Serum-free complete medium for human umbilical cord mesenchymal stem cells and preparation method of serum-free complete medium

Abstract

The invention relates to the technical field of culture media, in particular to a serum-free complete culture medium for human umbilical cord mesenchymal stem cells and a preparation method thereof.The serum-free complete culture medium takes DMEM / F12 as a basic culture medium and is matched with UltraGRO-Advanced, a penicillin-streptomycin double-antibody solution, a recombinant human epidermal growth factor and a basic fibroblast growth factor, and the serum-free complete culture medium for the human umbilical cord mesenchymal stem cells is obtained. The serum-free complete culture medium can replace a serum culture medium in a conventional culture system of mesenchymal stem cells, the characteristics of normal adherence, good growth state and stem cells of the cells are kept, the serum-free complete culture medium reduces the risks of heterologous contamination and immunological rejection to the maximum extent, the added components capable of promoting cell growth and the content can be determined and controlled, and the culture medium is suitable for large-scale popularization and application. The use is safer.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium, and in particular to a serum-free complete culture medium for human umbilical cord mesenchymal stem cells and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells are a type of adult stem cells with self-replication and multidirectional differentiation potential. They are widely present in various tissues throughout the body, can be cultured and expanded in vitro, and can differentiate into cells including fat cells, bone cells, chondrocytes, etc. under specific conditions. They are increasingly attracting attention due to their multiple differentiation potentials, hematopoietic support, promotion of stem cell implantation, immune regulation and self-replication.

[0003] Umbilical cord mesenchymal stem cells are derived from the umbilical cord of healthy full-term newborns. They are obtained by tissue scraping or enzyme digestion. They have strong immunomodulatory effects, can promote hematopoietic recovery function and repair diseased tissues and organs, and have high clinical application value in organ transplantation, autoimmune diseases, leukemia, bone and muscle degenerative diseases, etc.

[0004] At present, the in vitro culture method of stem cells is mainly based on DMEM / F12 culture medium, with the addition of fetal bovine serum and some other nutrients required by cells. However, animal serum also has the risk of inducing immune rejection and the risk of contamination by pathogenic microorganisms such as bacteria and viruses, so it is necessary to develop serum-free culture medium with clear chemical composition. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method for improving the stability of phenethylresorcinol whitening gel in view of the deficiencies in the prior art.

[0006] The purpose of the present invention is achieved through the following technical solutions: A serum-free complete culture medium for human umbilical cord mesenchymal stem cells consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

[0007] Wherein, the content of L-glutamine in the DMEM / F12 basic culture medium is 5 mM / L.

[0008] Wherein, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

[0009] The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 2-10%.

[0010] The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

[0011] Wherein, the concentration of the recombinant human epidermal growth factor in the serum-free complete culture medium is 1-10 ng / mL.

[0012] Wherein, the concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 1-10 ng / mL.

[0013] A method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells comprises adding UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor into a DMEM / F12 basal culture medium and stirring the culture medium to obtain the complete culture medium.

[0014] The DMEM / F12 basal medium used in the present invention is a DMEM / F12 basal medium containing L-glutamine, and the content of L-glutamine is 5 mM / L, preferably, further containing sodium pyruvate 1mM / L, more preferably, using Wuhan Pu Nuo Sai Life Science Technology Co., Ltd. DMEM / F12, L-glutamine is an essential amino acid for cell growth, it is an important energy source for cells, and participates in protein synthesis and nucleic acid metabolism. L-glutamine is essential for maintaining normal growth and proliferation of cells, but it is unstable in solution and will degrade after a period of time. The degradation product ammonia is toxic to some cells, especially to stem cells and primary cells. The impact is greater, so the amount of L-glutamine should be controlled at an optimal concentration.

[0015] UltraGRO™-Advanced uses Helios' product number: HPCFDCRL10. As a serum substitute, it is rich in growth factors and cytokines and can be used for research purposes or industrial cell growth and proliferation of multiple cell types (such as mesenchymal stem cells). 5% UltraGRO™-Advanced can replace 10%-20% fetal bovine serum. It is more effective than fetal bovine serum in primary and secondary culture, has high batch-to-batch stability, and costs less to culture every 1 million cells.

[0016] Recombinant human epidermal growth factor is a small peptide composed of 53 amino acid residues, which has a strong mitogenic effect on a variety of tissue cells both in vivo and in vitro.

[0017] Recombinant human basic fibroblast growth factor can mainly promote cell repair and cell regeneration. It has the effect of promoting repair and cell proliferation for cells derived from the ectoderm and mesoderm.

[0018] Beneficial effects of the present invention: The serum-free complete culture medium of the present invention uses DMEM / F12 as the basic culture medium, and is matched with UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor and basic fibroblast growth factor as raw materials to prepare a serum culture medium that can replace the conventional culture system of mesenchymal stem cells, maintain normal cell adhesion, good growth state, and the characteristics of stem cells. The serum-free complete culture medium of the present invention has simple raw materials, minimizes the risks of heterologous contamination and immune rejection, and the added components that can promote cell growth and their content can be determined and controlled, making it safer to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Microscope image of human umbilical cord mesenchymal stem cells cultured in commercially available serum-free complete medium.

[0020] Figure 2 This is a microscope picture of human umbilical cord mesenchymal stem cells cultured in the serum-free complete medium prepared in the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described in conjunction with the following examples. Embodiment 1

[0022] A serum-free complete culture medium for human umbilical cord mesenchymal stem cells consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

[0023] Wherein, the content of L-glutamine in the DMEM / F12 basic culture medium is 5 mM / L.

[0024] Wherein, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

[0025] The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 2%.

[0026] The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

[0027] Wherein, the concentration of the recombinant human epidermal growth factor in the serum-free complete culture medium is 10 ng / mL.

[0028] Wherein, the concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 10 ng / mL.

[0029] A method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells comprises adding UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor into a DMEM / F12 basal culture medium and stirring the culture medium to obtain the complete culture medium. Embodiment 2

[0030] A serum-free complete culture medium for human umbilical cord mesenchymal stem cells consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

[0031] Wherein, the content of L-glutamine in the DMEM / F12 basic culture medium is 5 mM / L.

[0032] Wherein, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

[0033] The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 4%.

[0034] The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

[0035] Wherein, the concentration of the recombinant human epidermal growth factor in the serum-free complete culture medium is 8 ng / mL.

[0036] Wherein, the concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 8 ng / mL.

[0037] A method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells comprises adding UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor into a DMEM / F12 basal culture medium and stirring the culture medium to obtain the complete culture medium. Embodiment 3

[0038] A serum-free complete culture medium for human umbilical cord mesenchymal stem cells consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

[0039] Wherein, the content of L-glutamine in the DMEM / F12 basic culture medium is 5 mM / L.

[0040] Wherein, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

[0041] The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 6%.

[0042] The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

[0043] Wherein, the concentration of the recombinant human epidermal growth factor in serum-free complete culture medium is 6 ng / mL.

[0044] Wherein, the concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 6 ng / mL.

[0045] A method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells comprises adding UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor into a DMEM / F12 basal culture medium and stirring the culture medium to obtain the complete culture medium. Embodiment 4

[0046] A serum-free complete culture medium for human umbilical cord mesenchymal stem cells consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

[0047] Wherein, the content of L-glutamine in the DMEM / F12 basic culture medium is 5 mM / L.

[0048] Wherein, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

[0049] The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 8%.

[0050] The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

[0051] Wherein, the concentration of the recombinant human epidermal growth factor in serum-free complete culture medium is 4 ng / mL.

[0052] Wherein, the concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 4 ng / mL.

[0053] A method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells comprises adding UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor into a DMEM / F12 basal culture medium and stirring the culture medium to obtain the complete culture medium. Embodiment 5

[0054] A serum-free complete culture medium for human umbilical cord mesenchymal stem cells consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

[0055] Wherein, the content of L-glutamine in the DMEM / F12 basic culture medium is 5 mM / L.

[0056] Wherein, in the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

[0057] The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 10%.

[0058] The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

[0059] Wherein, the concentration of the recombinant human epidermal growth factor in the serum-free complete culture medium is 2 ng / mL.

[0060] Wherein, the concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 2 ng / mL.

[0061] A method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells comprises adding UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor into a DMEM / F12 basal culture medium and stirring the culture medium to obtain the complete culture medium.

[0062] The culture conditions used in the embodiments of the present invention are all cultured in a CO2 incubator at 37°C, and the CO2 concentration is 5% (v / v).

[0063] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0064] Control group: Commercially available serum-free culture medium. This control group uses Youkang umbilical cord mesenchymal stem cell serum-free culture medium sold by Foshan Yunyayou Biotechnology Co., Ltd., product number NC0103+NC0103.S. Experimental part:

[0065] 1. Calculation of cell viability Experimental method: Trypan blue staining was used to compare the cell viability of cells cultured in the culture medium of Examples 1 to 5 and the control group.

[0066] 1) Inoculate human umbilical cord mesenchymal stem cells before P5 into 6 T25 culture flasks at the same density, add culture medium to 4 mL, and then place them in a cell culture incubator at 37°C and 5% CO2 for cell culture.

[0067] 2) After about 2-3 days, the cell density grows to more than 90%, and the cells in the cell culture flask are collected.

[0068] 3) The collected human umbilical cord mesenchymal stem cells were resuspended in culture medium and used for cell counting.

[0069] 4) Wipe the blood cell counting plate and cover slide clean, and place the cover slide on the counting plate.

[0070] 5) Take 20µL of cell suspension and add it to 20µL of trypan blue solution, dilute it 2 times, and mix the diluted cell samples.

[0071] 6) Pipette 10 µL of the suspension and drop it on the edge of the cover slip, so that the suspension fills the space between the cover slip and the counting plate.

[0072] 5) Under the microscope, dead cells are stained blue, while live cells are transparent and refractile (unstained).

[0073] 6) Cell density calculation method: Average number of viable cells in each square × dilution factor × 10000 = number of viable cells / mL 7) The cell number calculation method is as follows: Cell density × constant volume = number of cells 8) The cell viability calculation method is as follows: Live cell density / (live cell density+dead cell density)×100%=cell viability The experimental results of cell number and cell viability in each group are shown in Table 1:

[0074] Result analysis: The cell number and cell viability of Examples 1-5 were higher than those of the control group.

[0075] 2. Electron Microscope Scanning The umbilical cord mesenchymal stem cells of the present invention and the control group were scanned by electron microscope, and the representative electron microscope scans of Example 3 and the control group were selected. Figure 1 and Figure 2, Result analysis: Compared with the commercially available serum-free culture medium, the serum-free complete culture medium for human umbilical cord mesenchymal stem cells of the present invention has high cell activity and excellent growth state, and is more conducive to the culture of human umbilical cord mesenchymal stem cells.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A serum-free complete culture medium for human umbilical cord mesenchymal stem cells, characterized in that: It consists of DMEM / F12 basal culture medium, UltraGRO™-Advanced, penicillin-streptomycin double antibody solution, recombinant human epidermal growth factor, and basic fibroblast growth factor.

2. The serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The content of L-glutamine in the DMEM / F12 basal culture medium is 5 mM / L.

3. The serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: In the penicillin-streptomycin dual antibiotic solution, the content of penicillin is 10000U / mL, and the content of streptomycin is 10mg / mL.

4. The serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The volume proportion of UltraGRO™-Advanced in serum-free complete culture medium is 2-10%.

5. The serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The volume proportion of the penicillin-streptomycin dual antibody solution in the serum-free complete culture medium is 1%.

6. The serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The concentration of the recombinant human epidermal growth factor in the serum-free complete culture medium is 1-10 ng / mL.

7. The serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: The concentration of the recombinant human basic fibroblast growth factor in the serum-free complete culture medium is 1-10 ng / mL.

8. The method for preparing a serum-free complete culture medium for human umbilical cord mesenchymal stem cells according to any one of claims 1 to 7, characterized in that: UltraGRO™-Advanced, penicillin-streptomycin dual antibody solution, recombinant human epidermal growth factor, and recombinant human basic fibroblast growth factor were added to DMEM / F12 basal culture medium and stirred evenly to obtain the product.

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