Method for culturing human umbilical cord mesenchymal stem cells

Abstract

The invention provides a culture method of human umbilical cord mesenchymal stem cells, and belongs to the technical field of cell culture. The method comprises the following steps: firstly, constructing an activation culture medium aiming at an initial culture stage and an amplification culture medium aiming at an amplification culture stage; wherein the activation culture medium takes alpha-MEM containing 10% of fetal calf serum as a basic culture medium, and alpha-tocopherol, nicotinic acid and gypenoside are added as additives, so that the cell amplification efficiency in the initial stage can be fully improved; a serum-free scheme is adopted for an amplification culture medium, RPMI-1640 is taken as a basic culture medium, and glutamine, rhG-CSF, bFGF, VEGF, transferrin, vitamin C, 2-mercaptoethanol, zoledronic acid, lenalidomide, ethanolamine and sodium selenite with specific amounts are added, so that the cell amplification speed in an amplification culture stage is remarkably increased. On the basis, a three-stage amplification process is adopted, and large-scale culture of the umbilical cord mesenchymal stem cells is realized. The method is short in culture time and high in cell quantity accumulation speed, and the culture efficiency is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for culturing human umbilical cord mesenchymal stem cells. Background Technology

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a type of pluripotent stem cells extracted from the umbilical cord tissue of newborns. They possess functions such as self-renewal, multi-lineage differentiation, and immune regulation, and are widely used in regenerative medicine, disease treatment, and anti-aging research. These stem cells are mainly found in Wharton's jelly within the umbilical cord. After delivery, the umbilical cord is usually disposed of as medical waste, but mesenchymal stem cells can be extracted through aseptic collection, isolation, and in vitro expansion. Compared to stem cells derived from bone marrow, adipose tissue, etc., umbilical cord-derived stem cells are younger, have stronger proliferative capacity, and the extraction process is non-invasive and ethically uncontroversial.

[0003] Human umbilical cord mesenchymal stem cells possess multi-lineage differentiation capacity: they can differentiate into various cell types such as bone, cartilage, fat, and nerve cells, and are used to repair damaged tissues. They also have immunomodulatory effects: by secreting cytokines, they suppress excessive immune responses and are used to treat autoimmune diseases (such as rheumatoid arthritis) or rejection reactions after organ transplantation. Furthermore, they have low immunogenicity: low expression of surface antigens, resulting in a lower risk of rejection during allogeneic transplantation. Finally, they have paracrine effects: secreting growth factors, exosomes, and other substances that promote tissue repair and angiogenesis.

[0004] Cell culture is a technique that isolates cells from living organisms and grows and proliferates them in a sterile, artificial environment under laboratory conditions. This process typically requires specific culture media and suitable temperature and gas environments to support cell survival and growth. Cell culture technology is widely used in biomedical research, drug screening, and vaccine development. During cell culture, cells undergo stages such as adhesion, growth, and division, ultimately forming cell colonies. Different cell types have different requirements for culture conditions, thus necessitating adjustments to the culture medium composition and environmental parameters based on cell characteristics. Current conventional culture methods for human umbilical cord mesenchymal stem cells are relatively inefficient and struggle to achieve significant cell proliferation in a short time. Therefore, developing new and more efficient methods for culturing human umbilical cord mesenchymal stem cells remains a key technical challenge in this field. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to develop a more efficient method for culturing human umbilical cord mesenchymal stem cells.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: A method for culturing human umbilical cord mesenchymal stem cells, comprising: 1) Add the following additives to RPMI-1640 medium at final concentrations: glutamine 10-20 mg / mL, rhG-CSF 10-20 ng / mL, bFGF 20-40 ng / mL, VEGF 40-60 ng / mL, transferrin 30-60 μg / mL, vitamin C 2-8 mg / mL, 2-mercaptoethanol 10-20 μg / mL, zoledronic acid 25-40 μg / mL, lenalidomide 10-20 μg / mL, ethanolamine 1-5 μg / mL, and sodium selenite 10-45 μg / mL to obtain the amplification medium; 2) Add the following additives to α-MEM medium containing 10% fetal bovine serum to obtain the activation medium by adding the following final concentrations: α-tocopherol 10~30μg / mL, nicotinic acid 5~10μg / mL, and gypenosides 15~50μg / mL. 3) Use the activation medium obtained in step 2) to activate and culture umbilical cord mesenchymal stem cells to obtain an activated cell suspension; dilute the activated cell suspension with the amplification medium and continue culturing for 48 hours; add the amplification medium and continue culturing for 48 hours; transfer the obtained culture medium to a culture container, add the amplification medium, and continue culturing for 72 hours; collect the cells.

[0007] Preferably, the additives in step 1) further include cynarin and oleuropein, wherein the final concentration of cynarin is 1~5 μg / mL and the final concentration of oleuropein is 1~5 μg / mL.

[0008] Preferably, the additives in step 1) further include γ-aminobutyric acid and adenosine-5'-bisphosphate disodium, wherein the final concentration of γ-aminobutyric acid is 8~15 μg / mL and the final concentration of adenosine-5'-bisphosphate disodium is 20~30 μg / mL.

[0009] Preferably, in step 1), the following additives are added to the RPMI-1640 medium at final concentrations: glutamine 15 mg / mL, rhG-CSF 15 ng / mL, bFGF 30 ng / mL, VEGF 50 ng / mL, transferrin 45 μg / mL, vitamin C 5 mg / mL, 2-mercaptoethanol 15 μg / mL, zoledronic acid 32 μg / mL, lenalidomide 15 μg / mL, ethanolamine 3 μg / mL, and sodium selenite 28 μg / mL to obtain the amplification medium.

[0010] Preferably, in step 2), the additive further includes EGF and TGF-β, wherein the final concentration of EGF is 10~20 ng / mL and the final concentration of TGF-β is 25~35 ng / mL.

[0011] Preferably, in step 2), DMEM medium or RPMI-1640 medium is used instead of the α-MEM medium.

[0012] Preferably, in step 2), additives with the following final concentrations are added to the α-MEM medium containing 10% fetal bovine serum: α-tocopherol 20 μg / mL, nicotinic acid 9 μg / mL, and gypenosides 30 μg / mL to obtain the activation medium.

[0013] Preferably, in step 3), the pH of the culture environment at each stage is maintained at 7.2-7.4, and the osmotic pressure is maintained at 320-350 mOsm / kg.

[0014] Preferably, in step 3), the culture environment is maintained at 37°C and 5% CO2 at each stage.

[0015] Preferably, in step 3), the concentration of the amplification culture medium in the diluted and activated cell suspension is 0.3 to 0.5 times its original concentration.

[0016] This invention provides a method for culturing human umbilical cord mesenchymal stem cells. The technical solution first constructs an activation medium for the initial culture stage and an expansion medium for the scaling-up culture stage. The activation medium uses α-MEM containing 10% fetal bovine serum as the base medium, with added α-tocopherol, nicotinic acid, and gypenosides as additives, which can significantly improve the efficiency of cell expansion in the initial stage. The expansion medium uses a serum-free protocol, with RPMI-1640 as the base medium, and adds specific amounts of glutamine, rhG-CSF, bFGF, VEGF, transferrin, vitamin C, 2-mercaptoethanol, zoledronic acid, lenalidomide, ethanolamine, and sodium selenite, significantly improving the cell expansion rate in the scaling-up culture stage. Based on this, this invention employs a three-stage scale-up process to achieve large-scale culture of umbilical cord mesenchymal stem cells. This invention features short culture time, rapid cell accumulation, and significantly improved culture efficiency, possessing outstanding technical advantages and promising prospects for widespread application. Attached Figure Description

[0017] Figure 1 This is a graph showing the statistical results of the final cell concentration obtained in each embodiment of the present invention. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below. To avoid excessive and unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. The approximate language used in the following embodiments is for quantitative purposes, indicating that a certain degree of variation in quantity is permissible without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Example

[0019] Take the umbilical cord, separate Wharton's jelly, and cut it into pieces approximately 1 mm thick. 3 Small pieces of the cells were cultured in a cell culture medium containing albumin at 37°C and 5% CO2. The culture medium was changed every 5 days (DMEM + 2% Ultroser G). Primary mesenchymal stem cells were obtained when the cells reached 70%-80% confluence.

[0020] Add the following additives to α-MEM medium containing 10% fetal bovine serum to final concentrations: α-tocopherol 10 μg / mL, nicotinic acid 5 μg / mL, and gypenosides 15 μg / mL to obtain activation medium; collect the culture medium from the primary cell culture flasks, centrifuge, wash the flasks once with PBS, add trypsin containing EDTA for 1-3 min digestion, and observe continuously with an inverted microscope during digestion. When cytoplasmic retraction, increased intercellular spaces, and cell rounding are observed, immediately add the appropriate volume of supernatant to stop digestion. Repeatedly pipette the adherent cells to detach them, centrifuge at 210g for 10 min, resuspend the cells in cell culture medium, and repeat at 1×10⁻⁶. 6 The cells were seeded at a density of / flask into T-175 culture flasks, and activated culture medium was added. The cells were cultured at 37°C and 5% CO2 for 3 days to obtain primary cells.

[0021] Add the following additives to RPMI-1640 medium at final concentrations: glutamine 10 mg / mL, rhG-CSF 10 ng / mL, bFGF 20 ng / mL, VEGF 40 ng / mL, transferrin 30 μg / mL, vitamin C 2 mg / mL, 2-mercaptoethanol 10 μg / mL, zoledronic acid 25 μg / mL, lenalidomide 10 μg / mL, ethanolamine 1 μg / mL, and sodium selenite 10 μg / mL to obtain the amplification medium. Clean a 250 mL glass culture flask, dry it completely, add silanizing solution to the flask, slowly rotate the flask to wet the flask wall, remove the silanizing solution, place the flask in a ventilated place to air dry for 12 h, rinse with purified water, and autoclave.

[0022] Add amplification medium to the culture system to adjust the final concentration of primary cells to 0.5*10⁻⁶. 5The cells / mL were cultured for another 48 hours; then expansion medium was added, and the mixture was intermittently stirred for 4 hours (3 min at 70 rpm, 30 min incubation), and cultured for another 48 hours. The resulting culture was transferred to a culture vessel, the expansion medium was added, and the mixture was stirred at 70 rpm and cultured at 37°C and 5% CO2 for 72 hours. Then, stirring was stopped, the supernatant was aseptically collected, and digested with trypsin-EDTA. After 15 min, the supernatant was added to terminate the digestion, yielding human umbilical cord mesenchymal stem cells, with a concentration of 4.5*10⁻⁶. 6 cells / mL.

[0023] When umbilical cord mesenchymal stem cells reach 80-90% confluence in a square flask, the supernatant is aseptically collected and washed once with 0.9% physiological saline. Then, trypsin containing EDTA is added for 1-3 minutes to digest the cells. After all cells have detached, the supernatant is added to stop the digestion. The cells are centrifuged at 210g / min for 10 minutes, the supernatant is removed, and the cells are resuspended in stem cell cryopreservation medium. The resulting umbilical cord mesenchymal stem cell suspension is added to a cell cryopreservation tube and placed in a programmed cooling box containing isopropanol at -80°C overnight. The next day, the cells are transferred to liquid nitrogen at -196°C for long-term storage.

[0024] After thawing frozen cells at 37°C, they were added to umbilical cord mesenchymal stem cell culture. Cultured for 3 days at 37°C and 5% CO2, yielding 3*10 cells per cell. 6 The cell was used for flow cytometry detection of cell surface markers such as CD105, CD73, CD90, CD45, CD34, CD14, CD19, and HLA-DR. The results showed that the cells uniformly and highly expressed mesenchymal stem cell markers CD45, CD105, CD73, and CD90; and expressed little or no CD34, CD14, CD19, and HLA-DR (positive rate ≤5%). This is consistent with the results of mesenchymal stem cell surface antigen identification, indicating that revived umbilical cord mesenchymal stem cells still retain the surface characteristics of mesenchymal stem cells. Example

[0025] A method for culturing human umbilical cord mesenchymal stem cells, comprising: 1) Add the following additives to RPMI-1640 medium at final concentrations: glutamine 20 mg / mL, rhG-CSF 20 ng / mL, bFGF 40 ng / mL, VEGF 60 ng / mL, transferrin 60 μg / mL, vitamin C 8 mg / mL, 2-mercaptoethanol 20 μg / mL, zoledronic acid 40 μg / mL, lenalidomide 20 μg / mL, ethanolamine 5 μg / mL, and sodium selenite 45 μg / mL to obtain the amplification medium; 2) Add the following additives to α-MEM medium containing 10% fetal bovine serum to obtain the activation medium by adding the following final concentrations: α-tocopherol 30 μg / mL, nicotinic acid 10 μg / mL, and gypenosides 50 μg / mL. 3) Umbilical cord mesenchymal stem cells were activated and cultured using the activation medium obtained in step 2) to obtain an activated cell suspension; the activated cell suspension was diluted with the amplification medium and cultured for another 48 hours; the amplification medium was added and cultured for another 48 hours; the resulting culture medium was transferred to a culture vessel, the amplification medium was added, and the cells were cultured for another 72 hours; the cells were collected and their concentration was measured to be 7.3*10⁻⁶. 6 cells / mL. Example

[0026] A method for culturing human umbilical cord mesenchymal stem cells, comprising: 1) Add the following additives to RPMI-1640 medium at final concentrations: glutamine 10-20 mg / mL, rhG-CSF 10-20 ng / mL, bFGF 20-40 ng / mL, VEGF 40-60 ng / mL, transferrin 30-60 μg / mL, vitamin C 2-8 mg / mL, 2-mercaptoethanol 10-20 μg / mL, zoledronic acid 25-40 μg / mL, lenalidomide 10-20 μg / mL, ethanolamine 1-5 μg / mL, and sodium selenite 10-45 μg / mL to obtain the amplification medium; 2) Add the following additives to α-MEM medium containing 10% fetal bovine serum to obtain the activation medium by adding the following final concentrations: α-tocopherol 10~30μg / mL, nicotinic acid 5~10μg / mL, and gypenosides 15~50μg / mL. 3) Umbilical cord mesenchymal stem cells are activated and cultured using the activation medium obtained in step 2) to obtain an activated cell suspension; the activated cell suspension is diluted with the amplification medium and cultured for another 48 hours; the amplification medium is added and cultured for another 48 hours; the resulting culture medium is transferred to a culture vessel, the amplification medium is added, and the cells are cultured for another 72 hours; the cells are then collected. The additives mentioned in step 1) further include cynarin and oleuropein, with the final concentration of cynarin being 1-5 μg / mL and the final concentration of oleuropein being 1-5 μg / mL. The additives mentioned in step 1) also include γ-aminobutyric acid (GABA) and adenosine-5'-bisphosphate disodium, with the final concentration of GABA being 8-15 μg / mL and the final concentration of adenosine-5'-bisphosphate disodium being 20-30 μg / mL. In step 1), the following additives are added to RPMI-1640 medium at final concentrations: glutamine 15 mg / mL, rhG-CSF 15 ng / mL, bFGF 30 ng / mL, VEGF 50 ng / mL, transferrin 45 μg / mL, vitamin C 5 mg / mL, 2-mercaptoethanol 15 μg / mL, zoledronic acid 32 μg / mL, lenalidomide 15 μg / mL, ethanolamine 3 μg / mL, and sodium selenite 28 μg / mL to obtain the amplification medium. In step 2), the additives also include EGF and TGF-β, with the final concentration of EGF being 10-20 ng / mL and the final concentration of TGF-β being 25-35 ng / mL. In step 2), DMEM medium or RPMI-1640 medium is used instead of the α-MEM medium. In step 2), additives were added to α-MEM medium containing 10% fetal bovine serum at the following final concentrations: α-tocopherol 20 μg / mL, nicotinic acid 9 μg / mL, and gypenosides 30 μg / mL, to obtain the activation medium. In step 3), the pH of the culture environment was maintained at 7.2–7.4, and the osmotic pressure was maintained at 320–350 mOsm / kg at each stage. In step 3), the culture environment was maintained at 37°C and 5% CO2 at each stage. In step 3), the concentration of the amplification medium in the diluted activated cell suspension was 0.3–0.5 times its original concentration. The concentration of the obtained human umbilical cord mesenchymal stem cells was measured to be 1.25 × 10⁻⁶. 7 cells / mL.

[0027] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for culturing human umbilical cord mesenchymal stem cells, characterized in that, include: 1) Add the following additives to RPMI-1640 medium at final concentrations: glutamine 10-20 mg / mL, rhG-CSF 10-20 ng / mL, bFGF 20-40 ng / mL, VEGF 40-60 ng / mL, transferrin 30-60 μg / mL, vitamin C 2-8 mg / mL, 2-mercaptoethanol 10-20 μg / mL, zoledronic acid 25-40 μg / mL, lenalidomide 10-20 μg / mL, ethanolamine 1-5 μg / mL, and sodium selenite 10-45 μg / mL to obtain the amplification medium; 2) Add the following additives to α-MEM medium containing 10% fetal bovine serum to obtain the activation medium by adding the following final concentrations: α-tocopherol 10~30μg / mL, nicotinic acid 5~10μg / mL, and gypenosides 15~50μg / mL. 3) Use the activation medium obtained in step 2) to activate and culture umbilical cord mesenchymal stem cells to obtain an activated cell suspension; dilute the activated cell suspension with the amplification medium and continue culturing for 48 hours; add the amplification medium and continue culturing for 48 hours; transfer the obtained culture medium to a culture container, add the amplification medium, and continue culturing for 72 hours; collect the cells.

2. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The additives mentioned in step 1) also include cynarin and oleuropein, wherein the final concentration of cynarin is 1~5 μg / mL and the final concentration of oleuropein is 1~5 μg / mL.

3. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The additives described in step 1) further include γ-aminobutyric acid and adenosine-5'-bisphosphate disodium, wherein the final concentration of γ-aminobutyric acid is 8~15 μg / mL and the final concentration of adenosine-5'-bisphosphate disodium is 20~30 μg / mL.

4. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 1), the following additives were added to the RPMI-1640 medium at final concentrations: glutamine 15 mg / mL, rhG-CSF 15 ng / mL, bFGF 30 ng / mL, VEGF 50 ng / mL, transferrin 45 μg / mL, vitamin C 5 mg / mL, 2-mercaptoethanol 15 μg / mL, zoledronic acid 32 μg / mL, lenalidomide 15 μg / mL, ethanolamine 3 μg / mL, and sodium selenite 28 μg / mL to obtain the amplification medium.

5. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 2), the additives also include EGF and TGF-β, wherein the final concentration of EGF is 10~20 ng / mL and the final concentration of TGF-β is 25~35 ng / mL.

6. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 2), the α-MEM medium is replaced with DMEM medium or RPMI-1640 medium.

7. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 2), additives with the following final concentrations are added to α-MEM medium containing 10% fetal bovine serum: α-tocopherol 20 μg / mL, nicotinic acid 9 μg / mL, and gypenosides 30 μg / mL to obtain the activation medium.

8. The method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 3), the pH of the culture environment at each stage is maintained at 7.2-7.4, and the osmotic pressure is maintained at 320-350 mOsm / kg.

9. A method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 3), the culture environment is maintained at 37°C and 5% CO2 at each stage.

10. A method for culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, In step 3), the concentration of the amplification culture medium in the diluted and activated cell suspension is 0.3 to 0.5 times its original concentration.

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