Mesenchymal stem cell culture medium, preparation method and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a mesenchymal stem cell culture medium and a preparation method and application thereof.The mesenchymal stem cell culture medium comprises a basic culture medium, fetal bovine serum, basic fibroblast growth factor, polymyxin B and deionized water.The present application can simultaneously improve the proliferation activity of mesenchymal stem cells and the secretion performance of HGF by simultaneously adding the basic fibroblast growth factor and the polymyxin B in the basic culture medium; more stem cells can be in the active proliferation and division process by limiting the content of the basic fibroblast growth factor in the mesenchymal stem cell culture medium to 5-50 ng / mL; and the secretion performance of HGF can be improved while the cell proliferation activity is maintained by limiting the content of the polymyxin B in the mesenchymal stem cell culture medium to 50-100 mu g / mL.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a mesenchymal stem cell culture medium, its preparation method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal and multi-lineage differentiation potential. Among them, umbilical cord MSCs have become an important research subject in regenerative medicine and cell therapy due to their convenient sourcing, lack of ethical controversy, low immunogenicity, and strong proliferative capacity. The biological functions of umbilical cord MSCs are closely related to their paracrine cytokines. Various cytokines secreted by umbilical cord MSCs, such as epidermal growth factor (EGGF), basic fibroblast growth factor (BGF), and vascular endothelial growth factor (VEGF), can regulate cell signal transduction and promote tissue repair and regeneration, demonstrating significant application value in skin damage repair, angiogenesis, and immune regulation. Therefore, how to efficiently induce umbilical cord MSCs to secrete cytokines has become a research focus in this field.

[0003] Current research attempts to enhance cytokine secretion levels from umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells) by optimizing culture medium formulations and combining them with physical stimulation. For example, Chinese invention patent CN109852654B discloses a culture medium and method for inducing cytokine secretion from umbilical cord mesenchymal stem cells. This method involves preparing an induction culture medium by adding vitamin A, glutamine, lysophosphatidylcholine, and icariin to high-glucose DMEM, and then combining this with mechanical traction physical stimulation to enhance the cytokine secretion capacity of umbilical cord mesenchymal stem cells. However, this existing technology still has significant shortcomings. First, its induction system can only achieve an increase in the secretion of conventional cytokines, with limited targeted induction effects on specific functional cytokines such as vascular endothelial growth factor (VEGF), failing to meet the high demands of specific clinical applications such as vascular injury repair. Second, the mechanical traction parameters of this technology are fixed in frequency and amplitude, without dynamic regulation based on the physiological state of cell growth, which can easily cause mechanical damage to cells, affecting stem cell activity and subsequent secretion capacity. Third, the component ratio of its induction culture medium is only optimized within a fixed range, without considering the synergistic potential between different components, leaving considerable room for improvement in induction efficiency. Furthermore, most existing induction techniques suffer from complex operational procedures and difficulties in large-scale culture, limiting their industrial application. Therefore, developing a highly targeted, efficient, and minimally impactful method for inducing cytokines in umbilical cord mesenchymal stem cells suitable for large-scale production has significant clinical and industrial value. Summary of the Invention

[0004] The first aspect of the present invention provides a mesenchymal stem cell culture medium, comprising basal culture medium, fetal bovine serum, basic fibroblast growth factor, polymyxin B and deionized water.

[0005] Traditional in vitro expansion media for mesenchymal stem cells (MSCs) often fail to simultaneously achieve both high-efficiency enhancement of cell proliferation activity and high-level secretion of hepatocyte growth factor (HGF). This often results in insufficient HGF secretion after optimization of proliferation, or significant inhibition of cell proliferation after increased HGF secretion. This invention, however, achieves a synergistic enhancement of both by combining basic fibroblast growth factor (BGF) and polymyxin B in the basal culture medium. BGF regulates the cell cycle progression of MSCs, promoting the transformation from the quiescent G0 / G1 phase (resting phase / pre-DNA synthesis) to the active proliferative G2 / M phase (post-DNA synthesis / mitosis), reducing the proportion of quiescent cells and increasing the proportion of proliferating cells, thereby significantly enhancing the in vitro proliferation activity of stem cells. Polymyxin B works at the gene expression level, upregulating the gene expression level of HGF in MSCs, promoting HGF synthesis and expression at the transcriptional stage, and thus significantly increasing the amount of HGF secreted by cells. Ultimately, this invention achieves a dual enhancement of MSC proliferation activity and HGF secretion performance.

[0006] The volume content of fetal bovine serum in the mesenchymal stem cell culture medium is 6-15%.

[0007] Optionally, the volume content of fetal bovine serum in the mesenchymal stem cell culture medium is 8-12%.

[0008] The content of basic fibroblast growth factor in the mesenchymal stem cell culture medium is 5-50 ng / mL.

[0009] Optionally, the content of basic fibroblast growth factor in the mesenchymal stem cell culture medium is 5-20 ng / mL.

[0010] The polymyxin B content in the mesenchymal stem cell culture medium is 20-150 μg / mL.

[0011] Optionally, the polymyxin B content in the mesenchymal stem cell culture medium is 50-100 μg / mL.

[0012] The basal culture medium includes at least one of DMEM / F-12 medium, α-MEM medium, high-glucose DMEM medium, Ham's F-10 medium, and RPMI-1640 medium.

[0013] The second aspect of the present invention provides a method for preparing a mesenchymal stem cell culture medium, comprising the following steps: dissolving basic fibroblast growth factor and polymyxin B in deionized water to obtain a basic fibroblast growth factor solution and a polymyxin B solution; adding fetal bovine serum, basic fibroblast growth factor solution and polymyxin B solution to a basal culture medium, and mixing them evenly to obtain a mesenchymal stem cell culture medium.

[0014] A third aspect of the present invention provides an application of a mesenchymal stem cell culture medium for the in vitro expansion and culture of mesenchymal stem cells.

[0015] Beneficial effects 1. This invention improves the proliferative activity of mesenchymal stem cells and the secretory performance of HGF by simultaneously adding basic fibroblast growth factor and polymyxin B to the basal culture medium.

[0016] 2. By limiting the content of basic fibroblast growth factor in the mesenchymal stem cell culture medium to 5-50 ng / mL, more stem cells can be in an active proliferation and division process.

[0017] 3. By limiting the polymyxin B content in the mesenchymal stem cell culture medium to 50-100 μg / mL, the secretion performance of HGF can be further improved while maintaining cell proliferation activity.

[0018] 4. The mesenchymal stem cell culture medium prepared by this invention helps umbilical cord mesenchymal stem cell-related drug preparations to exert stronger tissue regeneration and repair functions.

[0019] 5. The umbilical cord mesenchymal stem cells cultured in the culture medium of this invention do not have a significantly affected multi-lineage differentiation potential of osteogenic, adipose, and cartilage cells, thus maintaining the core biological characteristics of mesenchymal stem cells. Attached Figure Description

[0020] Figure 1 The results of detecting the proliferation capacity of human umbilical cord mesenchymal stem cells (hUC-MSCs) using the culture media prepared in Examples 1-3 and Comparative Examples 1-4 are shown. A, B, and C represent the CCK8 assay results of P4, P5, and P6 generation umbilical cord mesenchymal stem cells, respectively; D, E, and F represent the PI staining results of P4, P5, and P6 generation umbilical cord mesenchymal stem cells, respectively; bFGF0 corresponds to the culture medium in Example 1, bFGF5 to the culture medium in Example 2, bFGF10 to the culture medium in Example 3, and bFGF20 to the culture medium in Example 1. The experimental results are the average values ​​obtained from three independent repeated assays. The error bar represents the standard deviation, and statistical differences were detected by two-way ANOVA. P <0.01; #: P<0.05, ##: P <0.01; &&: P <0.05, &&: P <0.01; ****: P <0.0001. G2 / M period: *: P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001; Period S: #: P <0.05, ##: P <0.01; G0 / G1 period: &: P <0.05, &&: P <0.01.

[0021] Figure 2 The results of detecting the proliferation activity, HGF secretion, and HGF gene expression of P6 generation hUC-MSCs in the culture media prepared in Examples 11 and 2-44-6 are shown. Figures A, B, C, and D correspond to typical images of P6 generation hUC-MSCs cultured in the culture media prepared in Examples 24, 1, 35, and 46, respectively, where bar (micrograph scale) = 100 μm; Figure E shows the CCK8 cell viability detection results of hUC-MSCs cultured in each group of culture media; Figure F shows the ELISA detection results of HGF concentration in the culture supernatant of hUC-MSCs cultured in each group of culture media; Figure G shows the qPCR detection results of HGF expression level of hUC-MSCs cultured in each group of culture media; the experimental results are the average values ​​of data obtained from four independent repeated experiments, with the error bar representing the standard deviation. Statistical differences were detected by one-way ANOVA. * P <0.05, **: P <0.01, ***: P <0.001, ****: P <0.0001.

[0022] Figure 3 The results of the multi-lineage differentiation test of hUC-MSCs cultured in the culture media prepared in Example 1 and Comparative Example 51 are shown. A and D are the results of Alizarin Red staining for osteogenic differentiation of P6 generation hUC-MSCs cultured in the culture media of Comparative Example 51 and Example 1, respectively (bar = 100 μm); B and E are the results of Oil Red-O staining for adipogenic differentiation of P6 generation hUC-MSCs cultured in the culture media of Comparative Example 51 and Example 1, respectively (bar = 200 μm); C and F are the results of Alixin Blue staining for chondrogenic differentiation of P6 generation hUC-MSCs cultured in the culture media of Comparative Example 51 and Example 1, respectively (bar = 1000 μm). Detailed Implementation

[0023] Example 1 A mesenchymal stem cell culture medium comprises: basal medium (DMEM / F-12 medium, sourced from Gibco, catalog number 12500-096), fetal bovine serum (sourced from ExCell, catalog number FND500), basic fibroblast growth factor (bFGF, sourced from PeproTech, catalog number AF-100-18B-1000), polymyxin B (PMB, sourced from Selleck, catalog number S1395), and deionized water (sterile deionized water); wherein the mesenchymal stem cell culture medium contains 10% fetal bovine serum by volume, 20 ng / mL basic fibroblast growth factor, and 100 μg / mL polymyxin B.

[0024] A method for preparing a mesenchymal stem cell culture medium comprises the following steps: 1. After centrifuging 1 mg of basic fibroblast growth factor lyophilized powder, use a micropipette to add 1 mL of sterile deionized water to the basic fibroblast growth factor lyophilized powder tube, mix by pipetting and let stand for 15 min to completely dissolve it, and obtain basic fibroblast growth factor solution. 2. Weigh 2.5g of bovine serum albumin and add it to a centrifuge tube. Add 50mL of sterile deionized water to the centrifuge tube to dissolve the bovine serum albumin and obtain a bovine serum albumin solution. 3. Using a micropipette, add 1 mL of basic fibroblast growth factor solution to 49 mL of bovine serum albumin solution. After filtration through a 0.22 μm filter membrane, dispense the solution into 1.5 mL centrifuge tubes and store at -80℃ for later use to obtain a diluted basic fibroblast growth factor solution. 4. Weigh 50 mg of polymyxin B into a 1.5 mL centrifuge tube, add 1 mL of sterile deionized water, mix well, and store at -80℃ for later use to obtain polymyxin B solution. 5. Add 450 mL of DMEM / F-12 medium and 50 mL of fetal bovine serum to a sterile bottle. Add 0.5 mL of basic fibroblast growth factor dilution and 1 mL of polymyxin B solution to the sterile bottle. Mix well to obtain mesenchymal stem cell culture medium, named PMB100.

[0025] Example 2 The specific implementation method is the same as in Example 1; the difference is that the content of the basic fibroblast growth factor is 5 ng / mL.

[0026] Example 3 The specific implementation method is the same as in Example 1; the difference is that the content of basic fibroblast growth factor in the mesenchymal stem cell culture medium is 10 ng / mL.

[0027] Comparative Example 1 bFGF0 group: conventional culture medium, specifically composed of DF-12 medium containing 10% (by volume) fetal bovine serum.

[0028] Comparative Example 2 bFGF5 group: standard culture medium + 5 ng / ml bFGF.

[0029] Comparative Example 23 bFGF10 group: standard culture medium + 10 ng / ml bFGF.

[0030] Comparative Example 34 bFGF20 group (PMB0): standard culture medium + 20 ng / ml bFGF.

[0031] Comparative Example 45 PMB200: Standard culture medium + 20 ng / ml bFGF + 200 μg / ml PMB Comparative Example 56 PMB300: Standard culture medium + 20 ng / ml bFGF + 300 μg / ml PMB.

[0032] Performance testing methods 1. The ability of hUC-MSCs (human umbilical cord mesenchymal stem cells) cultured in the culture media prepared in Example 1, Example 2, and Comparative Examples 2-44-6 to secrete HGF (hepatocyte growth factor) was detected by ELISA.

[0033] Prepare relevant reagents 1) Remove the hUC-MSCs culture supernatant and the kit (Xinbosheng EHC138) from the refrigerator 20 minutes in advance to allow them to equilibrate to room temperature; 2) Dilute the 20× concentrated washing solution with ultrapure water to make a 1× working solution. Store any unused portion at 4℃. 3) Preparation of Standards: Centrifuge the standard (Human HGF Elisa Kit, manufacturer: Xinbosheng, catalog number: EHC138) at 6000 rpm for 5 seconds at room temperature. Add 1 mL of the standard and universal sample diluent, let stand for 15 minutes until fully dissolved, then gently mix to prepare an HGF standard with a concentration of 8000 pg / mL. Then, perform serial dilutions to prepare six different concentrations of HGF standards: 4000 pg / mL, 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, and 125 pg / mL. 4) 20 minutes before use, dilute the 30× concentrated biotinylated antibody to 1× working solution using biotinylated antibody dilution buffer; 5) 20 minutes before use, dilute the 30× concentrated enzyme conjugate to 1× working solution using enzyme conjugate diluent.

[0034] ELISA testing 1) Add 100 μL of standard and the sample cultured in each group of culture medium to the corresponding wells. Add 100 μL of standard and universal sample diluent to the blank wells. Set up 2 and 3 parallel wells for each standard and each group of sample, respectively, for detection. Seal the reaction wells with sealing tape and incubate at 37℃ for 90 min; 2) Prepare the biotinylated antibody working solution 20 minutes before use; 3) Wash the plates using an automatic plate washer. Add 350 μL of washing solution to each well, with a 60-second interval between injection and aspiration. Repeat the washing process 5 times. 4) Add 100 μL of biotinylated antibody dilution solution to the blank wells, and add 100 μL of biotinylated antibody working solution to each of the remaining wells. Incubate at 37°C for 60 min. 5) Prepare the enzyme conjugate working solution 20 minutes before use; 6) Repeat step 3). 7) Add 100 μL of enzyme conjugate dilution to the blank wells, and 100 μL of enzyme conjugate working solution to each of the remaining wells. Incubate at 37°C for 30 min. 8) Turn on the microplate reader to preheat and set the detection program; 9) Repeat step 3). 10) Add 100 μL of chromogenic substrate (TMB) to each well and incubate at 37°C in the dark for 15 min; 11) Add 100 μL of stop solution to each well, mix well, and immediately test the OD450 value.

[0035] 2. The CCK8 assay was used to detect the in vitro proliferation capacity of hUC-MSCs cultured in different culture media.

[0036] After hUC-MSCs cells reached 80%–90% confluence (P6 generation), the cells cultured in the media prepared in Examples 1-3 and Comparative Examples 1-4 were digested and seeded into 96-well plates for cell proliferation assay. The main steps are as follows: (1) Adjust the concentration of the cells to be tested to 5×10⁻⁶. 4 Cells / mL, then seeded into 96-well plates, 100 μL per well.

[0037] (2) Add 10 μL of CCk8 (Tongren Chemical CK04) solution to each well at 4h, 24h, 48h, 72h and 96h after inoculation.

[0038] (3) After incubating in an incubator for 2 hours, the absorbance (OD450 value) at 450 nm was measured using an ELISA reader.

[0039] 3. Cell cycle detection.

[0040] (1) Resuspend the cells cultured in the previous step with D-hanks buffer and adjust the concentration to 1×10⁻⁶. 6 Cells / mL.

[0041] (2) Add 3 mL of pre-cooled anhydrous ethanol to 1 mL of cell suspension, mix well, and fix at 2-8℃ for 3 h.

[0042] (3) After centrifuging at 1000 rpm for 5 min, discard the supernatant, take 5 mL of D-hanks buffer to resuspend the cells, gently pipette to mix, and then centrifuge at 1000 rpm for 5 min.

[0043] (4) Discard the supernatant, add 0.5 mL of PI / RNase staining solution (Thermo, F10797) to the centrifuge tube, gently pipette to mix, and incubate at room temperature in the dark for 30 min.

[0044] (5) After incubation, gently mix the cell suspension and perform flow cytometry analysis.

[0045] 4. HGF gene expression level detection RNA extraction (kit: Thermo, K0731): (1) Collect 0.5~1×10 from the previous step 6 Transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 6000 rpm for 5 min, and then discard the supernatant.

[0046] (2) Add 600 μL of lysis buffer to each centrifuge tube and shake for 10 seconds to mix thoroughly.

[0047] (3) Add 360 μL of anhydrous ethanol to each 1.5 mL centrifuge tube and mix well by pipetting with a micropipette.

[0048] (4) Take 700 μL of the lysis buffer from the 1.5 mL centrifuge tube and add it to the RNA purification column. Centrifuge at 12000 g for 1 min. (5) Discard the liquid in the collection tube, take the remaining lysis buffer and add it to the RNA purification column, and centrifuge at 12000g for 1min.

[0049] (6) Discard the liquid in the collection tube, and draw 700 μL of Wash Buffer 1 into the RNA purification column. Heat at 12000g for 1 min.

[0050] (7) Discard the liquid in the collection tube, and aspirate 600 μL of Wash Buffer 2 into the RNA purification column. Centrifuge at 12000g for 1 min.

[0051] (8) Discard the liquid in the collection tube, and take 250 μL of Wash Buffer 2 into the RNA purification column. Centrifuge at 12000g for 2 min.

[0052] (9) Discard the collection tube and replace it with a 1.5mL RNase-free centrifuge tube. Add 50μL Nuclease-free water to the RNA purification column and centrifuge at 12000g for 1min.

[0053] (10) Discard the RNA purification column and take 2 μL to detect the RNA concentration.

[0054] Reverse transcription of RNA (kit: TOYOBO, FSQ-201): (1) Based on the measured value of RNA concentration, determine the volume of 2 μg RNA and add the reagent to the PCR reaction tube according to the following ratio.

[0055] The RNA reverse transcription system (40 μL) consists of: 5X reverse transcription buffer mixed with RNA and ddH2O. 8μL + 2μg to make up to 40μL (2) Place the PCR reaction tubes into the PCR instrument and set the following program: 37℃: 15min → 50℃: 5min → 98℃: 5min; (3) After PCR amplification, the obtained cDNA is stored at -20℃ for later use.

[0056] Real-time PCR (Thermo, A25742) reaction: (1) Add each component to the qPCR system as shown in Table 1.

[0057] Table 1

[0058] (2) Place the qPCR reaction tube in the ABI7500-Fast PCR instrument and set the program as shown in the figure below: heat up to 95℃ and hold for 20s → heat up to 95℃ and hold for 3s, cool down to 60℃ and hold for 30s, heat up to 95℃ and repeat this step 45 times → heat up to 95℃ and hold for 15s, cool down to 60℃ and hold for 60s → heat up to 95℃ and hold for 15s → cool down to 60℃ and hold for 15s.

[0059] (3) Record and save the data after the reaction is complete, and then turn off the machine.

[0060] Performance test results like Figure 1 China A Figure 1 China B and Figure 1 As shown in Figure C, the CCK8 cell proliferation assay results indicated that the addition of 5–20 ng / mL bFGF during the P4 to P6 generation umbilical cord mesenchymal stem cell culture promoted their proliferation to varying degrees, exhibiting a certain dose-dependent effect. Compared with the culture system without bFGF, the addition of 20 ng / mL bFGF significantly and sustainably enhanced the proliferative activity of umbilical cord mesenchymal stem cells during the P4 to P6 generation expansion process. P <0.05). For example... Figure 1 D, Figure 1 China E and Figure 1 As shown in Figure F, cell cycle analysis results also indicate that the addition of 20 ng / mL bFGF significantly increased the number of mesenchymal stem cells in the G2 / M phase. P <0.01), while the proportion of cells in the G0 / G1 phase was significantly reduced ( P <0.0001) indicates that more cells are in the process of active proliferation and division.

[0061] Different concentrations of PMB were added during the culture of P6 generation umbilical cord mesenchymal stem cells, and cell morphology, cell viability, and the level of HGF secreted by cells were examined after 48 hours of culture. Figure 2 China A Figure 2 B, Figure 2 C, Figure 2As shown in Figure D, in the culture system without PMB, the hUC-MSCs cells in the control group were tightly packed in a spindle shape with few suspended cells. The hUC-MSCs culture system with 100 μg / mL PMB showed no significant difference in cell morphology compared to the control group. The hUC-MSCs culture system with 200 μg / mL PMB showed significantly increased intercellular spacing and more suspended cells. The hUC-MSCs culture system with 300 μg / mL PMB showed further increased intercellular spacing and even more suspended cells. Consistent with this, as... Figure 2 As shown in Figure E, the CCK8 cell viability assay results also indicated that the cell viability of hUC-MSCs cultured with 200 μg / mL and 300 μg / mL PMB was significantly lower than that of the control group. P <0.05, P <0.001). The above results indicate that the proliferation activity of hUC-MSCs was inhibited to some extent with the increase of PMB addition in the culture system.

[0062] like Figure 2 As shown in Figure F, ELISA results of HGF concentration in the culture supernatant indicated that the addition of PMB significantly promoted HGF secretion by hUC-MSCs and exhibited a clear dose-dependent effect. Compared to the HGF concentration in the culture supernatant without PMB (9488.2 ± 1126.2 pg / mL), the addition of 100 μg / mL, 200 μg / mL, and 300 μg / mL PMB increased the HGF secretion by hUC-MSCs by 1.9 times (…). P <0.01), 2.2 times ( P <0.001) and 2.7 times ( P <0.0001). Meanwhile, such as Figure 2 As shown in Figure G, the qPCR results also indicate that adding PMB to the culture system can enhance the HGF expression level of hUC-MSCs, and this level increases with increasing PMB concentration. In summary, these results suggest that adding PMB can enhance the HGF secretion function of hUC-MSCs, but excessively high concentrations of PMB inhibit cell proliferation.

[0063] hUC-MSCs obtained using the culture media prepared in Example 1 and Comparative Example 51 were subjected to cell surface marker and multi-lineage differentiation potential detection. Table 2 shows the expression of hUC-MSCs surface markers (mean ± SD, n=3) after optimization of the culture system. (See Table 2 and...) Figure 3As shown, compared with hUC-MSCs obtained from the culture medium of Comparative Example 51, the culture system optimization (Example 1) did not significantly alter the surface markers and multi-lineage differentiation potential of mesenchymal stem cells. The hUC-MSCs cultured from the optimized culture system still met the identification criteria for mesenchymal stem cells.

[0064] Table 2

Claims

1. A mesenchymal stem cell culture medium, characterized in that, It includes basal culture medium, fetal bovine serum, basic fibroblast growth factor, polymyxin B, and deionized water.

2. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The volume content of fetal bovine serum in the mesenchymal stem cell culture medium is 6-15%.

3. The mesenchymal stem cell culture medium according to claim 2, characterized in that, The volume content of fetal bovine serum in the mesenchymal stem cell culture medium is 8-12%.

4. The mesenchymal stem cell culture medium according to claim 1 or 3, characterized in that, The content of basic fibroblast growth factor in the mesenchymal stem cell culture medium is 5-50 ng / mL.

5. The mesenchymal stem cell culture medium according to claim 4, characterized in that, The content of basic fibroblast growth factor in the mesenchymal stem cell culture medium is 5-20 ng / mL.

6. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The polymyxin B content in the mesenchymal stem cell culture medium is 20-150 μg / mL.

7. The mesenchymal stem cell culture medium according to claim 6, characterized in that, The polymyxin B content in the mesenchymal stem cell culture medium is 50-100 μg / mL.

8. The mesenchymal stem cell culture medium according to claim 1, characterized in that, The basal culture medium includes at least one of DMEM / F-12 medium, α-MEM medium, high-glucose DMEM medium, Ham's F-10 medium, and RPMI-1640 medium.

9. A method for preparing a mesenchymal stem cell culture medium according to any one of claims 1-8, characterized in that, Includes the following steps: Basic fibroblast growth factor and polymyxin B were dissolved in deionized water to obtain basic fibroblast growth factor solution and polymyxin B solution, respectively. Fetal bovine serum, basic fibroblast growth factor solution and polymyxin B solution were added to the basal culture medium and mixed evenly to obtain mesenchymal stem cell culture medium.

10. The application of a mesenchymal stem cell culture medium according to any one of claims 1-8, characterized in that, Used for in vitro expansion and culture of mesenchymal stem cells.

Images