CN118755667A - A culture medium for menstrual blood mesenchymal stem cells and a method for extracting and culturing the same - Google Patents

Abstract

The present invention belongs to the technical field of stem cell preparation, and specifically relates to a culture medium of menstrual blood mesenchymal stem cells and an extraction and culture method thereof. The extraction and culture method comprises collecting samples, extracting and separating MenSCs, collecting primary MenSCs, and subculture of MenSCs. The prepared menstrual blood mesenchymal stem cells can be stably subcultured to 15 generations or later. The present invention prepares menstrual blood mesenchymal stem cell raw materials by in vitro collection, using a culture medium prepared by adding jackfruit extract and screening primary cells in a better state, thereby overcoming the problem that menstrual blood mesenchymal stem cells cannot be stably subcultured to 15 generations or later. At the same time, the culture medium of the present application is serum-free, which greatly reduces the risk caused by exogenous substances. The present invention provides an operation process of MenSCs separation, collection and subculture that conforms to the standardized quality management of pharmaceutical production, and is suitable for industrial large-scale production.

Description

A culture medium for menstrual blood mesenchymal stem cells and a method for extracting and culturing the same

Technical Field

The invention belongs to the technical field of stem cell preparation, and particularly relates to a culture medium for menstrual blood mesenchymal stem cells and an extraction and culture method thereof.

Background Art

Normal menstruation in women is accompanied by periodic shedding and bleeding of the endometrium caused by cyclical changes in the ovaries. This is mainly because the ovaries periodically secrete estrogen and progesterone, which causes periodic changes in the endometrium. During a menstrual cycle, the endometrium gradually thickens from thin, and after reaching a certain thickness, the blood vessels under the endometrium become tortuous and filled, and eventually the endometrium sheds and causes bleeding.

In 2008, the Hida team in Japan successfully cultured stem cells with multiple differentiation functions using components of female menstrual blood - mesenchymal blood derived endomentrial stem cells (MenSCs). Endometrial stem cells are abundant in sources, easy to obtain, easy to separate, can be established, can be large-scale amplified, and have little loss of biological efficacy after freezing. They have self-renewal, multidirectional differentiation and high proliferation capabilities. They contain stem cell markers such as OCT-4, SSE-4 and CD117, and have extremely similar regenerative abilities to cord blood stem cells and bone marrow stem cells. They can double in 24 hours and be passaged up to 50 times. They have strong proliferation ability and differentiation potential and can be directed to differentiate into other types of healthy cells. A large number of basic and clinical research results have also confirmed that MenSCs transplantation can achieve good results in disease treatment, and the same donor can be periodically collected multiple times to obtain a large number of seed cells with consistent genetic backgrounds, which has the advantage of autologous transplantation, suggesting that MenSCs may be very promising seed cells in stem cell therapy.

Serum-free culture medium refers to a culture medium that does not require the addition of animal or human serum during cell culture. However, in order to meet the needs of cell growth, raw materials that replace the function of serum are usually added to the culture medium, mainly including binding proteins, growth factors, adhesion factors, hormones, trace elements and other enlarged categories. In the serum-free culture medium currently on the market, bovine serum albumin or human serum albumin is an essential raw material in the basic components. The addition ratio is large and the cost is high, but it has the disadvantages of insufficient cell passage and expansion capabilities. Invention patent CN 101525594B discloses a low serum concentration complete culture medium for culturing mesenchymal stem cells and a method for culturing mesenchymal stem cells using the culture medium. The complete culture medium includes a cell basal culture medium and a final concentration of fetal bovine serum, epidermal growth factor, and basic fibroblast growth factor. Although the low serum concentration complete culture medium of the invention can achieve the same or even better effect of promoting cell proliferation as the use of high serum concentration culture reagent, the culture medium contains fetal bovine serum, which has a complex composition and contains foreign proteins, and is easily infected with viruses or mycoplasma. In addition, there are large differences between production batches of fetal bovine serum, and the source is unstable, which has a great impact on the in vitro large-scale culture and expansion process of mesenchymal stem cells. The stem cell culture effect is not good, and the cells can often only be cultured to P3 before gradually aging, and there is no guarantee that they can be stably propagated for more than 15 generations. Therefore, based on the deficiencies of the prior art, a method for extracting and culturing mesenchymal stem cells from menstrual blood is designed.

Summary of the invention

The purpose of the present invention is to overcome the shortcoming of the prior art that the cells cannot be stably propagated beyond the 15th generation, and to provide a culture medium for menstrual blood mesenchymal stem cells and a method for extracting and culturing the culture medium.

The purpose of the present invention is achieved through the following technical solutions:

The embodiment of the first aspect of the present invention provides a culture medium for menstrual mesenchymal stem cells, the culture medium comprising DMEM/F12, 5-10ug/ml recombinant human serum albumin HSA, 2-3ng/ml recombinant human basic fibroblast growth factor bFGF, 50-120ng/ml recombinant human insulin growth factor-1 rhIGF-1 LR3, 5-20ng/ml recombinant human vascular endothelial growth factor OsrhVEGF, 0.5-100μg/ml recombinant human transferrin (Transferrin), 10μg/ml recombinant human insulin solution, 12-20ug/ml recombinant human epidermal growth factor, 0.006mM L-alanyl-L-glutamine solution, 10-15ng/ml wood bark extract, HEPES buffer adjusted the pH range to 7.2-7.5, the culture medium contains 5-10ug/ml recombinant human serum albumin HSA, 2-3ng/ml recombinant human basic fibroblast growth factor bFGF , 50-120ng/ml recombinant human insulin growth factor-1 rhIGF-1 LR3, 5-20ng/ml recombinant human vascular endothelial growth factor OsrhVEGF, 0.5-100μg/ml recombinant human transferrin (Transferrin), 10μg/ml recombinant human insulin solution and 12-20ug/ml recombinant human epidermal growth factor, the total content is less than 1.5mg/ml.

As a preferred embodiment, the method for extracting the Psoralea corylifolia extract comprises the following steps:

(1) Take the fruit of the Nanchuan Pampas grass, remove impurities, wash it, crush it, and soak it in ethyl acetate for 5 times at room temperature, each time for 3 days, and concentrate the extract under reduced pressure to obtain the ethyl acetate extract;

(2) Weigh 0.3 g of ethyl acetate extract, pass the ethyl acetate extract through an MCI column, and perform gradient elution through a methanol-water system at a ratio of 30% → 50% → 70% → 90% → 100%; separate the methanol-insoluble portion of the ethyl acetate extract by silica gel column chromatography, and perform elution through a petroleum ether-ethyl acetate system at a ratio of 30:1 → 1:1 to obtain five fractions Fr.1 to Fr.5, and perform elution through a petroleum ether-acetone system at a ratio of 50:1 → 1:1 to obtain four subfractions Fr.2-1 to Fr.2-4, wherein crystals precipitate from the Fr.2-2 fraction, and compound 4 is obtained by repeated recrystallization; the Fr.2-4 fraction is eluted through a developing solvent system of petroleum ether: acetone to obtain compound 4; Compound 5 was purified by PTLC thin layer chromatography with a developing solvent system of petroleum ether: ethyl acetate in a volume ratio of 15:1, and compound 6 was obtained by PTLC thin layer chromatography with a developing solvent system of petroleum ether: ethyl acetate in a volume ratio of 20:1. The Fr.5 fraction was eluted with petroleum ether-acetone at a ratio of 50:1→1:1 to obtain 4 subfractions Fr.5-1~Fr.5-4. Fr.5-1 was purified by PTLC thin layer chromatography with a developing solvent system of petroleum ether: acetone in a volume ratio of 10:1 to obtain compounds 1 and 2; the crystals precipitated from Fr.5-2 were washed by recrystallization to obtain compound 3, and compounds 1-6 were mixed in a ratio of 1:1:1:1:1:1 to obtain the wood borage extract.

The second aspect of the present invention provides a method for extracting and culturing menstrual blood mesenchymal stem cells, which are obtained by culturing the menstrual blood mesenchymal stem cells in a culture medium provided by the embodiment of the present invention. The culturing method comprises the following steps:

S1. Sample collection: using an in vitro tissue collection method, when meat-like or coagulated blood-like contents are found, the samples are placed in a first preservation tube containing an anticoagulant, antibiotics, mixed enzymes and the above-mentioned culture medium, wherein the volume ratio of the anticoagulant, antibiotics, mixed enzymes and culture medium is 1:1:1:80-120, the mixed enzymes include 1% type II collagenase and 1% trypsin, the 1% type II collagenase and the 1% trypsin are mixed in a ratio of 1:1, the anticoagulant is heparin sodium, and the antibiotic is 5 kU/ml penicillin-streptomycin-neomycin solution (PSN) (100×);

S2. Isolation and extraction of MenSCs:

(1) After thoroughly mixing the sample in the storage tube, filter it with a 100um cell strainer to remove blood clots and mucus. Then slowly add the filtered sample to the density gradient centrifugation liquid TBD (50ml centrifuge tube) with a sample to TBD ratio of 1:1. Centrifuge at room temperature at 2000r·min ^-1 for 10min. During the whole process, ensure that the separation interface is clear. After the centrifugation, extract the middle white film layer into a new centrifuge tube and keep the bottom red blood cell separation layer;

(2) Pipette the buffy coat layer into another clean centrifuge tube, add PBS (1% triple antibody) and repeat centrifugation and washing once;

(3) Resuspend the red blood cell pellet and the preserved tissue with an equal volume of red blood cell lysis solution to remove the red blood cells in the tissue fluid. Lyse the cells at room temperature for 5 minutes, and then centrifuge at 1200 r/min for 5 minutes.

(4) After centrifugation, add sterile PBS (1% antibiotics) to the pellet and resuspend it. Centrifuge horizontally at 1200 r/min ^-1 -5 min, repeat once, remove the supernatant, resuspend the pellet with digestion solution to remove collagen and nucleic acids in the tissue fluid, centrifuge again and discard the supernatant, resuspend the pellet with PBS, centrifuge at 1200 r/min -1 -5 min, wash the MenSCs solution with sterile D-Hanks solution, centrifuge at 1200 r/min ^-1 -5 min and discard the supernatant, repeat the washing twice to obtain the MenSCs pellet;

(5) Add 5 ml of adhesion promoter to each bottle of sample to completely soak the bottom of the bottle. The adhesion promoter includes 0.1 mg/mL poly-L-lysine solution (10×). Incubate at 37°C, 5% CO ₂ for 60 min, then discard the adhesion promoter solution and wash once with PBS.

(6) Adjust the concentration of the MenSCs suspension, 2 ml of no more than 0.3 ml of precipitation, inoculate into T25 culture flasks, and add about 2 ml of culture medium to each flask. Too much culture medium will affect cell adhesion.

(7) Spread the separated and extracted menstrual blood tissue evenly in a T25 culture bottle;

S3. Collection of primary MenSCs:

(1) Culture the cells in a medium supplemented with 400 ng/ml hydrocortisone at 37°C and 5% _CO2 , and observe the cell growth and contamination. Replace half of the medium on the fourth day to remove the non-adherent cells. Replace the medium every 48-72 hours after the adherent cells grow. The replaced medium does not contain hydrocortisone.

(2) When the degree of confluence reaches 80-90%, add 0.25% stem cell mild enzyme to the washed culture flask, about 1 ml per flask, and gently shake the flask to evenly infiltrate the bottom of each flask. Place it in the incubator for 2 minutes and observe. When the cells become round and most of the cells fall off, immediately add 2 times the volume of cell culture supernatant or complete culture medium used for 0.25% stem cell mild enzyme to dilute the cell suspension;

(3) Transfer the cell suspension in the culture flask to a 50 ml centrifuge tube, then wash the residual cells adhering to the culture flask with physiological saline, transfer the washing solution to the above centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant after centrifugation;

(4) Then add 3-5 ml of complete culture medium to resuspend the cells, take 1 ml of the cell suspension and make the volume to 10 ml for sampling and counting to calculate the cell survival rate;

S4. Subculture of MenSCs: In the subculture, further subculture is performed according to the actual number of cells, and the cell number is inoculated at a density of 5000-10000 cells/ ^cm2 . The primary MenSCs after harvest are placed in a culture bottle, and cultured at 37°C, 5% _CO2 , and saturated humidity until the confluence is 80-90%, and then subculture is performed; P4 and later cultures: After being digested into single cells with stem cell mild enzymes, MenSCs are irradiated with LIPUS at an intensity of 20P, an output power of 25mW/ ^cm2 , and a frequency of 10kHz. The intensity of LIPUS irradiation output for 1S is P. The stem cell mild enzyme is an artificial synthetic enzyme expressed by Escherichia coli, which specifically digests only the cell matrix between cells and between cells and culture bottles, thereby avoiding excessive digestion of cells; it does not contain any animal-derived components and is suitable for this culture system.

As a preferred technical solution, in step (2) of S2, the menstrual blood tissue is cut into 4-5 mm rectangular pieces using a sterile blade or forceps in a culture dish.

As a preferred technical solution, if the buffy coat layer obtained after filtration in S2 is small in number, the steps of first lysing at room temperature for 5 minutes and then centrifuging horizontally at 1200r for 5 minutes are omitted, and a multiple wash method is adopted.

As a preferred technical solution, the primary culture time of the MenSCs should not exceed 14 days to avoid the problems of primary cell death and low activity.

As a preferred technical solution, the primary cells are screened and then subcultured. The primary cells meet the conditions that there is no obvious halo around the cells, and they are circular, bipolar or tripole in the first three days; on the 10th to 15th day, the cells are spindle-shaped or fibrous, grow well, and form several cells that aggregate in the form of small islands with uniform morphology and close arrangement. After the above screening, the cells cultured in the above preservation solution/culture medium greatly improve the culture effect of stem cells, avoid waste of culture medium, improve the use efficiency of culture medium, and improve the culture efficiency of stem cells.

The present invention has the following advantages:

(1) The method for extracting and culturing menstrual blood mesenchymal stem cells of the present invention comprises adding a mulberry extract to the culture medium. The mulberry extract comprises ascorbyl dipalmitate, butyl linoleate, ethyl linolenate, ethyl palmitate, methyl linoleate, methyl palmitate and the like. In the present application, the mulberry extract has the effects of anti-aging and promoting cell growth, and can maintain the stability and proliferation of cells, effectively enabling MenSCs to be stably subcultured for 15 generations and beyond, and does not contain any animal-derived components, thereby greatly reducing the risk of exogenous substances. At the same time, compared with traditional culture media, the culture medium in the present application has the effects of promoting stem cell proliferation, inhibiting stem cell differentiation and maintaining stem cell phenotype, and increasing the number of stem cell subcultures, thereby reducing the need for the frequency of culture medium replacement.

(2) The present invention uses LIPUS to irradiate MenSCs after P4. LIPUS promotes the expression of growth factors in MenSCs, provides low-intensity mechanical stimulation, interacts with cells, induces intracellular biochemical effects, and ultimately leads to tissue repair and regeneration, thereby reducing the mortality rate of cells passaged after P4 and making the passage more stable.

(3) The present invention has found the optimal cell culture density, which avoids the problem that the cells cannot grow due to too low cell density, and the cells easily grow in layers and aggregate into balls due to too high cell density, and at the same time, cell contact inhibition occurs, leading to serious cell aging and differentiation.

(4) The present invention screens primary cells and then cultured the cells in the culture medium of the present application, which can be passaged to 15-20 generations, thereby greatly improving the culture effect of stem cells, avoiding the waste of culture medium, improving the use efficiency of culture medium, and improving the culture efficiency of stem cells.

(5) The present invention uses mixed proteases for digestion, which can better remove mucus and pass through the filter without damaging the cell membrane. The cells have good activity and can be passed to a higher generation. The use of mixed enzymes can purify them to a better purity while reducing cell consumption.

(6) The present invention uses an adhesion promoter for pre-coating, which can protect the adherent cells. In particular, when the adherent cells are about to adhere, frequent changes of the culture medium will have a great impact on them, thereby greatly affecting the subsequent stem cell culture effect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows, from left to right, the morphological images of cells of the P0, P1 and P4 generations cultured in a common medium;

FIG2 is a morphological diagram of cells of the P0, P1 and P4 generations cultured according to the method of Example 1 of the present application from left to right;

Figure 3 shows the death of cells that are not passaged at a high cell density;

Figure 4 shows a stacking situation where the cell density is too high;

FIG5 is a diagram of primary cells screened for subculture in Example 1 of the present application;

FIG6 is a diagram showing the screening of primary cells that cannot be subcultured;

FIG7 is a diagram of passaged cells of the same batch of primary cells in the comparative example of the present application at 24h, 48h, and 72h from left to right;

FIG8 is a schematic diagram of the unrepaired endometrium of a rat after injury;

Figure 9 is a schematic diagram of PRP repairing rat endometrium after injury;

FIG. 10 is a schematic diagram of MenSCs repairing rat endometrium in Example 1 after injury.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to the following description.

Unless otherwise specified, all professional and scientific terms used in this document have the same meanings as those familiar to those skilled in the art.

Introduction of some raw materials in this application:

DMEM/F12: Catalog No. SP032030500, provided by Sperlicon;

Recombinant human epidermal growth factor EGF cat: Y00801 Dongkang Biological;

Recombinant human basic fibroblast growth factor bEGE (activity>1.0x106IU/mg, catalog number: HYC005M01);

Recombinant human insulin growth factor-1 rhIGF-1 LR3 CAS No: 946870-92-4) Wuhan Heyuan Biotechnology Co., Ltd.;

Recombinant human vascular endothelial growth factor OsrhVEGF (monomer 19.2 kD, catalog number: HYC026M01) Wuhan Heyuan Biotechnology Co., Ltd.;

Recombinant human serum albumin HSA Catalog number: HYC002M01 Wuhan Heyuan Biotechnology Co., Ltd.;

Recombinant human transferrin (cat: 10-366A, molecular weight 80kDa, provided by PeproTech, USA;

Recombinant human insulin solution (10 mg/mL) (1 mL), SKU: SP002460001;

L-Alanyl-L-glutamine solution (200 mM), 100×, catalog number: SP002450100, provided by Sperlikon;

HEPES solution (1M), catalog number: SP002160100, provided by Spericon;

Trypsin, with phenol red, Catalog No.: 25200072; Collagenase type II, powder, Catalog No.: 17101015, provided by Gibco;

Hydrocortisone, product number 50-23-7; provided by Merck;

Stem cell mild enzyme: Youkang NC1004.1;

Preparation method of wood pine extract:

(1) Take 5.7 kg of Nanchuan Pampas grass fruit, remove impurities, wash and crush, and soak and extract with ethyl acetate at room temperature for 5 times, each time for 3 days, and concentrate the extract under reduced pressure to obtain ethyl acetate extract (260 g);

(2) Weigh 0.3 g of ethyl acetate extract, pass the ethyl acetate extract through an MCI column, and perform gradient elution through a methanol-water system at a ratio of 30% → 50% → 70% → 90% → 100%; separate the methanol-insoluble portion of the ethyl acetate extract by silica gel column chromatography, and perform elution through a petroleum ether-ethyl acetate system at a ratio of 30:1 → 1:1 to obtain five fractions Fr.1 to Fr.5, and perform elution through a petroleum ether-acetone system at a ratio of 50:1 → 1:1 to obtain four subfractions Fr.2-1 to Fr.2-4, wherein crystals precipitate from the Fr.2-2 fraction, and compound 4 is obtained by repeated recrystallization; the Fr.2-4 fraction is eluted through a developing solvent system of petroleum ether: acetone to obtain compound 4; Compound 5 was purified by PTLC thin layer chromatography with a developing solvent system of petroleum ether: ethyl acetate in a volume ratio of 15:1, and compound 6 was obtained by PTLC thin layer chromatography with a developing solvent system of petroleum ether: ethyl acetate in a volume ratio of 20:1. The Fr.5 fraction was eluted with petroleum ether-acetone at a ratio of 50:1→1:1 to obtain 4 subfractions Fr.5-1~Fr.5-4. Fr.5-1 was purified by PTLC thin layer chromatography with a developing solvent system of petroleum ether: acetone in a volume ratio of 10:1 to obtain compounds 1 and 2; the crystals precipitated from Fr.5-2 were washed by recrystallization to obtain compound 3, and compounds 1-6 were mixed in a ratio of 1:1:1:1:1:1 to obtain the wood borage extract.

Embodiment 1:

Provided is a culture medium for menstrual mesenchymal stem cells, the culture medium comprising 500ml of DMEM/F12, 2.75ng of recombinant human serum albumin HSA, 1.1mg of recombinant human basic fibroblast growth factor bFGF, 27.5mg of recombinant human insulin growth factor-1 rhIGF-1 LR3, 2.75mg of recombinant human vascular endothelial growth factor OsrhVEGF, 0.275mg/ml of recombinant human transferrin (Transferrin), 5.5mg of recombinant human insulin solution, 6.6mg of recombinant human epidermal growth factor, 0.006mM L-alanyl-L-glutamine solution, and 5.5mg of Xanthoceras chinensis extract, wherein the 2.75ng of recombinant human serum albumin HSA, 1.1mg of recombinant human basic fibroblast growth factor bFGF, 27.5mg of recombinant human insulin growth factor-1 rhIGF-1 LR3, LR3, 2.75mg recombinant human vascular endothelial growth factor OsrhVEGF, 0.275mg/ml recombinant human transferrin, 5.5mg recombinant human insulin solution, 6.6mg recombinant human epidermal growth factor, 0.006mM L-alanyl-L-glutamine solution, 5.5mg Xanthoceras chinensis extract were diluted to 50ml with deionized water, and the pH range was adjusted to 7.2-7.5 with HEPES buffer;

A method for extracting and culturing menstrual blood mesenchymal stem cells according to the above culture medium comprises the following steps:

S1. Sample collection: using an in vitro tissue collection method, when meat-like or coagulated blood-like contents are found, the samples are placed in a first preservation tube containing an anticoagulant, antibiotics, mixed enzymes and culture medium, wherein the volume ratio of the anticoagulant, antibiotics, mixed enzymes and culture medium solution is 1:1:1:80, including the mixed enzyme comprising 1% type II collagenase and 1% trypsin, the 1% type II collagenase and the 1% trypsin are mixed in a 1:1 ratio, the anticoagulant is heparin sodium, and the antibiotic is 5 kU/ml penicillin-streptomycin-neomycin solution (PSN) (100×);

S2. Isolation and extraction of MenSCs:

(1) After thoroughly mixing the sample in the storage tube, filter it with a 100um cell strainer to remove blood clots and mucus. Then slowly add the filtered sample to the density gradient centrifugation solution TBD (50ml centrifuge tube) with a sample to TBD ratio of 1:1. Centrifuge at room temperature at 2000r.min ^- 1 for 10min. During the whole process, ensure that the separation interface is clear. After the centrifugation, extract the middle white film layer into a new centrifuge tube and keep the bottom red blood cell separation layer;

(2) Pipette the buffy coat layer into another clean centrifuge tube, add PBS (1% triple antibody) and repeat centrifugation washing once. Use a sterile blade or forceps to cut the menstrual blood tissue into 4-5 mm rectangular pieces in a culture dish;

(3) Resuspend the red blood cell pellet and the preserved tissue with an equal volume of red blood cell lysis buffer to remove the red blood cells in the tissue fluid, first lyse at room temperature for 5 minutes, and then centrifuge horizontally at 1200r/min for 5 minutes;

(4) After centrifugation, add sterile PBS (1% antibiotics) to the pellet and resuspend it. Centrifuge horizontally at 1200 r.min-5 min, repeat once, remove the supernatant, resuspend the pellet with digestion solution to remove collagen and nucleic acid in the tissue fluid, centrifuge again and discard the supernatant, resuspend the pellet with PBS, centrifuge at 1200 r.min-5 min, wash the MenSCs solution with sterile D-Hanks solution, centrifuge at 1200 r.min-5 min and discard the supernatant, repeat twice to obtain MenSCs pellet;

(5) Coat the T25 culture flask with 0.1 mg/mL poly-L-lysine solution (10×), incubate at 37°C, 5% CO ₂ for 60 min, discard the coating solution, incubate at 37°C for 60 min, and wash once with PBS;

(6) Adjust the concentration of the MenSCs suspension, 2 ml of no more than 0.3 ml of precipitation, inoculate into T25 culture flasks, and add about 2 ml of culture medium to each flask. Too much culture medium will affect cell adhesion.

(7) Spread the separated and extracted menstrual blood tissue evenly in a pre-coated T25 culture bottle;

S3. Collection of primary MenSCs:

(1) Adherent culture at 37°C and 5% CO _2. To inhibit the proliferation of fibroblasts, 400 ng/ml hydrocortisone was added to the culture medium to promote the attachment and growth of menstrual blood stem cells. The growth of cells and the presence of contamination were observed. On the fourth day, half of the culture medium was replaced to remove cells that did not adhere to the wall. After the adherent cells grew, the culture medium was replaced every 48-72 hours. 400 ng/ml hydrocortisone was not added to the replaced culture medium.

(2) When the degree of confluence reaches 80-90%, add 0.25% stem cell mild enzyme to the washed culture flask, about 1 ml per flask, and gently shake the flask to allow the digestion solution in each flask to evenly infiltrate the bottom of the flask. Place it in the incubator for 2 minutes and observe. When the cells become round and most of the cells fall off, immediately add 2 times the volume of stem cell mild enzyme used in cell culture supernatant or complete culture medium to dilute the cell suspension;

(3) Transfer the cell suspension in the culture flask to a 50 ml centrifuge tube, then wash the residual cells adhering to the culture flask with physiological saline, transfer the washing solution to the above centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant after centrifugation;

(4) Then add 3-5 ml of complete culture medium to resuspend the cells, take 1 ml of the cell suspension and make the volume to 10 ml for sampling and counting to calculate the cell survival rate;

S4. Subculture of MenSCs: In the subculture, further subculture is performed according to the actual number of cells, and the cell number is inoculated at a density of 5000 cells/ ^cm2 . The harvested primary MenSCs are placed in a culture bottle. Since the screening of primary cells has high requirements, if the cell density is not enough, the cells cannot be subcultured and will die quickly. For example, please refer to Figure 3, which is a schematic diagram of cell death caused by subculture due to insufficient cell density provided in an embodiment of the present application; if the cell density is too high, the subculture effect will be greatly reduced due to cell stacking. For example, please refer to Figure 4, which is a situation where stacking occurs due to excessively high cell density provided in an embodiment of the present application. The primary cells meet the conditions that there is no obvious halo around the cells, and they are round, bipolar or tripole in the first three days; on the 10th to 15th day, the cells are spindle-shaped or fibrous, grow well, and form several cells that aggregate in an island-like manner with uniform morphology and close arrangement. Based on the above screening principles, the primary cells that can be subcultured screened out in this application are shown in Figure 5, and see Figure 6, which is a diagram of primary cells that cannot be subcultured based on the above screening principles provided in the embodiments of this application. The screened primary cells that can be subcultured are placed at 37°C, 5% CO ₂ , cultured under saturated humidity conditions to 80-90% confluence for subculture inoculation; referring to FIG2 , from left to right are the morphological diagrams of P0, P1 and P4 cells cultured by the method of Example 1 in the present application; as a comparative example, referring to FIG1 , from left to right are the morphological diagrams of P0, P1 and P4 cells cultured in ordinary culture medium; culture of P4 and later generations: after mild enzymatic digestion of stem cells into single cells, LIPUS was used to irradiate MenSCs with an intensity of 20P, an output power of 25 mW/cm ² , a frequency of 10kHz, and an intensity of P for LIPUS irradiation output for 1S.

In order to show that MenSCs in this application has the best repair effect on rat endometrium, three groups of effect diagrams are listed. Figures 8 to 10 are schematic diagrams of unrepaired rat endometrium after injury, schematic diagrams of PRP-repaired rat endometrium after injury, and schematic diagrams of MenSCs-repaired rat endometrium in this Example 1 after injury. The three groups of effect diagrams can clearly and intuitively show that MenSCs in this example has the best repair effect on rat endometrium.

Embodiment 2:

Provided is a culture medium for menstrual mesenchymal stem cells, the culture medium comprising 500 ml DMEM/F12, 3.85 ng recombinant human serum albumin HSA, 1.37 mg recombinant human basic fibroblast growth factor bFGF, 49.5 mg recombinant human insulin growth factor-1 rhIGF-1 LR3, 6.6 mg recombinant human vascular endothelial growth factor OsrhVEGF, 27.5 ng recombinant human transferrin (Transferrin), 5.5 ng recombinant human insulin solution, 8.25 ng recombinant human epidermal growth factor, 0.006 mM L-alanyl-L-glutamine solution, and 6.6 mg wood borage extract, wherein the 3.85 ng recombinant human serum albumin HSA, 1.37 mg recombinant human basic fibroblast growth factor bFGF, 49.5 mg recombinant human insulin growth factor-1 rhIGF-1 LR3, LR3, 6.6 mg recombinant human vascular endothelial growth factor OsrhVEGF, 27.5 ng recombinant human transferrin, 5.5 ng recombinant human insulin solution, 8.25 ng recombinant human epidermal growth factor, 0.006 mM L-alanyl-L-glutamine solution, 6.6 mg Xanthoceras chinensis extract were diluted to 50 ml with deionized water, and the pH range was adjusted to 7.2-7.5 with HEPES buffer;

A method for extracting and culturing menstrual blood mesenchymal stem cells according to the above culture medium comprises the following steps:

S1. Collecting samples: using an in vitro tissue collection method, when meat-like or coagulated blood-like contents are found, the samples are placed in a first preservation tube containing an anticoagulant, antibiotics, mixed enzymes and culture medium, wherein the volume ratio of the anticoagulant, antibiotics, mixed enzymes and culture medium solution is 1:1:1:100, the mixed enzyme includes 1% type II collagenase and 1% trypsin, the 1% type II collagenase and the 1% trypsin are mixed in a 1:1 ratio, the anticoagulant is heparin sodium, and the antibiotic is 5 kU/ml penicillin-streptomycin-neomycin solution (PSN) (100×);

S2. Isolation and extraction of MenSCs:

(1) After thoroughly mixing the sample in the storage tube, filter it with a 100um cell strainer to remove blood clots and mucus. Then slowly add the filtered sample to the density gradient centrifugation liquid TBD (50ml centrifuge tube) with a sample to TBD ratio of 1:1. Centrifuge at room temperature at 2000r.min ^- 1 for 10min. Ensure that the separation interface is clear during the whole process. After the centrifugation, extract the middle white film layer into a new centrifuge tube and keep the bottom red blood cell separation layer;

(2) Pipette the buffy coat layer into another clean centrifuge tube, add PBS (1% triple antibody) and repeat centrifugation washing once. Use a sterile blade or forceps to cut the menstrual blood tissue into 4-5 mm rectangular pieces in a culture dish;

(3) Resuspend the red blood cell pellet and the preserved tissue with an equal volume of red blood cell lysis buffer to remove the red blood cells in the tissue fluid, first lyse at room temperature for 5 minutes, and then centrifuge horizontally at 1200r/min for 5 minutes;

(4) After centrifugation, add sterile PBS (1% antibiotics) to the pellet and resuspend it. Centrifuge horizontally at 1200 r.min-5 min, repeat once, remove the supernatant, resuspend the pellet with digestion solution to remove collagen and nucleic acid in the tissue fluid, centrifuge again and discard the supernatant, resuspend the pellet with PBS, centrifuge at 1200 r.min-5 min, wash the MenSCs solution with sterile D-Hanks solution, centrifuge at 1200 r.min-5 min and discard the supernatant, repeat twice to obtain MenSCs pellet;

(5) Coat the T25 culture flask with 0.1 mg/mL poly-L-lysine solution (10×), incubate at 37°C, 5% CO ₂ for 60 min, discard the coating solution, incubate at 37°C for 60 min, and wash once with PBS;

(6) Adjust the concentration of the MenSCs suspension, 2 ml of no more than 0.3 ml of precipitation, inoculate into T25 culture flasks, and add about 2 ml of culture medium to each flask. Too much culture medium will affect cell adhesion.

(7) Spread the separated and extracted menstrual blood tissue evenly in a pre-coated T25 culture bottle;

S3. Collection of primary MenSCs:

(1) Adherent culture at 37°C and 5% _CO2 . To inhibit the proliferation of fibroblasts, 400 ng/ml hydrocortisone was added to the culture medium to promote the attachment and growth of menstrual blood stem cells. The growth of cells and the presence of contamination were observed. On the fourth day, half of the culture medium was replaced to remove non-adherent cells. After the adherent cells grew, the culture medium was replaced every 48-72 hours.

(2) When the degree of confluence reaches 80-90%, add 0.25% stem cell mild enzyme to the washed culture flask, about 1 ml per flask, and gently shake the flask to allow the digestion solution in each flask to evenly infiltrate the bottom of the flask. Place it in the incubator for 2 minutes and observe. When the cells become round and most of the cells fall off, immediately add 2 times the volume of stem cell mild enzyme used in cell culture supernatant or complete culture medium to dilute the cell suspension;

(3) Transfer the cell suspension in the culture flask to a 50 ml centrifuge tube, then wash the residual cells adhering to the culture flask with physiological saline, transfer the washing solution to the above centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant after centrifugation;

(4) Then add 3-5 ml of complete culture medium to resuspend the cells, take 1 ml of the cell suspension and make the volume to 10 ml for sampling and counting to calculate the cell survival rate;

S4. Subculture of MenSCs: The subculture is further subcultured according to the actual number of cells, and the cell number is seeded at a density of 5000-10000 cells/ ^cm2 , that is, 5000 cells/ ^cm2 are seeded for P1-P6, 8000 cells/ ^cm2 are seeded for P7-P11, and 10000 cells/cm2 are seeded for P12 and later generations. ^2. Inoculation of cells. The cell activity decreases with the increase of the passage number. The cell inoculation density can be adjusted according to the passage number during the passage process, thereby further improving the stability of the passage. The harvested primary MenSCs are placed in a culture bottle. The primary cells meet the requirement of no obvious halo around the cells. They are round, bipolar or tripole in the first three days. On the 10th to 15th day, the cells are spindle-shaped or fibrous, grow well, and form small islands with several cells aggregated in a uniform morphology and closely arranged. The selected primary cells that can be subcultured are placed at 37°C, 5% _CO2 , and saturated humidity. When the confluence is 80-90%, the operation is carried out for subculture inoculation. P4 and later cultures: After the stem cells are gently enzymatically digested into single cells, LIPUS is used to irradiate MenSCs with an intensity of 20P, an output power of 25 mW/ ^cm2 , a frequency of 10kHz, and an intensity of P for LIPUS irradiation output for 1S.

Embodiment 3:

Provided is a culture medium for menstrual mesenchymal stem cells, the culture medium comprising 500 ml DMEM/F12, 5.5 ng recombinant human serum albumin HSA, 1.5 mg recombinant human basic fibroblast growth factor bFGF, 66 mg recombinant human insulin growth factor-1 rhIGF-1 LR3, 11 mg recombinant human vascular endothelial growth factor OsrhVEGF, 55 ng recombinant human transferrin, 5.5 ng recombinant human insulin solution, 11 ng recombinant human epidermal growth factor, 0.006 mM L-alanyl-L-glutamine solution, and 8.25 mg Xanthoceras chinensis extract, wherein the 5.5 ng recombinant human serum albumin HSA, 1.5 mg recombinant human basic fibroblast growth factor bFGF, 66 mg recombinant human insulin growth factor-1 rhIGF-1 LR3, 11 mg recombinant human vascular endothelial growth factor OsrhVEGF, 55 ng recombinant human transferrin , 5.5 ng recombinant human insulin solution, 11 ng recombinant human epidermal growth factor, 0.006 mM L-alanyl-L-glutamine solution, 8.25 mg Xanthoceras chinensis extract were diluted to 50 ml with deionized water, and the pH range was adjusted to 7.2-7.5 with HEPES buffer;

A method for extracting and culturing menstrual blood mesenchymal stem cells according to the above culture medium comprises the following steps:

S1. Sample collection: using an in vitro tissue collection method, when meat-like or coagulated blood-like contents are found, the samples are placed in a first preservation tube containing an anticoagulant, antibiotics, mixed enzymes and culture medium, wherein the volume ratio of the anticoagulant, antibiotics, mixed enzymes and culture medium solution is 1:1:1:120, the mixed enzyme includes 1% type II collagenase and 1% trypsin, the 1% type II collagenase and the 1% trypsin are mixed in a 1:1 ratio, the anticoagulant is heparin sodium, and the antibiotic is 5 kU/ml penicillin-streptomycin-neomycin solution (PSN) (100×);

S2. Isolation and extraction of MenSCs:

(1) After thoroughly mixing the sample in the storage tube, filter it with a 100um cell strainer to remove blood clots and mucus. Then slowly add the filtered sample to the density gradient centrifugation solution TBD (50ml centrifuge tube) with a sample to TBD ratio of 1:1. Centrifuge at room temperature at 2000r.min ^- 1 for 10min. During the whole process, ensure that the separation interface is clear. After the centrifugation, extract the middle white film layer into a new centrifuge tube and keep the bottom red blood cell separation layer;

(2) Pipette the buffy coat layer into another clean centrifuge tube, add PBS (1% triple antibody) and repeat centrifugation washing once. Use a sterile blade or forceps to cut the menstrual blood tissue into 4-5 mm rectangular pieces in a culture dish;

(3) Resuspend the red blood cell pellet and the preserved tissue with an equal volume of red blood cell lysis buffer to remove the red blood cells in the tissue fluid, first lyse at room temperature for 5 minutes, and then centrifuge horizontally at 1200r/min for 5 minutes;

(4) After centrifugation, add sterile PBS (1% antibiotics) to the pellet and resuspend it. Centrifuge horizontally at 1200 r.min-5 min, repeat once, remove the supernatant, resuspend the pellet with digestion solution to remove collagen and nucleic acid in the tissue fluid, centrifuge again and discard the supernatant, resuspend the pellet with PBS, centrifuge at 1200 r.min-5 min, wash the MenSCs solution with sterile D-Hanks solution, centrifuge at 1200 r.min-5 min and discard the supernatant, repeat twice to obtain MenSCs pellet;

(5) Coat the T25 culture flask with 0.1 mg/mL poly-L-lysine solution (10×), incubate at 37°C, 5% CO ₂ for 60 min, discard the coating solution, incubate at 37°C for 60 min, and wash once with PBS;

(6) Adjust the concentration of the MenSCs suspension, 2 ml of no more than 0.3 ml of precipitation, inoculate into T25 culture flasks, and add about 2 ml of culture medium to each flask. Too much culture medium will affect cell adhesion.

(7) Spread the separated and extracted menstrual blood tissue evenly in a pre-coated T25 culture bottle;

S3. Collection of primary MenSCs:

(1) Adherent culture at 37°C and 5% _CO2 . To inhibit the proliferation of fibroblasts, 400 ng/ml hydrocortisone was added to the culture medium to promote the attachment and growth of menstrual blood stem cells. The growth of cells and the presence of contamination were observed. On the fourth day, half of the culture medium was replaced to remove non-adherent cells. After the adherent cells grew, the culture medium was replaced every 48-72 hours.

(2) When the degree of confluence reaches 80-90%, add 0.25% stem cell mild enzyme to the washed culture flask, about 1 ml per flask, and gently shake the flask to allow the digestion solution in each flask to evenly infiltrate the bottom of the flask. Place it in the incubator for 2 minutes and observe. When the cells become round and most of the cells fall off, immediately add 2 times the volume of stem cell mild enzyme used in cell culture supernatant or complete culture medium to dilute the cell suspension;

(3) Transfer the cell suspension in the culture flask to a 50 ml centrifuge tube, then wash the residual cells adhering to the culture flask with physiological saline, transfer the washing solution to the above centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant after centrifugation;

(4) Then add 3-5 ml of complete culture medium to resuspend the cells, take 1 ml of the cell suspension and make the volume to 10 ml for sampling and counting to calculate the cell survival rate;

S4. Subculture of MenSCs: In the subculture, further subculture is performed according to the actual number of cells, and the cell number is inoculated at a density of 8000 cells/ ^cm2 . The harvested primary MenSCs are placed in a culture bottle, and the primary cells meet the conditions that there is no obvious halo around the cells and they are round, bipolar or tripole in the first three days; on the 10th to 15th day, the cells are spindle-shaped or fibrous, grow well, and form several cells that aggregate in an island-like shape with uniform morphology and close arrangement. The selected primary cells that can be subcultured are placed at 37°C, 5% _CO2 , and saturated humidity. When the confluence is 80-90%, the operation is carried out for subculture inoculation; P4 and later cultures: After the stem cells are gently enzymatically digested into single cells, LIPUS is used to irradiate MenSCs with an intensity of 20P, an output power of 25 mW/ ^cm2 , a frequency of 10kHz, and the intensity of LIPUS irradiation output for 1S is P.

Comparative Example, referring to FIG. 7 , the cell passage diagrams of the primary cells cultured by the method after 24h, 48h, and 72h are obtained. The specific implementation method is as follows:

Collection, separation and culture process:

(1) Use a menstrual cup to collect menstrual blood during the first 3 days of the menstrual cycle. The menstrual cup should be kept in the body for an average of 3 hours (2-4 hours).

(2) Transfer the menstrual blood into phosphate-buffered saline containing 1% penicillin and streptomycin, store at 4°C, and return to the laboratory within 24 hours after collection.

(3) Sieve the menstrual blood (100 μm) and add Ficoll separation solution with a volume ratio of Ficoll to menstrual blood of 2:3.

(4) Centrifuge at 800 g for 15 min, up 5, down 3. Aspirate the white film layer after centrifugation, wash twice with PBS, add appropriate amount of culture medium to resuspend, and inoculate into a 6-well plate. Inoculate 7 ml of blood-separated cells into one well of the 6-well plate.

(5) The culture medium was changed every 72 hours. The culture medium included: basal medium DMEMFS 90%, serum 10%, Glutamine 1%, and double antibody 1%.

As can be seen intuitively from FIG7 , in this comparative example, the cells were cultured for 72 hours and the original generation performed well, but by the fourth generation they aged and grew slowly or could not grow.

The beneficial effects of the present invention are described below by experiments:

The data are obtained in Example 2.

The above data and the accompanying drawings clearly show that the present invention can stably propagate to the 15th generation, and the method of the present invention can stably cultivate more stable and longer generations. Unless otherwise specified in the text, it is understood and known by those skilled in the art based on the existing technology and common knowledge they master.

The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims (9)

1. A culture medium for menstrual mesenchymal stem cells, characterized in that the culture medium comprises DMEM/F12, 5-10ug/ml recombinant human serum albumin HSA, 2-3ng/ml recombinant human basic fibroblast growth factor bFGF, 50-120ng/ml recombinant human insulin growth factor-1 rhIGF-1 LR3, 5-20ng/ml recombinant human vascular endothelial growth factor OsrhVEGF, 0.5-100μg/ml recombinant human transferrin, 10μg/ml recombinant human insulin solution, 12-20ug/ml recombinant human epidermal growth factor, 0.006mM L-alanyl-L-glutamine solution, 10-15ng/ml wood bark extract, HEPES buffer is used to adjust the pH range to 7.2-7.5, and the wood bark extract comprises ascorbyl dipalmitate, butyl linoleate, ethyl linolenate, ethyl palmitate, methyl linoleate, and methyl palmitate. 2. The culture medium for menstrual blood mesenchymal stem cells according to claim 1, wherein the method for extracting the Xylocarpus chinensis extract comprises the following steps: (1) Take the fruit of the Nanchuan Pampas grass, remove impurities, wash it, crush it, and soak it in ethyl acetate for 5 times at room temperature, each time for 3 days, and concentrate the extract under reduced pressure to obtain the ethyl acetate extract; (2) Weigh 0.3 g of ethyl acetate extract, pass the ethyl acetate extract through an MCI column, and perform gradient elution through a methanol-water system at a ratio of 30% → 50% → 70% → 90% → 100%; separate the methanol-insoluble portion of the ethyl acetate extract by silica gel column chromatography, and perform elution through a petroleum ether-ethyl acetate system at a ratio of 30:1 → 1:1 to obtain five fractions Fr.1 to Fr.5, and perform elution through a petroleum ether-acetone system at a ratio of 50:1 → 1:1 to obtain four subfractions Fr.2-1 to Fr.2-4, wherein crystals precipitate from the Fr.2-2 fraction, and compound 4 is obtained by repeated recrystallization; the Fr.2-4 fraction is eluted through a developing solvent system of petroleum ether: acetone to obtain compound 4; Compound 5 was purified by PTLC thin layer chromatography with a developing solvent system of petroleum ether: ethyl acetate in a volume ratio of 15:1, and compound 6 was obtained by PTLC thin layer chromatography with a developing solvent system of petroleum ether: ethyl acetate in a volume ratio of 20:1. The Fr.5 fraction was eluted with petroleum ether-acetone at a ratio of 50:1→1:1 to obtain 4 subfractions Fr.5-1~Fr.5-4. Fr.5-1 was purified by PTLC thin layer chromatography with a developing solvent system of petroleum ether: acetone in a volume ratio of 10:1 to obtain compounds 1 and 2; the crystals precipitated from Fr.5-2 were washed by recrystallization to obtain compound 3, and compounds 1-6 were mixed in a ratio of 1:1:1:1:1:1 to obtain the wood borage extract. 3. A method for extracting and culturing menstrual blood mesenchymal stem cells, characterized in that the menstrual blood mesenchymal stem cells are obtained by culturing using the culture medium of any one of claims 1 to 2, and the culturing method specifically comprises the following steps: S1. Sample collection: When meat-like or coagulated blood-like contents are found by in vitro tissue collection, the samples are placed in a first preservation tube containing an anticoagulant, an antibiotic, a mixed enzyme, and a culture medium. The volume ratio of the anticoagulant, the antibiotic, the mixed enzyme, and the culture medium is 1:1:1:80-120. The mixed enzyme includes 1% type II collagenase and 1% trypsin. The 1% type II collagenase and the 1% trypsin are mixed in a ratio of 1:1. The anticoagulant is sodium heparin, and the antibiotic is 5 kU/ml penicillin-streptomycin-neomycin solution PSN, 100×; S2. Extraction and separation of MenSCs: After centrifugation and washing of the samples, 5 ml of an adhesion promoter was added to each bottle of samples, and the adhesion promoter completely soaked the bottom of the bottle. The adhesion promoter included 0.1 mg/mL poly-L-lysine solution 10×, incubated at 37°C, 5% CO ₂ for 60 min, then the adhesion promoter solution was discarded, washed once with PBS, cut into pieces and evenly spread in a T25 culture bottle with culture medium added; S3. Collection of primary MenSCs: Add 400ng/ml hydrocortisone to the culture medium at 37℃ and 5% _CO2 concentration, and culture the cells to adhere to the wall. After observing the growth of the cells and whether there is any contamination, replace half of the culture medium once on the 4th day to remove the cells that are not attached to the wall. After the attached cells grow, replace the culture medium every 48-72h. When the degree of confluence reaches 80-90%, the primary MenSCs can be digested and harvested. S4. Subculture of MenSCs: In the subculture, further subculture is performed according to the actual number of cells, and the cell number is inoculated at a density of 5000-10000 cells/ ^cm2 . The primary MenSCs after harvest are placed in a culture bottle, and cultured at 37°C, 5% _CO2 , and saturated humidity. When the confluence is 80-90%, subculture is performed; P4 and later cultures: After the stem cells are gently enzymatically digested into single cells, LIPUS is used to irradiate the daughter MenSCs with an intensity of 20P, an output power of 25 mW/ ^cm2 , a frequency of 10kHz, and the intensity of LIPUS irradiation output 1S is P. 4. The method for extracting and culturing menstrual blood mesenchymal stem cells according to claim 3 is characterized in that the sample centrifugation and washing in S2 comprises: sample mixing and filtration - centrifugation of the filtered sample in density gradient centrifugation solution TBD - taking the buffy coat layer - washing the buffy coat layer with PBS - resuspending the bottom red blood cell sediment and preserved tissue with an equal volume of red blood cell lysis separation solution - lysis centrifugation - taking the sediment, adding sterile PBS 1% antibiotics, resuspending and centrifuging to remove the supernatant - resuspending the sediment with digestion solution - centrifuging again to remove the supernatant - resuspending the sediment with PBS - centrifugation - repeating twice with sterile D-Hanks solution to wash the MenSCs solution - MenSCs sediment. 5 . The method for extracting and culturing menstrual blood mesenchymal stem cells according to claim 4 , wherein if the number of red blood cells in the buffy coat obtained after filtration in S2 is small, the lysis and centrifugation step is omitted and a multiple wash method is used. 6. The method for extracting and culturing menstrual blood mesenchymal stem cells according to claim 3, characterized in that the digestion in S3 is to add 0.25% stem cell mild enzyme to the washed culture bottle, and immediately add cell culture supernatant or complete culture medium with a volume twice the amount of stem cell mild enzyme used to dilute the cell suspension after the cells become round and most of the cells fall off, transfer the cell suspension in the culture bottle to a centrifuge tube, wash the residual cells adhering to the culture bottle with physiological saline, discard the supernatant after centrifugation, and then add complete culture medium to resuspend the cells, take the cell suspension to a fixed volume for sampling and counting, and calculate the cell survival rate. 7. The method for extracting and culturing menstrual blood mesenchymal stem cells according to claim 3, characterized in that the primary culture time of the MenSCs shall not exceed 14 days. 8. The method for extracting and culturing menstrual mesenchymal stem cells according to claim 3 is characterized in that the primary MenSCs are screened and then subcultured, and the primary MenSCs meet the conditions that there is no obvious halo around the cells, and they are round, bipolar or tripole in the first three days; on the 10th to 15th day, the cells are spindle-shaped or fibrous, grow well, and form small islands with several cells aggregated in a uniform morphology and closely arranged. 9 . The method for extracting and culturing menstrual blood mesenchymal stem cells according to claim 3 , characterized in that, in S2 , the concentration of the MenSCs suspension is adjusted to 2 ml so that no more than 0.3 ml of MenSCs precipitation is obtained, and the MenSCs suspension is inoculated into a T25 culture flask.