Serum-free culture medium for hypoxia culture of mesenchymal stem cells as well as preparation method and application of serum-free culture medium
By adding ginseng saponin Rg1, Centella asiaticin and other components to serum-free culture medium, the problem of proliferation and survival of mesenchymal stem cells under low oxygen conditions was solved, and efficient and stable cell proliferation and anti-apoptotic effects were achieved.
Patent Information
- Application Number
- CN202510457105.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-13
- Publication Date
- 2025-05-13
AI Technical Summary
Traditional serum-free culture media is difficult to effectively reduce the apoptosis rate of mesenchymal stem cells under low oxygen conditions and is difficult to promote their proliferation.
The cell cycle, antioxidant enzyme system and energy metabolism are regulated by adding ginseng saponin Rg1, Centella asiaticin, sodium pyruvate, recombinant human albumin and recombinant transferrin to the culture medium, providing stable nutrition and functional support.
It significantly improves the proliferation rate of mesenchymal stem cells in a low oxygen environment, reduces the apoptosis rate, maintains the stemness and differentiation ability of the cells, and achieves the efficiency and stability of the culture medium.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell culture, and in particular to a serum-free culture medium for low-oxygen culture of mesenchymal stem cells, and a preparation method and application thereof. Background Art
[0002] Mesenchymal stem cells are a type of extremely valuable pluripotent stem cells with the ability to differentiate in multiple directions. They can differentiate into various cell types under specific induction conditions, such as osteoblasts, adipocytes, chondrocytes, etc. They have shown great application potential in the field of regenerative medicine and have become the focus of research in areas such as tissue repair and regeneration, and disease treatment.
[0003] In recent years, studies have found that mesenchymal stem cells can survive in low oxygen (such as 2% O 2 ) environment, the survival, proliferation and differentiation potential of cells are improved. This discovery has opened up a new path for the large-scale culture of mesenchymal stem cells. Traditional cell culture mostly uses culture medium containing animal serum. Relatively speaking, serum-free culture medium has clear ingredients, stable properties and controllable risks. The use of serum-free culture medium is of great significance for the research and application of mesenchymal stem cells.
[0004] However, in practical applications, traditional serum-free culture media have obvious defects under hypoxic conditions. Hypoxic environment will disrupt the normal metabolic balance in cells, causing a significant increase in the generation of reactive oxygen species (ROS) in cells. Mesenchymal stem cells cultured in ordinary serum-free culture media are difficult to promptly and effectively remove excessive ROS. Excessive ROS attack various biological macromolecules in cells, easily causing damage to cell membranes, proteins and DNA chain structures, triggering oxidative stress responses in cells, activating a series of complex signaling pathways, and inducing cell apoptosis, ultimately leading to a significant increase in the apoptosis rate of cultured mesenchymal stem cells.
[0005] Therefore, developing a culture medium that can adapt to hypoxic environments, effectively reduce cell apoptosis rates and promote the proliferation of mesenchymal stem cells is a key issue that needs to be urgently addressed in this field and has extremely important practical significance. Summary of the invention
[0006] In order to solve the problems existing in the prior art, the present invention provides a serum-free culture medium for culturing mesenchymal stem cells under hypoxia, and a preparation method and application thereof. The serum-free culture medium of the present invention includes a basal culture medium, which contains nutritional factors required for cell growth, does not contain serum components, and can provide an excellent growth and proliferation environment for mesenchymal stem cells under a hypoxic environment. By adding ginsenoside Rg1 and asiaticoside, the expression and activity of cell cycle-related proteins can be regulated, cell division can be accelerated, and the proliferation rate of mesenchymal stem cells can be increased. The generation of ROS can also be inhibited through a variety of mechanisms, the activity of the intracellular antioxidant enzyme system can be regulated, and excessive ROS can be removed in time to reduce the risk of cell apoptosis. By adding sodium pyruvate, recombinant human albumin and recombinant transferrin, the additional needs of mesenchymal stem cells for energy and materials under hypoxia can be supplemented. Sodium pyruvate can enter the tricarboxylic acid cycle of cells as an energy supplement to provide cells with additional energy. Recombinant human albumin not only serves as a nutrient carrier, but also has certain antioxidant and cell protection effects. Recombinant transferrin is responsible for binding and transporting iron ions to ensure that cells obtain enough iron ions. In addition, the serum-free culture medium provided by the present invention has clear ingredients, stable effects, and no batch-to-batch differences, and is suitable for standardized process production.
[0007] The scheme of the present invention is to provide a serum-free culture medium for hypoxic culture of mesenchymal stem cells, characterized in that it comprises the following components: basal culture medium, ginsenoside Rg1, asiatica glycoside, sodium pyruvate, recombinant human albumin and recombinant transferrin.
[0008] The serum-free medium of the present invention is characterized in that, by adding basal medium, sodium pyruvate, recombinant human albumin and recombinant transferrin, the synergistic effect ensures the nutrient supply and function maintenance of mesenchymal stem cell growth and proliferation, the basal medium provides basic nutrients for mesenchymal stem cells, recombinant human albumin is used as a carrier protein to assist the nutrients in the basal medium to be better taken up and utilized by cells, recombinant transferrin transports iron ions, ensures the activity of key metabolic enzymes in cells, and maintains the normal metabolism of cells, and sodium pyruvate provides additional energy for cells under hypoxic conditions, ensuring that cells are guaranteed in terms of nutrient supply and energy metabolism, and these substances work together to provide a stable, good nutrient and function maintenance environment for the growth and proliferation of mesenchymal stem cells under hypoxic conditions. The components of the serum-free medium of the present invention jointly regulate the physiological activities of mesenchymal stem cells under hypoxic conditions from multiple aspects such as cell proliferation, anti-oxidation, energy metabolism, and nutrient supply, so that mesenchymal stem cells can maintain a higher proliferation rate and a lower apoptosis rate under hypoxic conditions, while maintaining higher stem cell characteristics and differentiation ability, and achieving the high efficiency and stability of mesenchymal stem cells cultured under hypoxic conditions.
[0009] The serum-free culture medium of the present invention is characterized in that, by adding ginsenoside Rg1 and centella asiatica, the proliferation, anti-oxidation and anti-apoptosis of mesenchymal stem cells are synergistically promoted; ginsenoside Rg1 specifically activates the PI3K / AKT signaling pathway, promotes the expression of cell cycle-related proteins (such as cyclin D, etc.), accelerates cell division and proliferation; centella asiatica regulates the activity of the intracellular antioxidant enzyme system, promptly removes excessive ROS in the cell, reduces the damage of oxidative stress to the cell, and inhibits the activation of apoptosis-related signaling pathways. In a hypoxic environment, ginsenoside Rg1 and Centella asiatica work together to promote cell division and reduce cell death, solving the proliferation-survival contradiction of mesenchymal stem cells in a hypoxic environment. At the same time, the activated PI3K / AKT pathway can upregulate the expression of some antioxidant enzymes, enhance the antioxidant capacity of the cells themselves, and complement the antioxidant effect of Centella asiatica, further reducing the cell apoptosis rate and improving the cell proliferation efficiency. After 5 days of culture, the cell proliferation rate can reach as high as 245.7±9.2%, and the cell apoptosis rate is lower than 7%. After 7 days of culture, the CD105, CD90 and CD73 positive cell rates are all greater than 99%, and the CD45, CD3, CD19 and HLA-DR positive cell rates are all lower than 1%.
[0010] Preferably, the basal culture medium can be DMEM / F12 culture medium. DMEM / F12 culture medium contains a variety of nutrients such as amino acids, vitamins, minerals, etc., which can provide a comprehensive and necessary material basis for the growth and metabolism of mesenchymal stem cells in a hypoxic environment.
[0011] Preferably, the serum-free medium for hypoxic culture of mesenchymal stem cells has the following final mass concentrations of the following components: ginsenoside Rg1 0.2~1.0μg / mL, asiaticoside 0.96~2.88μg / mL, sodium pyruvate 0.06~0.22mg / mL, recombinant human albumin 1~3mg / mL, recombinant transferrin 3~7μg / mL. It is worth noting that the final mass concentration described in the present invention refers to the ratio of the mass of each component in the solution to the total volume of the solution after the solution is prepared.
[0012] Preferably, the serum-free culture medium for hypoxic culture of mesenchymal stem cells has the following final mass concentrations of the components: ginsenoside Rg1 0.5 μg / mL, asiaticaside 2.88 μg / mL, sodium pyruvate 0.11 mg / mL, recombinant human albumin 2 mg / mL, and recombinant transferrin 5 μg / mL.
[0013] In addition, the present invention also provides a method for preparing a serum-free culture medium for hypoxic culture of mesenchymal stem cells, characterized in that it comprises the following steps: Step 1: Accurately weigh recombinant human albumin, sodium pyruvate and recombinant transferrin, add them to DMEM / F12 basal culture medium, and stir until completely dissolved to obtain a fortified basal culture medium.
[0014] Step 2: Accurately weigh ginsenoside Rg1 and asiaticoside, respectively, dissolve them in an organic solvent, and then slowly add them to the fortified basal medium obtained in step 1, stirring while adding, to obtain a serum-free medium stock solution. The organic solvent is a commonly used organic solvent in the art.
[0015] Step 3: Filter the serum-free culture medium obtained in step 2 through a 0.22 μm filter membrane to sterilize and obtain a serum-free culture medium for hypoxic culture of mesenchymal stem cells.
[0016] Preferably, the organic solvent in step 2 is dimethyl sulfoxide.
[0017] Preferably, the serum-free culture medium in step 3 is sealed and stored in a refrigerator at 4°C.
[0018] In addition, the present invention also provides the use of a serum-free medium for culturing mesenchymal stem cells under hypoxia in culturing mesenchymal stem cells. Using the serum-free medium prepared by the present invention, the proliferation rate, stem cell-specific surface marker expression, and differentiation ability of the cultured mesenchymal stem cells are significantly improved, and the cell apoptosis rate is significantly reduced.
[0019] In summary, the beneficial effects of the present invention are: (1) The present invention ensures the nutrient supply and functional maintenance of mesenchymal stem cells for growth and proliferation in a hypoxic environment by adding basal culture medium, sodium pyruvate, recombinant human albumin and recombinant transferrin to the culture medium. The basal culture medium provides basic nutrition, recombinant human albumin assists in nutrient uptake and has antioxidant and cell protection functions, recombinant transferrin transports iron ions to maintain cell metabolism, and sodium pyruvate provides additional energy. These components together provide cells with a stable and comprehensive growth environment, solving the problems of insufficient nutrient supply, imbalanced energy metabolism and obstruction of key metabolic processes in a hypoxic environment.
[0020] (2) The present invention adds ginsenoside Rg1 and asiaticoside to serum-free culture medium to synergistically promote the proliferation, anti-oxidation and anti-apoptosis of mesenchymal stem cells. Ginsenoside Rg1 activates the PI3K / AKT signaling pathway, and asiaticoside regulates the antioxidant enzyme system and inhibits cell apoptosis. The synergistic effect of the two solves the proliferation-survival contradiction of mesenchymal stem cells under hypoxic environment, enhances the cell's own antioxidant capacity, further reduces the apoptosis rate, and improves the proliferation efficiency, solving the problem that traditional serum-free culture medium cannot effectively promote cell proliferation and inhibit apoptosis under hypoxic conditions.
[0021] (3) The present invention achieves the purpose of preparing a serum-free culture medium with clear components and no microbial contamination by accurately setting the concentration range of each component in the serum-free culture medium and adopting a standardized preparation method, thereby ensuring the quality of the culture medium and providing a reliable guarantee for high-quality cell culture.
[0022] (4) The serum-free culture medium prepared by the present invention has clear components, stable properties, no batch-to-batch differences, meets the requirements of standardized production, and is conducive to the industrial production and wide application of the culture medium.
[0023] (5) The present invention achieves the effect of significantly improving the proliferation rate, expression of stem cell-specific surface markers and differentiation ability of the cultured mesenchymal stem cells and significantly reducing the cell apoptosis rate by applying the prepared serum-free culture medium to the culture of mesenchymal stem cells, thereby solving the problem that the existing culture medium for culturing mesenchymal stem cells is of low quality and cannot meet the needs of scientific research and clinical application. DETAILED DESCRIPTION
[0024] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by ordinary technicians in the field to which the present invention belongs.
[0025] In the present invention, the components and reagents involved are conventional commercial products, or can be obtained by conventional technical means in the art. Unless otherwise specified, the materials, methods and embodiments of the present invention are only exemplary and non-limiting.
[0026] Example 1: A serum-free culture medium for hypoxic culture of mesenchymal stem cells and its preparation This embodiment provides a serum-free medium for culturing mesenchymal stem cells under hypoxia. The basic medium is DMEM / F12 medium purchased from Wuhan Pronosai Life Science Technology Co., Ltd. The remaining components, measured by final mass concentration, are as follows: ginsenoside Rg1 0.2 μg / mL, asiaticoside 0.96 μg / mL, sodium pyruvate 0.06 mg / mL, recombinant human albumin 1 mg / mL, and recombinant transferrin 3 μg / mL. The medium configuration includes the following steps: Step 1: Accurately measure the DMEM / F12 basal medium, accurately weigh the corresponding mass of recombinant human albumin, sodium pyruvate and recombinant transferrin, add them to the DMEM / F12 basal medium, and stir until completely dissolved to obtain the fortified basal medium.
[0027] Step 2: Accurately weigh the corresponding masses of ginsenoside Rg1 and asiaticaoside, respectively, and dissolve them in dimethyl sulfoxide, and then slowly add them to the fortified basal culture medium obtained in step 1, stirring while adding to ensure that ginsenoside Rg1 and asiaticaoside are evenly dispersed in the culture medium to obtain a serum-free culture medium stock solution.
[0028] Step 3: Filter the serum-free medium obtained in step 2 with a 0.22 μm filter membrane to sterilize, thereby obtaining a serum-free medium for hypoxic culture of mesenchymal stem cells. Seal the prepared serum-free medium and store it in a 4°C refrigerator.
[0029] Example 2: A serum-free culture medium for hypoxic culture of mesenchymal stem cells and its preparation This embodiment provides a serum-free medium for hypoxic culture of mesenchymal stem cells. The basal medium is DMEM / F12 medium purchased from Wuhan Punosai Life Science Technology Co., Ltd. The remaining components, measured by final mass concentration, are as follows: ginsenoside Rg1 0.5 μg / mL, asiaticoside 2.88 μg / mL, sodium pyruvate 0.11 mg / mL, recombinant human albumin 2 mg / mL, and recombinant transferrin 5 μg / mL. The medium configuration method is the same as in Example 1.
[0030] Example 3: A serum-free culture medium for hypoxic culture of mesenchymal stem cells and its preparation This embodiment provides a serum-free medium for hypoxic culture of mesenchymal stem cells, which is purchased from Wuhan Punosai Life Science Technology Co., Ltd. The remaining components, measured by final mass concentration, are as follows: ginsenoside Rg1 1.0 μg / mL, asiaticoside 2.88 μg / mL, sodium pyruvate 0.22 mg / mL, recombinant human albumin 3 mg / mL, and recombinant transferrin 7 μg / mL. The medium preparation method is the same as in Example 1.
[0031] Comparative Example 1: A serum-free culture medium without ginsenoside Rg1 and its preparation This example provides a serum-free culture medium without ginsenoside Rg1, which is purchased from Wuhan Punosai Life Science Technology Co., Ltd. The remaining components, calculated by final mass concentration, are as follows: asiaticoside 2.88 μg / mL, sodium pyruvate 0.11 mg / mL, recombinant human albumin 2 mg / mL, and recombinant transferrin 5 μg / mL. The culture medium preparation method is the same as in Example 1.
[0032] The difference between the serum-free medium provided in Comparative Example 1 and Example 2 is that ginsenoside Rg1 is not added, and the other operations are the same.
[0033] Comparative Example 2: A serum-free culture medium without asiaticaside and its preparation This embodiment provides a serum-free culture medium without asiaticoside, which is purchased from Wuhan Punosai Life Science Technology Co., Ltd. The remaining components, calculated by final mass concentration, are as follows: ginsenoside Rg1 0.5 μg / mL, sodium pyruvate 0.11 mg / mL, recombinant human albumin 2 mg / mL, and recombinant transferrin 5 μg / mL. The culture medium preparation method is the same as in Example 1.
[0034] The serum-free medium provided in Comparative Example 2 differs from that in Example 2 in that no asiaticaside is added, and the remaining operations are the same.
[0035] Comparative Example 3: A serum-free culture medium without ginsenoside Rg1 and asiaticaside and its preparation This example provides a serum-free culture medium without ginsenoside Rg1 and asiaticoside, which is purchased from Wuhan Pronosai Life Science Co., Ltd. The remaining components, calculated by final mass concentration, are as follows: sodium pyruvate 0.11 mg / mL, recombinant human albumin 2 mg / mL, and recombinant transferrin 5 μg / mL. The culture medium is prepared as follows: Step 1. Accurately measure the DMEM / F12 basal medium, accurately weigh the recombinant human albumin, sodium pyruvate and recombinant transferrin, add them to the DMEM / F12 basal medium, stir until completely dissolved, and obtain the fortified basal medium.
[0036] Step 2: Filter the fortified basal medium obtained in step 1 with a 0.22 μm filter membrane to sterilize, thereby obtaining a serum-free medium that does not contain ginsenoside Rg1 and asiaticaside. Seal the prepared serum-free medium and store it in a refrigerator at 4°C.
[0037] The serum-free medium provided in Comparative Example 3 differs from that in Example 2 in that ginsenoside Rg1 and asiaticaside are not added, and the remaining operations are the same.
[0038] Comparative Example 4: DMEM / F12 basal medium This embodiment provides DMEM / F12 basal culture medium purchased from Wuhan Pronocell Life Science Co., Ltd. The difference from Example 2 is that it only contains basal culture medium components, and the rest of the operations are the same.
[0039] In order to verify the effect of the serum-free culture medium for culturing mesenchymal stem cells under hypoxia described in the present invention, experimental tests were conducted, as follows: Experiment 1: Proliferation rate of mesenchymal cells cultured under hypoxic conditions in serum-free medium In this experiment, the culture medium described in Examples 1 to 3 and Comparative Examples 1 to 4 was used to culture mesenchymal stem cells under hypoxic conditions. The specific operation was as follows: (1) Take out the cryopreservation tube containing mesenchymal stem cells from the liquid nitrogen tank, place it in a 37°C water bath for rapid thawing, wash it with PBS, and then collect the cell precipitate by centrifugation to obtain mesenchymal stem cells.
[0040] (2) Resuspend the mesenchymal stem cells in each culture medium and adjust the cell density to 5×10 3 Cells were cultured in a 96-well cell culture plate, 100 μL of cell suspension was added to each well, and 3 replicate wells were set for each culture medium. A blank control group was also set up, that is, only culture medium was added without inoculating cells.
[0041] (3) Place the 96-well plate in a hypoxic incubator and set the oxygen concentration to 2% and 5% CO 2 , cultured at 37℃.
[0042] (4) Perform the test on the first, third, and fifth days of culture. During the test, add 10 μL of CCK-8 solution to each well and continue incubation for 3 hours. Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm. Calculate the cell activity according to the following formula.
[0043] Proliferation rate (%) = (OD value of experimental group - OD value of blank group) / (OD value of experimental group on day 1 - OD value of blank group) × 100%.
[0044] Table 1 Proliferation rate of mesenchymal cells cultured in serum-free medium under hypoxic conditions From the results of Table 1, compared with Comparative Example 4, the culture medium provided by Examples 1 to 3 has a significant pro-proliferation effect, among which Example 2 has the best effect, and the proliferation rates on the 3rd and 5th days reach 178.4 ± 5.8% and 245.7 ± 9.2%, respectively, which are significantly higher than other groups, indicating that it can most effectively promote cell proliferation. Although the proliferation rates of Examples 1 and 3 are lower than those of Example 2, they are also significantly higher than those of the Comparative Example at each time point, indicating that the components in the culture medium of the present invention can effectively enhance the cell proliferation ability according to different concentration ratios. The proliferation rates of Comparative Examples 1 to 4 are significantly lower than those of the embodiments, wherein Comparative Example 1 lacks ginsenoside Rg1, Comparative Example 2 lacks asiaticoside, and Comparative Example 3 lacks both. The proliferation rates of these three groups are all lower than those of the embodiments, and the proliferation rate of Comparative Example 3 is the lowest, indicating that ginsenoside Rg1 and asiaticoside play an important role in promoting cell proliferation, and the synergistic effect of the two is better. Comparative Example 4 contained only basal culture medium, and had the lowest proliferation rate, indicating that the basal culture medium alone could not meet the proliferation requirements of mesenchymal stem cells in a hypoxic environment, and the cell proliferation on the 5th day was not obvious compared with that on the 3rd day, which may be due to cell apoptosis.
[0045] Experiment 2: Apoptosis rate of mesenchymal cells cultured under hypoxic conditions in serum-free medium In this experiment, the culture medium described in Examples 1 to 3 and Comparative Examples 1 to 4 was used to culture mesenchymal stem cells under hypoxic conditions. The specific operation was as follows: (1) Mesenchymal stem cells were inoculated into T25 culture flasks containing different culture media and placed in a hypoxic incubator (2% O 2 , 5%CO 2 , 37°C) for 5 days to obtain adherent cells.
[0046] (2) After the culture is completed, use an appropriate amount of trypsin to digest the adherent cells. After the cells begin to round up and the intercellular spaces increase under a microscope, gently blow the cells to evenly disperse them to form a single-cell suspension, which is then transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant is discarded to obtain the cultured cells.
[0047] (3) Wash the cultured cells twice with pre-cooled PBS, add 500 μL Binding Buffer to resuspend the cells, then add 5 μL Annexin V-FITC and 5 μL PI respectively, mix gently, and incubate at room temperature in the dark for 15 to 20 minutes.
[0048] (4) Transfer the stained cell suspension to a flow cytometer and immediately detect it using a flow cytometer to record the cell apoptosis data. Calculate the cell apoptosis rate according to the following formula. Set up 3 replicate experimental groups for each sample.
[0049] Early apoptotic cell rate (%) = number of early apoptotic cells / total number of cells × 100%.
[0050] Late apoptotic cell rate (%) = number of late apoptotic cells / total number of cells × 100%.
[0051] Total cell apoptosis rate (%) = (number of early apoptotic cells + number of late apoptotic cells) / total number of cells × 100%.
[0052] Table 2 Apoptosis rate of mesenchymal cells cultured in serum-free medium under hypoxic conditions From the results in Table 2, compared with Comparative Examples 1 to 4, the apoptosis rate of the cultured cells in Examples 1 to 3 was significantly reduced, wherein the early apoptotic cell rate in Example 2 was 3.1 ± 0.6%, the late apoptotic cell rate was 1.2 ± 0.4%, and the total apoptosis rate was only 4.4 ± 0.8%, the lowest apoptosis rate in all experimental groups, and the total apoptosis rate of Examples 1 and 3 was also at a low level, indicating that the culture medium of the present invention effectively regulates the intracellular antioxidant enzyme system and inhibits ROS production by adding key components such as ginsenoside Rg1 and asiaticoside, and significantly reduces the apoptosis rate. In the results of the comparative cultured cells, Comparative Example 1 lacks ginsenoside Rg1, Comparative Example 2 lacks asiaticoside, and Comparative Example 3 has neither ginsenoside Rg1 nor asiaticoside. The apoptosis rates of Comparative Examples 1 to 3 are significantly higher than those of the examples, indicating that ginsenoside Rg1 and asiaticoside play a positive role in inhibiting apoptosis, and the two synergistically inhibit apoptosis. Comparative Example 4 contains only basal culture medium, with an early apoptosis rate of 22.3±1.4%, a late apoptosis rate of 15.3±1.0%, and an apoptosis rate as high as 37.6±1.6%, indicating that the basal culture medium alone is difficult to meet the anti-apoptosis requirements of cells in a hypoxic environment. The culture medium of the present invention can effectively make up for this defect through the combined action of various components.
[0053] Experiment 3: Identification of surface markers of mesenchymal cells cultured under hypoxic conditions in serum-free medium In this experiment, the culture medium described in Examples 1 to 3 and Comparative Examples 1 to 4 was used to culture mesenchymal stem cells under hypoxic conditions. The specific operation was as follows: (1) Mesenchymal stem cells were inoculated into T25 culture flasks containing different culture media and placed in a hypoxic incubator (2% O 2 , 5%CO 2 , 37°C) for 7 days to obtain adherent cells.
[0054] (2) After the culture is completed, use an appropriate amount of trypsin to digest the adherent cells. After the cells begin to round up and the intercellular spaces increase under a microscope, gently blow the cells to evenly disperse them to form a single-cell suspension, which is then transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant is discarded to obtain the cultured cells.
[0055] (3) Adjust the cell concentration to 1×10 6 / mL, take 100μL of cell suspension to the flow tube, add appropriate amount of fluorescently labeled anti-CD73, CD90, CD105, CD34, CD45, CD19 and HLA-DR antibodies, react at room temperature in the dark for 30min. Add 500μL PBS to resuspend the cells, detect the expression of cell surface markers by flow cytometry, and analyze the percentage of positive cells. Set up 3 replicate experimental groups for each sample.
[0056] Table 3 Surface markers of mesenchymal cells cultured in serum-free medium under hypoxic conditions The results are shown in Table 3. In Examples 1 to 3, CD105, CD90, and CD73, as specific surface markers of mesenchymal stem cells, have positive cell percentages greater than 99%, indicating that the culture medium of the present invention can effectively maintain the stemness of mesenchymal stem cells and ensure cell purity and quality. In Comparative Examples 1 to 4, the percentages of CD105, CD90, and CD73 positive cells are slightly lower than those in the examples, indicating that the components added by the present invention are more conducive to maintaining the stemness of mesenchymal stem cells.
[0057] As shown in Table 3, in Examples 1 to 3, the percentages of CD45, CD34, CD19 and HLA-DR positive cells were all less than 1%, indicating that the culture medium of the present invention can effectively inhibit the proliferation or differentiation of non-mesenchymal stem cells, maintain the purity of the cell culture system, and maintain the characteristics of stem cells. In Comparative Examples 1 to 4, the percentages of CD45, CD34, CD19 and HLA-DR positive cells were all less than 3%, indicating that the ability of the culture medium containing the additives of the present invention to maintain the characteristics of mesenchymal stem cells is enhanced.
[0058] Experiment 4: Differentiation ability of mesenchymal cells cultured under hypoxic conditions in serum-free medium In this experiment, the culture medium described in Examples 1 to 3 and Comparative Examples 1 to 4 was used to culture mesenchymal stem cells under hypoxic conditions, and osteogenic induction medium and adipogenic induction medium were used to induce the cultured mesenchymal stem cells. The osteogenic induction medium was based on DMEM / F12 medium, and 1×10 -7 mol / L dexamethasone, 10mmol / L β-glycerophosphate sodium, 50μg / mL vitamin C, 100U / mL penicillin, and 100μg / mL streptomycin; adipogenic induction medium was based on DMEM / F12 medium, with 1×10 -6 mol / L dexamethasone, 0.5mmol / L 3-isobutyl-1-methylxanthine (IBMX), 10μg / mL insulin, 200μmol / L indomethacin, 100U / mL penicillin, and 100μg / mL streptomycin. The specific operation is as follows: (1) The mesenchymal stem cells cultured in Experiment 1 were cultured at 1×10 4 Pieces / cm 2 The cells were seeded at a density of 100 μg / mL in a 6-well plate and 2 mL of osteogenic induction medium or 2 mL of adipogenic induction medium was added to each well.
[0059] (2) Culture the confluent cells using osteogenic induction medium or adipogenic induction medium. Culture the cells for osteogenic induction for 21 days, changing the medium every 3 days; culture the cells for adipogenic induction for 14 days, changing the medium every 3 days.
[0060] (3) After induction, discard the culture medium and wash twice with PBS. Then add 4% paraformaldehyde, fix for 30 minutes, and wash three times with PBS. Add Alizarin Red dye to the osteogenic induction test group, stain at room temperature in the dark for 30 minutes, wash several times with distilled water, and remove excess dye. Add Oil Red O dye to the adipogenic induction test group, stain at room temperature in the dark for 60 minutes. Wash several times with 60% isopropanol to remove excess dye.
[0061] (4) Place the 6-well plate under an inverted microscope for observation, select multiple fields of view for counting, and calculate the cell differentiation efficiency according to the formula. Set up 3 replicate experimental groups for each sample.
[0062] Osteoblast differentiation efficiency = (total number of mineralized nodule cells / total number of cells) × 100%.
[0063] Adipocyte differentiation efficiency = (total number of adipogenic staining / total number of cells) × 100%.
[0064] Table 4 Differentiation ability of mesenchymal cells cultured under hypoxic conditions in serum-free medium As shown in Table 4, the osteoblast differentiation efficiency and adipocyte differentiation efficiency of Examples 1 to 3 are relatively high, which indicates that the serum-free culture medium of the present invention can effectively maintain the differentiation potential of mesenchymal stem cells under a hypoxic environment, promote their differentiation into osteoblasts and adipocytes, and reflect good cell culture performance. The osteoblast and adipocyte differentiation efficiencies of Comparative Examples 1 to 4 are at a conventional level, but are significantly lower than those of the Examples, indicating that the synergistic effect of the components in the culture medium of the present invention better maintains the cell differentiation ability of the cultured mesenchymal stem cells.
[0065] In summary, the serum-free culture medium for hypoxic culture of mesenchymal stem cells involved in the present invention significantly improves the proliferation rate of mesenchymal stem cells in a hypoxic environment, reduces the apoptosis rate, effectively maintains the stemness of cells, ensures the stable expression of cell surface markers, and enhances the differentiation ability of cells into osteoblasts and adipocytes by adding ginsenoside Rg1, asiaticoside, sodium pyruvate, recombinant human albumin and recombinant transferrin. The culture medium has clear ingredients, stable properties, no batch differences, meets standardized production requirements, and provides a reliable and efficient solution for large-scale culture of mesenchymal stem cells in scientific research and clinical applications.
[0066] At the same time, the present invention provides scientific ideas and methods for culturing mesenchymal stem cells using the serum-free culture medium. The technical scheme described provides new ideas and methods for technicians in this field. For ordinary technicians in this technical field, any improvements and innovations made based on the principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A serum-free culture medium for hypoxic culture of mesenchymal stem cells, characterized in that: The invention comprises the following components: basic culture medium, ginsenoside Rg1, asiatica glycoside, sodium pyruvate, recombinant human albumin and recombinant transferrin.
2. The serum-free medium according to claim 1, characterized in that The basic culture medium is DMEM / F12 culture medium.
3. The serum-free medium according to claim 1, characterized in that The final mass concentrations of each component were as follows: ginsenoside Rg1 0.2~1.0μg / mL, asiaticaside 0.96~2.88μg / mL, sodium pyruvate 0.06~0.22mg / mL, recombinant human albumin 1~3mg / mL, and recombinant transferrin 3~7μg / mL.
4. The serum-free medium according to claim 1, characterized in that The final mass concentrations of each component were as follows: ginsenoside Rg1 0.5 μg / mL, asiaticaside 2.88 μg / mL, sodium pyruvate 0.11 mg / mL, recombinant human albumin 2 mg / mL, and recombinant transferrin 5 μg / mL.
5. A method for preparing a serum-free medium according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: Accurately weigh recombinant human albumin, sodium pyruvate and recombinant transferrin, respectively, add them to DMEM / F12 basal medium, and stir until completely dissolved to obtain a fortified basal medium; Step 2: Accurately weigh ginsenoside Rg1 and asiaticaoside respectively, dissolve them in an organic solvent, and then slowly add them to the fortified basal medium obtained in step 1, stirring while adding, to obtain a serum-free medium stock solution; Step 3: Filter the serum-free culture medium obtained in step 2 through a 0.22 μm filter membrane to sterilize and obtain a serum-free culture medium for hypoxic culture of mesenchymal stem cells.
6. The preparation method according to claim 5, characterized in that: The organic solvent in step 2 is dimethyl sulfoxide.
7. The preparation method according to claim 5, characterized in that: The serum-free culture medium described in step 3 is sealed and stored in a 4°C refrigerator.
8. Use of the serum-free medium according to any one of claims 1 to 4 in culturing mesenchymal stem cells.