A serum-containing osteogenic induction differentiation medium, culture method and its application

Abstract

This invention relates to the field of cell culture technology, specifically to a serum-containing osteogenic induction differentiation medium, a culture method, and its application. The differentiation medium consists of a basal medium and supplementary components. The basal medium is MEMα, and the supplementary components consist of component A and component B. Component A includes fetal bovine serum, L-ascorbic acid-2-phosphate magnesium salt, β-glycerophosphate sodium, dexamethasone, resveratrol, estradiol, strontium chloride, vitamin K2, and sodium butyrate; component B is 1α,25-dihydroxyvitamin D3. This invention exhibits high differentiation efficiency. Mesenchymal stem cells induced using the differentiation medium of this invention show obvious calcium nodules within 7 days, and osteoblast differentiation is achieved within 10-12 days.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a serum-containing osteogenic induction differentiation medium, a culture method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from the mesoderm that have high self-renewal and multi-lineage differentiation potential. They are widely distributed in various tissues throughout the body and have the advantages of being easy to isolate and obtain, and can be differentiated and expanded in vitro.

[0003] For mesenchymal stem cells, regardless of their origin, in vitro testing should be performed to assess their differentiation capacity with various cell types (such as adipocytes, chondrocytes, osteoblasts, etc.) in order to determine their pluripotency in cell differentiation.

[0004] Currently, there are commercially available culture media with good results for identifying osteogenic differentiation of mesenchymal stem cells. However, most of these media have long differentiation times, usually requiring 14 to 21 days or even longer, which is a significant challenge, especially in scenarios where rapid research results or high-throughput screening are needed. Summary of the Invention

[0005] To address the technical problems mentioned in the background art, this invention provides a serum-containing osteogenic induction differentiation culture medium, a culture method, and its application. The technical solution is as follows:

[0006] A serum-containing osteogenic induction differentiation medium, comprising MEMα basal medium and additive components, wherein the additive components consist of component A and component B;

[0007] In component A, the volume percentage of fetal bovine serum is 1-10%, the content of sodium β-glycerophosphate is 10 mM, the content of magnesium L-ascorbic acid-2-phosphate is 50 μM-200 μM, the content of dexamethasone is 50-200 nM, the content of resveratrol is 0.1-10 μM, the content of estradiol is 1-100 nM, the content of strontium chloride is 0.1 mM-10 mM, the content of vitamin K2 is 0.1 μM-30 μM, and the content of sodium butyrate is 0.05 mM-5 mM.

[0008] In component B, the content of 1α,25-dihydroxyvitamin D3 is 1~30 nM.

[0009] Preferably, in component A, the volume percentage of fetal bovine serum is 5%, the content of L-ascorbic acid-2-phosphate magnesium salt is 150 μM, the content of dexamethasone is 100 nM, the content of resveratrol is 1 μM, the content of estradiol is 20 nM, the content of strontium chloride is 1.5 mM, the content of vitamin K2 is 2.25 μM, and the content of sodium butyrate is 0.4 mM;

[0010] In component B, the content of 1α,25-dihydroxyvitamin D3 is 3 nM.

[0011] Preferably, the fetal bovine serum is EcoSy FND500 fetal bovine serum.

[0012] The application of any of the preceding claims of a serum-containing osteogenic induction differentiation culture medium for osteogenic induction differentiation culture of mesenchymal stem cells.

[0013] Preferably, the osteogenic induction differentiation culture process of the mesenchymal stem cells is as follows:

[0014] S1: Recovery and expansion of mesenchymal stem cells: Mesenchymal stem cells were cultured in MEMα medium containing 10% fetal bovine serum and expanded for 2 generations to restore the cell state before being used for differentiation experiments.

[0015] S2: Starvation culture: Cells were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum for cell counting at a density of 4.5 × 10⁻⁶ cells / year. 4 / cm 2 Cells were seeded into well plates at a density and starved in MEMα medium containing 0.5% fetal bovine serum to culture mesenchymal stem cells.

[0016] S3: Induction of differentiation: After starvation culture for 24 hours, the starvation medium is replaced with osteogenic induction differentiation medium A composed of the MEMα basal medium and the component A. Fresh osteogenic induction differentiation medium A is replaced every 2 days.

[0017] S4: Induction of differentiation and maturation: After 7 days of differentiation, the osteogenic induction differentiation medium A was replaced with osteogenic induction differentiation medium B composed of the MEMα basal medium + component A + component B, and differentiation into mature osteoblasts was continued.

[0018] S5: Sample collection and identification: When the cells have differentiated to 10-12 days, the cells are stained and analyzed using the Alizarin Red staining method.

[0019] A method for osteogenic induction differentiation culture of bone marrow mesenchymal stem cells, using a serum-containing osteogenic induction differentiation culture medium as described above, wherein the culture process is as follows:

[0020] S1: Recovery and expansion of mesenchymal stem cells: Mesenchymal stem cells were cultured in MEMα medium containing 10% fetal bovine serum and expanded for 2 generations to restore the cell state before being used for differentiation experiments.

[0021] S2: Starvation culture: Cells were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum for cell counting at a density of 4.5 × 10⁻⁶ cells / year. 4 / cm 2 Cells were seeded into well plates at a density and starved in MEMα medium containing 0.5% fetal bovine serum to culture mesenchymal stem cells.

[0022] S3: Induction of differentiation: After starvation culture for 24 hours, the starvation medium is replaced with osteogenic induction differentiation medium A composed of the MEMα basal medium and the component A. Fresh osteogenic induction differentiation medium A is replaced every 2 days.

[0023] S4: Induction of differentiation and maturation: After 7 days of differentiation, the osteogenic induction differentiation medium A was replaced with osteogenic induction differentiation medium B composed of the MEMα basal medium + component A + component B, and differentiation into mature osteoblasts was continued.

[0024] S5: Sample collection and identification: When the cells have differentiated to 10-12 days, the cells are stained and analyzed using the Alizarin Red staining method.

[0025] The main beneficial effects of this invention are: it provides a serum-containing osteogenic induction differentiation culture medium and its application method. This culture medium contains a variety of substances that are conducive to the differentiation of mesenchymal stem cells into osteoblasts. Combined with the fetal bovine serum product unique to Icosai, the differentiation efficiency is high. Mesenchymal stem cells induced by the differentiation culture medium of this invention can show obvious calcium nodules within 7 days and can achieve osteoblast differentiation in 10-12 days.

[0026] The mechanism of action of this invention: During the differentiation of mesenchymal stem cells into osteoblasts, the process generally involves osteoblast activation, osteoblast proliferation, extracellular matrix maturation, and extracellular matrix mineralization. The differentiation culture medium in this invention targets this differentiation process, systematically identifying effective substances that can improve differentiation efficiency. Through screening a large number of substances and optimizing their concentrations, the optimal formula is finally determined. This shortens the time required to identify the differentiation potential of mesenchymal stem cells and solves the problems of long differentiation time and low differentiation efficiency of osteoblasts.

[0027] In this invention, L-ascorbic acid-2-phosphate magnesium salt promotes osteoblast proliferation and collagen synthesis; sodium β-glycerophosphate provides a phosphorus source for mineralized matrix formation; dexamethasone induces osteoblast differentiation and enhances alkaline phosphatase activity; and resveratrol has antioxidant and anti-inflammatory effects, which are beneficial for maintaining cell viability. Estradiol and strontium chloride synergistically promote bone formation; vitamin K2 participates in γ-carboxylation and enhances osteocalcin function; sodium butyrate affects gene expression by regulating histone deacetylase activity; and 1α,25-dihydroxyvitamin D3 regulates calcium and phosphorus metabolism, collectively promoting osteoblast functional maturation and mineralized nodule formation.

[0028] In addition, the differentiation culture medium of this invention optimizes the method of differentiating mesenchymal stem cells into osteoblasts. After mesenchymal stem cells are seeded, they are first cultured with low concentration of serum to starve the cells for 24 hours, so that most cells are arrested in the G0 / G1 phase, allowing the cells to be "prepared" in advance to enter the differentiation process.

[0029] Finally, by using Ekosei's unique fetal bovine serum, mesenchymal stem cells are prevented from dying due to insufficient nutrition during cell starvation, while also being kept in a better state to enter the differentiation phase. This is an advantage that other brands of fetal bovine serum do not have.

[0030] The above three points demonstrate that the differentiation culture medium used in this invention significantly shortens the time required for mesenchymal stem cells to differentiate into osteoblasts and improves the differentiation efficiency. Attached Figure Description

[0031] Figure 1 This is an electron micrograph taken on day 7 of the osteogenic induction differentiation medium A1+B1 of the present invention.

[0032] Figure 2 This is an electron micrograph taken on day 7 of the osteogenic induction differentiation medium A2+B2 of the present invention.

[0033] Figure 3 This is an electron micrograph taken on day 7 of the osteogenic induction differentiation medium A3+B3 of the present invention.

[0034] Figure 4 The electron micrograph of osteoblasts induced to differentiate using the osteogenic induction differentiation medium A1+B1 component of the present invention, stained with alizarin red on day 12.

[0035] Figure 5 The electron micrograph of osteoblasts induced to differentiate using the osteogenic induction differentiation medium A2+B2 component of the present invention, stained with alizarin red on day 12.

[0036] Figure 6The electron micrograph of osteoblasts induced to differentiate using the osteogenic induction differentiation medium A3+B3 component of the present invention, stained with alizarin red on day 12.

[0037] Figure 7 The electron micrograph of osteoblasts induced to differentiate using the osteogenic induction differentiation medium components of the present invention, stained with alizarin red on day 10.

[0038] Figure 8 Electron micrograph of osteoblasts induced to differentiate using commercially available osteogenic induction culture medium, stained with alizarin red on day 10.

[0039] Figure 9 The image shows the results of an electron microscope stained with Alizarin Red on day 12 of osteoblasts differentiated using osteogenic induction culture medium prepared with Gibberellic brand serum.

[0040] Figure 10 The image shows the results of an electron microscope stained with Alizarin Red on day 12, showing osteoblasts differentiated using the osteogenic induction differentiation medium prepared with EcoSy brand serum.

[0041] Figure 11 This is an electron micrograph of a calcium nodule. The image is from... Figure 2 The locations of the calcium nodules are marked with arrows on the basis of the image. Detailed Implementation

[0042] The technical solutions of the present invention will be further described below with reference to specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] This invention provides a serum-containing osteogenic induction differentiation medium, a culture method, and its application. The differentiation medium consists of a basal medium and supplementary components. The basal medium is MEMα, and the supplementary components consist of component A and component B. Component A includes fetal bovine serum, magnesium L-ascorbic acid-2-phosphate, sodium β-glycerophosphate, dexamethasone, resveratrol, estradiol, strontium chloride, vitamin K2, and sodium butyrate. Component B is 1α,25-dihydroxyvitamin D3.

[0044] In component A of this invention, the volume percentage of fetal bovine serum is 1-10%, the content of sodium β-glycerophosphate is 10 mM, the content of magnesium L-ascorbic acid-2-phosphate is 50 μM-200 μM, the content of dexamethasone is 50-200 nM, the content of resveratrol is 0.1-10 μM, the content of estradiol is 1-100 nM, the content of strontium chloride is 0.1 mM-10 mM, the content of vitamin K2 is 0.1 μM-30 μM, and the content of sodium butyrate is 0.05 mM-5 mM; in component B, the content of 1α,25-dihydroxyvitamin D3 is 1-30 nM.

[0045] Preferably, the fetal bovine serum contains 5% by volume, L-ascorbic acid-2-phosphate magnesium salt contains 150 μM, dexamethasone contains 100 nM, resveratrol contains 1 μM, estradiol contains 20 nM, strontium chloride contains 1.5 mM, vitamin K2 contains 2.25 μM, and sodium butyrate contains 0.4 mM; component B contains 3 nM of 1α,25-dihydroxyvitamin D3.

[0046] Preparation method of MEMα basal medium (prepared in 100 mL volumes):

[0047] S1: Add 80 mL of water for injection to a 100 mL beaker, and weigh out 1.01 g of MEMα powder and dissolve it in the solution;

[0048] S2: After stirring for 15 minutes, weigh out 0.22g of sodium bicarbonate powder and dissolve it in the solution;

[0049] S3: After stirring for 10 minutes, pour into a 100mL volumetric flask and make up to volume.

[0050] S4: After bringing the volume to a final volume, pour the mixture back into the beaker and stir for 5 minutes. Then filter and use.

[0051] Preparation method for adding component A (based on 100 mL of complete culture medium):

[0052] According to Table 1, first prepare the stock solution of all materials in component A, then mix all the stock solutions together according to the working volume, filter and store at -20℃ or below to prepare the bone induction differentiation culture medium additive component A.

[0053] Table 1

[0054]

[0055] Preparation method for adding component B:

[0056] Prepare a 30 μM stock solution of 1α,25-dihydroxyvitamin D3, then add 3.33 μL to 100 μL of the stock solution to 1 mL of 0.5% bovine serum albumin (BSA) solution, filter, and store at -80℃ to prepare osteogenic induction differentiation medium supplement component B.

[0057] Preparation method of osteogenic induction differentiation medium:

[0058] Osteogenic induction differentiation medium A: MEMα basal medium + supplementary component A;

[0059] Osteogenic induction differentiation medium B: MEMα basal medium + supplementary component A + supplementary component B;

[0060] The ratio of MEMα basal culture medium to added component A is 100:2.25 to 100:17.19 (corresponding to the total cumulative working volume in Tables 3 to 5 of the specific embodiments), with a preferred ratio of 100:6.84 (corresponding to the total cumulative working volume in Table 4 of the specific embodiments).

[0061] The ratio of MEMα basal medium to added component B is 100:1.

[0062] The application method of a serum-containing osteogenic induction differentiation medium is as follows:

[0063] S1: Recovery and expansion of human bone marrow mesenchymal stem cells (hMSCs): hMSCs were cultured in MEMα medium containing 10% fetal bovine serum and expanded for 2 generations to restore the cell state before being used for differentiation experiments.

[0064] S2: Starvation culture: Cells were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum for cell counting at a density of 4.5 × 10⁻⁶ cells / year. 4 / cm 2 Cells were seeded into 12-well plates and starved in MEMα medium containing 0.5% fetal bovine serum;

[0065] S3: Induction of differentiation: After starvation culture for 24 hours, replace the starvation medium with osteogenic induction differentiation medium A, and replace with fresh osteogenic induction differentiation medium A every 2 days;

[0066] S4: Induction of differentiation and maturation: 7 days after differentiation, replace osteogenic induction differentiation medium A with osteogenic induction differentiation medium B, and continue to induce differentiation into mature osteoblasts;

[0067] S5: Sample collection and identification: When the cells have differentiated to 10-12 days, the cells are stained and analyzed using the Alizarin Red staining method.

[0068] Alizarin Red Staining Method

[0069] 1. Washing: Discard the osteogenic induction differentiation medium in the 12-well plate, and gently wash twice with phosphate-buffered saline (PBS) for 2 minutes each time to remove residual medium;

[0070] 2. Cell fixation: Add 4% paraformaldehyde solution to a 12-well plate and fix the cells at room temperature for 15 minutes to stabilize the cell structure. After fixation, discard the paraformaldehyde and wash twice with PBS to completely remove the fixative.

[0071] 3. Alizarin Red staining: Prepare 0.1% alizarin red staining solution (pH 4.2), filter and use. Add 0.3 mL of 0.1% alizarin red staining solution to a 12-well plate and stain at room temperature for 15 minutes.

[0072] 4. Washing and observation: After staining, discard the staining solution, wash slowly with PBS 3-5 times, observe under an inverted microscope, the mineralized nodules will be stained orange-red or dark red, and take pictures for record.

[0073] The experimental materials used in the embodiments of this invention are shown in Table 2.

[0074] Table 2

[0075]

[0076] Example 1: Using bone marrow mesenchymal stem cells (BMSCs) (ATCC, PCS-500-012, Lot 70011720, Passage 7) to verify the optimal concentration of each component in the osteogenic induction differentiation medium of the present invention.

[0077] (1) Preparation of MEMα basal culture medium (prepared in 100 mL volumes):

[0078] S1: Add 80 mL of water for injection to a 100 mL beaker, and weigh out 1.01 g of MEMα powder and dissolve it in the solution;

[0079] S2: After stirring for 15 minutes, weigh out 0.22g of sodium bicarbonate powder and dissolve it in the solution;

[0080] S3: After stirring for 10 minutes, pour into a 100mL volumetric flask and make up to volume.

[0081] S4: After bringing the volume to a final volume, pour the mixture back into the beaker and stir for 5 minutes. Then filter and use.

[0082] (2) Preparation of Component A (prepared according to 100 mL of complete culture medium):

[0083] First, prepare stock solutions of all materials in additive component A. Then, mix all stock solutions together according to the addition volumes in Tables 3, 4 and 5. The fetal bovine serum used is the Icosai brand. After filtration, it is stored below -20℃ and prepared into bone induction culture medium additive components A1, A2 and A3 respectively.

[0084] Table 3

[0085]

[0086] Table 4

[0087]

[0088] Table 5

[0089]

[0090] (3) Preparation of Component B (prepared according to 100 mL culture medium volume):

[0091] Prepare a 30 μM stock solution of 1α,25-dihydroxyvitamin D3, then add 10 μL of the stock solution to 1 mL of 0.5% bovine serum albumin (BSA) solution, filter, and store at -80°C to prepare osteogenic induction differentiation medium supplement component B.

[0092] (4) Prepare the osteoinduction differentiation medium:

[0093] Osteogenic induction differentiation medium A1: 97.75 mL MEMα basal medium + 2.25 mL supplemented with component A1;

[0094] Osteogenic induction differentiation medium A2: 93.16 mL MEMα basal medium + 6.84 mL supplemented with component A2;

[0095] Osteogenic induction differentiation medium A3: 82.81 mL MEMα basal medium + 17.19 mL supplemented with component A3;

[0096] Osteogenic induction differentiation medium B1: 96.75 mL MEMα basal medium + 2.25 mL supplemented component A1 + 1 mL supplemented component B;

[0097] Osteogenic induction differentiation medium B2: 92.16 mL MEMα basal medium + 6.84 mL of supplemented component A2 + 1 mL of supplemented component B;

[0098] Osteogenic induction differentiation medium B3: 81.81 mL MEMα basal medium + 17.19 mL supplemented component A3 + 1 mL supplemented component B.

[0099] (5) The application method of a serum-containing osteogenic induction differentiation culture medium in bone marrow mesenchymal stem cells is as follows:

[0100] S1: Recovery and expansion of BMSCs: BMSCs were cultured in MEMα medium containing 10% fetal bovine serum and expanded for 2 generations to restore the cell state before being used for differentiation experiments.

[0101] S2: Starvation culture: Cells were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum for cell counting at a density of 4.5 × 10⁻⁶ cells / year. 4 / cm 2 Cells were seeded into three 12-well plates and starved in MEMα medium containing 0.5% fetal bovine serum.

[0102] S3: Induction of differentiation: After 24 hours of starvation culture, the starvation medium in the three 12-well plates was replaced with osteogenic induction differentiation mediums A1, A2 and A3, respectively. Fresh differentiation mediums A1, A2 and A3 were replaced every 2 days.

[0103] S4: Induction of differentiation and maturation: 7 days after differentiation, the osteogenic induction differentiation media A1, A2 and A3 in the three 12-well plates were replaced with osteogenic induction differentiation media B1, B2 and B3 respectively, and the differentiation was continued to induce mature osteoblasts.

[0104] S5: Sample collection and identification: After the cells differentiated to 12 days, the cells in 3 12-well plates were stained and analyzed using the Alizarin Red staining method.

[0105] Results Analysis: Among the three concentrations of osteogenic induction differentiation media, the combination of osteogenic induction differentiation media A2 and B2 showed the highest differentiation efficiency, with obvious calcium nodules appearing as early as day 7. The other two concentrations of differentiation media did not show calcium nodules, and their higher concentrations even exhibited some toxicity to cells. Furthermore, Alizarin Red staining results on day 12 clearly showed that the combination of osteogenic induction differentiation media A2 and B2 resulted in a larger staining area, indicating the best differentiation effect (see results below). Figures 1-6 , Figure 11 (As shown).

[0106] Example 2: Comparison of differentiation efficiency between the osteogenic induction differentiation medium of the present invention and a commercially available brand osteogenic induction differentiation medium.

[0107] (1) Preparation of MEMα basic culture medium (prepared in 100mL volume): Refer to Example 1.

[0108] (2) Preparation of Additive A (prepared according to 100 mL of complete culture medium): According to Table 4 of Example 1, first prepare the stock solution of all materials in Additive A, then mix all the stock solutions together according to the working volume, filter and store at -20℃ to prepare bone induction culture medium Additive A.

[0109] (3) Preparation of added component B (prepared according to 100 mL of complete culture medium): Refer to Example 1.

[0110] (4) Prepare the osteogenic induction differentiation medium of the present invention: refer to Example 1.

[0111] (5) Preparation of commercial Stemsell brand osteogenic induction differentiation medium: Prepare according to the instructions, 200mL of osteogenic induction differentiation basal medium + 50mL of osteogenic induction differentiation medium additives.

[0112] (6) The application method of a serum-containing osteogenic induction differentiation culture medium in bone marrow mesenchymal stem cells is as follows:

[0113] S1: Recovery and expansion of BMSCs: BMSCs were cultured in MEMα medium containing 10% fetal bovine serum and expanded for 2 generations to restore the cell state before being used for differentiation experiments.

[0114] S2: Starvation culture: Cells were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum for cell counting at a density of 4.5 × 10⁻⁶ cells / year. 4 / cm 2 Cells were seeded into 12-well plates and starved in MEMα medium containing 0.5% fetal bovine serum;

[0115] S3: Induction of differentiation: After 24 hours of starvation culture, the starvation medium was replaced with osteogenic induction differentiation medium A, and fresh differentiation medium A was replaced every 2 days.

[0116] S4: Induction of differentiation and maturation: 7 days after differentiation, replace osteogenic induction differentiation medium A in the 12-well plate with osteogenic induction differentiation medium B, and continue to induce differentiation into mature osteoblasts;

[0117] S5: Sample collection and identification: When the cells differentiate to day 10, the cells are stained and analyzed using the Alizarin Red staining method.

[0118] (7) The application method of commercial Stemcell brand osteogenic induction differentiation medium in bone marrow mesenchymal stem cells is as follows:

[0119] S1: Recovery and expansion of BMSCs: BMSCs were cultured in MEMα medium containing 10% fetal bovine serum and expanded for 2 generations to restore the cell state before being used for differentiation experiments.

[0120] S2: Cell seeding: Digest cells with trypsin, centrifuge, resuspend cells in MEMα medium containing 10% fetal bovine serum, count cells, and seed at a rate of 8 × 10⁶ cells / year. 3 / cm 2 Cells were seeded into 12-well plates at a density of [number missing].

[0121] S3: Induction of differentiation: When the cells have merged to 80%~90%, replace the culture medium with Stemsell brand osteogenic induction differentiation medium, and replace it with fresh osteogenic induction differentiation medium every 3~4 days.

[0122] S4: Sample collection and identification: Ten days after differentiation, cells were stained and analyzed using alizarin red staining.

[0123] Results analysis: The osteogenic induction differentiation medium of this invention produced more staining than the commercially available Stemsell osteogenic induction differentiation medium, indicating that the osteogenic induction differentiation medium of this invention has a better differentiation effect (see results). Figure 7 , Figure 8 (As shown).

[0124] Example 3: Comparison of differentiation efficiency of the osteogenic induction differentiation medium of the present invention prepared with Ekosei brand serum and Gibco brand serum.

[0125] (1) Preparation of MEMα basic culture medium (prepared in 100mL volume): Refer to Example 1.

[0126] (2) Preparation of Additive A (prepared according to 100 mL of complete culture medium): According to Table 4 of Example 1, first prepare the stock solution of all materials in Additive A, then mix all the stock solutions together according to the working volume, mix 2 portions, one portion is mixed entirely in 5% volume of Ekosai fetal bovine serum, and the other portion is mixed entirely in Gibco brand fetal bovine serum, filtered and stored below -20℃, to prepare Ekosai brand Additive A and Gibco brand Additive A.

[0127] (3) Preparation of component B (prepared according to 100 mL culture medium volume): Refer to Example 1.

[0128] (4) Prepare bone differentiation culture medium:

[0129] Ekosei brand osteogenic induction differentiation medium A: 93.16 mL MEMα basal medium + 6.84 mL Ekosei brand supplementary component A;

[0130] Gibbsco brand osteogenic induction differentiation medium A: 93.16 mL MEMα basal medium + 6.84 mL Gibbsco brand additive component A;

[0131] Ekosei brand osteogenic induction differentiation medium B: 92.16 mL MEMα basal medium + 6.84 mL Ekosei brand additive component A + 1 mL additive component B;

[0132] Gibbsco brand osteogenic induction differentiation medium B: 92.16 mL MEMα basal medium + 6.84 mL Gibbsco brand additive component A + 1 mL additive component B.

[0133] (5) The application method of a serum-containing osteogenic induction differentiation culture medium in bone marrow mesenchymal stem cells is as follows:

[0134] S1: Recovery and expansion of BMSCs: BMSCs were cultured in MEMα medium containing 10% EcoSy brand fetal bovine serum and 10% Gibbsco brand fetal bovine serum, respectively. After two generations of culture and expansion, the cells were used for differentiation experiments after their state was restored.

[0135] S2: Starvation culture: BMSCs amplified with two different brands of fetal bovine serum were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum from each brand. Cells were then counted at a density of 4.5 × 10⁻⁶ cells / mL. 4 / cm 2 Cells were seeded into two 12-well plates and starved in MEMα medium containing 0.5% fetal bovine serum from two different brands.

[0136] S3: Induction of differentiation: After starvation culture for 24 hours, the starvation medium in the two 12-well plates was replaced with osteogenic induction differentiation medium A of Ecosai brand and osteogenic induction differentiation medium A of Gibco brand, respectively. Fresh differentiation medium A was replaced every 2 days.

[0137] S4: Induction of differentiation and maturation: 7 days after differentiation, the osteogenic induction differentiation medium A of the two brands was replaced with osteogenic induction differentiation medium B respectively, and the differentiation was continued to induce the differentiation into mature osteoblasts.

[0138] S5: Sample collection and identification: After the cells differentiated to 12 days, the cells differentiated in the induction differentiation medium of this invention prepared with fetal bovine serum from two different brands were stained and analyzed using the Alizarin Red staining method.

[0139] Results analysis: Alizarin red staining of osteoblasts 12 days after differentiation showed that the osteogenic induction differentiation medium prepared by Ecosai (a brand of this invention) stained a significantly larger area with Alizarin Red than the osteogenic induction differentiation medium prepared by Gibraltar (a brand of this invention), indicating a significantly higher efficiency in inducing differentiation (see results). Figure 9 , Figure 10 (As shown).

[0140] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A serum-containing osteogenic induction differentiation medium, characterized in that, osteogenic induction differentiation of mesenchymal stem cells in bone marrow; consisting of MEMα basal medium and an additive component, which consists of component A and component B; the MEMα basal medium + the component A constitute osteogenic induction differentiation medium A; the MEMα basal medium + the component A + the component B constitute osteogenic induction differentiation medium B; in the component A, the volume ratio content of fetal bovine serum is 1-10%, the content of sodium β-glycerophosphate is 10 mM, the content of L-ascorbic acid-2-phosphate magnesium salt is 50-200 μM, the content of dexamethasone is 50-200 nM, the content of resveratrol is 0.1-10 μM, the content of estradiol is 1-100 nM, the content of strontium chloride is 0.1-10 mM, the content of vitamin K2 is 0.1-30 μM, and the content of sodium butyrate is 0.05-5 mM; in the component B, the content of 1α, 25-dihydroxyvitamin D3 is 1-30 nM; the fetal bovine serum is Eco-Serum FND500 fetal bovine serum. 2.The serum-containing osteogenic induction differentiation medium according to claim 1, characterized in that, in the component A, the volume ratio content of fetal bovine serum is 5%, the content of L-ascorbic acid-2-phosphate magnesium salt is 150 μM, the content of dexamethasone is 100 nM, the content of resveratrol is 1 μM, the content of estradiol is 20 nM, the content of strontium chloride is 1.5 mM, the content of vitamin K2 is 2.25 μM, and the content of sodium butyrate is 0.4 mM; in the component B, the content of 1α, 25-dihydroxyvitamin D3 is 3 nM.

3. Use of a serum-containing osteogenic induction differentiation medium according to any one of claims 1 to 2, characterized in that, osteogenic induction differentiation culture of mesenchymal stem cells in bone marrow. 4.The application of the serum-containing osteogenic induction differentiation medium according to claim 3, characterized in that, the osteogenic induction differentiation culture process of mesenchymal stem cells is as follows: S1: resuscitation and expansion of mesenchymal stem cells: mesenchymal stem cells are cultured in MEMα medium containing 10% fetal bovine serum, and expanded for 2 generations, and then used for differentiation experiments after the cell state is restored; S2: Starvation: Cells were trypsinized, resuspended in MEMa medium containing 0.5% fetal bovine serum after centrifugation, counted, and seeded at 4.5 x 10 4 / cm 2 Densities: Mesenchymal stem cells were seeded into the well plates and starved with MEMa medium containing 0.5% fetal bovine serum; S3: induction differentiation: after 24 hours of starvation culture, the starvation medium is replaced with osteogenic induction differentiation medium A consisting of the MEMα basal medium + the component A, and fresh osteogenic induction differentiation medium A is replaced every 2 days; S4: induction differentiation maturation: after 7 days of differentiation, the osteogenic induction differentiation medium A is replaced with osteogenic induction differentiation medium B consisting of the MEMα basal medium + the component A + the component B, and the induction differentiation into mature osteoblasts is continued; S5: sample collection and identification: when the cells are differentiated for 10-12 days, the cells are dyed and analyzed using alizarin red staining method. 5.A method for osteogenic induction differentiation culture of mesenchymal stem cells in bone marrow, characterized in that, a serum-containing osteogenic induction differentiation medium according to any one of claims 1-2 is used, and the culture process is as follows: S1: Resuscitation and expansion of mesenchymal stem cells: mesenchymal stem cells were cultured using MEMa medium containing 10% fetal bovine serum, and cultured and expanded for 2 generations, and after the cell state was restored, used for differentiation experiments; S2: Starvation culture: Cells were digested with trypsin, centrifuged, and resuspended in MEMα medium containing 0.5% fetal bovine serum for cell counting at a density of 4.5 × 10⁻⁶ cells / year. 4 / cm 2 Cells were seeded into well plates at a density and starved in MEMα medium containing 0.5% fetal bovine serum to culture mesenchymal stem cells. S3: Induced differentiation: after 24 hours of starvation culture, the starvation medium was replaced with osteogenic induction differentiation medium A consisting of the MEMa base medium + the component A, and fresh osteogenic induction differentiation medium A was replaced every 2 days; S4: Induced differentiation and maturation: after 7 days of differentiation, the osteogenic induction differentiation medium A was replaced with osteogenic induction differentiation medium B consisting of the MEMa base medium + the component A + the component B, and the induced differentiation into mature osteoblasts was continued; S5: Sample collection and identification: when the cells were differentiated to 10-12 days, the cells were stained and analyzed using alizarin red staining.

Images