until the cells cannot grow and cannot be passaged anymore, and record the number of passages.

[0082] Experimental Example: Repeat the above experimental process according to the same cell passage experimental steps under the culture conditions of Examples 4, 5, 6 and Comparative Examples 1, 2, 3, 4. The experimental results are as Figure 2 shown. Using the culture method of the medium combination of the present invention, umbilical cord blood, umbilical cord, and adipose mesenchymal stem cells can all be passaged to about 40 generations. The umbilical cord blood - derived mesenchymal stem cells cultured and expanded by referring to the culture method of the Chinese patent application with the publication number CN113774019A can also be passaged to about 40 generations. For cell culture using traditional media and traditional culture methods, umbilical cord - and adipose - derived mesenchymal stem cells can be passaged to about 15 - 18 generations, while umbilical cord blood mesenchymal stem cells can only be cultured to about 4 generations. Therefore, the culture method of the present invention can achieve large - scale expansion of mesenchymal stem cells.

[0083] To evaluate the consistency of gene expression profiles among umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and adipose mesenchymal stem cells under different culture conditions at multiple passages (the 5th, 15th, and 40th passages). We used CDM (the CDM1 - 3 medium combination of the present invention), CM (Comparative Examples 2, 3, and 4), and the Chinese patent application with the publication number CN113774019A (Comparative Example 1) to culture these stem cells, extracted RNA at different passages, and analyzed the gene expression levels through whole - gene expression profiling. The specific experimental procedures for cell culture are detailed in Examples 4, 5, 6 and Comparative Examples 1 - 4.

[0084] The Pearson correlation coefficient method was used to quantify gene expression, and the gene expression detection consistency of umbilical cord blood mesenchymal stem cells, umbilical cord mesenchymal stem cells (Comparative Example 2 and Comparative Example 4), and adipose mesenchymal stem cells (Comparative Example 3) cultured under the culture method of Comparative Example 1 with that of the P0 generation was determined; the gene expression consistency of the mesenchymal stem cells cultured by the method of the present invention was compared with that of the cell spheres. The calculation results were as shown in Figure 3 shown, where 1 indicates complete consistency, and the smaller the data, the greater the difference. The data were presented as bar charts. Figure 3 For the expression consistency analysis in , whole transcriptome sequencing (RNA-seq) was performed using the Illumina sequencing platform. Total RNA was extracted at the 5th, 15th, and 40th generations, and the Pearson correlation coefficient was calculated with the P0 generation as a reference. The results showed that the cells treated with the CDM series culture system (the CDM1-3 medium combination of the present invention) had highly consistent expression profiles (>0.9) among multiple generations, while the cells cultured in the traditional system showed a significant decrease (<0.7).

Claims

1. A serum-free medium, characterized in that, The serum-free medium includes α-MEM medium. Based on the α-MEM medium, it further includes 15-25 mg / L insulin, 10-20 μg / L selenium, 55-75 mg / L L-ascorbic acid, 5-25 μg / L basic fibroblast growth factor, 5-25 μg / L epidermal growth factor, 1-5 μg / L transforming growth factor β, 200-500 μg / L hydrocortisone, 10-30 μg / L insulin-like growth factor, 15-30 mg / L heparin, 450-500 mg / L NaHCO3, 250-400 mg / L transporter protein, 15-30 nM R-spondin 1, and 0.5-1 mM N-acetylcysteine.

2. The serum-free medium according to claim 1, wherein The serum-free medium further includes B27, N2, and methylcellulose.

3. A culture medium combination, characterized in that, The medium combination includes the serum-free medium described in claims 1 and 2, and further includes a conventional mesenchymal stem cell medium.

4. The culture medium combination according to claim 3, wherein The components of the conventional mesenchymal stem cell medium are as follows: including α-MEM medium. Based on the α-MEM medium, it further includes 15-25 mg / L insulin, 10-20 μg / L selenium, 55-75 mg / L L-ascorbic acid, 5-25 μg / L basic fibroblast growth factor, 5-25 μg / L epidermal growth factor, 1-5 μg / L transforming growth factor β, 200-500 μg / L hydrocortisone, 10-30 μg / L insulin-like growth factor, 15-30 mg / L heparin, 450-500 mg / L NaHCO3, and 250-400 mg / L transporter protein.

5. Use of the serum-free medium according to claim 1 or 2 or the medium combination according to claim 3 or 4 in stem cell clone culture.

6. A method for culturing stem cell clones, characterized in that, It includes the following steps: (1) Early culture of mesenchymal stem cells: The serum-free medium described in claim 1 is evenly dispersed to separate and expand mesenchymal stem cells from tissues or white membrane layer cells. After adherent culture, P0-P4 generation adherent cells are obtained. (2) Preparation of single-cell-derived cell spheres: Any generation of the obtained P0-P4 generation cells is digested and dissociated into a single-cell suspension, placed in a low-attachment culture well plate to ensure one cell per well, cultured in the serum-free medium described in claim 2, and the medium is changed every three days until cell spheres grow. After the cell spheres reach a diameter of 50-400 microns, they are digested and dissociated into a single-cell suspension. (3) Amplification of single-cell-derived cells: The single-cell suspension obtained in step (2) is inoculated into a conventional cell culture plate and cultured with a conventional mesenchymal stem cell medium. When the cells reach 85-90% confluence, continuous passage amplification is carried out.

7. The stem cell cloning and culturing method according to claim 6, wherein The mesenchymal stem cells described in step (1) include one or more of umbilical cord blood mesenchymal stem cells, umbilical cord mesenchymal stem cells, or adipose mesenchymal stem cells.

8. The stem cell cloning culture method according to claim 7, characterized in that, The separation and amplification steps of the umbilical cord blood mesenchymal stem cells in step (1) are as follows: The white membrane layer cells are obtained by density gradient centrifugation of umbilical cord blood, and then inoculated into a culture plate and cultured adherently in the serum-free medium described in claim 1.

9. The stem cell cloning and culturing method according to claim 7, characterized in that, The steps for the isolation and amplification of the umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells described in step (1) are as follows: The umbilical cord, adipose tissue obtained by liposuction, or subcutaneous adipose tissue collected under aseptic conditions is cut into pieces, diluted with PBS, centrifuged, the supernatant is removed, and fresh PBS is added to disperse and dilute it. It is washed again under the same conditions. The tissue pieces deposited at the bottom of the centrifuge tube can be seen. The collected tissue pieces are evenly dispersed using the serum-free medium described in claim 1, inoculated into a coated cell culture plate, and the medium is changed every 3 days until the cells crawl out and grow to 85-90% confluence.

10. The stem cell cloning and culturing method according to claim 6, wherein The low-attachment culture well plate described in step (2) is an agarose-coated low-attachment culture well plate, and its preparation method is as follows: 0.8-1% agarose is prepared using ultrapure water, autoclaved, and poured into plates while hot. Each plate is obtained after equilibration with the serum-free medium described in claim 2 containing gellan gum.

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