Bone marrow mesenchymal stem cell culture medium as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a bone marrow mesenchymal stem cell culture medium and a preparation method and application thereof. The mesenchymal stem cell culture medium comprises a basic culture medium, and betaine, glycyrrhizin, insulin, EGF (Epidermal Growth Factor), L-propyl dipeptide and naloxicol which are added into the basic culture medium. The bone marrow mesenchymal stem cell culture medium can effectively improve the multiplication capacity of bone marrow mesenchymal stem cells, delay cell senescence and improve the osteogenic differentiation capacity of the bone marrow mesenchymal stem cells, fetal calf serum is not added, the risk of introducing heterologous serum to carry viruses is avoided, and the safety of clinical application is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and particularly relates to a bone marrow mesenchymal stem cell culture medium and a preparation method and application thereof. BACKGROUND

[0002] Stem cells are a kind of cells with unlimited or immortal self-renewal capacity, and have great potential in regenerative cell therapy. Bone marrow mesenchymal stem cells are derived from the bone marrow tissue of adult individuals, similar to umbilical cord and adipose tissue mesenchymal stem cells, and have excellent self-renewal capacity and potential to differentiate into multiple cell types. Under suitable external conditions, bone marrow mesenchymal stem cells can differentiate into multiple cell types such as osteoblasts, chondrocytes and adipocytes. In addition, they are easy to isolate and culture, and exhibit strong self-renewal potential, high differentiation capacity and immunosuppressive properties. These properties make bone marrow mesenchymal stem cells have broad application potential and prospect in dealing with tissue and organ defect diseases, tissue and organ degenerative diseases, and cell therapy and tissue engineering.

[0003] The commonly used bone marrow mesenchymal stem cell culture medium at present is a synthetic culture medium prepared by adding nutrient substances fetal bovine serum to a basic culture medium such as DMEM bone marrow mesenchymal stem cell. Fetal bovine serum is the most common nutrient supplement in cell culture, which provides relatively complete nutrients for cell culture, but the culture medium containing fetal bovine serum has high cost and the risk of introducing bacteria and viruses carried by heterologous serum. In addition, the cell yield and passage number of serum culture are limited, which greatly limits the application. The research and application of bone marrow mesenchymal stem cells require a sufficient number of cells. Therefore, higher requirements are put forward for the culture medium of bone marrow mesenchymal stem cells to improve the activity and proliferation rate of bone marrow mesenchymal stem cells in the culture process.

[0004] Bone marrow mesenchymal stem cells (BMSCs) have broad application prospects in basic research and clinical application, but the number of bone marrow mesenchymal stem cells directly isolated is limited, which is the main problem restricting its large-scale clinical application. SUMMARY

[0005] The first object of the present application is to provide a bone marrow mesenchymal stem cell culture medium.

[0006] The first object of the present application is achieved by the following technical solution: A bone marrow mesenchymal stem cell culture medium, which comprises a basic culture medium, and betaine, glycyrrhizin, insulin, EGF, L-proline, naloxegol added in the basic culture medium.

[0007] Further, the concentration of betaine in the bone marrow mesenchymal stem cell culture medium is 250-400 ng / mL, the concentration of glycyrrhizin is 40-100 ng / mL, the concentration of insulin is 5-15 μg / mL, the concentration of EGF is 5-20 ng / mL, the concentration of L-arginine is 20-55 ng / mL, and the concentration of naloxegol is 35-60 ng / mL.

[0008] Further, the concentration of betaine in the bone marrow mesenchymal stem cell culture medium is 325 ng / mL, the concentration of glycyrrhizin is 70 ng / mL, the concentration of insulin is 10 μg / mL, the concentration of EGF is 12 ng / mL, the concentration of L-arginine is 37 ng / mL, and the concentration of naloxegol is 47 ng / mL.

[0009] Further, the base culture medium is DMEM / F12 culture medium containing penicillin and streptomycin.

[0010] Further, the concentration of penicillin in the bone marrow mesenchymal stem cell culture medium is 90-110 U / mL, and the concentration of streptomycin in the bone marrow mesenchymal stem cell culture medium is 90-110 μg / mL.

[0011] The second object of the present application is to provide a preparation method of the bone marrow mesenchymal stem cell culture medium.

[0012] The second object of the present application is achieved by the following technical solution: The preparation method of the bone marrow mesenchymal stem cell culture medium is as follows: betaine, glycyrrhizin, insulin, EGF, L-arginine, and naloxegol are added to the base culture medium according to the respective usage amounts corresponding to the respective concentrations, fully dissolved and mixed, then filtered and sterilized, and the bone marrow mesenchymal stem cell culture medium is obtained.

[0013] The third object of the present application is to provide the application of the bone marrow mesenchymal stem cell culture medium in the culture of bone marrow mesenchymal stem cells.

[0014] The object of the present application is achieved by the following technical solution: The application of the bone marrow mesenchymal stem cell culture medium is used for the culture of bone marrow mesenchymal stem cells.

[0015] Compared with the prior art, the present application has the following beneficial effects: The application adds betaine, glycyrrhizin, L-proline-glycine dipeptide and naloxegol to a basic medium to prepare the mesenchymal stem cell culture medium of the application. The medium can effectively improve the proliferation ability of mesenchymal stem cells, delay the aging of cells, improve the osteogenic differentiation ability of mesenchymal stem cells, and does not add fetal bovine serum, avoiding the risk of introducing heterologous serum carrying viruses and improving the safety of clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 P3 generation of mesenchymal stem cells morphological chart; Figure 2 Different mesenchymal stem cell culture medium on the effect of mesenchymal stem cell proliferation result chart; Figure 3 The effect of mesenchymal stem cell culture medium prepared in examples 1-3 on the osteogenic differentiation of mesenchymal stem cells result chart; Figure 4 The effect of mesenchymal stem cell culture medium prepared in comparative examples 1-4 on the osteogenic differentiation of mesenchymal stem cells result chart. DETAILED DESCRIPTION

[0017] The technical solutions of the application are further described below in combination with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the application and should not be regarded as limiting the application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, such as those not specifically mentioned, are conventional products obtained through market channels.

[0018] Example 1 A mesenchymal stem cell culture medium, based on the final volume of the mesenchymal stem cell culture medium, comprises DMEM / F12 medium containing penicillin and streptomycin, and betaine 325 ng / mL, glycyrrhizin 70 ng / mL, insulin 10 μg / mL, EGF 12 ng / mL, L-proline-glycine dipeptide 37 ng / mL, and naloxegol 47 ng / mL added in the DMEM / F12 medium; wherein the final concentration of penicillin is 100 U / mL and the final concentration of streptomycin is 100 μg / mL.

[0019] The specific preparation steps of the mesenchymal stem cell culture medium are as follows: Take betaine, glycyrrhizin, insulin, EGF, L-proline-glycine dipeptide and naloxegol, and add them to the basic medium according to the respective concentration usage amount, fully dissolve and mix, then filter sterilize with a filter membrane to obtain the mesenchymal stem cell culture medium.

[0020] Example 2 A bone marrow mesenchymal stem cell culture medium, comprising DMEM / F12 culture medium containing penicillin and streptomycin, and betaine 250 ng / mL, glycyrrhizin 40 ng / mL, insulin 5 μg / mL, EGF 5 ng / mL, L-arginine 20 ng / mL, naloxegol 35 ng / mL added in the DMEM / F12 culture medium, wherein the final concentration of penicillin is 90 U / mL, and the final concentration of streptomycin is 90 μg / mL, based on the final volume of the bone marrow mesenchymal stem cell culture medium.

[0021] The specific preparation steps of the bone marrow mesenchymal stem cell culture medium are as follows: Take betaine, glycyrrhizin, insulin, EGF, L-arginine, and naloxegol, and add them into the base medium according to the respective concentration usage amount, fully dissolve and mix uniformly, and then filter sterilize with a filter membrane to obtain the bone marrow mesenchymal stem cell culture medium.

[0022] Example 3 A bone marrow mesenchymal stem cell culture medium, comprising DMEM / F12 culture medium containing penicillin and streptomycin, and betaine 400 ng / mL, glycyrrhizin 100 ng / mL, insulin 15 μg / mL, EGF 20 ng / mL, L-arginine 55 ng / mL, and naloxegol 60 ng / mL added in the DMEM / F12 culture medium, wherein the final concentration of penicillin is 110 U / mL, and the final concentration of streptomycin is 110 μg / mL, based on the final volume of the bone marrow mesenchymal stem cell culture medium.

[0023] The specific preparation steps of the bone marrow mesenchymal stem cell culture medium are as follows: Take betaine, glycyrrhizin, insulin, EGF, L-arginine, and naloxegol, and add them into the base medium according to the respective concentration usage amount, fully dissolve and mix uniformly, and then filter sterilize with a filter membrane to obtain the bone marrow mesenchymal stem cell culture medium.

[0024] Comparative Example 1 Comparative Example 1 and Example 1 differ in that L-arginine is omitted from the components of the bone marrow mesenchymal stem cell culture medium, and the others are the same as in Example 1.

[0025] Comparative Example 2 Comparative Example 2 and Example 1 differ in that naloxegol is omitted from the components of the bone marrow mesenchymal stem cell culture medium, and the others are the same as in Example 1.

[0026] Comparative Example 3 Comparative Example 3 and Example 1 differ in that the concentration of naloxegol in the bone marrow mesenchymal stem cell culture medium is 20 ng / mL, and the others are the same as in Example 1.

[0027] Comparative Example 4 Comparative Example 4 and Example 1 are different in that the concentration of naloxegol in the bone marrow mesenchymal stem cell culture medium is 80 ng / mL, and the others are the same as Example 1.

[0028] Comparative Example 5 Comparative Example 5 is a DMEM / F12 culture medium added with fetal bovine serum, penicillin and streptomycin, wherein the volume concentration of fetal bovine serum is 10%, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 100 μg / mL.

[0029] Test Example 1 The purchased primary bone marrow mesenchymal stem cells were placed in a DMEM / F12 culture medium added with fetal bovine serum, penicillin and streptomycin for subculture, wherein the volume concentration of fetal bovine serum is 10%, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 100 μg / mL, to obtain P3 generation bone marrow mesenchymal stem cells. The morphological chart of P3 generation bone marrow mesenchymal stem cells is as shown in Figure 1 .

[0030] The P3 generation bone marrow mesenchymal stem cells were inoculated in a 96-well culture plate at 1×10 4 / well, and were cultured with the bone marrow mesenchymal stem cell culture medium prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively, and 3 replicate wells were set in each group, and were placed in a cell culture incubator at 37°C and 5% CO2. After 48h of culture, CCK-8 reagent was added, and an enzyme marker was used to detect the reading value of each group at 450nm wavelength, and the results are shown in Figure 2 .

[0031] Figure 2 The results chart of the influence of different bone marrow mesenchymal stem cell culture media on the proliferation of bone marrow mesenchymal stem cells. Compared with Comparative Examples 1-5, the bone marrow mesenchymal stem cell culture medium prepared in Examples 1-3 can significantly improve the proliferation activity of bone marrow mesenchymal stem cells. Among them, the bone marrow mesenchymal stem cell culture medium prepared in Example 1 has the highest proliferation activity of bone marrow mesenchymal stem cells, and the OD value is (3.851±0.072). It shows that by adding betaine, glycyrrhizin, L-proline and naloxegol to the basic culture medium, the proliferation activity of bone marrow mesenchymal stem cells can be effectively improved.

[0032] Test Example 2 The P3 generation bone marrow mesenchymal stem cells obtained in Test Example 1 were taken out at 1×10 4The bone marrow mesenchymal stem cells were inoculated in 96-well culture plates and cultured with the bone marrow mesenchymal stem cell culture medium prepared by Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively, and three replicate wells were set in each group and placed in a 37℃, 5% CO2 cell incubator for passage to P15 generation. The P15 generation cells were collected, and the p21 and p16 protein expression levels in the senescent bone marrow mesenchymal stem cells were detected by Western Blot method, and the specific steps were as follows: (1) The cells were digested and centrifuged, and an appropriate amount of RIPI lysis buffer containing protease inhibitors was added to the cell precipitate and mixed thoroughly, and then centrifuged at 10000g at 4℃ for 5 minutes, and the supernatant was collected.

[0033] (2) Protein quantification was performed by BCA kit.

[0034] (3) Before loading, blow away the glycerol in each well of the pre-prepared gel, and load the cell protein sample at a protein amount of 40ug / well, run the gel at a voltage of 160V for about 1 hour, and then transfer the membrane by automatic membrane transfer machine for 15min.

[0035] (4) Then incubate p21 primary antibody and p16 primary antibody in 5% BSA for about 1 hour, and then incubate on a rocking bed at 4℃ overnight.

[0036] (5) Wash the membrane with TBST for 3 times, each for 10 minutes; then incubate with HRP-labeled secondary antibody for 1 hour, and then wash the membrane with TBST for 3 times, each for 10 minutes.

[0037] (6) Finally, expose with ECL. After scanning the bands by ChemiScope Western Blot imaging system, analyze the gray scale ratio by Image J software.

[0038] Table 1 Effect of different bone marrow mesenchymal stem cell culture media on the relative expression level of p21 and p16 proteins in bone marrow mesenchymal stem cells The results are shown in Table 1, which are the effects of different bone marrow mesenchymal stem cell culture mediums on the relative expression levels of p21 and p16 proteins in bone marrow mesenchymal stem cells. The expression levels of p21 and p16 proteins are closely related to cell aging and are often used to characterize the degree of cell aging. Compared with Comparative Examples 1-4, the bone marrow mesenchymal stem cell culture mediums prepared in Example 1-3 can significantly reduce the relative expression levels of p21 and p16 proteins in bone marrow mesenchymal stem cells. Among them, the bone marrow mesenchymal stem cell culture medium prepared in Example 1 has the lowest relative expression levels of p21 and p16 proteins in bone marrow mesenchymal stem cells. It is shown that by adding betaine, glycyrrhizin, L-carnosine and naloxegol to the basic medium, the relative expression levels of p21 and p16 proteins in bone marrow mesenchymal stem cells can be effectively reduced, and the aging of bone marrow mesenchymal stem cells can be inhibited.

[0039] Further analysis shows that, compared with Example 1, the cell culture medium of Comparative Example 1 omits L-carnosine; the cell culture medium of Comparative Example 2 omits naloxegol; the cell culture medium of Comparative Example 3 adds 20 ng / mL of naloxegol; and the cell culture medium of Comparative Example 4 adds 80 ng / mL of naloxegol. The proliferation activities of the bone marrow mesenchymal stem cells in the four groups are all decreased, indicating that the addition of L-carnosine and naloxegol to the basic medium both has the effect of promoting cell proliferation and inhibiting the aging of bone marrow mesenchymal stem cells. When the concentration of naloxegol is 35-60 ng / mL, the effect of promoting cell proliferation and inhibiting the aging of bone marrow mesenchymal stem cells is the best.

[0040] Test Example 3 The P3 bone marrow mesenchymal stem cells obtained in Test Example 1 were taken and diluted to 2x10 4The bone marrow mesenchymal stem cells were inoculated in 6-well plates and cultured with the bone marrow mesenchymal stem cell culture medium prepared by Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, respectively, and 3 wells were set in each group. When the bone marrow mesenchymal stem cells were about 70% confluent, the supernatant was discarded, and the bone marrow mesenchymal stem cells were induced to differentiate into bone cells in the osteogenic induction medium (DMEM / F12 medium containing dexamethasone, β-glycerophosphate sodium, vitamin C, sodium ascorbate, fetal bovine serum, penicillin, and streptomycin), wherein the concentration of dexamethasone was 0.15 μmol / L, the concentration of β-glycerophosphate sodium was 10 mmol / L, the concentration of vitamin C was 40 μmol / L, and the concentration of sodium ascorbate was 50 mg / L. The osteogenic induction medium was replaced every 2 days. On the 10th day of osteogenic induction culture, the 6-well plate was taken out from the incubator and placed on a clean bench, and the original medium in the wells was removed and washed with PBS for 3 times. Then, 40 g / L paraformaldehyde solution was added to each well for fixation, and after 30 min, the fixing solution was discarded and washed with PBS for 3 times. The obtained bone marrow mesenchymal stem cells differentiated into bone cells were dyed according to the instructions of the alkaline phosphatase kit, and the dyeing was observed under a fluorescence microscope. The bone marrow mesenchymal stem cell osteogenic differentiation ability was evaluated according to the dyeing results.

[0041] The results are shown in Table 1, and are shown in the following table as the effect of different bone marrow mesenchymal stem cell culture media on the osteogenic differentiation of bone marrow mesenchymal stem cells. Figures 3-4 As shown in Table 1, compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, the bone marrow mesenchymal stem cell culture medium prepared by Examples 1-3 can significantly improve the osteogenic differentiation ability of bone marrow mesenchymal stem cells. This indicates that the addition of betaine, glycyrrhizin, L-proline and naloxegol to the basic medium can effectively improve the osteogenic differentiation ability of bone marrow mesenchymal stem cells.

[0042] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application, but not to limit them. The basic principles and main features of the present application have been described above with specific embodiments, and some modifications or replacements can be made on the basis of the present application, but these modifications or replacements do not make the corresponding technical solutions deviate from the scope of the present application.

Claims

1. A bone marrow mesenchymal stem cell culture medium, characterized by, The bone marrow mesenchymal stem cell culture medium comprises a basic culture medium and betaine, glycyrrhizin, insulin, EGF, L-proline, naloxegol added in the basic culture medium.

2. The bone marrow mesenchymal stem cell culture medium of claim 1, wherein, The concentration of betaine in the bone marrow mesenchymal stem cell culture medium is 250-400 ng / mL, the concentration of glycyrrhizin is 40-100 ng / mL, the concentration of insulin is 5-15 μg / mL, the concentration of EGF is 5-20 ng / mL, the concentration of L-proline is 20-55 ng / mL, and the concentration of naloxegol is 35-60 ng / mL.

3. The bone marrow mesenchymal stem cell culture medium of claim 2, wherein, The concentration of betaine in the bone marrow mesenchymal stem cell culture medium is 325 ng / mL, the concentration of glycyrrhizin is 70 ng / mL, the concentration of insulin is 10 μg / mL, the concentration of EGF is 12 ng / mL, the concentration of L-proline is 37 ng / mL, and the concentration of naloxegol is 47 ng / mL.

4. The bone marrow mesenchymal stem cell culture medium of claim 1, wherein, The basic culture medium is DMEM / F12 culture medium containing penicillin and streptomycin.

5. The bone marrow mesenchymal stem cell culture medium of claim 4, wherein, The concentration of penicillin in the bone marrow mesenchymal stem cell culture medium is 90-110 U / mL, and the concentration of streptomycin in the bone marrow mesenchymal stem cell culture medium is 90-110 μg / mL.

6. The method of claim 1-5, wherein the bone marrow mesenchymal stem cell culture medium is prepared by the following steps: Betaine, glycyrrhizin, insulin, EGF, L-proline and naloxegol are taken, and added into the basic culture medium according to the respective usage amount corresponding to the concentration, dissolved and mixed uniformly, then filtered and sterilized to obtain the bone marrow mesenchymal stem cell culture medium. ​ 7. Use of a bone marrow mesenchymal stem cell culture medium according to any one of claims 1 to 5, characterized in that, The bone marrow mesenchymal stem cell culture medium is used for culturing bone marrow mesenchymal stem cells. The bone marrow mesenchymal stem cell culture medium is used for culturing bone marrow mesenchymal stem cells.

Images