Bone marrow mesenchymal stem cell culture medium combination and culture method

Abstract

The present invention belongs to the field of cell culture technology, and specifically relates to a combination of bone marrow mesenchymal stem cell culture media and a culture method. The bone marrow mesenchymal stem cell culture media combination of the present invention includes culture media I and culture media II; the culture media I is a DMEM / F12 culture media with added asterone, melatonin, linoleic acid, L-glutamine, bFGF, insulin, and astragalus polysaccharide; the culture media II is a DMEM / F12 culture media with added ACE2, linoleic acid, L-glutamine, bFGF, insulin, and astragalus polysaccharide. The present invention provides a method for culturing bone marrow mesenchymal stem cells, improves the culture media used for bone marrow mesenchymal stem cells, and specifically designs primary culture media and passage culture media for different cell culture processes, and the two cooperate with each other, and the bone marrow mesenchymal stem cells obtained by culture have a good growth state and better proliferation ability and osteogenic differentiation potential.

Description

Technical Field

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

[0002] Bone marrow mesenchymal stem cells (BMSCs) are a type of multipotent stem cells with self-renewal ability and multidirectional differentiation potential, and can differentiate into a variety of cell types, including osteoblasts, chondrocytes, adipocytes, etc. These characteristics make BMSCs have broad application prospects in the fields of cell therapy, tissue engineering and regenerative medicine. Therefore, in order to effectively culture, expand and apply BMSCs, it is necessary to develop a culture medium suitable for its growth and differentiation characteristics to provide it with a more suitable growth environment.

[0003] With the continuous development of technology, more and more researchers have begun to focus on the optimization of BMSCs culture medium to improve the proliferation efficiency and differentiation potential of BMSCs. For example, by adding specific growth factors, cytokines or signaling molecules; or by using gene editing technology to further promote the proliferation, differentiation or maintain the performance of BMSCs. At the same time, for BMSCs from different sources or health conditions, the formula of the culture medium can also be adjusted to meet their specific growth needs.

[0004] Through in vitro culture technology, a sufficient number of high-quality BMSCs can be obtained for use in cell therapy, tissue engineering and other fields. In addition, with a deeper understanding of the biological characteristics and differentiation mechanisms of BMSCs, personalized treatment strategies can be customized for specific diseases or tissue damage to improve the treatment effect. However, BMSCs from different sources have different growth and differentiation characteristics, and therefore, the requirements for culture media are also different. Most of the current bone marrow mesenchymal stem cell culture media are difficult to meet the specific needs of different BMSCs, and the cell culture effect is poor. In particular, some culture media containing animal serum contain some substances that are toxic to cells, which have a negative effect on cell growth and adhesion, and can also cause cell morphological changes and reduced differentiation ability.

[0005] Therefore, it is urgent to develop a more efficient, safe and cell growth and differentiation-friendly method for culturing bone marrow mesenchymal stem cells, so as to provide new ideas for the development of bone marrow mesenchymal stem cells. Summary of the invention

[0006] The first purpose of the present invention is to provide a bone marrow mesenchymal stem cell culture medium combination, in which primary culture medium and passage culture medium are specifically designed in different processes of cell culture. The bone marrow mesenchymal stem cells obtained by culture with the above culture medium combination have a good growth state and better proliferation activity and osteogenic differentiation potential.

[0007] The second object of the present invention is to provide a method for culturing bone marrow mesenchymal stem cells. The bone marrow mesenchymal stem cell culture medium combination provided by the present invention is used to culture bone marrow mesenchymal stem cells, and the operation is simple.

[0008] In order to achieve the above object, the technical solution adopted by the first object of the present invention is:

[0009] A bone marrow mesenchymal stem cell culture medium combination, comprising culture medium I and culture medium II;

[0010] The culture medium I is a DMEM / F12 culture medium supplemented with 3-10 μg / mL asterone, 3-10 μM melatonin, 5-12 μg / mL linoleic acid, 4-10 mM L-glutamine, 10-18 ng / mL bFGF, 22-30 μg / mL insulin, and 42-50 μg / mL astragalus polysaccharide;

[0011] The culture medium II is a DMEM / F12 culture medium supplemented with 45-70 ng / mL ACE2, 5-12 μg / mL linoleic acid, 4-10 mM L-glutamine, 10-18 ng / mL bFGF, 22-30 μg / mL insulin, and 42-50 μg / mL astragalus polysaccharide.

[0012] Further, the culture medium I is a DMEM / F12 culture medium supplemented with 6 μg / mL asterone, 6 μM melatonin, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide;

[0013] The culture medium II is a DMEM / F12 culture medium supplemented with 60 ng / mL ACE2, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide.

[0014] The technical solution adopted in the second object of the present invention is:

[0015] The present invention provides a method for culturing bone marrow mesenchymal stem cells, which adopts the culture medium combination for culturing, wherein the culture medium I is used for primary culture, and the culture medium II is used for subculture culture.

[0016] Furthermore, the method for culturing bone marrow mesenchymal stem cells specifically comprises the following steps: (1) separation and extraction of bone marrow mesenchymal stem cells: bone marrow mesenchymal stem cells are separated and obtained by density gradient centrifugation;

[0017] (2) Primary culture of bone marrow mesenchymal stem cells: inoculating the bone marrow mesenchymal stem cells obtained in step (1) into culture medium I for primary culture;

[0018] (3) Subculture of bone marrow mesenchymal stem cells: When the primary culture of bone marrow mesenchymal stem cells in step (1) reaches a confluence of 80-90%, remove medium I and digest the bone marrow mesenchymal stem cells with 0.25% trypsin containing 0.02% EDTA. After digestion, centrifuge the cells and inoculate the resulting bone marrow mesenchymal stem cells into medium II for subculture.

[0019] Furthermore, the seeding density of bone marrow mesenchymal stem cells in the primary culture of step (2) is 2×10 5 -6×10 5 Pieces / mL.

[0020] Furthermore, the seeding density of bone marrow mesenchymal stem cells during subculture in step (3) is 1×10 4 -5×10 4 Pieces / mL.

[0021] Furthermore, the primary culture and subculture are both carried out in an incubator at 37°C, 5% CO2, and saturated humidity.

[0022] Furthermore, the medium is changed every 2 days during the primary culture and subculture process.

[0023] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0024] 1. The present invention provides a bone marrow mesenchymal stem cell culture medium combination, which mainly improves the culture medium used for bone marrow mesenchymal stem cells. The present invention specifically designs a primary culture medium and a passage culture medium according to the differences in the cell culture process. The two cooperate with each other, and the bone marrow mesenchymal stem cells obtained by culture have a good growth state, and the obtained bone marrow mesenchymal stem cells have better proliferation ability and osteogenic differentiation potential.

[0025] 2. The present invention provides a method for culturing bone marrow mesenchymal stem cells. The bone marrow mesenchymal stem cell culture medium combination provided by the present invention is used to culture bone marrow mesenchymal stem cells. The operation is simple, and it provides strong support for the development and application of bone marrow mesenchymal stem cells in the fields of regenerative medicine and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a morphological picture of the P3 bone marrow mesenchymal stem cells cultured in Example 1 (magnification is 20 times);

[0027] Figure 2 This is a morphological picture of the P3 bone marrow mesenchymal stem cells cultured in Example 2 (magnification is 20 times);

[0028] Figure 3This is a morphological picture of the P3 bone marrow mesenchymal stem cells cultured in Example 3 (magnification is 20 times);

[0029] Figure 4 The results of the ALP activity test of bone marrow mesenchymal stem cells in Examples 1 to 3 and Comparative Examples 1 to 4 are shown;

[0030] Figure 5 The graph shows the expression of proteins related to osteogenic differentiation of bone marrow mesenchymal stem cells in Examples 1 to 3 and Comparative Examples 1 to 4. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0032] Example 1

[0033] A bone marrow mesenchymal stem cell culture medium combination, consisting of culture medium I and culture medium II, wherein culture medium I is a DMEM / F12 culture medium supplemented with 6 μg / mL asterone, 6 μM melatonin, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL basic fibroblast growth factor (bFGF), 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide;

[0034] The culture medium II is a DMEM / F12 culture medium supplemented with 60 ng / mL angiotensin converting enzyme 2 (ACE2), 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide.

[0035] The method for culturing bone marrow mesenchymal stem cells specifically comprises the following steps:

[0036] (1) Isolation and extraction of bone marrow mesenchymal stem cells: First, select 2-week-old SD rats, kill them by cervical dislocation, and soak them in 75% alcohol. After disinfection for 5 minutes, rinse them with PBS buffer three times. Then, take out the tibia and femur of the rat on the clean bench and soak them in PBS buffer solution containing double antibodies. Use sterile scissors to cut off both ends of the rat's tibia and femur to expose the bone marrow cavity. Then use a syringe to absorb DMEM culture medium to repeatedly rinse the bone marrow cavity until the bone marrow cavity turns white, and collect the bone marrow suspension. Add an equal amount of PBS buffer to dilute it, then place it in a centrifuge tube for centrifugation (1500rpm, 10min), and discard the fat layer. Dilute the precipitate with an equal volume of PBS buffer to form a cell suspension, slowly add it on top of an equal volume of Ficoll separation solution, place it in a centrifuge tube for centrifugation (3000rpm, 20min), and absorb the bone marrow mesenchymal stem cells on the interface of the white film layer, and wash it twice with PBS buffer.

[0037] (2) Primary culture of bone marrow mesenchymal stem cells: The bone marrow mesenchymal stem cells obtained in step (1) were cultured at a rate of 4 × 10 5 The cells were inoculated into medium I at a density of 100 / mL and cultured in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every two days. The cells obtained from the primary culture were labeled as P0 generation bone marrow mesenchymal stem cells.

[0038] (3) Subculture of bone marrow mesenchymal stem cells: When the cell fusion degree of bone marrow mesenchymal stem cells reaches 80% under a microscope, discard the original culture medium and use 0.25% trypsin containing 0.02% EDTA at 37°C to digest the bone marrow mesenchymal stem cells and remove them from the bottom of the culture dish. After digestion for 2 minutes, add an equal volume of DMEM / F12 culture medium to terminate the digestion. Collect the above cells into a centrifuge tube and centrifuge (2000rpm, 8min), discard the supernatant, rinse the precipitate twice with saline to remove the residual trypsin, and centrifuge the bone marrow mesenchymal stem cells at 3×10 4 The cells were inoculated into medium II at a density of 100 / mL and subcultured in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every 2 days during the culture period, and P3 bone marrow mesenchymal stem cells were collected.

[0039] Example 2

[0040] A bone marrow mesenchymal stem cell culture medium combination, consisting of culture medium I and culture medium II, wherein culture medium I is a DMEM / F12 culture medium supplemented with 3 μg / mL asterone, 3 μM melatonin, 5 μg / mL linoleic acid, 4 mM L-glutamine, 10 ng / mL bFGF, 22 μg / mL insulin, and 42 μg / mL astragalus polysaccharide;

[0041] The culture medium II is a DMEM / F12 culture medium supplemented with 45 ng / mL ACE2, 5 μg / mL linoleic acid, 4 mM L-glutamine, 10 ng / mL bFGF, 22 μg / mL insulin, and 42 μg / mL astragalus polysaccharide.

[0042] The method for culturing bone marrow mesenchymal stem cells specifically comprises the following steps:

[0043] (1) The separation and extraction of bone marrow mesenchymal stem cells is the same as in Example 1.

[0044] (2) Primary culture of bone marrow mesenchymal stem cells: The bone marrow mesenchymal stem cells obtained in step (1) were cultured at a rate of 6×10 5 The cells were inoculated into medium I at a density of 100 / mL and cultured in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every two days. The cells obtained from the primary culture were labeled as P0 generation bone marrow mesenchymal stem cells.

[0045] (3) Subculture of bone marrow mesenchymal stem cells: When the cell fusion degree of bone marrow mesenchymal stem cells reaches 80% under a microscope, the original culture medium is discarded and the bone marrow mesenchymal stem cells are digested with 0.25% trypsin containing 0.02% EDTA at 37°C to remove them from the bottom of the culture dish. After digestion for 2 minutes, an equal volume of DMEM culture medium is added to terminate the digestion. The above cells are collected in a centrifuge tube and centrifuged (2000rpm, 8min), the supernatant is discarded, and the precipitate is rinsed twice with physiological saline to remove the residual trypsin. The bone marrow mesenchymal stem cells are plated at 5×10 4 The cells were inoculated into medium II at a density of 100 / mL and subcultured in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every 2 days during the culture period, and P3 bone marrow mesenchymal stem cells were collected.

[0046] Example 3

[0047] A bone marrow mesenchymal stem cell culture medium combination, consisting of culture medium I and culture medium II, wherein culture medium I is a DMEM / F12 culture medium supplemented with 10 μg / mL asterone, 10 μM melatonin, 12 μg / mL linoleic acid, 10 mM L-glutamine, 18 ng / mL bFGF, 30 μg / mL insulin, and 50 μg / mL astragalus polysaccharide;

[0048] The culture medium II is a DMEM / F12 culture medium supplemented with 70 ng / mL ACE2, 12 μg / mL linoleic acid, 10 mM L-glutamine, 18 ng / mL bFGF, 30 μg / mL insulin, and 50 μg / mL astragalus polysaccharide.

[0049] The method for culturing bone marrow mesenchymal stem cells specifically comprises the following steps:

[0050] (1) The separation and extraction of bone marrow mesenchymal stem cells is the same as in Example 1.

[0051] (2) Primary culture of bone marrow mesenchymal stem cells: The bone marrow mesenchymal stem cells obtained in step (1) were cultured at a rate of 2×10 5 The cells were inoculated into medium I at a density of 100 / mL and cultured in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every two days. The cells obtained from the primary culture were labeled as P0 generation bone marrow mesenchymal stem cells.

[0052] (3) Subculture of bone marrow mesenchymal stem cells: When the cell fusion degree of bone marrow mesenchymal stem cells reaches 80% under a microscope, the original culture medium is discarded and the bone marrow mesenchymal stem cells are digested with 0.25% trypsin containing 0.02% EDTA at 37°C to remove them from the bottom of the culture dish. After digestion for 2 minutes, an equal volume of DMEM culture medium is added to terminate the digestion. The above cells are collected in a centrifuge tube and centrifuged (2000rpm, 8min), the supernatant is discarded, and the precipitate is rinsed twice with saline to remove the residual trypsin. The bone marrow mesenchymal stem cells are plated at 1×10 4 The cells were inoculated into medium II at a density of 100 / mL and subcultured in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every 2 days during the culture period, and P3 bone marrow mesenchymal stem cells were collected.

[0053] Comparative Example 1

[0054] The bone marrow mesenchymal stem cell culture medium combination of this comparative example 1 is composed of culture medium I and culture medium II, wherein the culture medium I is a DMEM / F12 culture medium supplemented with 6 μM melatonin, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide;

[0055] The medium II is a DMEM / F12 medium supplemented with 60 ng / mL ACE2, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide. The other steps are the same as those in Example 1, that is, the difference from Example 1 is that asterone is omitted in the medium I.

[0056] Comparative Example 2

[0057] The bone marrow mesenchymal stem cell culture medium combination of this comparative example 2 is composed of culture medium I and culture medium II, wherein the culture medium I is a DMEM / F12 culture medium supplemented with 6 μg / mL asterone, 6 μM melatonin, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide;

[0058] The medium II is a DMEM / F12 medium supplemented with 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide. The other steps are the same as those in Example 1, that is, the difference from Example 1 is that ACE2 is omitted in the medium II.

[0059] Comparative Example 3

[0060] In this comparative example 3, medium I was used to replace medium II for subculture of bone marrow mesenchymal stem cells, and the rest was consistent with that in example 1.

[0061] Comparative Example 4

[0062] In this comparative example 4, medium II was used to replace medium I for primary culture of bone marrow mesenchymal stem cells, and the rest was consistent with that in example 1.

[0063] Test Example 1

[0064] Cell morphology characterization: The P3 generation bone marrow mesenchymal stem cells obtained by subculture in Examples 1 to 3 were placed under an inverted microscope and photographed to record the changes in cell morphology and quantity of bone marrow mesenchymal stem cells after culture in different culture media. Figures 1 to 3 The morphological microscope images of bone marrow mesenchymal stem cells obtained in Examples 1 to 3 correspond respectively.

[0065] Depend on Figures 1 to 3 It can be seen that the bone marrow mesenchymal stem cells obtained in Examples 1 to 3 have a relatively uniform morphology, a large number of cells, and are all short spindle-shaped, indicating that the culture medium combination used in the culture method of the present invention can culture bone marrow mesenchymal stem cells with good growth status.

[0066] Test Example 2

[0067] Cell proliferation ability detection: P3 bone marrow mesenchymal stem cells of Examples 1-3 and Comparative Examples 1-4 were taken respectively, digested with 0.25% trypsin to completely separate the cells, prepared into a cell suspension, counted with a cell counting plate, and the cell density was adjusted to 1×10 4The cells were inoculated in 24-well plates and cultured in medium II at 37°C and 5% CO2 for 7 consecutive days. The medium was replaced every other day during the culture period. The proliferation times of bone marrow mesenchymal stem cells were calculated, and the results are shown in Table 1.

[0068] The formula for proliferation multiple is as follows: proliferation multiple = total number of cells obtained by final culture / initial inoculation number.

[0069] Table 1

[0070]

[0071] Table 1 shows the results of the proliferation multiples of bone marrow mesenchymal stem cells. It can be seen from the table that the proliferation multiples of bone marrow mesenchymal stem cells obtained by culture in Examples 1 to 3 are the highest, all above 52.7 times. In Comparative Example 1, asterone was omitted from the culture medium I, and in Comparative Example 2, the addition of ACE2 was omitted from the culture medium II. Compared with Examples 1 to 3, the proliferation multiples of the cells were significantly reduced, indicating that asterone and ACE2 have different degrees of promoting effects on cell proliferation, among which ACE2 has a better effect on promoting the proliferation of bone marrow mesenchymal stem cells.

[0072] Comparative Example 3 uses medium I for primary and subculture, and Comparative Example 4 uses medium II for primary and subculture. Compared with Examples 1 to 3, the proliferation multiples of bone marrow mesenchymal stem cells are significantly reduced, which shows that the proliferation ability of cells obtained by culturing with only one medium is not as good as that of two mediums. Therefore, the medium I and medium II involved in the present invention can cooperate with each other in primary cell culture and subculture, better improve cell proliferation activity, and enable bone marrow mesenchymal stem cells to achieve rapid expansion in a short time.

[0073] Test Example 3

[0074] Osteogenic differentiation experiment: The osteogenic differentiation potential of the P3 generation bone marrow mesenchymal stem cells cultured in Example 1-3 and Comparative Example 1-4 was detected by an alkaline phosphatase kit. The specific steps are as follows: the P3 generation bone marrow mesenchymal stem cells cultured in Example 1-3 and Comparative Example 1-4 were taken and 2×10 5The cells were inoculated into the culture plate at a density of 100 / mL and placed in a cell culture incubator at 37°C and 5% CO2 for culture. When the cell confluence reached 85%, the culture medium was removed, the cells were rinsed with PBS, and osteogenic induction medium (DMEM culture medium, containing 10% fetal bovine serum, 100U / mL penicillin, 100mg / L streptomycin, 50μg / L vitamin C, 100nM dexamethasone, 10mM sodium β-glycerophosphate) was added and placed in the incubator for culture. The medium was changed every 2 days. After 14 days of osteogenic induction, the original culture medium was discarded, the cells were rinsed with PBS, and Triton X-100 lysis buffer was added. The cells were placed on ice for 20 minutes. The lysed cells were collected in a centrifuge tube for centrifugation at a speed of 10,000rpm. The supernatant was collected after centrifugation for 5 minutes. The ALP activity of the cells was tested using an ALP kit, and the results were as follows: Figure 4 shown.

[0075] Depend on Figure 4 It can be seen that the ALP activity of bone marrow mesenchymal stem cells in Examples 1 to 3 is significantly higher than that in Comparative Examples 1 to 4, which is mainly attributed to the fact that Comparative Examples 1 to 4 respectively changed the composition and culture method of the cell culture medium, Comparative Examples 1 and 2 respectively omitted asterone and ACE2 in the culture medium, Comparative Example 3 was cultured only with culture medium I, and Comparative Example 4 was cultured only with culture medium II. The above results not only illustrate that the bone marrow mesenchymal stem cells obtained by primary and subculture of cells using culture medium I and culture medium II alone have poor osteogenic differentiation effects. On the other hand, it also illustrates that asterone in culture medium I is beneficial to primary culture of cells; ACE2 in culture medium II is beneficial to subculture of cells, and the two culture media can work together to better promote cell growth and improve the osteogenic differentiation potential of bone marrow mesenchymal stem cells.

[0076] Test Example 4

[0077] Western Blot detection: Western Blot technology is used to detect the protein expression of bone marrow mesenchymal stem cells after osteogenic induction differentiation. The P3 generation bone marrow mesenchymal stem cells of the above examples 1-3 and comparative examples 1-4 were induced to differentiate into osteogenics for 14 days, and then the differentiated bone marrow mesenchymal stem cells were collected and rinsed with PBS, and RIPA cell lysis solution was added to fully lyse the cells, and the proteins in the cells were extracted. After lysis for 30 minutes, the cells were collected, centrifuged at 4°C (12000rpm, 10min), and the supernatant was collected. The protein concentration was measured using a BCA kit; the protein was mixed with 5× loading buffer at a ratio of 1:4, and then boiled in a 100°C water bath for 8min to denature the protein sample to obtain the loading solution. A 10% SDS-PAGE gel was prepared in advance, 20μL of the above loading solution was taken, and added to the gel loading well for electrophoresis separation. Under the action of the electric field, proteins of different molecular weights were effectively separated. The protein was transferred to the PVDF membrane by wet transfer. This step was performed in the transfer buffer to ensure the integrity and activity of the protein. The PVDF membrane was washed with TBST buffer for 5 min × 3 times; then the PVDF membrane was blocked with 5% skim milk and incubated at room temperature for 1 h, and then incubated with the primary antibody (ALP, Runx2, GAPDH) at 4°C overnight to allow the primary antibody to fully bind to the target protein. The membrane was removed and washed with TBST buffer for 5 min × 3 times; the next day, the membrane was incubated with the secondary antibody at 37°C for 40 min, and the membrane was removed and washed with TBST buffer for 5 min × 3 times; finally, the ultrasensitive ECL luminescent solution was used for color development, and the expression levels of different proteins were observed. The results are shown in Figure 5 shown.

[0078] Depend on Figure 5 It can be seen that the induction effects of different embodiments and comparative examples show obvious differences in protein expression levels, indicating that after changes in different culture media and culture methods, the osteogenic differentiation performance of bone marrow mesenchymal stem cells will also be different. Compared with Comparative Examples 1 and 2, the protein expression levels of osteogenic differentiation genes ALP and Runx2 in bone marrow mesenchymal stem cells cultured in Examples 1-3 are upregulated, indicating that the addition of asterone and ACE2 in the culture medium can significantly affect the osteogenic differentiation potential of bone marrow mesenchymal stem cells, and compared with Comparative Examples 3-4, it is shown that culture media I and II can cooperate with each other in the primary culture and subculture of cells to enhance the osteogenic differentiation process of bone marrow mesenchymal stem cells.

[0079] Therefore, the bone marrow mesenchymal stem cells obtained in Examples 1-3 have better osteogenic differentiation potential, and asterone and ACE2 in culture medium I and culture medium II can greatly increase the protein expression level of osteogenic differentiation genes in bone marrow mesenchymal stem cells, which is consistent with the above-mentioned ALP activity detection results.

[0080] In summary, the present invention provides a bone marrow mesenchymal stem cell culture medium combination and a culture method thereof, and a large amount of experimental data proves that the cells obtained by primary and subculture of cells using only culture medium I and culture medium II have poor osteogenic differentiation effects. On the other hand, it also shows that asterone in culture medium I is beneficial to primary culture of cells; ACE2 in culture medium II is beneficial to subculture of cells, and the two culture media can work together to better promote cell growth and improve the proliferation ability and osteogenic differentiation potential of bone marrow mesenchymal stem cells.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A method for culturing bone marrow mesenchymal stem cells, characterized in that: Medium I was used for primary culture, and medium II was used for subculture; The culture medium I is a DMEM / F12 culture medium supplemented with 3-10 μg / mL asterone, 3-10 μM melatonin, 5-12 μg / mL linoleic acid, 4-10 mM L-glutamine, 10-18 ng / mL bFGF, 22-30 μg / mL insulin, and 42-50 μg / mL astragalus polysaccharide; The culture medium II is a DMEM / F12 culture medium supplemented with 45-70 ng / mL ACE2, 5-12 μg / mL linoleic acid, 4-10 mM L-glutamine, 10-18 ng / mL bFGF, 22-30 μg / mL insulin, and 42-50 μg / mL astragalus polysaccharide.

2. The method for culturing bone marrow mesenchymal stem cells according to claim 1, characterized in that: The culture medium I is a DMEM / F12 culture medium supplemented with 6 μg / mL asterone, 6 μM melatonin, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide; The culture medium II is a DMEM / F12 culture medium supplemented with 60 ng / mL ACE2, 9 μg / mL linoleic acid, 7 mM L-glutamine, 14 ng / mL bFGF, 26 μg / mL insulin, and 46 μg / mL astragalus polysaccharide.

3. The method for culturing bone marrow mesenchymal stem cells according to claim 1 or 2, characterized in that: The specific steps include: (1) Isolation and extraction of bone marrow mesenchymal stem cells: Bone marrow mesenchymal stem cells were isolated by density gradient centrifugation; (2) Primary culture of bone marrow mesenchymal stem cells: inoculating the bone marrow mesenchymal stem cells obtained in step (1) into culture medium I for primary culture; (3) Subculture of bone marrow mesenchymal stem cells: When the primary culture of bone marrow mesenchymal stem cells in step (2) reaches a confluence of 80-90%, remove medium I and digest the bone marrow mesenchymal stem cells with 0.25% trypsin containing 0.02% EDTA. After digestion, centrifuge the cells and inoculate the resulting bone marrow mesenchymal stem cells into medium II for subculture.

4. The method for culturing bone marrow mesenchymal stem cells according to claim 3, characterized in that: The seeding density of bone marrow mesenchymal stem cells in the primary culture of step (2) is 2×10 5 -6×10 5 Pieces / mL.

5. The method for culturing bone marrow mesenchymal stem cells according to claim 3, characterized in that: The seeding density of bone marrow mesenchymal stem cells in the subculture in step (3) is 1×10 4 -5×10 4 Pieces / mL.

6. The method for culturing bone marrow mesenchymal stem cells according to claim 3, characterized in that: The primary culture and subculture were both carried out in an incubator at 37° C., 5% CO 2 , and saturated humidity.

7. The method for culturing bone marrow mesenchymal stem cells according to claim 3, characterized in that: The medium was changed every 2 days during the primary culture and subculture.

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