Culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells and preparation method thereof

Abstract

The invention discloses a culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells and a preparation method of the culture medium. The culture medium comprises a serum-free basic culture medium, a solvent and the following components according to the final volume of the culture medium: 0.7-1.3 mM of aesculin, 0.7-1.3 mM of apigenin, 0.7-1.3 mM of baicalin and 7-13 mM of vanillin. The culture medium provided by the invention optimizes proliferation of human endometrial mesenchymal stem cells in primary culture and early culture.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell, a self-renewing multipotent progenitor cell. According to the International Society for Cellular Therapy (ISCT), the criteria for mesenchymal stem cells are their ability to adhere to plastics; high expression of CD105, CD73, and CD90 (≥95%), and no expression of CD45, CD34, CD14 / CD11b, CD79α / CD19, and HLA class II (≤2%); and the ability to differentiate into adipocytes, osteocytes, and chondrocytes under appropriate induction conditions.

[0003] Mesenchymal stem cells can be isolated from a variety of sources, the most relevant being bone marrow, adipose tissue, umbilical cord tissue, placental tissue, umbilical cord blood, and menstrual blood. Among them, bone marrow is the most common source of MSCs and has been widely used in clinical trials. However, the isolation of mesenchymal stem cells from bone marrow and adipose tissue is an invasive collection process that may cause harm to the donor. In addition, the proliferation capacity of bone marrow mesenchymal stem cells in patients aged 59-75 years is significantly reduced with age compared to patients aged 0-18 years.

[0004] In contrast, menstrual blood is considered the most suitable source because it has no ethical issues and is a simple source of MSCs. In addition, menstrual blood-derived endometrial MSCs can be extensively maintained in culture, are easy to expand on a large scale, have the characteristics of delayed aging, and exhibit more effective anti-inflammatory effects than others.

[0005] Despite the recognized advantages and clear benefits, the use of EMSCs as the primary source for cultured MSCs has been hampered by their low isolation and expansion success rates, including low isolation efficiency, poor culture stability, and low colony forming unit (CFU) frequency.

[0006] Another important characteristic of MSCs is the ability to adhere, so the use of factors that support the adhesion process of MSCs can help improve isolation yield and cell proliferation. So far, several strategies have been developed to support cell attachment. The traditional approach is to use fetal bovine serum, a highly enriched supplement that provides a wide range of cell attachment proteins, growth factors, and other important biomolecules. In addition to fetal bovine serum, different xeno-free protein-based coating agents or modified culture surfaces for MSC culture have been developed and are currently available on the market. In many research applications, proteins and other adhesion-supporting protein components are believed to help enhance cell adhesion and proliferation of MSCs. However, the use of fetal bovine serum as a coating fluid also has limitations in terms of the risk of transferring animal factors to humans, and the differences between each MSC line depend on different commercial products.

[0007] Therefore, the isolation and expansion culture process of menstrual blood-derived endometrial mesenchymal stem cells needs to be optimized.

[0008] It should be noted that the information disclosed in the above background technology section is only used for understanding the background of the present application, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the invention

[0009] In order to make up for the deficiencies of the prior art, the present invention provides a culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells and a preparation method thereof.

[0010] The present invention adopts the following technical solution:

[0011] In a first aspect, a culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells is provided, comprising a serum-free basal culture medium, a solvent, and the following components in the final volume of the culture medium: 0.7-1.3 mM fraxin, 0.7-1.3 mM apigenin, 0.7-1.3 mM baicalin, and 7-13 mM costunolide.

[0012] In the second aspect, a method for preparing the culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells as described in the first aspect is provided, comprising the following steps: preparing the components of the freezing solution at the required concentrations and mixing them with a serum-free basal culture medium.

[0013] The present invention has the following beneficial effects: the culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells of the present invention significantly enhances the proliferation growth and colony formation of hEMSCs, and verifies the practicability of the culture medium of the present invention from aspects such as immunophenotype, tri-lineage differentiation ability and karyotype detection. Therefore, the culture medium provided by the present invention optimizes the proliferation of human endometrial mesenchymal stem cells in primary and early culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A It is a positive marker for hEMSCs analyzed by flow cytometry in a specific embodiment of the present invention;

[0015] Figure 1B It is a negative marker for hEMSCs analyzed by flow cytometry in a specific embodiment of the present invention;

[0016] Figure 1C is a statistical analysis of flow cytometry results in a specific embodiment of the present invention;

[0017] Figure 2A In a specific embodiment of the present invention, the cell morphology of cells of different generations in different culture media is observed under a microscope;

[0018] Figure 2B is the proliferation time of cells of different generations in different culture media in a specific embodiment of the present invention;

[0019] Figure 2C is the number of cell colonies at different generations in different culture media in a specific embodiment of the present invention;

[0020] Figure 3A In a specific embodiment of the present invention, staining of adipogenesis, osteoblastogenesis and chondrogenesis of cells of different generations in different culture media is observed under a microscope;

[0021] Figure 3B is the expression level of adipogenic, osteogenic and chondrogenic genes in cells of different generations in different culture media in a specific embodiment of the present invention;

[0022] Figure 4A In a specific embodiment of the present invention, flow cytometry is used to analyze the cell cycle of cells cultured in different culture media.

[0023] Figure 4B In a specific embodiment of the present invention, the G1 phase of cells of different generations in different culture media is analyzed;

[0024] Figure 4C In a specific embodiment of the present invention, the S / G2 / M phases of cells of different generations in different culture media are analyzed;

[0025] Figure 4D is the β-galactosidase method for aging analysis under a microscope in a specific embodiment of the present invention;

[0026] Figure 4E It is a statistical β-galactosidase aging analysis in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0027] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope and application of the present invention. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other.

[0028] A specific embodiment of the present invention provides a culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells, which includes a serum-free basal culture medium, a solvent and the following components in the final volume of the culture medium: 0.7-1.3mM Esculin, 0.7-1.3mM Apigenin, 0.7-1.3mM Baicalin and 7-13mM Costunolide.

[0029] The culture medium of the present invention is a serum-free culture medium, with DMEM / F12 as the serum-free basal culture medium, and four natural active ingredients, Esculin, Apigenin, Baicalin and Costunolide, are added. Among them, Esculin is a fluorescent coumarin glucoside with an antioxidant stress effect. Apigenin (4',5,7-Trihydroxyflavone) is a natural flavonoid with an antioxidant effect. Baicalin, as a flavonoid glycoside, is an activator of allosteric carnitine palmitoyltransferase 1 (CPT1). Baicalin reduces NF-κB expression. Costunolide (NSC 106404) is a natural sesquiterpenoid compound with antioxidant properties. The culture medium of the present invention has the characteristics of promoting proliferation and anti-oxidation.

[0030] In a preferred embodiment, the serum-free basal culture medium is DMEM / F12 (such as DMEM / F12 culture medium with brand GIBCO product number C11330500BT).

[0031] In a preferred embodiment, the solvent is DMSO.

[0032] In a preferred embodiment, the culture medium comprises the following components in amounts based on the final volume of the culture medium: 1 mM aesculin, 1 mM apigenin, 1 mM baicalin and 10 mM costunolide.

[0033] The specific embodiment of the present invention also provides a method for preparing the culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells, which comprises the following steps: preparing the components of the freezing solution at the required concentration and mixing them with the serum-free basal culture medium.

[0034] In a preferred embodiment, the preparation method comprises the following steps:

[0035] S1, dissolving the aesculin in a solvent to prepare an aesculin mother solution;

[0036] S2, dissolving the apigenin in a solvent to prepare an apigenin mother solution;

[0037] S3, dissolving the baicalin in a solvent to prepare a baicalin mother solution;

[0038] S4, dissolving the costunolide in a solvent to prepare a costunolide mother solution;

[0039] S5. Dilute the aesculin mother solution, the apigenin mother solution, the baicalin mother solution and the costunolide mother solution to desired concentrations respectively, and then mix them with serum-free basal culture medium to form the culture medium.

[0040] In a further preferred embodiment, in step S1, the aesculin mother solution is prepared with DMSO and stored at -20°C for future use, and in step S5, it is diluted with DMSO to a desired concentration.

[0041] In a further preferred embodiment, in step S2, the apigenin mother solution is prepared with DMSO and stored at -20°C for future use, and in step S5, it is diluted with DMSO to a desired concentration.

[0042] In a further preferred embodiment, in step S2, the baicalin mother solution is prepared with DMSO and stored at -20°C for future use, and in step S5, it is diluted with DMSO to a desired concentration.

[0043] In a further preferred embodiment, in step S4, the costunolide mother solution is prepared with DMSO and stored at -20°C for future use, and in step S5, it is diluted with DMSO to a desired concentration.

[0044] Specific embodiments of the present invention are further described below.

[0045] The culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells is referred to as HMC.

[0046] 1. Preparation of human endometrial mesenchymal stem cells (hEMSCs)

[0047] According to the Declaration of Helsinki, the Ethics Committee of Shenzhen Second People's Hospital reviewed and approved the technical protocol of the ethics statement. All methods were performed in accordance with relevant guidelines and regulations. The donors were fully informed of the purpose of the study, and their voluntary consent was obtained in written form. In this study, hEMSCs were isolated and cultured from menstrual blood samples of 6 people.

[0048] Preparation method of hEMSCs: After collecting menstrual blood on the second day with a sterile menstrual cup, immediately store it on ice and quickly transfer it to an ultra-clean bench for operation; dilute it with the same volume of PBS in the ultra-clean bench. Then take 2 ml of the mixed solution, add 2 ml PBS (containing 1% penicillin-streptomycin solution) to resuspend it; then add 4 ml of lymphocyte separation solution in a centrifuge tube to fully shake and mix, centrifuge at 800g for 20 minutes, and take the middle white film layer after centrifugation. Then add 10 ml of PBS to the centrifuge tube for washing, centrifuge at 400g for 5 minutes, wash twice, discard the supernatant, add 1 ml of red blood cell lysis solution, place it at room temperature in the dark for 4 minutes, centrifuge at 400g for 5 minutes, discard the supernatant, add 2 ml of complete culture medium, resuspend the cell mass at the bottom of the centrifuge tube and wash it again. Count the obtained cells and calculate 3×10 4 Pieces / cm 2 Plant in a 10 cm culture dish (0.5% collagen was plated at least half an hour in advance), add 10 ml of complete medium (commercial common complete medium preparation: DMEM / F12 basal medium + 10% FBS + 10 μM Y-27632; the complete medium (HMC) of the present invention is prepared: DMEM / F12 basal medium + 1 mM Esculin + 1 mM Apigenin + 1 mM Baicalin + 10 mM Costunolide)) and culture. Replace the complete medium once every 3 days and observe the cell growth.

[0049] 2. HMC preparation

[0050] The complete culture medium (HMC) of the present invention is prepared, and the components of HMC except the solvent are as follows: DMEM / F12+1mM Esculin+1mM Apigenin+1mM Baicalin+10mM Costunolide.

[0051] Preparation of Esculin: Dissolve in DMSO to prepare a 100 mg / ml (293.88 mM) stock solution, dilute to a working solution when used, store in a -20°C refrigerator, and dilute with DMSO to the required concentration when used.

[0052] Preparation of Apigenin: Dissolve in DMSO to prepare 83.33 mg / ml (308.36 mM) stock solution, dilute to working solution when used, store in a -20°C refrigerator, and dilute with DMSO to the required concentration when used.

[0053] Preparation of Baicalin: Dissolve it in DMSO to prepare a 100 mg / ml (224.03 mM) stock solution, dilute it to a working solution when using it, store it in a -20°C refrigerator, and dilute it with DMSO to the required concentration when using it.

[0054] Costunolide preparation: Dissolve in DMSO to prepare a 49 mg / ml (210.92 mM) stock solution, dilute to a working solution when used, store in a -20°C refrigerator, and dilute with DMSO to the required concentration when used.

[0055] 3. Flow cytometry analysis

[0056] hEMSCs immunophenotyping was performed using BD StemFow TM Assay kit, markers are as follows: CD90, CD73 and CD105 positive markers; CD45, CD34, CD11b, CD19 and HLA-DR negative markers.

[0057] 4. Fibroblast colony forming unit (CFU-F) determination and population doubling time determination

[0058] To determine the frequency of fibroblast colony-forming units (CFU-F) grown under different conditions, hEMSCs were plated at 100 cells / cm at passage 3. 2 The cells were inoculated at a density of . The culture medium was changed every three days. After 2 weeks, the cells were frozen with 100% ethanol, stained with Giemsa 1×, incubated at room temperature for 10 min, then Giemsa was removed and washed with deionized water. CFU-F colonies were observed under a microscope and the number of colonies per 100 cells at the time of inoculation was determined.

[0059] In order to determine the proliferation capacity of hEMSCs under different conditions, the number of hEMSCs was counted and the viability test of each subculture was performed. The formula for determining the population doubling time (PDT) of each generation is: PDT = CT / CPDs, where CPDs (cumulative population doubling) = log (N / No) × 3.31; N is the number of cells at the delay time point; N0 is the initial number of cells when inoculated, and CT is the culture time between the two time points.

[0060] 5. Three-lineage differentiation ability

[0061] The tri-lineage differentiation capacity of hEMSCs was tested at passage 3. TMDifferentiation kit, initiate osteogenesis, adipogenesis and chondrogenesis, differentiated cells were stained with Alizarin Red, Oil Red O and Alcian Blue respectively. Commercially available complete medium (DMEM / F12 basal medium + 10% FBS + 10μM Y-27632) was used as the control group. Cells were cultured in each differentiation medium for 14 days, then fixed with 4% paraformaldehyde and stained as described above, as follows.

[0062] Use two different complete culture media to resuspend the hEMSCs in the centrifuge tubes, dilute them and inoculate them into 24-well plates, with 2×10 per well. 4 The hEMSCs were placed in a cell culture incubator and cultured for 3 wells in the induction group and 6 wells in the control group with a final volume of 1 ml in each well.

[0063] In the adipogenic induction group: on the second day, the original culture medium was replaced with adipogenic induction medium, which was replaced with adipogenic maintenance medium (DMEM / F12 basal medium + 10% FBS + 10 μg / ml Insulin) after 3 days of induction, which was replaced with adipogenic induction medium again after 3 days of induction, which was replaced with adipogenic maintenance medium again after 3 days of induction, and the induction was completed after 3 days of maintenance culture;

[0064] Osteogenic induction group: On the second day, the original culture medium was replaced with osteogenic induction medium, and half the amount of induction medium was replaced every 3 days. After two weeks of culture, the induction was completed;

[0065] Chondrogenic induction group: On the second day, the original culture medium was replaced with chondrogenic induction medium, and half the amount of induction medium was replaced every three days. After two weeks of culture, the induction was completed;

[0066] Control group: The medium was completely replaced at the same time as the induced group, and DMEM / F12 complete medium was used each time.

[0067] All induction medium uses DMEM / F12 medium as the basic medium, and the induction components are added according to the concentrations in the table.

[0068] Adipogenic medium composition list:

[0069] Element Preparation concentration Final concentration Adding ratio FBS N / A 10％ 10％ IBMX 500mM (DMSO) 500μM 1 / 1000 Dexamethasone 1mM 1μm 1 / 1000 Insulin 1816μg / ml 10μg / ml 5.5μl / ml Indomethacin 358mg / 15ml(DMSO) 10μM 1.5μl / ml

[0070] Osteogenic induction medium composition list:

[0071] Element Preparation concentration Final concentration Adding ratio FBS N / A 10％ 10％ Vc 0.1M 0.2mM 1 / 500 Dexamethasone 1mM 0.1μm 1 / 10000 β-Glycerol 1M 10mM 1 / 100 BMP-2 20μg / ml 50ng / ml 2.5 / 1000

[0072] Composition of chondrogenic induction medium:

[0073] Element Preparation concentration Final concentration Adding ratio FBS N / A 1％ 1％ Vc 0.1M 50μM 1 / 2000 TGF-β1 20μg / ml 10ng / ml 1 / 2000 Insulin 1816μg / ml 6.25μg / ml 3.4μl / ml

[0074] Oil Red O staining steps for adipogenic induced cells:

[0075] A. Aspirate and discard all the supernatant, and gently wash the cells once with 1 ml of PBS. After aspirating and discarding all the supernatant, add 1 ml of 4% paraformaldehyde to each well to fix the cells for 30 minutes at room temperature.

[0076] B. Aspirate and discard all the fixative solution, add 1 ml of PBS to each well, and wash cells three times, each wash for 1 min at room temperature.

[0077] C. Aspirate and discard all supernatant, add 500 μl of 2% Oil Red O dye solution to each well, and let it stand for 30 minutes at room temperature.

[0078] D. After staining, discard all the dye solution, add 1 ml PBS to each well to wash the cells, wash 3-5 times until the supernatant is colorless and transparent, add 1 ml PBS again and take pictures under a microscope.

[0079] Steps for osteogenic cell staining with Alizarin Red:

[0080] A. Aspirate and discard all the supernatant, and gently wash the cells once with 1 ml of PBS. After aspirating and discarding all the supernatant, add 1 ml of 4% paraformaldehyde to each well to fix the cells for 30 minutes at room temperature.

[0081] B. Aspirate and discard all the fixative solution, add 1 ml of PBS to each well and wash three times, 1 minute each time, at room temperature.

[0082] C. Aspirate and discard all the washing solution, add 500 μl of 0.1% Alizarin Red staining solution to each well, and let it stand for staining for 30 minutes at room temperature.

[0083] D. After staining, discard all the dye solution, add 1 ml PBS to each well to wash the cells, wash 3-5 times until the supernatant is colorless and transparent, add 1 ml PBS again and take pictures under a microscope.

[0084] Alcian blue staining steps for chondrogenic cells:

[0085] A. Aspirate and discard all the supernatant, and gently wash the cells once with 1 ml of PBS. After aspirating and discarding all the supernatant, add 1 ml of 4% paraformaldehyde to each well to fix the cells for 30 minutes at room temperature.

[0086] B. Aspirate and discard all the fixative solution, add 1 ml of PBS to each well and wash three times, 1 minute each time, at room temperature.

[0087] C. Aspirate and discard all the wash solution, add 500 μl of Alcian blue dye to each well, and let it stand for staining for 30 minutes at room temperature.

[0088] D. After staining, discard all the dye solution, add 1 ml PBS to each well to wash the cells, wash 3-5 times until the supernatant is colorless and transparent, add 1 ml PBS again and take pictures under a microscope.

[0089] 6. Determination of cell viability, proliferation and senescence

[0090] Prepare cells for flow cytometry by washing with 10× PBS and centrifuging at 300 g for 5 min. Stain cells with 7-AAD and fix for 30 min. Wash cells with 10× PBS and resuspend in appropriate PBS to assess cell cycle by flow cytometry.

[0091] hEMSCs senescence analysis. hEMSCs at passages 3 and 7 were subjected to senescence analysis using a senescent cell histochemical staining kit according to the manufacturer’s instructions. hEMSCs were plated at 4 × 10 5 Cells / cm 2 The cells were seeded at a density of 100 μg / cm2 in a 6-well plate and incubated at 37°C and 5% CO2 for 48 h for cell attachment. Then, the culture medium was discarded, the cells were washed twice with PBS, and then fixed with fixative at room temperature for 6 min. The cells were then rinsed 3 times with PBS, and the staining mixture was incubated at 37°C overnight. The stained cells were then washed twice with PBS, and then stained for the second time with DAPI staining solution at room temperature for 5 min. After DAPI staining, the cells were washed twice with PBS, and the images were observed with an inverted microscope. The images were observed and semi-qualitatively analyzed using Image J software.

[0092] 7. Statistical analysis

[0093] All statistical analyses were performed using R software version 3.4.4. T-test, TwoWay ANOVA, and Tukey HSD tests were used to determine if there were statistically significant differences between the groups. p < 0.05 was considered statistically significant. All data are presented as mean ± SD.

[0094] Experimental Results

[0095] 1. Characterization of human endometrial mesenchymal stem cells

[0096] The hEMSCs positive markers CD73, CD90 and CD105 and negative or negative expression markers CD11b, CD19, CD34, CD45 and HLA-DR were analyzed. The results of immunophenotyping showed that compared with the common complete medium (DMEM / F12 basal medium + 10% FBS + 10μM Y-27632) on the market, the hEMSCs cells cultured in the medium of the present invention showed obvious stem cell surface markers, among which CD90, CD73 and CD105 were all above 95% ( Figure 1A The average value of negative markers such as CD45, CD34, CD11b, CD19, and HLA-DR was 4.10% ( Figure 1B), statistical analysis p < value is 0.05 ( Figure 1C ).

[0097] 2. Effect of HMC on the growth of hEMSCs

[0098] To determine the effect of the culture medium on cell growth, hEMSCs were cultured to the 7th passage (n=7). hEMSCs expanded in HMC were smaller in size and spindle-shaped, and could achieve uniformity during the analysis period. In contrast, hEMSCs expanded in the Control group (using commercially available complete culture medium: DMEM / F12 basal medium + 10% FBS + 10μM Y-27632) also showed small size and spindle-shaped cells in the early passages, but became larger in higher passages and failed to achieve uniformity ( Figure 2A ). Therefore, the cell proliferation rates of the 6th and 7th generation Control groups were lower than those of HMC (p = 0.04 for both generations) ( Figure 2B ). The CFU frequency of cells in the Control group was 4.31±1.13% at the third passage and 0.69±0.61% at the seventh passage, which was significantly lower than that of HMC, which was 11.03±2.85% at the third passage (p<0.001) and 9.00±3.40% at the seventh passage (p<0.001). The CFU of hEMSCs expanded in the Control group decreased in the later passages (p=0.03), while the number of cell colonies cultured in HMC remained high (p=0.37) ( Figure 2C ).

[0099] 3. Effect of HMC on the differentiation ability of hEMSCs

[0100] To test the multipotency of HMCs for hEMSCs culture, hEMSCs were differentiated into adipogenic, osteogenic, and chondrogenic gene lineages. Oil Red O, Alizarin Red S, and Alcian Blue ( Figure 3A ) staining showed that hEMSCs in HMC could differentiate into three cell lineages. Compared with the Control group, PPARγ and Leptin for adipogenesis, PTH-R and ALP for osteogenesis, and SOX9 for chondrogenesis in HMC differentiated samples were significantly higher than those in the Control group. Figure 3B ) expression was increased. The expression level of PPARγ in the culture medium of the Control group was lower (p = 0.002), while the expression level of PTH-R was higher than that in the cells in HMC (p = 0.009). There was no difference in the expression of other genes analyzed.

[0101] 4. Cell viability, proliferation and senescence of hEMSCs

[0102] The 7-AAD method was used to analyze the cell viability of hEMSCs in the control group and HMC culture. hEMSCs were stained with 7-AAD and their cell cycle ( Figure 4A ). Compared with living cells, the DNA content of apoptotic cells was reduced. Compared with the 3rd and 7th generation HMC-cultured hEMSCs, the G1 cell cycle frequency of the control group cultured hEMSCs increased ( Figure 4B In addition, compared with HMC cultured cells, the proportion of proliferating cells in the S / G2 / M phase of the hEMSCs cell cycle in the 3rd and 7th generation Control groups decreased ( Figure 4C ). The above results indicate that the proliferation capacity of cells in the control group decreased during the higher passages of cells. In addition, we used the β-galactosidase assay for senescence analysis ( Figure 4D ). In the 3rd and 7th passages, the cells cultured in the control group showed a higher frequency of senescent cells compared with those cultured in HMC ( Figure 4E ).

[0103] Therefore, the results of cell cycle and cell senescence indicate that the culture medium of the present invention can maintain the proliferation state of hEMSCs for a longer period of time.

[0104] The above content is a further detailed description of the present invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, without departing from the concept of the present invention, it can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be regarded as belonging to the protection scope of the present invention. In the description of this specification, the description of the reference terms "an embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily target the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In the absence of mutual contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope of protection of the patent application.

Claims

1. A culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells, characterized in that: The culture medium includes a serum-free basal medium, a solvent, and the following contents of each component based on the final volume of the culture medium: 0.7-1.3 mM aesculin, 0.7-1.3 mM apigenin, 0.7-1.3 mM baicalin, and 7-13 mM caryophyllene.

2. The culture medium according to claim 1, characterized in that: The serum-free basal culture medium is DMEM / F12 culture medium.

3. The culture medium according to claim 1, wherein: The solvent is DMSO.

4. The culture medium according to claim 1, characterized in that: The culture medium includes the following components in amounts based on the final volume of the culture medium: 1 mM aesculin, 1 mM apigenin, 1 mM baicalin, and 10 mM costunolide.

5. The method for preparing a culture medium for promoting rapid proliferation of human endometrial mesenchymal stem cells according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: preparing each component of the freezing solution according to the required concentration and mixing the components with a serum-free basic culture medium.

6. The preparation method according to claim 5, characterized in that: The steps include: S1, dissolving the aesculin in a solvent to prepare an aesculin mother solution; S2, dissolving the apigenin in a solvent to prepare an apigenin mother solution; S3, dissolving the baicalin in a solvent to prepare a baicalin mother solution; S4, dissolving the costunolide in a solvent to prepare a costunolide mother solution; S5. Dilute the aesculin mother solution, the apigenin mother solution, the baicalin mother solution and the costunolide mother solution to desired concentrations respectively, and then mix them with serum-free basal culture medium to form the culture medium.

7. The preparation method according to claim 6, characterized in that: In step S1, the aesculin mother solution is prepared with DMSO and stored at -20°C for future use. In step S5, it is diluted with DMSO to a desired concentration.

8. The preparation method according to claim 6, characterized in that: In step S2, the apigenin mother solution is prepared with DMSO and stored at -20°C for future use. In step S5, it is diluted with DMSO to a desired concentration.

9. The preparation method according to claim 6, characterized in that: In step S2, the baicalin mother solution is prepared with DMSO and stored at -20°C for future use. In step S5, it is diluted with DMSO to a desired concentration.

10. The preparation method according to claim 6, characterized in that: In step S4, the costunolide mother solution is prepared with DMSO and stored at -20°C for future use. In step S5, it is diluted with DMSO to a desired concentration.

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