A bone marrow mesenchymal stem cell culture medium, its preparation method and application

Abstract

This invention belongs to the field of cell culture technology, specifically relating to a bone marrow mesenchymal stem cell culture medium, its preparation method, and its application. The bone marrow mesenchymal stem cell culture medium includes a basal culture medium, and betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine added to the basal culture medium. This bone marrow mesenchymal stem cell culture medium can effectively improve the proliferation capacity of bone marrow mesenchymal stem cells, delay cell senescence, and enhance the osteogenic differentiation capacity of bone marrow mesenchymal stem cells. Furthermore, it does not contain fetal bovine serum, avoiding the risk of introducing xenobiotic serum carrying viruses and improving the safety of clinical applications.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a bone marrow mesenchymal stem cell culture medium, its preparation method, and its application. Background Technology

[0002] Stem cells are a type of cell with unlimited or even immortal self-renewal capacity, holding immense potential in regenerative cell therapy. Bone marrow mesenchymal stem cells (BMSCs), derived from the bone marrow tissue of adult individuals, are similar to umbilical cord and adipose-derived mesenchymal stem cells (ADMSCs), possessing excellent self-renewal capacity and the potential to differentiate into various cell types. Under suitable external conditions, BMSCs can differentiate into various cell types such as osteoblasts, chondrocytes, and adipocytes. Furthermore, they are easily isolated and cultured, exhibiting strong self-renewal potential, high differentiation capacity, and immunosuppressive properties. These characteristics make BMSCs demonstrate broad application potential and prospects in multiple fields, including addressing organ and tissue defect diseases, degenerative diseases, cell therapy, and tissue engineering.

[0003] Currently, commonly used bone marrow mesenchymal stem cell (BMSC) culture media are synthetic media prepared by adding fetal bovine serum (FBS) to a basal medium with defined components, such as DMEM. FBS is the most common nutrient supplement in cell culture, providing relatively complete nutrients. However, media containing FBS are expensive and carry the risk of introducing bacteria and viruses from heterologous serum. Furthermore, serum-cultured cells have limited yield and passage times, greatly restricting their application. Research and application of BMSCs require a sufficient number of cells. Therefore, higher requirements are placed on BMSC culture media to improve the activity and proliferation rate of BMSCs during culture.

[0004] Bone marrow mesenchymal stem cells (BMSCs) have broad application prospects in both basic research and clinical applications, but the limited number of directly isolated BMSCs has become a major problem restricting their large-scale clinical application. Summary of the Invention

[0005] The primary objective of this invention is to provide a bone marrow mesenchymal stem cell culture medium.

[0006] The first objective of this invention is achieved through the following technical solution:

[0007] A bone marrow mesenchymal stem cell culture medium, comprising a basal culture medium and betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine added to the basal culture medium.

[0008] Furthermore, 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-propanedipeptide is 20-55 ng / mL, and the concentration of naloxetine is 35-60 ng / mL.

[0009] Furthermore, the concentrations of betaine, glycyrrhizin, insulin, EGF, L-propanediol, and naloxetine in the bone marrow mesenchymal stem cell culture medium are 325 ng / mL, 70 ng / mL, 10 μg / mL, 12 ng / mL, 37 ng / mL, and 47 ng / mL, respectively.

[0010] Furthermore, the basal culture medium is DMEM / F12 medium containing penicillin and streptomycin.

[0011] Furthermore, 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.

[0012] A second objective of this invention is to provide a method for preparing the above-mentioned bone marrow mesenchymal stem cell culture medium.

[0013] The second objective of this invention is achieved through the following technical solution:

[0014] The above-described method for preparing bone marrow mesenchymal stem cell culture medium involves taking betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine, and adding them to the basal culture medium according to their respective concentrations and dosages, dissolving and mixing thoroughly, and then filtering to remove bacteria to obtain the bone marrow mesenchymal stem cell culture medium.

[0015] A third objective of this invention is to provide the application of the above-mentioned bone marrow mesenchymal stem cell culture medium in the culture of bone marrow mesenchymal stem cells.

[0016] The objective of this invention is achieved through the following technical solution:

[0017] The above-described application of bone marrow mesenchymal stem cell culture medium is used for the culture of bone marrow mesenchymal stem cells.

[0018] Compared with the prior art, the main advantages of the present invention are:

[0019] This invention prepares a bone marrow mesenchymal stem cell culture medium by adding betaine, glycyrrhizin, L-propanediopeptide, and naloxixol to a basal culture medium. This culture medium can effectively improve the proliferation capacity of bone marrow mesenchymal stem cells, delay cell senescence, and enhance the osteogenic differentiation capacity of bone marrow mesenchymal stem cells. Furthermore, it does not contain fetal bovine serum, avoiding the risk of introducing heterologous serum carrying viruses and improving the safety of clinical applications. Attached Figure Description

[0020] Figure 1 Morphological diagram of P3 generation bone marrow mesenchymal stem cells;

[0021] Figure 2 Figure showing the effect of different bone marrow mesenchymal stem cell culture media on the proliferation of bone marrow mesenchymal stem cells;

[0022] Figure 3 The graph shows the effect of the bone marrow mesenchymal stem cell culture medium prepared in Examples 1-3 on osteogenic differentiation of bone marrow mesenchymal stem cells.

[0023] Figure 4 The figure shows the effect of the bone marrow mesenchymal stem cell culture media prepared in Comparative Examples 1-4 on osteogenic differentiation of bone marrow mesenchymal stem cells. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0025] Example 1

[0026] A bone marrow mesenchymal stem cell culture medium, based on the final volume of the bone marrow mesenchymal stem cell culture medium, includes 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-propanedipeptide 37 ng / mL, and naloxetine 47 ng / mL added to the DMEM / F12 medium; wherein the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / mL.

[0027] The specific preparation steps of the bone marrow mesenchymal stem cell culture medium are as follows:

[0028] Take betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine, add them to the basal culture medium according to their respective concentrations and dosages, dissolve and mix thoroughly, and then filter and sterilize with a filter membrane to obtain bone marrow mesenchymal stem cell culture medium.

[0029] Example 2

[0030] A bone marrow mesenchymal stem cell culture medium, based on the final volume of the bone marrow mesenchymal stem cell culture medium, includes DMEM / F12 medium containing penicillin and streptomycin, and betaine 250 ng / mL, glycyrrhizin 40 ng / mL, insulin 5 μg / mL, EGF 5 ng / mL, L-propanedipeptide 20 ng / mL, and naloxetine 35 ng / mL added to the DMEM / F12 medium; wherein the final concentration of penicillin is 90 U / mL and the final concentration of streptomycin is 90 μg / mL.

[0031] The specific preparation steps of the bone marrow mesenchymal stem cell culture medium are as follows:

[0032] Take betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine, add them to the basal culture medium according to their respective concentrations and dosages, dissolve and mix thoroughly, and then filter and sterilize with a filter membrane to obtain bone marrow mesenchymal stem cell culture medium.

[0033] Example 3

[0034] A bone marrow mesenchymal stem cell culture medium, based on the final volume of the bone marrow mesenchymal stem cell culture medium, includes DMEM / F12 medium containing penicillin and streptomycin, and betaine 400 ng / mL, glycyrrhizin 100 ng / mL, insulin 15 μg / mL, EGF 20 ng / mL, L-propanedipeptide 55 ng / mL, and naloxetine 60 ng / mL added to the DMEM / F12 medium; wherein the final concentration of penicillin is 110 U / mL, and the final concentration of streptomycin is 110 μg / mL.

[0035] The specific preparation steps of the bone marrow mesenchymal stem cell culture medium are as follows:

[0036] Take betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine, add them to the basal culture medium according to their respective concentrations and dosages, dissolve and mix thoroughly, and then filter and sterilize with a filter membrane to obtain bone marrow mesenchymal stem cell culture medium.

[0037] Comparative Example 1

[0038] The difference between Comparative Example 1 and Example 1 is that L-propanediopeptide is omitted from the composition of the bone marrow mesenchymal stem cell culture medium, while the rest are the same as in Example 1.

[0039] Comparative Example 2

[0040] The difference between Comparative Example 2 and Example 1 is that naloxol is omitted from the bone marrow mesenchymal stem cell culture medium, while the rest are the same as in Example 1.

[0041] Comparative Example 3

[0042] The difference between Comparative Example 3 and Example 1 is that the concentration of naloxetine in the bone marrow mesenchymal stem cell culture medium is 20 ng / mL, while all other aspects are the same as in Example 1.

[0043] Comparative Example 4

[0044] The difference between Comparative Example 4 and Example 1 is that the concentration of naloxetine in the bone marrow mesenchymal stem cell culture medium is 80 ng / mL, while all other aspects are the same as in Example 1.

[0045] Comparative Example 5

[0046] Comparative Example 5 was DMEM / F12 medium supplemented with fetal bovine serum, penicillin, and streptomycin, with a volume concentration of 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0047] Experimental Example 1

[0048] The purchased primary bone marrow mesenchymal stem cells were passaged in DMEM / F12 medium supplemented with fetal bovine serum (FBS), penicillin, and streptomycin. The FBS concentration was 10%, the penicillin concentration was 100 U / mL, and the streptomycin concentration was 100 μg / mL, yielding P3 generation bone marrow mesenchymal stem cells. The morphology of the P3 generation bone marrow mesenchymal stem cells is shown in the image below. Figure 1 .

[0049] P3 generation bone marrow mesenchymal stem cells were used at a rate of 1×10⁻⁶. 4 Cells were seeded in 96-well plates and cultured using bone marrow mesenchymal stem cell culture media prepared according to Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, and 5, respectively. Each group had three replicates. The plates were incubated at 37°C with 5% CO2. After 48 hours of culture, CCK-8 reagent was added, and the cells were read at 450 nm using a microplate reader. The results are shown below. Figure 2 As shown.

[0050] Figure 2The figure shows the effect of different bone marrow mesenchymal stem cell (BMSC) culture media on the proliferation of BMSCs. Compared with Comparative Examples 1-5, the BMSC culture media prepared in Examples 1-3 of this invention significantly improved the proliferation activity of BMSCs. Among them, the BMSC culture media prepared in Example 1 showed the highest proliferation activity of BMSCs, with an OD value of (3.851±0.072). This indicates that adding betaine, glycyrrhizin, L-propanediopeptide, and naloxetine to the basal culture medium can effectively improve the proliferation activity of BMSCs.

[0051] Experimental Example 2

[0052] P3 generation bone marrow mesenchymal stem cells obtained from Experiment 1 were used at a concentration of 1×10⁻⁶. 4 Cells were seeded in 96-well plates and cultured in bone marrow mesenchymal stem cell culture media prepared according to Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, and 5, with three replicates per group. Cells were passaged to passage P15 in a 37°C, 5% CO2 cell culture incubator. P15 cells were collected, and the expression levels of p21 and p16 proteins in senescent bone marrow mesenchymal stem cells were detected by Western blotting. The specific steps are as follows:

[0053] (1) Digest and centrifuge the cells, add an appropriate amount of RIPI lysis buffer containing protease inhibitor to the cell pellet, mix thoroughly, centrifuge at 10000g and 4℃ for 5 minutes, and collect the supernatant.

[0054] (2) Protein quantification was performed using the BCA kit.

[0055] (3) Before loading the sample, blow away the glycerol in each well of the precast gel. Load the cell protein sample at a protein amount of 40ug / well. Run the gel at 160V for about 1 hour, and then transfer the membrane for 15 minutes using an automatic transfer machine.

[0056] (4) Block in 5% BSA for about 1 hour, then incubate with p21 primary antibody and p16 primary antibody overnight at 4°C on a shaker.

[0057] (5) Wash the membrane 3 times with TBST for 10 minutes each time; then incubate with HRP-labeled secondary antibody for 1 hour, and then wash the membrane 3 times with TBST for 10 minutes each time.

[0058] (6) Finally, ECL exposure was used. After the strips were scanned using the ChemiScope Western Blot imaging system, the grayscale ratio was analyzed using ImageJ software.

[0059] Table 1. Effects of different bone marrow mesenchymal stem cell culture media on the relative expression levels of p21 and p16 proteins in bone marrow mesenchymal stem cells.

[0060]

[0061] The results are shown in Table 1, illustrating the effects of different bone marrow mesenchymal stem cell (BMSC) culture media on the relative expression levels of p21 and p16 proteins in BMSCs. The expression levels of p21 and p16 proteins are closely related to cell senescence and are often used to characterize the degree of cell senescence. Compared with Comparative Examples 1-4, the BMSC culture media prepared in Examples 1-3 of this invention significantly reduced the relative expression levels of p21 and p16 proteins in BMSCs. Among them, the BMSC culture media prepared in Example 1 showed the lowest relative expression levels of p21 and p16 proteins in BMSCs. This indicates that adding betaine, glycyrrhizin, L-propanediopeptide, and naloxetine to the basal culture medium can effectively reduce the relative expression levels of p21 and p16 proteins in BMSCs and inhibit BMSC senescence.

[0062] Further analysis revealed that, compared to Example 1, Comparative Example 1 omitted L-propionic acid dipeptide from its cell culture medium; Comparative Example 2 omitted naloxetine from its cell culture medium; Comparative Example 3 added naloxetine at a concentration of 20 ng / mL to its cell culture medium; and Comparative Example 4 added naloxetine at a concentration of 80 ng / mL to its cell culture medium. The proliferative activity of bone marrow mesenchymal stem cells decreased in all four groups, indicating that both L-propionic acid dipeptide and naloxetine, when added to the basal culture medium, have the effect of promoting cell proliferation and inhibiting the senescence of bone marrow mesenchymal stem cells. The concentration of naloxetine at 35-60 ng / mL showed the best effect in promoting cell proliferation and inhibiting the senescence of bone marrow mesenchymal stem cells.

[0063] Experimental Example 3

[0064] P3 bone marrow mesenchymal stem cells obtained from Experiment 1 were used at a rate of 2 × 10⁻⁶. 4Bone marrow mesenchymal stem cell (BMSC) cells were seeded into 6-well plates and cultured using bone marrow mesenchymal stem cell culture media prepared according to Examples 1, 2, 3, Comparative Examples 1, 2, 3, and 4, with 3 replicates per group. When the BMSCs reached approximately 70% confluence, the supernatant was discarded, and the cells were placed in osteogenic induction differentiation medium (DMEM / F12 medium containing dexamethasone, sodium β-glycerophosphate, vitamin C, sodium ascorbate, fetal bovine serum, penicillin, and streptomycin) to induce osteogenic differentiation. The concentrations of dexamethasone (0.15 μmol / L), sodium β-glycerophosphate (10 mmol / L), vitamin C (40 μmol / L), and sodium ascorbate (50 mg / L) were all specified. The osteogenic induction medium was changed every 2 days. On day 10 of osteogenic induction culture, the 6-well plates were removed from the incubator and placed in a clean bench. After removing the original culture medium from the wells, the cells were washed three times with PBS. Each well was fixed with 40 g / L paraformaldehyde solution. After 30 min, the fixative was discarded, and the cells were washed three times with PBS. The osteogenic differentiation cells obtained from bone marrow mesenchymal stem cells were stained according to the alkaline phosphatase kit instructions. After staining, images were taken under a fluorescence microscope, and the osteogenic differentiation capacity of bone marrow mesenchymal stem cells was assessed based on the staining results.

[0065] The results are as follows Figure 3-4 The figure shows the effect of different bone marrow mesenchymal stem cell culture media on osteogenic differentiation of bone marrow mesenchymal stem cells. Compared with Comparative Examples 1, 2, 3, and 4, the bone marrow mesenchymal stem cell culture media prepared in Examples 1-3 of this invention can significantly improve the osteogenic differentiation capacity of bone marrow mesenchymal stem cells. This indicates that adding betaine, glycyrrhizin, L-propanediopeptide, and naloxixol to the basal culture medium can effectively improve the osteogenic differentiation capacity of bone marrow mesenchymal stem cells.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A bone marrow mesenchymal stem cell culture medium, characterized in that, The bone marrow mesenchymal stem cell culture medium includes a basal culture medium and betaine, glycyrrhizin, insulin, EGF, L-propionyl dipeptide, and naloxoxol added to the basal culture medium; 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-propionyl dipeptide is 20-55 ng / mL, and the concentration of naloxoxol is 35-60 ng / mL. The basal culture medium is DMEM / F12 medium containing penicillin and streptomycin; 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.

2. The bone marrow mesenchymal stem cell culture medium according to claim 1, characterized in that, The bone marrow mesenchymal stem cell culture medium contained betaine at a concentration of 325 ng / mL, glycyrrhizin at a concentration of 70 ng / mL, insulin at a concentration of 10 μg / mL, EGF at a concentration of 12 ng / mL, L-propanedipeptide at a concentration of 37 ng / mL, and naloxetine at a concentration of 47 ng / mL.

3. The method for preparing bone marrow mesenchymal stem cell culture medium according to any one of claims 1-2, characterized in that, Take betaine, glycyrrhizin, insulin, EGF, L-propanediopeptide, and naloxetine, and add them to the basal culture medium according to their respective concentrations and dosages. Dissolve and mix thoroughly, then filter to remove bacteria to obtain bone marrow mesenchymal stem cell culture medium.

4. The application of the bone marrow mesenchymal stem cell culture medium according to any one of claims 1-2, characterized in that, Used for the culture of bone marrow mesenchymal stem cells.

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