A stem cell cloning culture method and serum-free culture medium

Abstract

The present invention discloses a stem cell cloning culture method and a serum-free culture medium. The present invention reduces the heterogeneity of the genetic phenotype within the cell population from the source, avoids the quality instability problem caused by the change in the proportion of cells with different phenotypes in the traditional culture method, greatly improves the safety and controllability of the culture process, reduces the risk of exogenous contamination and adverse reactions, and makes it more suitable for clinical application. The present invention can culture and subculture stem cells to more than 40 generations, realizes large-scale amplification, and ensures the large-scale supply of cells required for clinical treatment. Since the obtained cell population is more uniform, its characteristics and functions are more stable, which significantly improves the effect and safety of cell therapy and helps to reduce the uncertainty and unpredictability of the treatment effect. The present invention provides a simpler, more reliable and cost-effective solution, and also opens up new prospects for the clinical application of stem cells.

Description

Technical Field

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

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell with multipotential differentiation and immunomodulatory functions, and are widely used in regenerative medicine and tissue engineering. However, existing methods for isolating and culturing umbilical cord MSCs lack standardization, particularly for MSC suspension culture and long-term adherent culture in chemically defined culture media.

[0003] Stem cell culture methods primarily include the following common techniques and strategies. First, adherent culture is one of the most common methods, particularly suitable for mesenchymal stem cells (MSCs). In this method, stem cells are isolated from a tissue sample and allowed to adhere to the surface of a specific culture dish or flask. Once adhered, they begin to proliferate and form a monolayer of cells. Second, suspension culture is suitable for stem cell types that do not require adherent growth, such as hematopoietic stem cells. In suspension culture, cells are grown suspended in culture medium, sometimes using shaker flasks or bioreactors to maintain this suspended state. Three-dimensional culture techniques are another approach, involving the use of scaffolds, colloids, or microcarriers to grow stem cells in a three-dimensional structure. This method better mimics the in vivo environment and promotes stem cell proliferation and differentiation. Furthermore, microcarrier culture involves attaching cells to tiny particles (microcarriers) and culturing them in a stirred bioreactor. This increases the culture surface area and thus improves cell yield. Finally, hypoxic culture mimics the in vivo microenvironment of stem cells, such as the environment of hematopoietic stem cells in the bone marrow, by culturing them under low oxygen conditions. This helps maintain the undifferentiated state of stem cells and enhances their proliferation capacity. Culture medium optimization is also crucial, using specially designed culture media containing specific growth factors and nutrients to promote the proliferation of stem cells and maintain their undifferentiated state.

[0004] However, none of the above methods screen and identify the initial adherent cells, but instead indiscriminately culture all cells that can survive in a specific environment. This leads to a significant problem, namely the presence of cells with different genetic phenotypes in the initial cell population. As the cells are expanded and passaged, this genetic phenotype heterogeneity will lead to changes in the proportion of cells with different genetic phenotypes, resulting in cell populations with significant differences in characteristics and functions. This quality instability not only affects the reproducibility of basic research, but is also likely to lead to uncertainty and unpredictability in therapeutic effects in clinical applications, thereby limiting the widespread application of stem cell therapy.

[0005] Currently, there are several culture techniques that improve the uniformity of cell populations: Limited dilution: This method dilutes the stem cell population to only one cell per culture dish, achieving monoclonal culture. This method can theoretically produce a uniform cell population. However, in practice, this method is complex and time-consuming, with a low success rate, making it difficult to widely apply. Fluorescence-activated cell sorting (FACS): FACS technology uses fluorescent labeling and flow cytometry to sort cells based on specific cell surface markers. This method can accurately select cell populations expressing specific markers, thereby improving uniformity. However, FACS equipment is expensive and complex to operate, and the high-speed sorting process may cause some damage to cells. Magnetic bead sorting: This method uses magnetic labeling and magnetic field separation technology to isolate cells with specific surface markers from a mixed cell population. Compared to FACS, magnetic bead sorting is less expensive and easier to operate, but its purity and selectivity are generally lower than FACS, making it difficult to completely eliminate phenotypic heterogeneity.

[0006] While FACS and magnetic bead sorting improve the uniformity of cell populations to some extent, they both have limitations, such as complex operation, expensive equipment, and potential cell damage. Furthermore, these methods still cannot fundamentally avoid the cumulative effects of phenotypic heterogeneity during long-term culture, resulting in an inability to fully guarantee the stability of cell quality. The limiting dilution method often results in low survival rates, poor cell status, and ultimately low yields during the isolation and culture of mesenchymal stem cells. It also fails to provide a sufficient number of cells, making it difficult to achieve cell proliferation beyond 15 generations. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a serum-free culture medium that can achieve continuous cell passage and expansion for more than 40 generations.

[0008] The technical problem that the present invention also aims to solve is to provide the application of the serum-free culture medium in stem cell cloning culture.

[0009] The final technical problem solved by the present invention is to provide a stem cell cloning and culture method that can achieve continuous cell passage for more than 40 generations and uniform cell gene phenotype.

[0010] Technical solution: To solve the above technical problems, the present invention provides a serum-free culture medium, which includes α-MEM culture medium, which is based on α-MEM culture medium and 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 transport protein and 15-30 nM R-spondin 1, 0.5-1 mM N-acetylcysteine ​​(NAC), and is named CDM1 culture medium.

[0011] The serum-free culture medium further comprises B27, N2, and methylcellulose, and is named CDM2 culture medium. Preferably, the CDM2 culture medium comprises 1×B27, 1×N2, and 2-4% (w / v) methylcellulose; preferably, the methylcellulose content is 3%.

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

[0013] The conventional mesenchymal stem cell culture medium comprises α-MEM medium, which, based on the α-MEM medium, 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-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 transport protein.

[0014] The present invention also includes the use of the serum-free culture medium or the culture medium combination in stem cell cloning culture.

[0015] The present invention also includes a stem cell cloning and culture method, comprising the following steps:

[0016] (1) Early culture of mesenchymal stem cells: The serum-free medium is used to evenly disperse the mesenchymal stem cells separated and expanded from tissue or buffy coat cells, and the P0 to P4 generations of adherent cells are obtained after adherent culture;

[0017] (2) Preparation of single-cell derived cell spheres: digest and dissociate any of the P0 to P4 cell generations obtained into a single-cell suspension, place it in a low-adsorption culture plate, ensure that there is one cell per well, and culture it in the serum-free medium. Change the medium every three days until the cell spheres grow and the diameter of the cell spheres reaches 50 to 400 microns, then digest and dissociate it into a single-cell suspension;

[0018] (3) Cell expansion from single cells: The single cell suspension obtained in step (2) was inoculated into a conventional cell culture plate at a density of 50,000 cells per ml and cultured in a conventional mesenchymal stem cell culture medium. After the cells reached 85-90% fusion, they were serially passaged and expanded.

[0019] Wherein, the mesenchymal stem cells in step (1) include umbilical cord blood mesenchymal stem cells, umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells.

[0020] The separation and expansion of umbilical cord blood mesenchymal stem cells in step (1) is as follows: obtaining buffy coat cells from umbilical cord blood by density gradient centrifugation or other methods, and then inoculating the cells in the serum-free culture medium on LVF culture plates for adherent culture.

[0021] Preferably, the serum-free culture medium in step (1) is CDM1 culture medium.

[0022] The isolation and expansion steps of the umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells in step (1) are as follows: the umbilical cord, adipose tissue or subcutaneous adipose tissue collected under sterile conditions is cut into pieces, diluted with PBS, centrifuged for 10 minutes, the supernatant is removed, and new PBS is added to break it up, diluted, and washed again under the same conditions. The tissue fragments deposited at the bottom of the centrifuge tube are visible, and the collected tissue fragments are evenly broken up using the serum-free culture medium, and inoculated into a coated cell culture plate. The culture medium is replaced every 3 days until the cells crawl out and grow to 85-90% fusion.

[0023] Wherein, the low adsorption culture plate in step (2) is preferably a serum-free culture medium in step (2) is CDM2 culture medium.

[0024] Among them, the conventional mesenchymal stem cell culture medium in step (3) is named CDM3 culture medium, and its ingredients are: including α-MEM culture medium, based on α-MEM culture medium, and also including 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 transport protein.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention addresses the problem of genotypic heterogeneity in stem cell culture and proposes an innovative single-cell screening and culture method, which has many significant innovations and advantages. First, the core of the present invention lies in the application of single-cell screening technology, which ensures that only uniform stem cells are amplified, reduces the heterogeneity of genotypic types within the cell population from the source, and avoids the problem of quality instability caused by changes in the proportion of cells with different phenotypes in traditional culture methods. Secondly, the present invention adopts culture medium and digestive enzymes with clear chemical composition, which greatly improves the safety and controllability of the culture process, reduces the risk of exogenous contamination and adverse reactions, and makes it more suitable for clinical application. In addition, the present invention can culture stem cells for more than 40 generations, realizes large-scale amplification, and ensures the large-scale supply of cells required for clinical treatment. Since the obtained cell population is more uniform, its characteristics and functions are more stable, which significantly improves the effect and safety of cell therapy and helps to reduce the uncertainty and unpredictability of the treatment effect. Finally, compared to existing methods such as limiting dilution, fluorescence-activated cell sorting (FACS), and magnetic bead sorting, this invention overcomes the complex operation, expensive equipment, and potential cell damage associated with these techniques, providing a simpler, more reliable, and cost-effective solution. Therefore, this invention represents a significant 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of passage number and cell proliferation capacity of different types of stem cells cultured with different culture medium combinations;

[0027] Figure 2 Comparison of cell passage capacity under the culture conditions of the single cell culture method of the present invention and the traditional culture method: the vertical axis represents the maximum number of cell passages, and the horizontal axis represents different culture systems;

[0028] Figure 3This figure compares the phenotypic uniformity of cells cultured using the present method and traditional culture methods. The vertical axis represents the Pearson correlation coefficient (0–1, with 1 indicating perfect gene expression uniformity) at different passages (5, 15, and 40). The horizontal axis represents the stem cell types treated using different culture systems. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0030] The sources of the reagents of the present invention are shown in Table 1.

[0031] Table 1 Sources of reagents of the present invention

[0032]

[0033] The "homogeneity of cell phenotype" in this invention refers to the consistency of gene expression patterns across multiple generations of cells. The evaluation indicator is the Pearson correlation coefficient of the RNA expression profile of the entire transcriptome. The formula is:

[0034]

[0035] Where r represents the gene expression similarity between a certain generation of cells and P0 generation of cells at the whole transcriptome level (i.e., “cell gene phenotypic homogeneity”), x i represents the expression level of gene i in P0 cells (such as TPM or FPKM value), y i Indicates the expression level of the same gene in the ith generation (such as the 5th, 15th, and 40th generations). represents the average expression value of all genes in the P0 generation, It represents the average expression value of all genes in the comparison generations.

[0036] Example 1 Preparation of CDM1 culture medium

[0037] The present invention proposes the use of CDM1 culture medium and a protein matrix-coated culture plate therewith. Within the scope of the present invention, the specific concentrations of the culture medium components are adjusted to prepare a CDM1 culture medium: α-MEM medium and supplemental ingredients. The supplemental amounts (by volume) based on the α-MEM medium 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, 250 mg / L Transferrin, 30 nM R-spondin 1, and 0.8 mM NAC.

[0038] This embodiment also simultaneously configures the culture medium with different concentrations of the following components:

[0039] CDM1.A medium: α-MEM medium and supplements, calculated by volume based on α-MEM medium, include 15 mg / L insulin, 10 μg / L selenium, 75 mg / L L-ascorbic acid, 5 μg / L b-FGF, 5 μg / L L-EGF, 1 μg / L TGF-β, 500 μg / L hydrocortisone, 30 μg / L IGF, 15 mg / L heparin, 450 mg / L NaHCO₃, 400 mg / L Transferrin, 15 nM R-spondin 1, and 0.5 mM NAC.

[0040] CDM1.B medium: α-MEM medium and supplements. Calculated by volume based on α-MEM medium, the supplements 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, 300 mg / L Transferrin, 20 nM R-spondin 1, and 1 mM NAC.

[0041] CDM1.C medium: α-MEM medium and supplements. Calculated by volume based on α-MEM medium, the supplements 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; and 0.2 mM NAC.

[0042] CDM1.D medium: α-MEM medium and supplements. Calculated by volume based on α-MEM medium, the supplements 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, 300 mg / L Transferrin, 50 nM R-spondin 1, and 2 mM NAC.

[0043] Example 2 Preparation of CDM2 medium

[0044] CDM2 medium was prepared: α-MEM medium and supplementary components. Based on the α-MEM medium, the added components (volume) 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., 1:50 volume dilution), 1× N2 (i.e., 1:100 volume dilution), and 3% (w / v) methylcellulose.

[0045] This embodiment also simultaneously configures the culture medium with different concentrations of the following components:

[0046] CDM2.A: α-MEM medium and supplements, calculated by volume based on α-MEM medium, including 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 (1:25 volume dilution), 2× N₂ (1:50 volume dilution), and 2% (w / v) methylcellulose.

[0047] CDM2.B: α-MEM medium and supplements, calculated based on α-MEM medium (volume), include 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 (1:100 volume dilution), 0.5× N₂ (1:200 volume dilution), and 4% methylcellulose.

[0048] CDM2.C: α-MEM medium and supplements, calculated by volume based on α-MEM medium, including 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.

[0049] CDM2.D: α-MEM medium and supplements, calculated by volume based on α-MEM medium, including 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.

[0050] Example 3 Preparation of CDM3 culture medium

[0051] Prepare CDM3: α-MEM medium and supplements. Based on α-MEM medium, the following supplements (by volume) are added: 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.

[0052] The present invention also provides a culture medium with different concentrations of various components:

[0053] CDM3.A: α-MEM medium and supplements, calculated based on α-MEM medium (volume): 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.

[0054] CDM3.B: α-MEM medium and supplements, calculated based on α-MEM medium (volume): 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.

[0055] CDM3.C: α-MEM medium and supplements, calculated based on α-MEM medium (volume): 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.

[0056] CDM3.D: α-MEM medium and supplements, calculated by volume based on α-MEM medium, including 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.

[0057] Example 4 Single Cell Culture of Umbilical Cord Blood Mesenchymal Stem Cells

[0058] Obtain 100 mL of umbilical cord blood from a normal or premature fetus under sterile conditions and anticoagulate with heparin. Isolate and purify umbilical cord blood mesenchymal stem cells within 24 hours of delivery. The following preferred procedures are for the CB cell culture group.

[0059] (1) Isolation and expansion of umbilical cord blood mesenchymal stem cells: 50 ml of umbilical cord blood collected under sterile conditions was diluted with α-MEM medium (50 ml) at a volume ratio of 1:1 to obtain 100 ml, which was then mixed with 5 g / L methylcellulose at a volume ratio of 4:1. The mixture was allowed to stand for 30 minutes to allow the red blood cells to settle. The supernatant was aspirated and centrifuged. A single cell suspension was prepared using PBS (Gibco, Cat. 10010023) and superimposed on Ficoll-Hypaque (GE Healthcare, 17-1440-02), a lymphocyte separation medium with a relative density of 1.077. The suspension was centrifuged at 2500 rpm for 20 minutes. The interface layer was taken and PBS was added to prepare a single cell suspension. The suspension was then centrifuged and washed. Because the specific gravity of Ficoll-Hypaque is 1.077 g / ml, it is heavier than monocytes but lighter than red blood cells, so monocytes can be separated from residual red blood cells. Relatively pure monocytes can be collected by collecting the interface layer. The mononuclear cells were diluted with PBS and centrifuged at 2000 rpm for 10 minutes. The supernatant was removed and fresh PBS was added to disperse and dilute the cells. The cells were washed again under the same conditions. The cells were deposited at the bottom of the centrifuge tube.

[0060] Subsequently, 10 mL of the CDM1 medium prepared in Example 1 was used to evenly disperse the collected cells. 6 Cells were seeded at a density of 1000 μg / ml onto coated cell culture plates. The culture medium was changed after 7 days. The culture plates were washed to remove non-adherent cells. Cells were trypsinized to 90% confluent and seeded onto culture plates (Corning 96-well standard clear plates (3596, the same below). Cultured in the same CDM1 medium as the primary culture, changing the medium every 3 days. The cells were cultured until confluent and then passaged again, until they reached the second passage.

[0061] (2) After digestion of P2 cells, the cells were dispersed into a cell suspension using CDM2 medium. 5 The cells were seeded at a density of 1 / ml in a low-adhesion culture plate, ensuring one cell per well. The medium was changed by half every three days with CDM2 medium until the cell spheres grew to a diameter of 200 μm.

[0062] (3) Digest the cell spheres one by one with 1 mL of 0.25% trypsin and evenly disperse them into a single cell suspension with CDM3 medium. Inoculate the cells into a culture plate (Corning 3596) at a density of 50,000 cells per mL and culture them with CDM3 medium. Change the medium every three days and passage the cells after 85-90% fusion. The cells can be passaged for 42 generations.

[0063] The present invention also tests the culture effect of culture medium with different concentrations of each component in culturing umbilical cord blood mesenchymal stem cells.

[0064] Parallel experimental group CB-A: During the experiment, CDM1.A, CDM2.A, CDM3.A were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0065] Parallel experimental group CB-B: During the experiment, CDM1.B, CDM2.B, CDM3.B were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0066] Parallel experimental group CB-C: During the experiment, CDM1.C, CDM2.C, CDM3.C were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0067] Parallel experimental group CB-D: During the experiment, CDM1.D, CDM2.D, and CDM3.D were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0068] The results are as follows Figure 1 .from Figure 1As can be seen, the highest number of cell passages in the CB group reached 42. The CB-A and CB-B experimental groups were second, but both reached more than 35 passages. The CB-C and CB-D experimental groups had lower passage numbers, less than 10. This indicates that compared with the CB-C and CB-D experimental groups, CB, CB-A, and CB-B can significantly maintain cell viability and have a higher passage number and cell proliferation capacity.

[0069] Example 5 Single-cell expansion of umbilical cord mesenchymal stem cells

[0070] Umbilical cords from normal or premature fetuses were obtained under sterile conditions. Umbilical cord mesenchymal stem cells were isolated and purified within 24 hours of delivery. The following preferred procedures are for cell culture in the UC group.

[0071] (1) Isolation and expansion of umbilical cord mesenchymal stem cells: The umbilical cord collected under sterile conditions was cut into 0.5 mm pieces. 3 The fragments were diluted with PBS, centrifuged at 2000rpm for 10min, the supernatant was removed, and new PBS was added to break up and dilute. Wash again under the same conditions, and tissue fragments deposited at the bottom of the centrifuge tube were visible. Subsequently, 10mL of CDM1 culture medium configured in Example 1 was used to evenly break up the collected tissue fragments and inoculate them on the coated cell culture plate. The culture medium was changed every 3 days until the cells crawled out and grew to 85-90% fusion. The 85-90% fused cells were treated with 1mL of 0.25% trypsin.

[0072] Digest the cells and seed them onto standard culture plates (Corning 3596). Culture them using the same CDM1 medium used for the original strip culture, changing the medium every 3 days until they are confluent. Then, proceed to the next passage, until they reach the second generation.

[0073] (2) After digestion of the P2 cells, they were dispersed into a cell suspension using CDM2 medium and inoculated into low-adsorption culture plates, ensuring that there was one cell per well. The medium was changed by half every three days until the cell spheres grew to a diameter of 200 μm.

[0074] (3) Digest the cell spheres one by one with trypsin and evenly break them up into single cell suspension with CDM3. Inoculate the cells into culture plates at a density of 50,000 cells per ml and culture them with CDM3. Change the medium every three days. Passage the cells after 85-90% fusion. The cells can be passed for 35 generations.

[0075] In order to fully illustrate the protection scope of the present invention, the present invention also simultaneously tests the culture effect of culture medium with different concentrations of each component in culturing umbilical cord blood mesenchymal stem cells.

[0076] Parallel experimental group UC-A: During the experiment, CDM1.A, CDM2.A, CDM3.A were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0077] Parallel experimental group UC-B: During the experiment, CDM1.B, CDM2.B, CDM3.B were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0078] Parallel experimental group UC-C: During the experiment, CDM1.C, CDM2.C, CDM3.C were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0079] Parallel experimental group UC-D: During the experiment, CDM1.D, CDM2.D, and CDM3.D were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0080] The results are as follows Figure 1 .from Figure 1 As can be seen, the highest number of cell passages in the UC group reached 35. The UC-A and UC-B experimental groups were next highest, but both reached more than 30 passages. The UC-C and UC-D experimental groups had a lower number of passages, only around 5. This indicates that compared with the UC-C and UC-D experimental groups, UC, UC-A, and UC-B can significantly maintain cell viability and have a higher number of passages and cell proliferation capacity.

[0081] Example 6 Single-cell expansion of adipose-derived mesenchymal stem cells

[0082] Obtain adipose tissue after liposuction under sterile conditions. Adipose-derived mesenchymal stem cells were isolated and purified within 24 hours of liposuction. The following optimal procedures were performed, considered the AD experimental group.

[0083] (1) Isolation and expansion of adipose-derived mesenchymal stem cells: adipose tissue was collected under sterile conditions and minced into 0.5 mm pieces. 3 The fragments were diluted with PBS, centrifuged at 2000rpm for 10min, the supernatant was removed, and new PBS was added to break them up and diluted. Washed again under the same conditions, the tissue fragments deposited at the bottom of the centrifuge tube were visible. Subsequently, 10mL of CDM1 culture medium configured using Example 3 were evenly broken up the collected tissue fragments and inoculated into the coated cell culture plates. The culture medium was changed every 3 days until the cells crawled out and grew to 85-90% fusion. The 85-90% fused cells were digested with 1mL of trypsin, inoculated into a general culture plate or LVF culture plate, and the same CDM1 culture as the original band was used to cultivate. The solution was changed every 3 days. Until fusion, and the next passage was carried out until the second generation was passed on.

[0084] (2) After digestion of the P2 cells, they were dispersed with CDM2 culture medium to form a cell suspension, which was then inoculated into a low-absorbency culture plate, ensuring that there was one cell per well. The medium was changed by half every three days until the cell spheres grew to a diameter of 200 μm.

[0085] (3) Digest the cell spheres one by one with trypsin and evenly break them up into a single cell suspension with CDM3. Inoculate the cells into a regular culture plate at a density of 50,000 cells per ml and culture them with CDM3. Change the medium every three days and passage them after 85-90% fusion. The suspension can reach up to 32 generations.

[0086] The present invention also tests the culture effect of culture medium with different concentrations of each component in culturing umbilical cord blood mesenchymal stem cells.

[0087] Parallel experimental group AD-A: During the experiment, CDM1.A, CDM2.A, CDM3.A were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0088] Parallel experimental group AD-B: During the experiment, CDM1.B, CDM2.B, CDM3.B were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0089] Parallel experimental group AD-C: During the experiment, CDM1.C, CDM2.C, CDM3.C were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0090] Parallel experimental groups AD-D: During the experiment, CDM1.D, CDM2.D, and CDM3.D were used to replace CDM1, CDM2, and CDM3 used in the above operation.

[0091] The results are as follows Figure 1 .from Figure 1 As can be seen, the highest number of cell passages in the AD group reached 32. The AD-A and AD-B experimental groups were second, but both reached more than 25 passages, while the AD-C and AD-D experimental groups had lower passage numbers, less than about 10. This indicates that compared with the AD-C and AD-D experimental groups, AD, AD-A, and AD-B can significantly maintain cell viability and have a higher passage number and cell proliferation capacity.

[0092] Comparative Example 1 Isolation and Culture of Umbilical Cord Blood Mesenchymal Stem Cells

[0093] Umbilical cord blood (100 mL) was obtained from normal or premature fetuses under sterile conditions and anticoagulated with heparin. Umbilical cord blood-derived mesenchymal stem cells were isolated and purified within 24 hours of delivery.

[0094] Isolation and Expansion of Umbilical Cord Blood Mesenchymal Stem Cells: Aseptically collected umbilical cord blood (including cells) was diluted in α-MEM medium at a volume ratio of 1:1. The mixture was then mixed with 5g / L methylcellulose at a ratio of 4:1 and allowed to stand for 30 minutes to allow the red blood cells to settle. The supernatant was aspirated, centrifuged, and a single-cell suspension was prepared in PBS. The suspension was then overlaid with Ficoll-Hypaque (Sigma, USA), a lymphocyte separation medium with a relative density of 1.077. The suspension was centrifuged at 2500 rpm for 20 minutes. The interphase layer was removed, added with PBS to prepare a single-cell suspension, and washed by centrifugation.

[0095] The subsequent steps refer to the culture method of Example 4 in the Chinese patent application with publication number CN113774019A for culture and amplification.

[0096] Comparative Example 2 Isolation and Culture of Umbilical Cord Mesenchymal Stem Cells

[0097] Umbilical cords from normal or premature fetuses are obtained under sterile conditions. Umbilical cord mesenchymal stem cells are isolated and purified within 24 hours of delivery.

[0098] Isolation and expansion of umbilical cord mesenchymal stem cells: The umbilical cord collected under sterile conditions was cut into 0.5 mm pieces. 3 The fragments were diluted with PBS and centrifuged at 2000 rpm for 10 min. The supernatant was removed and fresh PBS was added to break them up and dilute. The tissue fragments were then washed again under the same conditions. The tissue fragments were deposited at the bottom of the centrifuge tube.

[0099] Subsequently, the collected tissue fragments were evenly dispersed using a commercial conventional mesenchymal stem cell culture medium (Gibco™ MSC Growth Medium Kit; Gibco, A1082901) and inoculated onto coated cell culture plates. The commercial conventional mesenchymal stem cell culture medium was replaced every 3 days until the cells crawled out. The cells were grown to 85-90% confluence and then passaged.

[0100] Comparative Example 3 Isolation and Culture of Adipose-Derived Mesenchymal Stem Cells

[0101] Adipose tissue was obtained under sterile conditions after liposuction. Adipose-derived mesenchymal stem cells were isolated and purified within 24 hours after liposuction.

[0102] Isolation and expansion of adipose-derived mesenchymal stem cells: The umbilical cord collected under sterile conditions was cut into 0.5 mm pieces. 3 The fragments were diluted with PBS and centrifuged at 2000 rpm for 10 min. The supernatant was removed and fresh PBS was added to break them up and dilute. The tissue fragments were then washed again under the same conditions. The tissue fragments were deposited at the bottom of the centrifuge tube.

[0103] Subsequently, the collected tissue fragments were evenly dispersed using commercial conventional mesenchymal stem cell culture medium and inoculated into coated cell culture plates. The commercial conventional mesenchymal stem cell culture medium was replaced every 3 days until the cells crawled out and were passaged after growing to 85-90% confluence.

[0104] Comparative Example 4 Isolation and Culture of Umbilical Cord Blood Mesenchymal Stem Cells

[0105] After isolating PBMCs from umbilical cord blood, culture them in a universal medium, changing the medium every 3-5 days until the cells adhere. The cells are then passaged in a universal medium, passaged every 3-5 days until they cease to grow and cannot be passaged further. The number of passages is recorded.

[0106] Experimental example:

[0107] The above experimental process was repeated according to the same cell passage experimental steps under the culture conditions of Examples 4, 5, 6 and Comparative Examples 1, 2, 3, and 4. The experimental results are as follows: Figure 2 As shown. Using the culture method of the culture medium combination of the present invention, umbilical cord blood, umbilical cord, and adipose tissue-derived mesenchymal stem cells can all be propagated to about 40 generations. Umbilical cord blood-derived mesenchymal stem cells cultured and expanded using the culture method described in Chinese patent application publication number CN113774019A can also be propagated to about 40 generations. Using traditional culture medium and traditional culture methods, umbilical cord and adipose tissue-derived mesenchymal stem cells can be cultured to about 15-18 generations, while umbilical cord blood-derived 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.

[0108] To evaluate the consistency of gene expression profiles across multiple passages (5, 15, and 40) of umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and adipose-derived mesenchymal stem cells (AMSCs) cultured under different culture conditions, we cultured these stem cells using CDM (the CDM 1-3 medium combination of the present invention), CM (Comparative Examples 2, 3, and 4), and reference to Chinese patent application publication number CN113774019A (Comparative Example 1). RNA was extracted at different passages and gene expression levels analyzed using global gene expression profiling. The specific cell culture experimental procedures are detailed in Examples 4, 5, and 6 and Comparative Examples 1-4.

[0109] The Pearson correlation coefficient method was used to quantify gene expression. The gene expression of umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cells (Comparative Examples 2 and 4), and adipose mesenchymal stem cells (Comparative Example 3) cultured in the manner of Example 1 was detected to be consistent with that of P0. The gene expression consistency of mesenchymal stem cells cultured by the method of the present invention was compared with that of cell spheres. The results were calculated according to the above formula. Figure 3 As shown, 1 is completely consistent, and the smaller the data, the greater the difference. The data is displayed in a bar graph. Figure 3Expression consistency analysis was performed using whole-transcriptome sequencing (RNA-seq) on the Illumina sequencing platform. Total RNA was extracted at passages 5, 15, and 40, and Pearson correlation coefficients were calculated using passage 0 as a reference. The results showed that cells treated with the CDM series culture system (the CDM1-3 medium combination of the present invention) maintained highly consistent expression profiles across multiple passages (>0.9), while cells cultured with the traditional system showed a significant decrease (<0.7).

Claims

1. A method for cloning and culturing mesenchymal stem cells, characterized in that: The following steps are involved: (1) Early culture of mesenchymal stem cells: The mesenchymal stem cells isolated and expanded from tissues or buffy coat cells were evenly dispersed in CDM1 medium, and adherent cells from P0 to P4 were obtained after adherent culture. (2) Preparation of single-cell derived cell spheres: Digest and dissociate any of the P0 to P4 cell generations obtained into a single-cell suspension, place it in a low-adsorption culture plate, ensure that there is one cell per well, and culture it in CDM2 medium. Change the medium every three days until the cell spheres grow and the diameter of the cell spheres reaches 50-400 μm, then digest and dissociate it into a single-cell suspension; (3) Cell expansion from single cells: The single cell suspension obtained in step (2) is inoculated into a conventional cell culture plate and cultured in CDM3 medium. When the cells are 85-90% confluent, continuous passage and expansion are performed; The CDM1 culture medium includes α-MEM culture medium, which, based on the α-MEM culture medium, 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 β1, 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; The CDM2 culture medium includes α-MEM culture medium, which is based on the α-MEM culture medium and 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 β1, 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 and 15-30 nM R-spondin 1, 0.5-2×B27, 0.5-2×N2, and 2-4% (w / v) methylcellulose; The CDM3 culture medium includes α-MEM culture medium, which, based on the α-MEM culture medium, 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 β1, 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 transport protein.

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

3. The method for cloning and culturing mesenchymal stem cells according to claim 2, wherein: The separation and expansion steps of the umbilical cord blood mesenchymal stem cells in step (1) are as follows: the umbilical cord blood is subjected to density gradient centrifugation to obtain the buffy coat cells, which are then inoculated into culture plates in the CDM1 culture medium described in claim 1 for adherent culture.

4. The method for cloning and culturing mesenchymal stem cells according to claim 2, wherein: The isolation and expansion steps of the umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells in step (1) are as follows: the umbilical cord, adipose tissue or subcutaneous adipose tissue collected under sterile conditions is cut into fragments, diluted with PBS, centrifuged, the supernatant is removed, and new PBS is added to break it up, diluted, and washed again under the same conditions. Tissue fragments deposited at the bottom of the centrifuge tube are visible. The collected tissue fragments are evenly broken up using the CDM1 culture medium described in claim 1, and are inoculated into coated cell culture plates. The culture medium is replaced every 3 days until the cells crawl out and grow to 85-90% fusion.

5. The method for cloning and culturing mesenchymal stem cells according to claim 1, wherein: The low-adsorption culture plate in step (2) is an agarose-coated low-adsorption culture plate, and its preparation method is as follows: 0.8-1% agarose is prepared using ultrapure water, sterilized under high pressure, poured into the plate while hot, and each plate is added with the CDM2 culture medium described in claim 1 containing gellan gum and balanced.

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