A method for preparing high-purity menstrual blood-derived stem cells

Abstract

The present invention provides a method for preparing high-purity menstrual blood-derived stem cells: using a menstrual blood collection kit to collect menstrual blood; collecting middle layer mononuclear cells; adding magnetic bead buffer, adding FcR reagent after fully mixing, adding micromagnetic bead reagent of cross-linked anti-human antibody after incubation, mixing and incubating; then adding magnetic bead buffer, washing, centrifuging and resuspending in magnetic bead buffer; adding to a sorting column pre-placed in a magnetic sorting rack, using magnetic bead buffer to wash the column; taking out the sorting column, adding 1.5-2ml magnetic bead buffer, collecting cell suspension; culturing the obtained suspension with menstrual blood stem cell primary culture medium, changing the liquid once every 2-3 days; digesting and collecting adherent cells after 5-7 days of culture, continuing to expand and culture with menstrual blood stem cell passage culture medium; digesting the obtained cells, adding freezing solution for freezing. The menstrual blood-derived stem cells obtained by this method have high purity, short growth cycle, strong self-renewal ability and low contamination rate.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell separation and purification, and in particular to a method for preparing, culturing and amplifying in vitro, and freezing high-purity menstrual blood-derived stem cells. Background Art

[0002] Menstrual stem cells are derived from the endometrial tissue that is shed with the menstrual blood during women's menstruation. They are a type of adult stem cells and have multidirectional differentiation potential close to that of embryonic stem cells. They have broad clinical application prospects in major diseases such as bone injury, spinal cord injury, nerve injury, liver disease, diabetes, and cardiovascular disease. Studies have found that menstrual blood-derived stem cells have incomparable advantages over embryonic, bone marrow, and umbilical cord blood stem cells, such as: the collection method is simple and safe, non-invasive, and can be collected completely by yourself without causing harm to the donor; the number of isolated stem cells is 30 times that of bone marrow-derived stem cells, which can provide a rich source of stem cells for disease treatment; the in vitro proliferation ability is strong, generally doubling in 24-36 hours, and can be passaged up to 50 times; the proliferation ability is more than 10 times that of bone marrow stem cells, and can provide enough cells required for treatment in a short time; the differentiation potential is large, in addition to the general characteristics of mesenchymal stem cells, it can also express embryonic stem cell surface markers such as Oct-4, SSEA-4 and c-Kit / CD117, indicating the great potential of menstrual blood-derived stem cells in regenerative medicine; after multiple passages, there is no abnormal change in the chromosome karyotype, and animal experiments show no tumorigenicity, indicating the safety of menstrual blood-derived stem cells in clinical applications; menstrual blood stem cells also have the advantages of low immunogenicity and can be used for allogeneic transplantation without the risk of immune rejection. In addition, menstrual stem cells are widely available, and theoretically most healthy premenopausal women can serve as potential donors without any ethical controversy.

[0003] However, under the existing collection and separation culture technology conditions, due to the various types of cells and different functions in the collected menstrual blood samples, the cells show diverse morphology and poor uniformity in the primary culture stage, indicating that there are many mixed cells and low purity of stem cells. It is necessary to culture them for 3-5 generations to obtain menstrual blood stem cells with higher purity. At the same time, there is a greater risk of contamination by pathogens and mycoplasma when culturing cells under this technical method. The above limitations will affect the further application of the obtained stem cells in experimental research and clinical applications. Summary of the invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and to provide a method for preparing high-purity menstrual blood-derived stem cells. The menstrual blood-derived stem cells obtained by this method have the characteristics of high purity, short growth cycle, strong self-renewal ability, low contamination rate, etc. Within 20 generations of in vitro culture, the stem cells do not show obvious aging and differentiation phenomena, and can maintain a relatively fast proliferation rate, stable morphology and phenotypic characteristics.

[0005] The technical solution adopted by the present invention is:

[0006] A method for preparing high-purity menstrual blood-derived stem cells, the method comprising the following steps:

[0007] (1) Using a menstrual blood collection kit, collect menstrual blood into a menstrual blood collection tube containing an antibiotic preservation solution, wherein the mixed volume ratio of the antibiotic preservation solution to the menstrual blood is (1-1.5):1, and the menstrual blood is stored at a low temperature of 2-8°C for a storage period of ≤72 hours;

[0008] (2) centrifuging the menstrual blood collection tube, discarding the supernatant, and collecting the cell pellet;

[0009] (3) After the cell pellet is fully mixed with saline at a volume ratio of 1:(1-1.5), it is slowly added to the interface of an equal volume of lymphocyte separation fluid, centrifuged, the middle layer mononuclear cells are collected, and then washed with saline and centrifuged;

[0010] (4) The obtained mononuclear cells were sampled and the number of cells was counted. 8 Cells: Add 100-200 μl magnetic bead buffer, mix thoroughly, add 50-100 μl FcR reagent, incubate at 2-8°C for 5-10 minutes, then add 50-100 μl cross-linked anti-human antibody micromagnetic bead reagent, mix thoroughly, and incubate at 2-8°C for 10-20 minutes;

[0011] (5) Add 1-1.5 ml of magnetic bead buffer to the liquid obtained in step (4), wash, centrifuge and resuspend in magnetic bead buffer. The concentration of the cell suspension is 1×10 8 cells / ml;

[0012] (6) Add all of the above cell suspension to a separation column pre-placed in a magnetic separation rack, and wash the column with a magnetic bead buffer;

[0013] (7) Take the sorting column out of the sorting rack, place it in a sterile collection tube, add 1.5-2 ml of magnetic bead buffer, and collect the cell suspension;

[0014] (8) The suspension was sampled and the number of cells was counted. After diluting with primary culture medium of menstrual stem cells, the concentration was 5000-7000 cells / cm. 2 The cells were inoculated into cell culture bottles at a density of 100 μg / mL and placed in a 37°C, 5% CO2 incubator for culture. The medium was changed by half every 2-3 days.

[0015] (9) After 5-7 days of cell culture, the cells reached a density of 70-80% under an inverted microscope. The adherent cells were digested and collected and plated at 3000-5000 cells / cm 2The cells were inoculated into cell culture flasks at a density of 100 μg / cm2 and then continued to be expanded and cultured using blood stem cell culture medium;

[0016] (10) The digested cells were mixed with menstrual stem cell cryopreservation solution precooled at 2-8°C and added to a cryopreservation tube at a cell density of 0.1-1×10 7 cells / ml, put them into a freezing box, place them in a -80℃ refrigerator for 12-24 hours, and then transfer them to -196℃ liquid nitrogen for freezing.

[0017] The antibiotic storage solution in step (1) is based on HBSS (Hank's balanced salt solution) and contains 420 units of heparin sodium, 250 μg / mL of cephalexin, 120 μg / mL of gentamicin and 3 μg / mL of amphotericin B.

[0018] The centrifugation in step (2) is specifically: centrifuging at (400-600)×g for 10 minutes.

[0019] The anti-human antibody in step (4) is one of anti-human CD73, CD105, CD166, CD90, CD29, CD44, CD117, CD34, and CD45 monoclonal antibodies.

[0020] The specific formula of the magnetic bead buffer is: phosphate buffered saline as the base liquid, containing 0.5% by volume of fetal bovine serum (FBS) or bovine serum albumin (BSA) or human serum albumin (HSA), and containing ethylenediaminetetraacetic acid (EDTA) with a final concentration of 2 mM.

[0021] The sorting column in step (6) is one of an MS sorting column, an LS sorting column and an XS sorting column.

[0022] The primary culture medium for menstrual stem cells in step (8) contains: 30% by volume of α-MEM culture medium, 40% by volume of Chang Amnio culture medium, 29% by volume of UltraCULTURE culture medium, and 1% by volume of blue / chain dual antibody.

[0023] In the step (8), the cell culture flask is a T25 square flask or a T75 square flask; in the step (9), the cell culture flask is a T150 square flask or a T75 square flask.

[0024] The menstrual stem cell culture medium in step (9) contains: 30% by volume of α-MEM medium, 35% by volume of Chang Amnio medium, 35% by volume of UltraCULTURE medium, 10-20 ng / ml of EGF (epidermal growth factor), and 10-20 ng / ml of bFGF (basic fibroblast growth factor).

[0025] In the step (9), TrypLE Express or 0.25% trypsin is used to digest the cells.

[0026] The menstrual stem cell freezing solution in step (10) contains: 32-40% by volume of cell culture supernatant, 8-10% by volume of dimethyl sulfoxide (DMSO), 25-30% by volume of Chang Amnio culture medium, and 25-30% by volume of UltraCULTURE culture medium.

[0027] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0028] (1) The present invention obtains menstrual blood stem cell precursor cells with multidirectional differentiation potential surface antigen markers by magnetic bead sorting, and then subcultures them in vitro in a serum-free medium, which not only removes a large number of foreign cells but also reduces the risk of contamination. At the same time, since the purity of the initial stem cells in the culture is high, the stem cell amplification rate is fast, the culture cycle is significantly shortened, and the cells obtained by culture have the characteristics of strong multidirectional differentiation ability and high safety in clinical application, and have broad prospects for clinical treatment application;

[0029] (2) The menstrual blood stem cell culture medium obtained is prepared in a certain proportion using commercial culture medium as raw material, without adding foreign serum, with relatively clear ingredients, without the risk of exogenous bacterial and viral contamination, and is suitable for clinical treatment applications;

[0030] (3) The cryopreservation solution used is beneficial to the maintenance and recovery of cell activity, and is beneficial to the functional research and further application of the menstrual blood-derived stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figures show the cell morphology of the menstrual stem cells of Example 1 of the present invention and the comparative example when cultured to the P1 generation under an inverted microscope; wherein (A) is the P1 generation cells of the comparative example, and (B) is the P1 generation cells of Example 1.

[0032] Figure 2 Shown are the cell proliferation curves of Example 1 of the present invention and the comparative example when the menstrual blood stem cells were cultured to the P5 generation.

[0033] Figure 3 The telomerase activity of the blood stem cells of Example 1 of the present invention and the comparative example when cultured to the P5 generation is shown. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to the embodiments, but is not limited thereto.

[0035] Example 1: Preparation of high-purity menstrual blood-derived stem cells

[0036] The preparation of high-purity menstrual blood-derived stem cells in this embodiment specifically includes the following steps:

[0037] (1) Use a menstrual blood collection kit to collect menstrual blood into a menstrual blood collection tube containing an antibiotic preservation solution, with the mixed volume ratio of the antibiotic preservation solution to the menstrual blood being 1:1, and store at a low temperature of 2-8°C for a storage period of ≤72 hours;

[0038] The antibiotic storage solution uses HBSS (Hank's balanced salt solution) as the base solution, and contains 420 units of heparin sodium, 250 μg / mL of cephalexin, 120 μg / mL of gentamicin, and 3 μg / mL of amphotericin B.

[0039] (2) Centrifuge the collection tube at 600×g for 10 minutes, discard the supernatant, and collect the cell pellet.

[0040] (3) After the cell pellet is fully mixed with saline in a volume ratio of 1:1, it is slowly added to the interface of an equal volume of lymph separation fluid, centrifuged, and the middle layer mononuclear cells are collected, and then washed with saline and centrifuged.

[0041] (4) The obtained mononuclear cells were sampled and the number of cells was counted. 8 Cells: Add 100 μl magnetic bead buffer, mix thoroughly, then add 100 μl FcR reagent (Miltenyi Biotec, catalog number 130-091-332), incubate at 2-8°C for 5-10 minutes, then add 100 μl cross-linked anti-human antibody (one of the anti-human CD73, CD105, CD166, CD90, CD29, CD44, CD117, CD34, CD45 monoclonal antibodies) micromagnetic bead reagent (Miltenyi Biotec, catalog number 130-091-332, particle size 50 nm), mix thoroughly, and incubate at 2-8°C for 15 minutes;

[0042] (5) Add 1 ml of magnetic bead buffer, wash, centrifuge and resuspend in magnetic bead buffer. The concentration of the cell suspension is 1×10 8 cells / ml;

[0043] The specific formula of the magnetic bead buffer of this embodiment is: phosphate buffered saline (PBS) as the base liquid, containing 0.5% by volume of fetal bovine serum (FBS) or bovine serum albumin (BSA) or human serum albumin (HSA), and containing ethylenediaminetetraacetic acid (EDTA) with a final concentration of 2mM.

[0044] (6) Add all of the above cell suspension to a MACS separation column placed on a magnetic separation rack, and wash the column with a magnetic bead buffer.

[0045] (7) Remove the sorting column from the sorting rack, place it in a sterile collection tube, add 2 ml of magnetic bead buffer, and collect the cell suspension.

[0046] (8) The suspension was sampled and the number of cells was counted. After diluting with primary culture medium of menstrual stem cells, the suspension was plated at 6000 cells / cm 2 The cells were inoculated into T25 square bottles at a density of 1.50 μg / mL and placed in a 37°C, 5% CO2 incubator for culture. The medium was changed by half every 2-3 days.

[0047] The menstrual stem cell primary culture medium contains: 30% by volume of α-MEM culture medium, 40% by volume of Chang Amnio culture medium, 29% by volume of UltraCULTURE culture medium, and 1% by volume of blue / chain double antibody.

[0048] (9) After 5-7 days of cell culture, the cells reached a density of 70-80% under an inverted microscope. The adherent cells were digested and collected using TrypLE Express and plated at 5000 cells / cm 2 The cells were inoculated into T75 flasks at a density of 100 μg / cm2 and then cultured using menstrual stem cell culture medium.

[0049] The menstrual stem cell passage culture medium contains: 30% by volume of α-MEM culture medium, 35% by volume of Chang Amnio culture medium, 35% by volume of UltraCULTURE culture medium, 10-20 ng / ml of EGF (epidermal growth factor), and 10-20 ng / ml of bFGF (basic fibroblast growth factor).

[0050] (10) The digested cells were mixed with menstrual stem cell cryopreservation solution and added to a cryopreservation tube at a cell density of 1-10×10 7 cells / ml, placed at -80°C for 12-24 hours, and then frozen in -196°C liquid nitrogen.

[0051] The menstrual stem cell freezing solution contains: 32% by volume of cell culture supernatant, 8% by volume of dimethyl sulfoxide (DMSO), 30% by volume of Chang Amnio culture medium, and 30% by volume of UltraCULTURE culture medium.

[0052] Comparative Example:

[0053] (1) Use a menstrual blood collection kit to collect menstrual blood into a menstrual blood collection tube containing an antibiotic preservation solution, with the volume ratio of the antibiotic preservation solution to the menstrual blood being 1:1, and store at a low temperature of 2-8°C for a storage period of ≤72 hours;

[0054] The antibiotic storage solution uses HBSS (Hank's balanced salt solution) as the base solution, and contains 420 units of heparin sodium, 250 μg / mL of cephalexin, 120 μg / mL of gentamicin, and 3 μg / mL of amphotericin B.

[0055] (2) Centrifuge the collection tube at 600×g for 10 minutes, discard the supernatant, and collect the cell pellet.

[0056] (3) After the cell pellet is fully mixed with saline in a volume ratio of 1:1, it is slowly added to the interface of an equal volume of lymph separation fluid, centrifuged, and the middle layer mononuclear cells are collected, and then washed with saline and centrifuged.

[0057] (4) The obtained mononuclear cells were sampled and the number of cells was calculated at 6000 cells / cm 2 The cells were inoculated into T25 square bottles at a density of 100 μg / cm2 and placed in a 37°C, 5% CO2 incubator for culture. The medium was changed by half every 2-3 days.

[0058] The menstrual stem cell primary culture medium contains: 30% by volume of α-MEM culture medium, 40% by volume of Chang Amnio culture medium, 29% by volume of UltraCULTURE culture medium, and 1% by volume of blue / chain double antibody.

[0059] (5) After 10-15 days of cell culture, the cells reached a density of 70-80% under an inverted microscope. The adherent cells were digested and collected using TrypLE Express and plated at 5000 cells / cm 2 The cells were inoculated into T75 flasks at a density of 100 μg / cm2 and continued to be expanded and cultured using menstrual stem cell culture medium.

[0060] The menstrual stem cell passage culture medium contains: 30% by volume of α-MEM culture medium, 35% by volume of Chang Amnio culture medium, 35% by volume of UltraCULTURE culture medium, 10-20 ng / ml of EGF (epidermal growth factor), and 10-20 ng / ml of bFGF (basic fibroblast growth factor).

[0061] (6) The digested cells were mixed with menstrual stem cell cryopreservation solution and added to cryopreservation tubes at a cell density of 1-10×10 7 cells / ml, placed at -80°C for 12-24 hours, and then frozen in -196°C liquid nitrogen.

[0062] The menstrual stem cell freezing solution contains: 32% by volume of cell culture supernatant, 8% by volume of dimethyl sulfoxide (DMSO), 30% by volume of Chang Amnio culture medium, and 30% by volume of UltraCULTURE culture medium.

[0063] Detection method:

[0064] (I) Cell morphology observation:

[0065] Place the cell culture bottle to be tested under an inverted microscope with a camera function for observation. Menstrual blood stem cells are spindle-shaped, refractive, and arranged in clusters under high-density growth conditions. Generally, the more uniform the cell morphology, the higher the purity.

[0066] Figure 1 The cell morphology of the menstrual stem cells of Example 1 and the comparative example observed under an inverted microscope when they were cultured to the P1 generation is shown; wherein (A) is the P1 generation cell of the comparative example, and (B) is the P1 generation cell of the example. From (A), it can be seen that the P1 generation cells of the comparative example have various morphologies, including elliptical, half-moon, polygonal and spindle-shaped, while the P1 generation cells of Example 1 have a more uniform morphology, mainly in the shape of a long spindle (B). It can be seen that the purity of the stem cells in the P1 generation cells of Example 1 is significantly higher than that of the comparative example, and has typical mesenchymal stem cell morphological characteristics.

[0067] (II) Cell proliferation curve:

[0068] When the cells grow to 70-80% density, take out the cells from the incubator and digest them. Take 0.5-1 ml of the cell suspension sample and count it with a cell counter. The obtained cell density multiplied by the total volume of the cell suspension is the total cell volume. The total cell volume is counted at each passage from P1 to P5 of the cell culture. According to the cell counting results, the cell expansion curve is drawn with the culture time (days) as the horizontal axis and the expansion multiple as the vertical axis (the expansion multiple of the P1 generation cells is set to 1).

[0069] Figure 2 The cell proliferation curves of the blood stem cells of Example 1 and the comparative example when cultured to the P5 generation are shown; Figure 2 It can be seen that the cell passage time of each generation in Example 1 is earlier than that in the comparative example, the expansion speed during culture is also significantly faster, and the growth period required to achieve the same expansion multiple is also shorter.

[0070] (III) Telomerase activity analysis:

[0071] During the cell subculture, TRAP (telomerase repeat amplification program) analysis method was used to detect the telomerase activity. 5 The cells to be tested were lysed with lysis buffer and 1 μg of supernatant was taken for telomerase repeat sequence PCR reaction. The reaction product was hybridized with a DIG-labeled probe and the absorbance was detected with an ELISA instrument. In this experiment, human embryonic stem cells (hESC) were used as a positive control (100%).

[0072] Figure 3 The telomerase activity of blood stem cells cultured to P5 in Example 1 of the present invention and the comparative example is shown. Figure 3 It can be seen that the telomerase activity of the menstrual stem cells in Example 1 at each passage time is higher than that of the control example, indicating that the stem cells in Example 1 have stronger self-renewal and proliferation capabilities.

[0073] (IV) Flow cytometric analysis of cell surface markers:

[0074] Take (0.5-1)×10 6 P5 generation menstrual blood stem cells were washed twice with antibody diluent, and then 5 μl of PE (phycoerythrin) or FITC (fluorescein isothiocyanate) labeled anti-human CD73, CD105, CD166, CD90, CD29, CD44, CD117, CD34, CD45, HLA-DR antibodies were added. They were incubated at 4-8°C in the dark for 30 minutes, washed twice with antibody diluent, and then resuspended in 500ul antibody storage solution. The positive rate of the above antibodies in the cell samples was detected by flow cytometry.

[0075] Table 1 shows the flow cytometry analysis results of cell surface markers of blood stem cells in Example 1 of the present invention and the comparative example when cultured to the P5 generation. As can be seen from Table 1, the expression rates of CD73, CD105, CD166, CD90, CD29, and CD44 of the P5 generation cells of Example 1 are all higher than those of the comparative example cells, indicating that they maintain stronger mesenchymal-like stem cell characteristics and higher stem cell purity. In addition, the stem cells in Example 1 also maintain a higher level of CD117 expression rate, indicating that they have stronger survival, proliferation, and multi-lineage differentiation capabilities.

[0076] Table 1

[0077]

[0078] The above embodiments of the present invention are for illustration of the present invention and cannot be used to limit the present invention. Any changes within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims.

Claims

1. A method for preparing high-purity menstrual blood-derived stem cells, characterized in that The steps include: (1) Using a menstrual blood collection kit, collect menstrual blood into a menstrual blood collection tube containing an antibiotic preservation solution, wherein the mixing ratio of the antibiotic preservation solution to the menstrual blood is (1-1.5):1, and the menstrual blood is stored at a low temperature of 2-8°C for a storage period of ≤72 hours; (2) centrifuging the menstrual blood collection tube, discarding the supernatant, and collecting the cell pellet; (3) After the cell pellet is fully mixed with saline at a volume ratio of 1:(1-1.5), it is slowly added to the interface of an equal volume of lymphocyte separation fluid, centrifuged, the middle layer mononuclear cells are collected, and then washed with saline and centrifuged; (4) The obtained mononuclear cells were sampled and the number of cells was counted. 8 Cells: add 100-200 μl magnetic bead buffer, mix thoroughly, add 50-100 μl FcR reagent, incubate at 2-8°C for 5-10 minutes, then add 50-100 μl cross-linked anti-human antibody micromagnetic bead reagent, mix well, and incubate at 2-8°C for 10-20 minutes; the anti-human antibody is anti-human CD117 monoclonal antibody; the micromagnetic bead reagent is a Miltenyi Biotec product, item number 130-091-332, with a particle size of 50 nm; (5) Add 1-1.5 ml of magnetic bead buffer to the liquid obtained in step (4), wash, centrifuge and resuspend in magnetic bead buffer. The concentration of the cell suspension is 1×10 8 cells / ml; (6) Add all of the above cell suspension to a separation column pre-placed in a magnetic separation rack, and wash the column with a magnetic bead buffer; (7) Take the sorting column out of the sorting rack, place it in a sterile collection tube, add 1.5-2 ml of magnetic bead buffer, and collect the cell suspension; (8) The suspension was sampled and the number of cells was counted. After diluting with primary culture medium of menstrual stem cells, the concentration was 5000-7000 cells / cm. 2 The cells were inoculated into cell culture bottles at a density of 100 μg / mL and placed in a 37°C, 5% CO2 incubator for culture. The medium was changed by half every 2-3 days. (9) After 5-7 days of cell culture, the cells reached a density of 70-80% under an inverted microscope. The adherent cells were digested and collected and plated at 3000-5000 cells / cm 2 The cells were inoculated into cell culture flasks at a density of 100 μg / cm2 and then continued to be expanded and cultured using blood stem cell culture medium; (10) The digested cells were mixed with menstrual stem cell cryopreservation solution precooled at 2-8°C and added to a cryopreservation tube at a cell density of 0.1-1×10 7 cells / ml, placed in a freezing box, placed in a -80°C refrigerator for 12-24 hours, and then transferred to -196°C liquid nitrogen for freezing; In step (1), the antibiotic storage solution is based on HBSS and contains 420 units of heparin sodium, 250 μg / mL of cephalexin, 120 μg / mL of gentamicin, and 3 μg / mL of amphotericin B; The specific formula of the magnetic bead buffer is: phosphate buffered saline as the base liquid, containing 0.5% by volume of fetal bovine serum or bovine serum albumin or human serum albumin, and containing ethylenediaminetetraacetic acid with a final concentration of 2mM; The primary culture medium for menstrual stem cells in step (8) comprises: 30% by volume of α-MEM culture medium, 40% by volume of Chang Amnio culture medium, 29% by volume of UltraCULTURE culture medium, and 1% by volume of blue / chain dual antibody; The menstrual stem cell passage medium in step (9) comprises: 30% by volume of α-MEM medium, 35% by volume of Chang Amnio medium, 35% by volume of UltraCULTURE medium, 10-20 ng / ml of EGF, and 10-20 ng / ml of bFGF; The menstrual stem cell freezing solution in step (10) contains: 32-40% by volume of cell culture supernatant, 8-10% by volume of dimethyl sulfoxide, 25-30% by volume of Chang Amnio culture medium, and 25-30% by volume of UltraCULTURE culture medium.

2. The method for preparing high-purity menstrual blood-derived stem cells according to claim 1, characterized in that: The sorting column in step (6) is one of an MS sorting column, an LS sorting column and an XS sorting column.

3. The method for preparing high-purity menstrual blood-derived stem cells according to claim 1, characterized in that: In the step (8), the cell culture flask is a T25 square flask or a T75 square flask; in the step (9), the cell culture flask is a T150 square flask or a T75 square flask.

4. The method for preparing high-purity menstrual blood-derived stem cells according to claim 1, characterized in that: In the step (9), TrypLE Express or 0.25% trypsin is used to digest the cells.

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