Mesenchymal stem cell culture medium and method of use thereof
By combining aescin IB pretreatment with low-dose dexamethasone osteogenic induction medium, the cytotoxicity problem caused by high concentrations of dexamethasone was solved, achieving safe and efficient osteogenic differentiation of mesenchymal stem cells and improving bone matrix mineralization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SINUO BIOTECHNOLOGY (SHENZHEN) CO LTD
- Filing Date
- 2026-03-10
- Publication Date
- 2026-07-03
AI Technical Summary
In the existing technology, dexamethasone, as an osteogenic inducer, is cytotoxic to mesenchymal stem cells when used at high concentrations or for a long period of time, leading to a decline in the quality of bone matrix mineralization in the later stages of differentiation, making it difficult to achieve safe and efficient osteogenic differentiation.
By using aescin IB pretreatment medium combined with low-dose dexamethasone and other osteogenic induction medium, the osteogenic differentiation potential of mesenchymal stem cells was significantly enhanced through the pretreatment stage, and the expression of core osteogenic genes RUNX2 and OCN was activated.
At a halved dexamethasone concentration, the osteogenic differentiation capacity of mesenchymal stem cells was significantly improved, cytotoxicity issues were avoided, a safer and more stable cell preparation strategy was provided, and the level of bone matrix mineralization was enhanced.
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Figure CN122326518A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of stem cell culture technology, and particularly relates to a mesenchymal stem cell culture medium and its application method. Background Technology
[0002] Mesenchymal stem cells (MSCs), especially human bone marrow-derived MSCs (hBMSCs), have attracted significant attention in regenerative medicine and tissue engineering due to their unique biological characteristics. These cells possess multipotent differentiation potential, capable of transforming into various cell types such as osteoblasts, chondrocytes, and adipocytes. Furthermore, they are easily expanded on a large scale in vitro and can suppress inflammatory responses and promote tissue repair. These advantages make MSCs a promising candidate for clinical applications in bone tissue engineering, bone defect repair, fracture healing, osteoporosis treatment, and other bone-related diseases.
[0003] Achieving efficient and targeted differentiation of MSCs into osteoblasts is a core prerequisite for the success of these clinical applications. However, current conventional osteogenic induction systems heavily rely on glucocorticoids, particularly dexamethasone (Dex). Dexamethasone, a synthetic glucocorticoid, is widely considered a key regulator of osteogenic gene transcription, promoting MSC differentiation towards osteogenic outcomes by regulating the expression of transcription factors such as SOX9 and PPARG. While dexamethasone acts as a central switch for initiating osteogenic gene transcription, long-term or high-concentration use has significant negative effects on stem cells. Studies have shown that dexamethasone exhibits significant cytotoxicity, leading to senescent phenotypes and significantly reducing bone matrix mineralization quality in late differentiation stages. Therefore, achieving efficient, stable, and safe osteogenic differentiation of MSCs while reducing dexamethasone usage, and providing a better cell preparation protocol for bone tissue engineering and regenerative medicine, is a current challenge. Summary of the Invention
[0004] The purpose of this invention is to provide a mesenchymal stem cell culture medium and its application method, thereby achieving efficient and safe osteogenic differentiation of mesenchymal stem cells.
[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a pretreatment culture medium for promoting osteogenic differentiation of mesenchymal stem cells, characterized in that the pretreatment culture medium contains aescin IB, wherein the CAS number of aescin IB is 26339-90-2.
[0006] Preferably, the pretreatment culture medium further comprises: DMEM / F12 basal culture medium, 2%-10% fetal bovine serum, and 1% penicillin / streptomycin.
[0007] Preferably, the concentration of aescin IB is 50-100 μmol / L.
[0008] Preferably, the pretreatment culture medium is used to improve the osteogenic differentiation capacity of mesenchymal stem cells at a low dose of dexamethasone concentration; The mesenchymal stem cells mentioned are human bone marrow mesenchymal stem cells.
[0009] In a second aspect, the present invention provides a culture medium for promoting the osteogenic differentiation ability of stem cells, the culture medium being composed of the above-mentioned pretreatment culture medium and a low-dose osteogenic induction culture medium; The low-dose osteogenic induction medium consists of sodium β-glycerophosphate, ascorbic acid, dexamethasone, and DMEM medium.
[0010] Preferably, in the low-dose osteogenic induction culture medium, the concentration of sodium β-glycerophosphate is 10 mM, the concentration of ascorbic acid is 50 μg / ml, and the concentration of dexamethasone is 50 nM.
[0011] Preferably, the stem cells are human bone marrow mesenchymal stem cells.
[0012] Thirdly, the present invention provides a method for promoting the differentiation of mesenchymal stem cells into osteoblasts, the method comprising the following steps: a) Seed mesenchymal stem cells into a culture vessel and culture them in basal culture medium until they reach 60%-80% confluence; b) Pretreatment of the mesenchymal stem cells using the above-described pretreatment culture medium; c) After pretreatment, discard the pretreatment medium, wash the cells, and add a low dose of osteogenic induction medium for osteogenic induction culture.
[0013] Preferably, the pretreatment time is 24 hours; The mesenchymal stem cells mentioned are human bone marrow mesenchymal stem cells.
[0014] Preferably, the low-dose osteogenic induction medium consists of 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, 50 nM dexamethasone, and DMEM medium.
[0015] The beneficial effects of this invention are as follows: This invention introduces a pretreatment step containing aescin IB before formal osteogenic induction, which significantly enhances the osteogenic differentiation potential of mesenchymal stem cells. Experimental data show that this method can efficiently activate the expression of core osteogenic genes RUNX2 and OCN, and its enhanced ALP activity and bone matrix mineralization level are significantly better than the traditional high-dose dexamethasone induction system.
[0016] More importantly, this invention achieves better osteogenic effects even with a halved dexamethasone concentration through the synergistic effect of pretreatment, effectively avoiding the cytotoxicity and aging phenotype problems caused by long-term high-concentration use of glucocorticoids.
[0017] Furthermore, this invention clarifies that aescin IB has a significant time window dependence and unexpected technical effects. Intervention during the pretreatment stage can maximize the activation of osteogenic gene networks, providing a safer, more efficient, and stable cell preparation strategy for bone tissue engineering and regenerative medicine, with extremely high clinical application value. Attached Figure Description
[0018] Figure 1 ALP staining results of human bone marrow mesenchymal stem cells 7 days after induction by different treatments; Figure 2 Alizarin Red S staining results of human bone marrow mesenchymal stem cells induced 14 days after different treatments; Figure 3 This is a graph showing the quantitative analysis results of the relative cell mineralization level of human bone marrow mesenchymal stem cells 14 days after induction by different treatment methods. Figure 4 The relative expression levels of RUNX2, the core osteogenic gene in human bone marrow mesenchymal stem cells, after different treatments. Figure 5 The relative expression levels of the core osteogenic gene OCN in human bone marrow mesenchymal stem cells after different treatments. Detailed Implementation
[0019] Cells and drugs used in this invention: Human bone marrow mesenchymal stem cells were purchased from Shanghai Yubo Biotechnology Co., Ltd. They were cultured in DMEM / F12 containing 10% FBS, 2 mL glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2 until the third generation.
[0020] Aesculin IB is a saponin isolated from the seed coat and endosperm of Aesculus hippocastanum, with CAS number 26339-90-2. Its chemical structure is as follows:
[0021] Existing research mainly focuses on its anti-inflammatory and antioxidant functions, and there are no reports on its use in stem cell osteogenic processes.
[0022] Common osteogenic induction medium: DMEM / F12 medium, 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, 100 nM dexamethasone.
[0023] Example 1 (1) Third-generation human bone marrow-derived mesenchymal stem cells were seeded in 6-well plates at a density of 5×10^4 cells / well, and 2 mL of basal culture medium (DMEM / F12 + 10% FBS + 1% penicillin / streptomycin) was added to each well. (2) The cells were placed in a saturated humidity incubator at 37°C and 5% CO2 and cultured statically until they were completely adhered to the wall. (3) After the cells adhere to the wall and grow to about 60-70% confluence, gently remove the original culture medium and wash the cells twice with sterile PBS to remove residual serum and metabolic waste. (4) Add pretreatment medium: 50 μmol / L aescin IB, 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, and pretreatment culture for 24 hours; (5) After the pretreatment is completed, remove the pretreatment medium and add low-dose osteogenic induction medium: 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 50 nM dexamethasone. Continue osteogenic induction culture for 14 days, and change the low-dose osteogenic induction medium every 3 days.
[0024] Example 2 (1) Third-generation human bone marrow-derived mesenchymal stem cells were seeded in 6-well plates at a density of 5×10^4 cells / well, and 2 mL of basal culture medium (DMEM / F12 + 10% FBS + 1% penicillin / streptomycin) was added to each well. (2) The cells were placed in a saturated humidity incubator at 37°C and 5% CO2 and cultured statically until they were completely adhered to the wall. (3) After the cells adhere to the wall and grow to about 60-70% confluence, gently remove the original culture medium and wash the cells twice with sterile PBS to remove residual serum and metabolic waste. (4) Add pretreatment medium: 100 μmol / L aescin IB, 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, and pretreatment culture for 24 hours; (5) After the pretreatment is completed, remove the pretreatment medium and add low-dose osteogenic induction medium: 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 50 nM dexamethasone. Continue osteogenic induction culture for 7 days and 14 days, and change the low-dose osteogenic induction medium every 3 days.
[0025] Comparative Example 1 (1) Third-generation human bone marrow-derived mesenchymal stem cells were seeded in 6-well plates at a density of 5×10^4 cells / well, and 2 mL of basal culture medium (DMEM / F12 + 10% FBS + 1% penicillin / streptomycin) was added to each well. (2) The cells were placed in a saturated humidity incubator at 37°C and 5% CO2 and cultured statically until they were completely adhered to the wall. (3) After the cells adhere to the wall and grow to about 60-70% confluence, gently remove the original culture medium and wash the cells twice with sterile PBS to remove residual serum and metabolic waste. (4) Add pretreatment medium: 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, pretreatment culture for 24 hours; (5) After the pretreatment is completed, remove the pretreatment medium and add low-dose osteogenic induction medium: 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 50 nM dexamethasone. Continue osteogenic induction culture for 7 days and 14 days, and change the low-dose osteogenic induction medium every 3 days.
[0026] Comparative Example 2 (1) Third-generation human bone marrow-derived mesenchymal stem cells were seeded in 6-well plates at a density of 5×10^4 cells / well, and 2 mL of basal culture medium (DMEM / F12 + 10% FBS + 1% penicillin / streptomycin) was added to each well. (2) The cells were placed in a saturated humidity incubator at 37°C and 5% CO2 and cultured statically until they were completely adhered to the wall. (3) After the cells adhere to the wall and grow to about 60-70% confluence, gently remove the original culture medium and wash the cells twice with sterile PBS to remove residual serum and metabolic waste. (4) Add pretreatment medium: 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, pretreatment culture for 24 hours; (5) After the pretreatment is completed, remove the pretreatment medium and add low-dose osteogenic induction medium: 10 mM β-glycerophosphate sodium, 50 μg / ml ascorbic acid, 100 nM dexamethasone. Continue osteogenic induction culture for 7 days and 14 days, and change the low-dose osteogenic induction medium every 3 days.
[0027] Example 3 (1) Remove the cells from the incubator after osteogenic induction culture for 7 days (Examples 1, 2, Comparative Example 1 and Comparative Example 2) and gently aspirate the culture medium. (2) Wash the cells twice with pre-cooled PBS, add 4% PFA fixative, and fix at room temperature for 20 minutes; (3) After fixation, wash the cells three times with PBS for 5 minutes each time to remove residual fixative; (4) Dissolve the BCIP / NBT substrate in staining buffer according to the kit instructions, add 1 mL of working solution to each well, and incubate in a dark chamber at 37°C for 50 minutes; (5) Remove the staining solution, wash three times with PBS for 5 minutes each time, and observe and photograph under a microscope.
[0028] The results show that, compared with control group 2, the ALP staining intensity was significantly reduced, indicating that half the dose of dexamethasone significantly reduces the osteogenic capacity of bone marrow mesenchymal stem cells.
[0029] The staining intensity of Example 1 was significantly better than that of Comparative Example 1, but slightly worse than that of Comparative Example 2, while that of Example 2 was significantly better than that of Comparative Example 2. This indicates that bone marrow mesenchymal stem cells pretreated with a culture medium containing aescin IB exhibit a significantly enhanced osteogenic differentiation capacity when cultured in osteogenic differentiation medium, and this enhancement is concentration-dependent.
[0030] Example 4 (1) Remove the cells from the incubator after 14 days of osteogenic induction culture of Examples 1, 2, Comparative Example 1 and 2, and gently aspirate the culture medium; (2) Wash the cells twice with pre-cooled PBS, add 4% PFA fixative, and fix at room temperature for 20 minutes; (3) After fixation, wash the cells three times with PBS for 5 minutes each time to remove residual fixative; (4) Add 1 mL of freshly prepared 1% Alizarin Red S staining solution to each well and incubate in a dark chamber at 37°C for 30 minutes; (5) Remove the staining solution, wash three times with PBS for 5 minutes each time, observe and photograph under a microscope, and obtain the results as follows. Figure 2 As shown; (6) After removing the PBS, add 1 mL of 10% hexadecyl chloride pyridine extraction solution to each well and incubate at room temperature on a shaker with slow shaking for 1 hour; (7) After incubation, gently blow and mix, then transfer the extract to a new 1.5 mL centrifuge tube and centrifuge at 12,000 g for 5 minutes to remove residue; (8) Transfer 200 μL of supernatant per well to a 96-well plate and measure the absorbance at 562 nm using a microplate reader. Using control group 2 as a baseline, calculate the relative cell mineralization level. The results are as follows: Figure 3 As shown.
[0031] from Figure 2 and Figure 3The results show that, in terms of cell mineralization, compared with control group 2, the cell mineralization level of control group 1 after culture with low-dose osteogenic induction medium was significantly reduced, while the cell mineralization level of Examples 1 and 2 after treatment with pretreatment medium was significantly improved. Among them, the effect of Example 2 was significantly better than that of control group 2, which further proves that pretreatment with pretreatment medium containing aescin IB can effectively improve the osteogenic capacity of bone marrow mesenchymal stem cells.
[0032] Example 5 (1) To further study the main mechanism of the present invention, the present invention selected the following groups to treat cells and detected the expression of core osteogenic genes in cells cultured under different groups: Control group: After the third-generation human bone marrow-derived mesenchymal stem cells adhered and grew to about 60-70% confluence, they were added to the pretreatment medium: 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, and pretreated for 24 hours. After pretreatment, the pretreatment medium was removed, and a low dose of osteogenic induction medium was added. The culture was continued for 7 days.
[0033] Experimental Group 1: After third-generation human bone marrow-derived mesenchymal stem cells adhered and grew to about 60-70% confluence, they were added to a pretreatment medium: 100 μmol / L aescin IB, 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, and pretreated for 24 hours. After pretreatment, the pretreatment medium was removed and a low dose of osteogenic induction medium was added.
[0034] Experimental Group 2: After third-generation human bone marrow-derived mesenchymal stem cells adhered and grew to about 60-70% confluence, they were added to a pretreatment medium: 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium, and pretreated for 24 hours. After pretreatment, the pretreatment medium was removed, and a low-dose osteogenic induction medium containing 100 μmol / L aescin IB was added for treatment for 1 day. After washing with PBS, the medium was replaced with a new low-dose osteogenic induction medium and cultured for another 6 days.
[0035] Experimental Group 3: After third-generation human bone marrow-derived mesenchymal stem cells adhered to the culture vessel and grew to about 60-70% confluence, low-dose osteogenic induction medium was added, and the cells were cultured for another 7 days. After the culture was completed, the culture was continued for 1 day using the pretreatment medium: 100 μmol / L aescin IB, 2% FBS, 1% penicillin / streptomycin, DMEM / F12 medium. (2) Remove the cell plate from the incubator, remove the culture medium, wash the cells twice with ice-cold PBS, add 1 mL of TRIzol reagent to each well, and let stand at room temperature for 5 minutes; (3) Repeatedly pipette the lysed cells, transfer the lysate to a 1.5 mL RNase-free centrifuge tube, add 0.2 mL chloroform, vortex vigorously for 15 seconds, and let stand at room temperature for 3 minutes; (4) Centrifuge at 12,000 g for 15 minutes at 4°C, transfer the upper aqueous phase to a new tube, add an equal volume of isopropanol, mix well, and precipitate at -20°C for 30 minutes. (5) Centrifuge at 4℃ and 12,000 g for 10 minutes, discard the supernatant and wash the precipitate twice with 75% ethanol, centrifuging at 4℃ and 7,500 g for 5 minutes each time; (6) Discard the ethanol, air dry for about 5 minutes, and add 30 μL of RNase-free water to resuspend the RNA; (7) After determining the RNA concentration, purity and integrity, the RNA was reverse transcribed into cDNA using a reverse transcription kit; (8) Set up the reaction in a 96-well qPCR plate (3 technical replicates per sample, including template-free control NTC and reverse transcription-free control NRT): Each well contains: SYBR Green Master Mix (10 μL 2×), forward / reverse primer mixture (1 μL, final concentration 0.2 μM), cDNA template (2 μL, 10 ng), and RNase-free water to bring the total volume to 20 μL. The primer sequences designed in this invention are as follows: RUNX2 forward primer: 5'-AACCCACGAATGCACTATCCA-3'; RUNX2 reverse primer: 5'-CGGACATACCGAGGGACATG-3'; OCN forward primer: 5'-TGCATAGGGTTCTTGTCTCT-3'; OCN reverse primer: 5'-CTCCACCACTCCTACTGTGT-3'; GAPDH forward primer: 5'-GAAGGTGAAGGTCGGAGTC-3'; GAPDH reverse primer: 5'-GAAGATGGTGATGGGATTTC-3'; (9) Perform PCR testing according to the following procedure: 95℃ for 10 minutes (initial denaturation); Cycling: 95℃ for 15 seconds (denaturation), 60℃ for 60 seconds (annealing / extension), 40 cycles; Melting curve: 95℃ for 15 seconds, 60℃ for 60 seconds, 95℃ for 15 seconds (in 0.5°C increments); (10) Using GAPDH as a baseline, the relative expression levels of RUNX2 and OCN were calculated, and the results are as follows: Figure 4 and Figure 5 As shown.
[0036] exist Figure 4 In the study, the relative expression level of RUNX2 in experimental group 1 was 2.711±0.150, the relative expression level of RUNX2 in experimental group 2 was 1.463±0.114, and the relative expression level of RUNX2 in experimental group 3 was 1.088±0.061.
[0037] exist Figure 5 In the study, the relative expression level of OCN in experimental group 1 was 3.163±0.195, the relative expression level of OCN in experimental group 2 was 1.566±0.101, and the relative expression level of OCN in experimental group 3 was 1.058±0.057.
[0038] from Figure 4 and Figure 5 The results showed that, compared with the control group, the relative expression levels of RUNX2 and OCN in experimental group 3 did not increase significantly, indicating that after osteogenic induction culture, the treatment of bone marrow mesenchymal stem cells with a pretreatment medium containing aescin IB did not significantly enhance the osteogenic capacity of the cells.
[0039] The relative expression levels of RUNX2 and OCN in experimental groups 1 and 2 were higher than those in the control group, indicating that both treatments in groups 1 and 2 could promote the expression of the core osteogenic genes RUNX2 and OCN in bone marrow mesenchymal stem cells. Further observation revealed that the relative expression levels of RUNX2 and OCN in experimental group 1 were significantly higher than those in group 2, with the overall expression level nearly doubled. This indicates that the use of aescin IB in the pretreatment stage resulted in the strongest activation effect on the core osteogenic genes, and its effect exhibited a clear time-window dependence. Therefore, the method of this invention for inducing bone marrow mesenchymal stem cells can efficiently activate core osteogenic genes, providing a safer and more stable cell preparation strategy for bone tissue engineering and regenerative medicine.
Claims
1. A pre-treatment medium for promoting osteogenic differentiation of mesenchymal stem cells, characterized in that, The pretreatment culture medium contains aescin IB, and the CAS number of aescin IB is 26339-90-2.
2. The pretreatment culture medium according to claim 1, characterized in that, The pretreatment culture medium further comprises: DMEM / F12 basal medium, 2%-10% fetal bovine serum, and 1% penicillin / streptomycin.
3. The pretreatment culture medium according to claim 2, characterized in that, The concentration of aescin IB is 50-100 μmol / L.
4. The pretreatment culture medium according to claim 3, characterized in that, The pretreatment culture medium is used to improve the osteogenic differentiation capacity of mesenchymal stem cells at low-dose dexamethasone concentrations. The mesenchymal stem cells mentioned are human bone marrow mesenchymal stem cells.
5. A culture medium that promotes the osteogenic differentiation ability of stem cells, characterized in that, The culture medium is composed of the pretreatment culture medium according to any one of claims 1-4 and the low-dose osteogenic induction culture medium; The low-dose osteogenic induction medium consists of sodium β-glycerophosphate, ascorbic acid, dexamethasone, and DMEM medium.
6. The culture medium according to claim 5, characterized in that, In the low-dose osteogenic induction medium, the concentration of sodium β-glycerophosphate is 10 mM, the concentration of ascorbic acid is 50 μg / ml, and the concentration of dexamethasone is 50 nM.
7. The culture medium according to claim 6, characterized in that, The stem cells mentioned are human bone marrow mesenchymal stem cells.
8. A method for promoting the differentiation of mesenchymal stem cells into osteoblasts, characterized in that, The method includes the following steps: a) Seed mesenchymal stem cells into a culture vessel and culture them in basal culture medium until they reach 60%-80% confluence; b) Pretreatment of the mesenchymal stem cells using the pretreatment culture medium according to any one of claims 1-4; c) After pretreatment, discard the pretreatment medium, wash the cells, and add a low dose of osteogenic induction medium for osteogenic induction culture.
9. The method according to claim 8, characterized in that, The preprocessing time is 24 hours; The mesenchymal stem cells mentioned are human bone marrow mesenchymal stem cells.
10. The method according to claim 9, characterized in that, The low-dose osteogenic induction medium consisted of 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, 50 nM dexamethasone, and DMEM medium.