Endometrial stem cell preparation as well as preparation method and application thereof

Abstract

The invention provides an endometrial stem cell preparation as well as a preparation method and application thereof, and belongs to the technical field of biological preparations. According to the preparation method of the endometrial stem cell preparation, the safety risk is controlled through an autoserum source, the cell function is optimized in combination with a low-oxygen environment, and a high-stability and low-immune-risk standardized solution is provided for clinical transformation of the endometrial stem cell preparation. Three batches of culture data show that the cell viability is gt; the mesenchymal stem cell culture medium is stable in form and meets the mesenchymal stem cell verification standard. The flow detection verification shows that the positive rate of CD73 / CD90 / CD105 is greater than or equal to 95%, the negative markers such as CD34 / CD45 and the like are less than or equal to 2%, and the high purity is proved; the cell cycle shows that the G0 / G1 period accounts for 74-80%, and the resting state and the genome stability are maintained. After the preparation provided by the invention is stored for a short time or frozen and unfrozen for a long time, the cell morphology and proliferation capacity are not obviously attenuated.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to an endometrial stem cell preparation, a preparation method thereof, and an application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent stem cell derived from tissues such as bone marrow, umbilical cord, and adipose tissue. They possess the ability to self-renew and differentiate into various mesenchymal tissue types, including osteoblasts, adipocytes, and chondrocytes. They exhibit low immunogenicity, immunomodulatory abilities, and excellent tissue repair capabilities. They are widely used in tissue engineering, immune disease treatment, and regenerative medicine research, and are a key research target in the field of cell therapy. Currently, mainstream sources of MSCs include bone marrow, adipose tissue, umbilical cord, and placenta. These cells possess excellent proliferation capacity and multipotential differentiation potential, and are widely used in clinical research and regenerative medicine. Endometrial stem cells (eMSCs) are a new type of MSCs derived from the endometrium. They have significant advantages such as periodic regeneration and relatively non-invasive sampling (such as through hysteroscopy or menstrual blood collection). They have shown unique application potential in the fields of gynecological disease repair (such as intrauterine adhesions and thin endometrium), reproductive medicine (improving endometrial receptivity) and tissue regeneration (such as pelvic floor repair).

[0003] However, the in vitro culture and expansion of MSCs, including endometrial stem cells, for clinical application still faces key challenges. First, traditional culture systems often rely on the addition of fetal bovine serum (FBS) or other xenogeneic sera as a crucial source of nutrients and growth factors. While FBS effectively supports cell growth, its heterologous nature carries the risk of introducing unknown pathogens (such as viruses and prions), potentially triggering immune rejection and raising potential ethical concerns. These factors severely limit the safety of cultured stem cell preparations for autologous or allogeneic clinical applications, refusing to meet the increasingly stringent regulatory requirements for the clarity and safety of cell therapy products. Furthermore, conventional cell culture is typically performed in a normoxic environment (approximately 21% O₂), which can lead to decreased proliferation rates, altered differentiation potential, increased genomic instability, accelerated senescence, and altered secretion profiles of key paracrine factors, ultimately compromising post-transplant survival, homing ability, and therapeutic efficacy. Summary of the Invention

[0004] The purpose of the present invention is to provide an endometrial stem cell preparation, its preparation method and application, which can eliminate the risk of heterologous components and simulate the in vivo microenvironment to optimize cell quality and function. It is of decisive significance for fully tapping the therapeutic potential of endometrial stem cells and promoting their safe and effective clinical transformation.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing an endometrial stem cell preparation, comprising the following steps: Menstrual blood samples were collected and endometrial stem cells were isolated by density gradient centrifugation; The obtained endometrial stem cells were inoculated into a complete culture medium containing fetal bovine serum and cultured for expansion at an oxygen concentration of 1%-5%; The expanded endometrial stem cells are resuspended in autologous serum to prepare a preparation; The autologous serum is derived from the patient's peripheral blood.

[0006] Preferably, the conditions for the expansion culture also include 3-8% CO2 and a temperature of 35-38°C.

[0007] Preferably, the complete culture medium containing fetal bovine serum includes a low-glucose DMEM medium containing 1-10% fetal bovine serum; The expansion culture includes cell passaging, which is completed in a low-oxygen workstation.

[0008] Preferably, the autologous serum is sterilized by 0.22 μm filtration before use; The autologous serum was activated by incubation at 35-38° C. for 20-40 min before use.

[0009] Preferably, the expanded endometrial stem cells are mixed with autologous serum in a volume ratio of 1:0.5-5.

[0010] Preferably, the cells are passaged to P3 to P6.

[0011] Preferably, the cell concentration of the endometrial stem cell preparation is 1×10 6 ~5×10 7 cells / mL.

[0012] Preferably, the cell viability of the endometrial stem cell preparation is ≥95%.

[0013] The present invention also provides an endometrial stem cell preparation prepared by the above preparation method.

[0014] The present invention also provides the use of the above-mentioned endometrial stem cell preparation in the preparation of a drug for treating intrauterine adhesions, thin endometrium, pelvic floor tissue defect repair, primary ovarian insufficiency, decompensated cirrhosis, diabetes, myocardial ischemic damage, idiopathic pulmonary fibrosis, aging-related dysfunction of the female reproductive system, vulvovaginal atrophy or elasticity loss, adjuvant treatment of ovarian cancer, or reproductive tract tissue lesions secondary to HPV infection.

[0015] Beneficial effects of the present invention: The preparation method of the endometrial stem cell preparation provided by this invention significantly improves the safety and stability of the preparation. It completely replaces fetal bovine serum with autologous serum extracted from the patient's peripheral blood, eliminating the risks of allogeneic immune rejection and pathogen transmission. By simulating the in vivo physiological microenvironment, it activates the HIF-1α pathway, enhances cell stemness (upregulating pluripotent genes), and inhibits oxidative damage. Culture data from three batches showed cell viability >90% and stable morphology, meeting the standards for mesenchymal stem cell identification. Flow cytometry validation demonstrated a positive rate of ≥95% for CD73 / CD90 / CD105 and a negative rate of ≤2% for markers such as CD34 / CD45, confirming high purity. The cell cycle showed a 74%–80% G0 / G1 phase, maintaining a quiescent state and genomic stability. The preparation provided by this invention demonstrated stable viability within 4 hours of short-term storage at room temperature or within 12 hours of refrigeration at 4–8°C. Long-term storage showed no significant decrease in cell morphology or proliferation after thawing, even after 3 months of storage at -80°C or over a year of storage in liquid nitrogen at -196°C. The present invention controls safety risks through the source of autologous serum and optimizes cell function in a hypoxic environment, providing a standardized solution with high stability and low immune risk for the clinical transformation of endometrial stem cell preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the growth graph of P1 cells in batch 2024082301 on the third day; Figure 2 This is the growth graph of P2 cells in batch 2024082301 on the third day; Figure 3 This is the growth graph of P3 cells in batch 2024082301 on the third day; Figure 4 This is the growth graph of P4 cells in batch 2024082301 on the third day; Figure 5 This is the growth graph of P5 cells in batch 2024082301 on the third day; Figure 6 This is the growth graph of P1 cells in batch 2024082901 on the third day; Figure 7 This is the growth graph of P2 cells in batch 2024082901 on the third day; Figure 8 This is the growth graph of P3 cells in batch 2024082901 on the third day; Figure 9 This is the growth graph of P4 cells in batch 2024082901 on the third day; Figure 10 This is the growth graph of P5 cells in batch 2024082901 on the third day; Figure 11 This is the growth graph of P1 cells in batch 2024090601 on the third day; Figure 12 This is the growth graph of P2 cells in batch 2024090601 on the third day; Figure 13 This is the growth graph of P3 cells from batch 2024090601 on the third day; Figure 14 This is the growth graph of P4 cells in batch 2024090601 on the third day; Figure 15 This is the growth graph of P5 cells in batch 2024090601 on the third day; Figure 16 This is the result of the detection of cell surface markers of P2 cells in batch 2024082301; Figure 17 This is the result of the detection of cell surface markers of P4 cells in batch 2024082301; Figure 18 This is the result of the detection of cell surface markers of P5 cells in batch 2024082301; Figure 19 This is the result of the detection of cell surface markers of P2 cells in batch 2024082901; Figure 20 This is the result of the detection of cell surface markers of P3 cells in batch 2024082901; Figure 21 This is the result of the detection of cell surface markers of P4 cells in batch 2024082901; Figure 22 This is the result of the detection of cell surface markers of P5 cells in batch 2024082901; Figure 23 This is the result of the detection of cell surface markers of P2 cells in batch 2024090601; Figure 24 This is the result of the detection of cell surface markers of P3 cells in batch 2024090601; Figure 25 This is the result of the detection of cell surface markers of P4 cells in batch 2024090601; Figure 26 This is a summary diagram of the cell cycle of endometrial mesenchymal stem cells; Figure 27 This is the cell cycle detection result of P4 cells in batch 2024082901; Figure 28 This is the cell cycle detection result of P3 cells in batch 2024082301; Figure 29 This is the cell cycle detection result of P3 cells in batch 2024090601; Figure 30 Storing test results for room temperature; Figure 31 Graph of test results for refrigerated storage environment. DETAILED DESCRIPTION

[0017] The present invention provides a method for preparing an endometrial stem cell preparation, comprising the following steps: Menstrual blood samples were collected and endometrial stem cells were isolated by density gradient centrifugation; The obtained endometrial stem cells were inoculated into a complete culture medium containing fetal bovine serum and cultured for expansion at an oxygen concentration of 1%-5%; The expanded endometrial stem cells are resuspended in autologous serum to prepare a preparation; The autologous serum is derived from the patient's peripheral blood.

[0018] Preferably, the expansion culture conditions also include 3-8% CO2 and a temperature of 35-38°C. Preferably, the complete culture medium containing fetal bovine serum includes low-glucose DMEM medium containing 1-10% fetal bovine serum; the expansion culture includes cell passaging, which is completed in a hypoxic workstation. Preferably, the autologous serum is sterilized by 0.22 μm filtration before use; the autologous serum is activated by incubation at 35-38°C for 20-40 minutes before use. Preferably, the expanded endometrial stem cells are mixed with autologous serum in a volume ratio of 1:0.5-5. Preferably, the cells are passaged to passages P3-P6. Preferably, the cell concentration of the endometrial stem cell preparation is 1×106-5×107 cells / mL. Preferably, the cell viability of the endometrial stem cell preparation is ≥95%.

[0019] The present invention also provides an endometrial stem cell preparation prepared by the above preparation method.

[0020] The present invention also provides the use of the above-mentioned endometrial stem cell preparation in the preparation of a drug for treating intrauterine adhesions, thin endometrium, pelvic floor tissue defect repair, primary ovarian insufficiency, decompensated cirrhosis, diabetes, myocardial ischemic damage, idiopathic pulmonary fibrosis, aging-related dysfunction of the female reproductive system, vulvovaginal atrophy or elasticity loss, adjuvant treatment of ovarian cancer, or reproductive tract tissue lesions secondary to HPV infection.

[0021] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] The autologous serum preparation process refers to the following scheme: Preparation of autologous serum begins with the collection of peripheral blood from the patient. Typically, 50–100 mL of blood is collected (the specific volume can be adjusted based on needs). Using sterile venipuncture, the blood is placed into an anticoagulant-free blood collection tube (such as a standard dry tube or a coagulant-promoting tube), strictly avoiding contamination with anticoagulants such as heparin or EDTA. After blood collection, the tube is left at room temperature for 30–60 minutes to allow the blood to naturally clot.

[0023] Subsequently, the clot is encouraged to retract to release more serum. The clotted blood can be left to rest at 4°C for 2–4 hours or at room temperature for 1–2 hours. Next, perform the first centrifugation at 2000–3000 × g for 10–15 minutes (this can be done at room temperature or 4°C). After centrifugation, carefully transfer the supernatant (i.e., serum) to a new sterile centrifuge tube, taking care to avoid aspirating blood cells or fibrin clots.

[0024] If red blood cells or particulate matter are still visible in the serum after transfer, an optional second centrifugation step can be performed to improve purity, again at 3000 × g for 5 minutes. To ensure sterility, the serum must be filtered through a 0.22 μm pore size sterile filter to remove potential microbial contamination. After filtration, the serum should be immediately aliquoted into sterile cryovials (e.g., 1 mL per vial) to minimize freeze-thaw cycles during subsequent use.

[0025] Depending on the specific application requirements (e.g., certain immunology studies), an optional complement inactivation step may be required. Place the serum in a 56°C water bath for 30 minutes, then immediately cool it in an ice bath. Prepared serum can be stored at 4°C if it is to be used within a short period (≤7 days). For long-term storage, it should be stored at -20°C or -80°C for 1–2 years. Repeated freezing and thawing should also be avoided to prevent loss of growth factor activity.

[0026] Quality control is an important step in ensuring the safety and efficacy of serum, and usually includes: performing bacterial / fungal culture or PCR testing for mycoplasma to confirm sterility; using the Limulus amebocyte lysate test to detect endotoxins (required to be <0.5EU / mL); determining the total protein concentration by BCA or Bradford assay (usually 40–80mg / mL); if the serum will be used for stem cell culture, it may be necessary to test the levels of specific growth factors (such as EGF, FGF, PDGF).

[0027] Precautions during the operation include: the blood collection process must be gentle and the centrifugation speed must be controlled to avoid hemolysis; all steps should be performed in a biological safety cabinet to ensure sterility throughout the process; it should be noted that the serum components (such as hormones and antibodies) of different patients may have individual differences, which may affect the behavior of stem cells, and preliminary experimental verification is required if necessary; if the serum is intended for clinical use, it must comply with GMP or local regulatory requirements (such as FDA 21 CFR Part 1271).

[0028] Advantages of autologous serum: Compared to other serum substitutes (such as fetal bovine serum (FBS) or human platelet lysate (hPL), autologous serum offers significant advantages. Its core advantage lies in its safe source and lack of immune risk, as it is obtained directly from the patient, completely avoiding the potential immune rejection reactions associated with allogeneic or xenogeneic serum. Secondly, autologous serum provides a personalized growth factor profile, its composition naturally matching the patient's own physiological environment. Furthermore, the use of autologous serum is free of animal ethics concerns (compared to FBS) and eliminates the need to consider donor matching (compared to hPL).

[0029] Example 1 Menstrual blood collection Use a sterile menstrual cup placed directly into the vagina to collect spontaneous menstrual blood. After 2–4 hours, remove the collected blood and transfer it to a sterile centrifuge tube containing penicillin-streptomycin PBS to prevent blood clotting. The entire process should be performed under sterile conditions, and the collected sample should be transported to the laboratory at 4°C to ensure cell viability and subsequent isolation and culture.

[0030] Collection and isolation of endometrial stem cells Dilute with PBS at a ratio of 1:1, and slowly overlay the dilution on a Ficoll-Paque density gradient solution with a density of 1.077 g / mL to avoid liquid mixing.

[0031] Centrifuge at 400 to 800×g for 20 to 30 minutes at room temperature with the centrifuge brake turned off to maintain the integrity of the liquid layers.

[0032] After centrifugation, carefully recover the mononuclear cell layer at the interface of the layers, transfer it to a new centrifuge tube, and wash twice with PBS, centrifuging at 300 to 400 × g for 5 to 10 minutes each time to remove residual density gradient fluid and plasma impurities.

[0033] The obtained cells were perfused with 10 ml of complete culture medium (low-glucose DMEM, 1% double antibody, 10% fetal bovine serum).

[0034] Count and inoculate cells into T75 cell culture flasks according to the cell quantity. Add 13 ml of complete culture medium to each flask and place the cells in a hypoxic incubator (three-gas incubator O2-CO2-N2). Culture at 37°C, 5% CO2 + 3% O2. Regularly change the medium and perform subculture operations in a hypoxic workstation to avoid exposure to a normoxic environment for more than 5 minutes.

[0035] Expansion and culture of endometrial stem cells: During the medium exchange process, non-adherent cells are gradually removed from the adherent cells. After several days of culture, a cell population with typical mesenchymal stem cell morphology is formed. Further phenotypic characterization (e.g., CD73⁺, CD90⁺, CD105⁺, and CD45⁻, CD34⁻) confirms that these cells are endometrial-derived mesenchymal stem cells. When the cells cover approximately 90% of the bottom of the culture flask, they are trypsinized and passaged. The culture medium is aspirated and discarded. After gently rinsing the cells with normal saline, 2 ml each of normal saline and 0.25% trypsin are added. The culture flask is placed flat and gently shaken to spread the trypsin over the bottom. Observe under an inverted microscope while gently tapping the flask walls. Once most cells have detached from the flask, dissipate the cells with complete culture medium. After digestion, transfer the cell suspension into a 50ml centrifuge tube. Wash the culture flask with a small amount of physiological saline and transfer it into a centrifuge tube. Add saline to resuspend the suspension to 50ml to mix the cells. Centrifuge at a fast speed of 1300 rpm for 5 minutes. Tilt 45° and slowly pour the supernatant into the waste bottle; discard the supernatant, resuspend the cells with 10ml complete culture medium, inoculate into 2 culture flasks (the culture flask number is determined by the cell growth situation), and add culture medium to 13ml / flask. Repeat this process until P5. Count the cells before freezing, add cell freezing solution according to the cell concentration, and resuspend them into 1.2*10 6 cells / ml, gently pipette to make the cells uniform, and then divide them into sterile cryopreservation tubes, add 1ml of liquid to each cryopreservation tube, and the number of cells in each tube is 1.2*10 6 Place the sealed cryovials in a programmed cooling box at room temperature, then place them in a -80°C freezer. After 12 hours overnight, place the cryovials in the programmed cooling box into a liquid nitrogen tank.

[0036] Preparation of uterine endometrial stem cell drugs: Mesenchymal stem cells (MSCs) stored in liquid nitrogen were rapidly revived in a 37°C water bath and immediately transferred to sterile PBS containing 10% AB human serum. The cells were centrifuged (300–400 × g, 5 minutes) to remove the cryoprotectant and washed 1–2 times to obtain purified stem cell pellets. Cell viability was determined by trypan blue staining or flow cytometry to ensure that the cell viability was not less than 98%, and the cell concentration was adjusted to 1 × 10 6 –1×10 7cells / mL. Use AB type human serum with a clear source and inactivated viruses, filter it using a 0.22 μm filter membrane under sterile conditions, and incubate it at 37°C for 30 minutes before use to restore its biological activity. Resuspend the treated stem cells in pre-warmed AB type human serum (extracted autologous serum), mix them in a 1:1 ratio, and mix gently. If needed, add appropriate amount of buffer or excipients to adjust the osmotic pressure and pH to ensure the biocompatibility and stability of the final preparation. The final mixture is dispensed into sterile syringes or ampoules under a Class A clean environment, sealed and stored at 2–8°C for clinical injection or topical application.

[0037] Example 2 Compared with Example 1, the only difference is that the treated stem cells are resuspended in pre-warmed AB type human serum (extracted autologous serum), mixed at a ratio of 1:2, and gently mixed.

[0038] Example 3 Compared with Example 1, the only difference is that the treated stem cells are resuspended in pre-warmed AB type human serum (extracted autologous serum), mixed at a ratio of 1:3, and gently mixed.

[0039] Example 4 Compared with Example 1, the only difference is that the treated stem cells are resuspended in pre-warmed AB type human serum (extracted autologous serum), mixed at a ratio of 1:4, and gently mixed.

[0040] Experimental example Based on Example 1, three batches of endometrial mesenchymal stem cells obtained by this scheme were cultured and observed: The main observation objects include cell growth rate, microscopic morphology, cell viability, etc.

[0041] The culture and microscopic examination data of batch 2024082301 are shown in Table 1 below: Table 1 Culture and microscopic examination data of batch 2024082301

[0042] Figures 1 to 5 This is a growth chart of the endometrial cells of batch 2024082901 of the present invention on the third day of each generation. As can be seen from the picture, the endometrial cells grow evenly, have a basically stable morphology, are spindle-shaped, and the cell viability is greater than 90%, which meets the requirements of the inspection procedures.

[0043] The culture and microscopic examination data of batch 2024082901 are shown in Table 2 below: Table 2 Culture and microscopic examination data of batch 2024082901

[0044] Figures 6-10 The images show the growth of endometrial cells from batch 2024082901 on day three of each passage. As can be seen, the endometrial cells grew uniformly, exhibited a generally stable spindle-shaped morphology, and maintained a viability exceeding 90%. Cells from later passages exhibited stringing, differing from the typical spindle shape but otherwise meeting the requirements of the assay.

[0045] The culture and microscopic examination data of batch 2024090601 are shown in Table 3 below: Table 3 Culture and microscopic examination data of batch 2024090601

[0046] Figures 11 to 15 The images show the growth of endometrial cells at various passages cultured in MSCM. As can be seen, the cells grow uniformly and have a generally stable, spindle-shaped morphology. The cell viability is greater than 90%, meeting the requirements of the assay.

[0047] Phenotypic detection of three batches of endometrial mesenchymal stem cells: The present invention completed the phenotypic result detection of three batches of endometrial mesenchymal stem cells. The detection method adopted flow cytometry, and the cell surface markers detected included CD34, CD11b, CD19, CD45, HLA-DR, CD73, CD90, and CD105.

[0048] Phenotypic test results for batch 2024082301: CD34, CD11b, CD19, CD45, and HLA-DR were negative, with a negative rate of ≤2%; CD73, CD90, and CD105 were positive, with a positive rate of ≥95%. The results showed that passages 2-5 of endometrial mesenchymal stem cells had a high purity. See the original data for more information. Figures 16-18 .

[0049] Phenotypic test results for batch 2024082901: CD34, CD11b, CD19, CD45, and HLA-DR were negative, with a negative rate of ≤2%; CD73, CD90, and CD105 were positive, with a positive rate of ≥95%. The results showed that passages 2-5 of endometrial mesenchymal stem cells had a high purity. See the original data for more information. Figures 19-22 .

[0050] The phenotypic test results of batch 2024090601 showed that the expression of CD34, CD11b, CD19, CD45, and HLA-DR was negative, with a negative rate of ≤2%; the expression of CD73, CD90, and CD105 was positive, with a positive rate of ≥95%. The results showed that the 2nd to 5th generation endometrial mesenchymal stem cells had a high purity. The original data can be found in Figures 23-25 .

[0051] The cell cycle of three batches of cultured endometrial mesenchymal stem cells was detected and summarized as follows: Figure 26 shown.

[0052] The specific results are as follows: Batch: 2024082901 Generation: 4th Generation Inspection Date: September 7, 2024 Cell cycle analysis: sub0G1(%) G0 / G1(%) reported result 76.47 S(%) reported result 14.3 G2 / M(%) reported result 8.95 like Figure 27 shown.

[0053] Batch: 2024082301 Generation: 3rd Generation Inspection Date: September 15, 2024 Cell cycle analysis: sub0G1(%) G0 / G1(%) reported result 80.05 S(%) reported result 10.15 G2 / M(%) reported result: 9.8 like Figure 28 shown.

[0054] Batch: 2024090601 Generation: 3rd Generation Inspection Date: September 21, 2024 Cell cycle analysis: sub0G1(%) G0 / G1(%) report result 74 S(%) reported result 7.86 G2 / M(%) reported result 18.15 like Figure 29 shown.

[0055] In order to confirm the storage conditions and effective storage time of the cell preparation in the external environment after preparation, the present invention tested the mesenchymal stem cell preparation prepared in Example 1.

[0056] The test conditions are: storage conditions (room temperature or 4-8°C), storage time (2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours).

[0057] The test contents are: cell viability and cell morphology in cell preparations.

[0058] Test results: The prepared cell preparation can be stored at room temperature for about 4 hours; it can be stored in a refrigerated environment of 4-8℃ for about 12 hours.

[0059] like Figures 30-31 shown.

[0060] In order to confirm the long-term storage of cell preparations (including intermediates and finished preparations) under cell storage conditions after preparation, our hospital tested the existing mesenchymal stem cell preparations.

[0061] The test conditions are: storage conditions (-80℃, -196℃), storage time (1 month, 3 months, 6 months, 1 year).

[0062] The test contents include: cell viability, cell morphology, and cell proliferation after thawing of the cell preparation.

[0063] Test results: Intermediates and finished preparations of cell preparations can be stored at -80°C for 3 months without affecting cell viability, cell morphology, and cell proliferation ability; they can be stored at -196°C in liquid nitrogen for more than 1 year (currently tested up to 1 year) without affecting cell viability, cell morphology, and cell proliferation ability.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing an endometrial stem cell preparation, characterized in that: The following steps are involved: Menstrual blood samples were collected and endometrial stem cells were isolated by density gradient centrifugation; The obtained endometrial stem cells were inoculated into a complete culture medium containing fetal bovine serum and cultured for expansion at an oxygen concentration of 1%-5%; The expanded endometrial stem cells are resuspended in autologous serum to prepare a preparation; The autologous serum is derived from the patient's peripheral blood.

2. The method according to claim 1, characterized in that The expansion culture conditions also include 3-8% CO2 and a temperature of 35-38°C.

3. The method according to claim 1, characterized in that The complete culture medium containing fetal bovine serum includes a low-glucose DMEM culture medium containing 1-10% fetal bovine serum; The expansion culture includes cell passaging, which is completed in a low-oxygen workstation.

4. The method according to claim 1, wherein The autologous serum was sterilized by 0.22 μm filtration before use; The autologous serum was activated by incubation at 35-38° C. for 20-40 min before use.

5. The method according to claim 1, wherein The expanded endometrial stem cells are mixed with autologous serum at a volume ratio of 1:0.5-5.

6. The method according to claim 3, characterized in that The cells were passaged to P3-P6.

7. The method according to claim 1, characterized in that The cell concentration of the endometrial stem cell preparation is 1×10 6 ~5×10 7 cells / mL.

8. The method according to claim 1, characterized in that The cell viability of the endometrial stem cell preparation is ≥95%.

9. An endometrial stem cell preparation prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the endometrial stem cell preparation according to claim 9 in the preparation of a medicament for treating intrauterine adhesions, thin endometrium, pelvic floor tissue defect repair, primary ovarian insufficiency, decompensated cirrhosis, diabetes, myocardial ischemic injury, idiopathic pulmonary fibrosis, aging-related dysfunction of the female reproductive system, vulvovaginal atrophy or loss of elasticity, adjuvant treatment of ovarian cancer, or reproductive tract tissue lesions secondary to HPV infection.

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