Mesenchymal stem cell culture medium and culture method

Abstract

The invention relates to the technical field of mesenchymal stem cell culture, in particular to a mesenchymal stem cell culture medium and a culture method. The mesenchymal stem cell culture medium is a serum-free culture medium formed by taking a DMEM / F12 culture medium as a basic culture medium and adding supplemental factors, the components in the culture medium are clear, adverse factors such as batch-to-batch quality difference and serum-derived pollution caused by a traditional serum-containing culture medium can be effectively avoided, and the culture medium has the advantages that the culture medium is suitable for large-scale popularization and application. Therefore, the influence of serum components on experimental research is avoided, and a positive effect on ensuring the accuracy of experimental research is achieved; meanwhile, corn glycopeptide is added into the culture medium, on one hand, a nitrogen source needed by cell proliferation is supplemented, on the other hand, rich amino acid and glucose can be provided, and the positive and effective effects on stable and rapid growth and proliferation of cells are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cell culture, and in particular to a mesenchymal stem cell culture medium and a culture method. Background Art

[0002] Mesenchymal stem cells are an important member of the stem cell family. They originate from the early mesoderm of development and are pluripotent stem cells. Mesenchymal stem cells were originally discovered in the bone marrow. They are increasingly attracting attention because of their multidirectional differentiation potential, hematopoietic support, promotion of stem cell engraftment, immune regulation and self-replication.

[0003] Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into a variety of tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc. They still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathology. The existing cell culture method is used to culture and proliferate mesenchymal stem cells. During the culture process, culture medium is required. Traditional culture medium generally contains animal serum, such as fetal bovine serum. The components in the serum play an active and effective role in the stable growth and proliferation of cells. However, when using serum, the quality difference between serum batches is uncontrollable and it is also susceptible to viral infection, which affects the accuracy of experimental research.

[0004] In order to avoid the influence of serum on the accuracy of experimental research, serum-free culture medium came into being. Serum-free culture medium is composed of basal culture medium with supplementary factors with clear ingredients. By discarding serum, it fundamentally avoids the uncontrollability of experimental research caused by differences between serum batches and source pollution. However, due to the complex composition of serum, its effect in cell culture is still unclear. After research, people have proposed many serum-free culture media, but none of them can completely replace serum-containing culture media. Therefore, research on serum-free culture media has been carried out tirelessly. Compared with animal-derived serum, plant-derived proteins have smaller differences between batches and are less susceptible to contamination. Therefore, through research, the present invention proposes a serum-free culture medium and a cell culture method based thereon, which adds plant-derived corn glycopeptide, which plays a positive and effective role in the rapid and stable proliferation and culture of cells, and provides a feasible direction for the development of a more excellent serum-free culture medium. Summary of the invention

[0005] The purpose of the present invention is to provide a mesenchymal stem cell culture medium and a culture method based on plant-derived protein to overcome the problems of large batch differences and susceptibility to contamination of traditional animal-derived serum, and to provide a feasible direction for the development of a more excellent serum-free culture medium.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a mesenchymal stem cell culture medium, comprising a basal culture medium and supplementary factors; the supplementary factors include penicillin, streptomycin, transferrin, recombinant human serum albumin, leukemia inhibitory factor, insulin, platelet-derived growth factor, corn glycopeptide, collagen, soybean trypsin inhibitor, and sodium selenite.

[0007] Furthermore, the basic culture medium is DMEM / F12 culture medium; DMEM / F12 serum-free culture medium is a mixture of DMEM culture medium and F12 culture medium in a volume ratio of 1:1, and the DMEM culture medium is a DMEM low-glucose culture medium.

[0008] Furthermore, the concentrations of the supplementary factors are: penicillin 50-100 U / mL, streptomycin 50-100 μg / mL, transferrin 40-60 μg / mL, recombinant human serum albumin 5-10 μg / mL, leukemia inhibitory factor 10-50 ng / mL, insulin 10-20 μg / mL, platelet-derived growth factor 20-30 μg / mL, corn glycopeptide 100-200 μg / mL, collagen 200-300 μg / mL, soybean trypsin inhibitor 10-20 μg / mL, and sodium selenite 20-30 μg / mL.

[0009] Furthermore, the corn glycopeptide is a product of covalent bonding of low molecular weight corn peptide obtained by protease hydrolysis of zein and D-glucosamine.

[0010] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0011] The method for culturing mesenchymal stem cells according to the above culture medium comprises the following steps:

[0012] S1. Take the frozen umbilical cord mesenchymal stem cells and put them into a 37°C water bath to thaw quickly;

[0013] S2, adding the culture medium of the present invention to the thawed cells in S1, washing them thoroughly and centrifuging them, and discarding the supernatant;

[0014] S3, adding the culture medium of the present invention to the cells obtained in S2 again, mixing by pipetting to prepare a cell suspension, inoculating the cell suspension into a culture dish, and culturing in a cell culture incubator;

[0015] S4. When the cell density reaches 80-90%, remove the old culture medium, rinse thoroughly with PBS buffer, and then add trypsin for digestion;

[0016] S5. After digestion is completed, the culture medium of the present invention is added to terminate digestion, and the culture medium of the present invention is added after sufficient rinsing and centrifugation with PBS buffer, and the culture medium of the present invention is added and blown evenly. After counting, steps S3-S5 are repeated to continue culturing.

[0017] Furthermore, in S3, the culture conditions are 37°C and 5% volume fraction CO 2 , relative humidity 95%.

[0018] Furthermore, in the S4, trypsin is 0.25% trypsin-EDTA digestion solution.

[0019] Furthermore, in the S4, the digestion temperature is 37° C. and the digestion time is 2 to 3 minutes.

[0020] Beneficial effects of the present invention:

[0021] 1. The culture medium of the present invention is composed of a basic culture medium and supplementary factors. By adding various supplementary factors during the process, stable and rapid cell proliferation can be achieved, which can significantly improve the efficiency of cell culture;

[0022] 2. The method of the present invention adds corn glycopeptide to the culture medium, which can provide a variety of nutrients required for cell production, such as nitrogen source, amino acids and glucose, and on the other hand, helps to avoid mechanical damage to the cells, helps the cells to grow adherently, and plays a positive role in the stable and rapid proliferation of the cells. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0024] Example 1

[0025] In this embodiment, the culture medium of the present invention comprises a basal culture medium and supplementary factors; wherein the basal culture medium is a DMEM / F12 culture medium; the DMEM / F12 serum-free culture medium is a mixture of a DMEM culture medium and a F12 culture medium in a volume ratio of 1:1, and the DMEM culture medium is a DMEM low-glucose culture medium (D-glucose content 1000 mg / L); the supplementary factors are 60 U / mL of penicillin, 80 μg / mL of streptomycin, 50 μg / mL of transferrin, 8 μg / mL of recombinant human serum albumin, 30 ng / mL of leukemia inhibitory factor, 15 μg / mL of insulin, 25 μg / mL of platelet-derived growth factor, 180 μg / mL of corn glycopeptide, 250 μg / mL of collagen, 15 μg / mL of soybean trypsin inhibitor, and 25 μg / mL of sodium selenite.

[0026] The cryopreserved umbilical cord mesenchymal stem cells were taken and revived, and then subcultured using the culture medium of the present invention. The subculture was continued until P10, and the time required for each subculture was recorded. According to the records, the average time required for each subculture was about 11 days.

[0027] P3, P5 and P10 were taken respectively, and the obtained passage cells were measured and observed. After observation, the cell morphology was normal, and the survival rates were 93.82%, 88.66% and 72.20%, respectively. It can be seen that the culture medium of the present invention can effectively ensure the survival rate of the cells, and at the same time, the culture time is short and the efficiency is high.

[0028] P10 cells were taken for induced differentiation. During the process, the P10 cells were tested for adipogenic differentiation and all showed excellent differentiation ability. The differentiation efficiency could reach 90% within 5 days.

[0029] Comparative Example 1

[0030] In this comparative example, the corn glycopeptide in the culture medium of the present invention was replaced with fetal bovine serum (10%), and the other conditions were the same as those in Example 1.

[0031] Under the same conditions as in Example 1, the umbilical cord mesenchymal stem cells were cultured using the culture medium of this comparative example. The culture steps were the same as those of the present invention, and the seeding density was 8000 cells / cm 2 , and cultured to P10 during the process.

[0032] According to observations and records, the average culture time of each generation of cells is 16 days. During the process, P3, P5 and P10 cells were taken. It was observed that the cell morphology was normal and the cell survival rates were measured, which were 93.81%, 88.68% and 75.43% respectively. P10 cells were taken for adipogenic differentiation and the cells had excellent differentiation ability, and the differentiation efficiency could reach 90% within 5 days.

[0033] By comparison, it can be found that the corn glycopeptide of the present invention has a similar effect to that of fetal bovine serum. After multiple passages, the cell survival rate of the culture medium using fetal bovine serum is better than that of the culture medium of the present invention. However, in comparison, the culture efficiency of the present invention is better than that of the culture medium using fetal bovine serum. Although the cell survival rate of the culture medium of the present invention is slightly lower than that of the culture medium using fetal bovine serum, the difference is not obvious. At the same time, the cells after proliferation culture have a good differentiation effect, indicating that the culture medium of the present invention can completely replace the traditional serum-containing culture medium.

[0034] Comparative Example 2

[0035] In this comparative example, the corn glycopeptide in the culture medium of the present invention was replaced with glutamine dipeptide (L-alanyl-L-glutamine dipeptide), and the rest was the same as in Example 1.

[0036] Under the same conditions as in Example 1, the umbilical cord mesenchymal stem cells were cultured using the culture medium of this comparative example, and the culture steps were the same as the specific steps of the present invention, and the cells were cultured to the P10 generation.

[0037] According to observations and records, the average culture time of each generation of cells is 22 days. During the process, P3, P5 and P10 cells were taken. It was observed that the cell morphology was normal. The cell survival rates were measured, which were 90.18%, 81.84% and 62.25% respectively. P10 cells were taken for adipogenic differentiation. The cells had good differentiation ability and the differentiation efficiency could reach 80% within 5 days.

[0038] By comparison, it can be found that the culture efficiency of the medium of the present invention is significantly lower when glutamine dipeptide is used to replace the corn glycopeptide of the present invention, indicating that corn glycopeptide has the effect of promoting the improvement of cell culture efficiency. At the same time, the cell survival rate is also significantly lower than that of the medium of the present invention, indicating that corn glycopeptide provides more sufficient and comprehensive nutrients and plays a positive role in cell survival. In addition, the activity and differentiation ability of the cells using the medium of the present invention are better than those using glutamine dipeptide.

[0039] Comparative Example 3

[0040] In this comparative example, the corn glycopeptide in the culture medium of the present invention was replaced with corn peptide, and the rest was the same as in Example 1.

[0041] Under the same conditions as in Example 1, the umbilical cord mesenchymal stem cells were cultured using the culture medium of this comparative example, and the culture steps were the same as the specific steps of the present invention, and the cells were cultured to the P10 generation.

[0042] According to observations and records, the average culture time of each generation of cells is 16 days. During the process, P3, P5 and P10 cells were taken. It was observed that the cell morphology was normal. The cell survival rates were measured, which were 91.31%, 82.74% and 65.36% respectively. P10 cells were taken for adipogenic differentiation. The cells had good differentiation ability and the differentiation efficiency could reach 85% within 5 days.

[0043] By comparison, it can be seen that although replacing the corn glycopeptide of the present invention with corn peptide can also achieve a good culture effect, the culture effect is not as good as that of Example 1 of the present invention. During the process, on the one hand, the culture efficiency is significantly reduced, which is only equivalent to the efficiency of using fetal bovine serum. At the same time, the cell survival rate and differentiation ability are also reduced to a certain extent, which is not the optimal technical solution.

[0044] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims

1. A mesenchymal stem cell culture medium, characterized in that: The invention comprises a basic culture medium and supplementary factors; the supplementary factors comprise penicillin, streptomycin, transferrin, recombinant human serum albumin, leukemia inhibitory factor, insulin, platelet-derived growth factor, corn glycopeptide, collagen, soybean trypsin inhibitor and sodium selenite.

2. A mesenchymal stem cell culture medium according to claim 1, characterized in that: The basic culture medium is DMEM / F12 culture medium; DMEM / F12 serum-free culture medium is prepared by mixing DMEM culture medium and F12 culture medium in a volume ratio of 1:1, and the DMEM culture medium is a DMEM low-glucose culture medium.

3. A mesenchymal stem cell culture medium according to claim 1, characterized in that: The concentrations of the supplementary factors are as follows: penicillin 50-100 U / mL, streptomycin 50-100 μg / mL, transferrin 40-60 μg / mL, recombinant human serum albumin 5-10 μg / mL, leukemia inhibitory factor 10-50 ng / mL, insulin 10-20 μg / mL, platelet-derived growth factor 20-30 μg / mL, corn glycopeptide 100-200 μg / mL, collagen 200-300 μg / mL, soybean trypsin inhibitor 10-20 μg / mL, and sodium selenite 20-30 μg / mL.

4. The mesenchymal stem cell culture medium according to claim 1, characterized in that: The corn glycopeptide is a product of covalent bonding of low-molecular-weight corn peptide obtained by protease hydrolysis of zein and D-glucosamine.

5. The mesenchymal stem cell culture medium according to claim 1, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

6. The method for culturing mesenchymal stem cells in the culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Take the frozen umbilical cord mesenchymal stem cells and put them into a 37°C water bath to thaw quickly; S2, adding the culture medium of the present invention to the thawed cells in S1, washing them thoroughly and centrifuging them, and discarding the supernatant; S3, adding the culture medium of the present invention to the cells obtained in S2 again, mixing by pipetting to prepare a cell suspension, inoculating the cell suspension into a culture dish, and culturing in a cell culture incubator; S4. When the cell density reaches 80-90%, remove the old culture medium, rinse thoroughly with PBS buffer, and then add trypsin for digestion; S5. After digestion is completed, the culture medium of the present invention is added to terminate digestion, and the culture medium of the present invention is added after sufficient rinsing and centrifugation with PBS buffer, and the culture medium of the present invention is added and blown evenly. After counting, steps S3-S5 are repeated to continue culturing.

7. The method for culturing mesenchymal stem cells according to claim 6, characterized in that: In the S3, the culture conditions are 37° C., 5% volume fraction CO 2 , and 95% relative humidity.

8. The method for culturing mesenchymal stem cells according to claim 6, characterized in that: In the S4, trypsin is 0.25% trypsin-EDTA digestion solution.

9. The method for culturing mesenchymal stem cells according to claim 6, characterized in that: In the S4, the digestion temperature is 37° C. and the digestion time is 2 to 3 minutes.