Preparation of mesenchymal stem cell agent and application thereof in repair of thin endometrium
Patent Information
- Application Number
- CN202610283461.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-03-10
- Publication Date
- 2026-05-29
Abstract
Description
Technical Field
[0001] This invention relates to the preparation of a mesenchymal stem cell agent and its application in the repair of thin endometrium, belonging to the field of biomedicine. Background Technology
[0002] Thin endometrium refers to a pathological condition where the endometrial thickness is significantly lower than the normal range during specific physiological periods (such as the period when the endometrial thickness should reach its normal range during the menstrual cycle). Currently, in assisted reproductive technology, an endometrial thickness of less than 7 mm is generally defined as thin endometrium. With the increase in induced abortions, intrauterine procedures, and older women trying to conceive in recent years, the incidence of thin endometrium has been rising annually. According to relevant studies, the incidence of thin endometrium is high among infertile women. A receptive endometrium is a key factor in ensuring a woman's successful conception and maintaining a pregnancy. When the endometrium thins, its receptivity decreases, which can lead to a series of serious health problems for women of childbearing age, such as infertility, ectopic pregnancy, recurrent miscarriage, and low birth weight infants.
[0003] Studies have found that factors affecting endometrial regeneration may be related to the absence or dysfunction of endometrial stem cells. Mesenchymal stem cells (MSCs) possess the potential for self-renewal and multi-lineage differentiation, enabling them to migrate to damaged sites and promote tissue regeneration and angiogenesis by secreting growth factors and anti-inflammatory molecules, thereby increasing endometrial thickness and improving its function. Various treatment methods have been explored in research on the treatment of thin endometrium. Drug treatments such as oral estrogen and aspirin have drawbacks including long treatment duration, significant side effects, and limited efficacy. While studies have confirmed that mesenchymal stem cells can effectively promote the repair of thin endometrium in vivo, this treatment requires stem cell transplantation, which is technically demanding and its effectiveness is inconsistent. Mice, as commonly used laboratory animals, share certain similarities with humans in their physiological structure and reproductive system, and possess advantages such as short reproductive cycles, relatively low cost, and relatively simple experimental procedures. Constructing a mouse model of thin endometrium and conducting therapeutic research using mouse-derived mesenchymal stem cells can provide an important theoretical basis and experimental evidence for the treatment of thin endometrium in humans.
[0004] The present invention aims to provide a method for preparing a mesenchymal stem cell agent and its application in the repair of thin endometrium, so as to solve the problems existing in the prior art and provide strong support for further in-depth research on the mechanism of human mesenchymal stem cell therapy for thin endometrium and its clinical translation. Summary of the Invention
[0005] The present invention aims to provide a method for preparing a mesenchymal stem cell preparation. The stem cell preparation obtained by this method can effectively restore the endometrial thickness of mice, increase the number of glands, and improve the pregnancy rate of mice with thin endometrium.
[0006] All strains described in this article were commercially available.
[0007] The *Enterococcus durans* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 1.3249.
[0008] The *Enterococcus durans* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 1.2023.
[0009] The Bacillus subtilis strain is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC 1.15792.
[0010] The Bacillus subtilis strain is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC 1.8886.
[0011] The technical problem to be solved by this invention can be achieved through the following technical solutions.
[0012] Preparation of probiotic supernatant: Preparation of sterile supernatant of Enterococcus durableis CGMCC1.3249: One loopful of Enterococcus durableis was inoculated into MRS solid medium and streaked, and incubated at 37°C for 24 h; 1% of the best-growing colonies from the plate were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h, centrifuged at 5000 rpm for 5 min to obtain the supernatant, the pH of the supernatant was adjusted to 7.0 using NaOH, and then the obtained supernatant was sterilized by passing it through a 0.22 μm membrane to obtain sterile supernatant for use.
[0013] Bacillus subtilis CGMCC1.15792: One loopful of Bacillus subtilis was inoculated into nutrient agar medium and streaked, and cultured at 37°C for 24 h; 1% of the well-growing colonies from the plate were picked and inoculated into nutrient agar liquid medium and cultured at 37°C for 24 h; the supernatant was obtained by centrifugation at 5000 rpm for 5 min; the pH of the supernatant was adjusted to 7.0 using NaOH; the supernatant was then sterilized by passing it through a 0.22 μm membrane to obtain sterile supernatant for later use.
[0014] Preparation of sterile supernatant for durable enterococci CGMCC1.2023: same as for durable enterococci CGMCC1.3249.
[0015] Preparation of sterile supernatant of Bacillus subtilis CGMCC1.8886: same as Bacillus subtilis CGMCC1.15792 above.
[0016] Preparation of mouse endometrial mesenchymal stem cells: SPF-grade C57BL / 6J multiparous weaned mice were selected. The mice were euthanized by cervical dislocation, and the uterus was harvested using routine aseptic surgery. The uterus was washed in 1×PBS, and the mesentery was removed. The uterus was cut into approximately 1mm × 1mm pieces in PBS, and twice the volume of 0.25% trypsin-EDTA solution was added. The mixture was incubated at 37°C for 30 minutes, gently pipetting every 10 minutes to disperse the tissue pieces. When a large number of single cells or small cell clusters were observed under a microscope, an equal volume of DMEM / F12 medium containing 10% FBS was added to terminate the digestion. The liquid was filtered through a 70μm cell filter. The filtered liquid was centrifuged at 1000 r / min for 10 minutes. The supernatant was discarded, and the cells were resuspended in culture medium and centrifuged again. The resulting precipitate contained endometrial cells. After resuspending and counting the cells, they were ready for culture. The single-cell suspension was seeded into 25cm² cell culture flasks, and the cell density was adjusted to 1×10⁶ cells / year. 5 -5×10 5 Cells were cultured at 37℃ in a 5% CO2 incubator at a density of 1 / mL. When the cell confluence reached 80%-90%, the cells were passaged, the culture medium was discarded, and the cells were washed twice with PBS. 2 mL of 0.25% trypsin-EDTA solution was added, and the cells were incubated at 37℃ for 2-3 min. When the cells became rounded and the intercellular spaces increased under a microscope, FBS-containing medium was added to stop the digestion. The cells were pipetted to form a suspension, centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in fresh culture medium and seeded into new culture flasks at a 1:3 ratio. By passage 3-5, the passaged cells were relatively flat and arranged in a polar pattern, yielding relatively pure mesenchymal stem cells. A positivity rate of ≥95% for the mesenchymal stem cell markers CD29, CD44, and CD105 was considered acceptable.
[0017] Qualified P3-P5 generation mesenchymal stem cells were added to 2 mL of 0.25% trypsin-EDTA solution and incubated at 37°C for 2-3 min. The cells were then pipetted to form a suspension, centrifuged at 1000 rpm for 5 min, and the precipitate was mixed with liquid to prepare a cell mixture with a concentration of 5 × 10⁻⁶. 5The solution consists of PBS, sterile supernatant of durable enterococci CGMCC 1.3249, and sterile supernatant of Bacillus subtilis CGMCC 1.15792, in a ratio of 8:0.5-1:0.5-1.
[0018] Advantages of this invention: This invention first adds the supernatant of *Enterococcus durableis* CGMCC 1.3249 and *Bacillus subtilis* CGMCC 1.15792 as active ingredients to cultured mesenchymal stem cells. The stem cell preparation obtained by this method can effectively restore endometrial thickness, increase glandular number, and improve the pregnancy rate in mice with thin endometrium. It is speculated that these two bacteria produce products dependent on stem cell proliferation during their proliferation process and can have a synergistic effect, possibly acting on multiple proliferation signaling pathways. This method is relatively simple to prepare and has significant importance and broad prospects. Detailed Implementation
[0019] The embodiments of the present invention are described in detail below. These embodiments are only used to explain the present invention and should not be construed as limiting the present invention.
[0020] Specific embodiments of the present invention are described below. Example
[0021] Preparation of probiotic supernatant: Preparation of sterile supernatant of Enterococcus durableis CGMCC1.3249: One loopful of Enterococcus durableis was inoculated into MRS solid medium and streaked, and incubated at 37°C for 24 h; 1% of the best-growing colonies from the plate were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h, centrifuged at 5000 rpm for 5 min to obtain the supernatant, the pH of the supernatant was adjusted to 7.0 using NaOH, and then the obtained supernatant was sterilized by passing it through a 0.22 μm membrane to obtain sterile supernatant for use.
[0022] Bacillus subtilis CGMCC1.15792: One loopful of Bacillus subtilis was inoculated into nutrient agar medium and streaked, and cultured at 37°C for 24 h; 1% of the well-growing colonies from the plate were picked and inoculated into nutrient agar liquid medium and cultured at 37°C for 24 h; the supernatant was obtained by centrifugation at 5000 rpm for 5 min; the pH of the supernatant was adjusted to 7.0 using NaOH; the supernatant was then sterilized by passing it through a 0.22 μm membrane to obtain sterile supernatant for later use.
[0023] Preparation of sterile supernatant for durable enterococci CGMCC1.2023: same as for durable enterococci CGMCC1.3249.
[0024] Preparation of sterile supernatant of Bacillus subtilis CGMCC1.8886: same as Bacillus subtilis CGMCC1.15792 above.
[0025] Preparation of mouse endometrial mesenchymal stem cells: SPF-grade C57BL / 6J multiparous weaned mice were selected. The mice were euthanized by cervical dislocation, and the uterus was harvested using routine aseptic surgery. The uterus was washed in 1×PBS, and the mesentery was removed. The uterus was cut into approximately 1mm × 1mm pieces in PBS, and twice the volume of 0.25% trypsin-EDTA solution was added. The mixture was incubated at 37°C for 30 minutes, gently pipetting every 10 minutes to disperse the tissue pieces. When a large number of single cells or small cell clusters were observed under a microscope, an equal volume of DMEM / F12 medium containing 10% FBS was added to terminate the digestion. The liquid was filtered through a 70μm cell filter. The filtered liquid was centrifuged at 1000 r / min for 10 minutes. The supernatant was discarded, and the cells were resuspended in culture medium and centrifuged again. The resulting precipitate contained endometrial cells. After resuspending and counting the cells, they were ready for culture. The single-cell suspension was seeded into 25cm² cell culture flasks, and the cell density was adjusted to 1×10⁶ cells / year. 5 -5×10 5 Cells were cultured at 37℃ in a 5% CO2 incubator at a density of 1 / mL. When the cell confluence reached 80%-90%, the cells were passaged, the culture medium was discarded, and the cells were washed twice with PBS. 2 mL of 0.25% trypsin-EDTA solution was added, and the cells were incubated at 37℃ for 2-3 min. When the cells became rounded and the intercellular spaces increased under a microscope, FBS-containing medium was added to stop the digestion. The cells were pipetted to form a suspension, centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in fresh culture medium and seeded into new culture flasks at a 1:3 ratio. By passage 3-5, the passaged cells were relatively flat and arranged in a polar pattern, yielding relatively pure mesenchymal stem cells. A positivity rate of ≥95% for the mesenchymal stem cell markers CD29, CD44, and CD105 was considered acceptable.
[0026] Qualified P3-P5 generation mesenchymal stem cells were added to 2 mL of 0.25% trypsin-EDTA solution and incubated at 37°C for 2-3 min. The cells were then pipetted to form a suspension and centrifuged at 1000 rpm for 5 min. The precipitate was used as mouse endometrial mesenchymal stem cells for the experiment.
[0027] Preparation Example 1 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P3 generation mouse endometrial mesenchymal stem cells are mixed with a liquid to form a cell mixture with a concentration of 5 × 10⁻⁶. 5The solution consists of PBS, sterile supernatant of durable enterococci CGMCC1.3249, and sterile supernatant of Bacillus subtilis CGMCC1.15792 in a ratio of 8:0.5:1.
[0028] Preparation Example 2 This preparation example provides a mouse mesenchymal stem cell preparation. P4 generation mouse endometrial mesenchymal stem cells were mixed with a liquid to prepare a cell mixture with a concentration of 5 × 10⁻⁶. 5 The solution consists of PBS, sterile supernatant of durable enterococci CGMCC1.3249, and sterile supernatant of Bacillus subtilis CGMCC1.15792 in a ratio of 8:1:0.5.
[0029] Preparation Example 3 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P5 generation mouse endometrial mesenchymal stem cells were mixed with a liquid to prepare a cell mixture with a concentration of 5 × 10⁻⁶. 5 The solution consists of PBS, sterile supernatant of durable enterococci CGMCC1.3249, and sterile supernatant of Bacillus subtilis CGMCC1.15792 in a ratio of 8:0.8:0.7.
[0030] Comparative Preparation Example 1 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P3 generation mouse endometrial mesenchymal stem cells were mixed with PBS to form a cell mixture with a concentration of 5 × 10⁻⁶. 5 per mL.
[0031] Comparative Preparation Example 2 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P3 generation mouse endometrial mesenchymal stem cells are mixed with a liquid to form a cell mixture with a concentration of 5 × 10⁻⁶. 5 The solution consists of PBS and sterile supernatant of durable enterococci CGMCC1.3249 in a ratio of 8.5:1.
[0032] Comparative preparation example 3 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P3 generation mouse endometrial mesenchymal stem cells are mixed with a liquid to form a cell mixture with a concentration of 5 × 10⁻⁶. 5 The solution consists of PBS and sterile supernatant of Bacillus subtilis CGMCC 1.15792 in a ratio of 8.5:1.
[0033] Comparative preparation example 4 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P3 generation mouse endometrial mesenchymal stem cells are mixed with a liquid to form a cell mixture with a concentration of 5 × 10⁻⁶. 5 The solution consists of PBS, sterile supernatant of durable enterococci CGMCC1.2023, and sterile supernatant of Bacillus subtilis CGMCC1.15792 in a ratio of 8:0.5:1.
[0034] Comparative preparation example 5 This preparation example provides a mouse mesenchymal stem cell preparation. The prepared P3 generation mouse endometrial mesenchymal stem cells are mixed with a liquid to form a cell mixture with a concentration of 5 × 10⁻⁶. 5 The solution consists of PBS, sterile supernatant of durable enterococci CGMCC1.3249, and sterile supernatant of Bacillus subtilis CGMCC1.8886 in a ratio of 8:0.5:1.
[0035] Practice Example 1 Establishment of a thin endometrial animal model: Six- to eight-week-old SPF-grade, sexually mature, unmated C57BL / 6J female mice were selected. After anesthesia, the abdominal cavity was opened layer by layer. A V-shaped uterus was visible behind the bladder. The upper V segment was selected on both sides, exposing the vaginal and ovarian ends of the uterus for modeling. The proximal and distal ends of the uterus were clamped with hemostatic forceps. A syringe needle was gently inserted into the proximal end of the uterus on one side of the mouse's vagina. The surrounding tissue was protected with sterile saline gauze to prevent leakage of anhydrous ethanol. The syringe needle was inserted into the uterine cavity, parallel to the longitudinal axis of the uterus. 0.1 ml of 95% ethanol was slowly injected into the uterine cavity near the vaginal end, filling the uterine cavity. After the needle remained in place for 5 minutes, the ethanol was slowly aspirated. Keeping the syringe needle in the uterine cavity, residual ethanol was gently squeezed out, and the cavity was rinsed twice with physiological saline. The other uterus was treated in the same way, and the abdomen was closed layer by layer.
[0036] Grouping: Six- to eight-week-old SPF-grade sexually mature and unmated female C57BL / 6J mice were randomly divided into a normal group (negative control group), a model group (positive control group), preparation cases 1-3, and control preparation cases 1-5. Each group consisted of 11 mice.
[0037] Normal group: The experimental mice were fed a normal diet without any treatment. After the estrus period, one mouse was sacrificed to collect its uterus for the experiment, and the other 10 mice were put together in the same cage. Model group: After successful modeling, the experimental mice were fed a normal diet without any other treatment; on day 20, one mouse was sacrificed to collect its uterus for the experiment, and the other 10 mice were put together in the same cage; Preparation group and control group: Ten days after modeling, the mice were anesthetized and operated on via the abdomen. 0.1 ml of mesenchymal stem cells were injected into the uterine cavity of each mouse and the abdomen was closed routinely. This was recorded as day 0. On day 10 (the end of the second estrus cycle), one mouse from each group was sacrificed to collect sample uteri for subsequent experiments, and the other 10 mice were put together.
[0038] Uterine thickness measurement: Endometrial morphological observation (HE staining): The fixed uterus was removed, dehydrated and cleared, and then vertically embedded in paraffin. After embedding, a circular uterine cavity was visible, and the section thickness was 5 μm. After HE staining, the endometrial tissue structure was observed under a microscope, and the image was acquired using the Leica Application Suite imaging system. The endometrial thickness value in any of the four quadrants of the same section was randomly measured, and the four values were recorded and the average value was calculated.
[0039] Under a 200x microscope, four fields of view were randomly selected from each slice to count the number of glands and calculate the average value. The statistical results are shown below: Table 1 Results of HE staining microscopy Group Endometrial thickness (um) Number of glandular cells (individuals) Preparation Example 1 <![CDATA[324.1±31.6 1 ]]> <![CDATA[15.6±3.1 1 ]]> Preparation Example 2 <![CDATA[332.5±28.3 1 ]]> <![CDATA[16.8±2.8 1 ]]> Preparation Example 3 <![CDATA[327.2±33.4 1 ]]> <![CDATA[15.8±4.2 1 ]]> Comparative Preparation Example 1 <![CDATA[180.5±21.1 1 ]]> <![CDATA[7.5±3.7 1 ]]> Comparative Preparation Example 2 <![CDATA[248.1±11.9 1 ]]> <![CDATA[11.0±5.5 1 ]]> Comparative preparation example 3 <![CDATA[186.6±21.8 1 ]]> <![CDATA[8.2±3.2 1 ]]> Comparative preparation example 4 <![CDATA[190.4±24.0 1 ]]> <![CDATA[8.5±3.9 1 ]]> Comparative preparation example 5 <![CDATA[255.6±21.9 1 ]]> <![CDATA[11.5±4.5 1 ]]> Model group 122.2±20.2 4.6±2.4 normal group 359.5±29.3 18.2±4.5 Note: t-test, l: P < 0.05 (compared with the model group) Practice Example 2 The fertility of the mice in Example 1 was assessed by mating them with male mice of confirmed reproductive capacity at a 1:1 female-to-male ratio at 17:30 daily. Vaginal plugs were observed daily at 08:00 to determine successful mating. If mating was successful, the female and male mice were separated and tagged. If mating was unsuccessful, the mice were kept together, but this was stopped after approximately two weeks. Approximately 16 days after observing vaginal plugs, the female mice were euthanized by cervical dislocation to determine pregnancy. The uterus was exposed, and the pregnancy status was observed. The results were recorded and analyzed as follows: Table 2 Pregnancy Results Group Number of pregnancies (individuals) Number of mice Pregnancy rate (%) Preparation Example 1 7 10 <![CDATA[70 1 ]]> Preparation Example 2 8 10 <![CDATA[80 1 ]]> Preparation Example 3 7 10 <![CDATA[70 1 ]]> Comparative Preparation Example 1 2 10 <![CDATA[20 1 ]]> Comparative Preparation Example 2 3 10 <![CDATA[30 1 ]]> Comparative preparation example 3 2 10 <![CDATA[20 1 ]]> Comparative preparation example 4 2 10 <![CDATA[20 1 ]]> Comparative preparation example 5 4 10 <![CDATA[40 1 ]]> Model group 0 10 0 normal group 8 10 80 Note: t-test, l: P < 0.05 (compared with the model group) Data showed that mesenchymal stem cells cultured by co-culturing supernatants of *Enterococcus durableis* CGMCC 1.3249 and *Bacillus subtilis* CGMCC 1.15792 significantly improved pregnancy outcomes in mice. The results were statistically significant.
[0040] In summary, this invention first adds the supernatant of *Enterococcus durableis* CGMCC 1.3249 and *Bacillus subtilis* CGMCC 1.15792 as active ingredients to cultured mesenchymal stem cells. The stem cell preparation obtained by this method can effectively restore thin endometrium in mice. It is speculated that these two bacteria produce products dependent on stem cell proliferation during their proliferation process and can have a synergistic effect, possibly acting on multiple proliferation signaling pathways. This method is relatively simple to prepare and has significant importance and broad prospects.
[0041] It should be noted that the above description is only a preferred embodiment of the present invention. Any changes made according to the concept of the present invention that do not exceed the spirit covered by the specification should be within the scope of the present invention.
[0042] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0043] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims
1. A method for preparing a mouse endometrial mesenchymal stem cell agent, characterized in that, Includes the following steps: Step 1: Prepare sterile supernatant of durable enterococci CGMCC 1.3249 and sterile supernatant of Bacillus subtilis CGMCC 1.15792; Step 2: Prepare mouse endometrial mesenchymal stem cells, wherein the stem cells are cells passaged to the 3rd-5th generation; Step 3: Resuspend the qualified mesenchymal stem cells obtained in Step 2 in liquid to prepare a solution with a concentration of 5×10⁻⁶. 5 The mesenchymal stem cell agent is prepared by mixing PBS, the sterile supernatant of durable enterococcus CGMCC 1.3249 obtained in step 1, and the sterile supernatant of Bacillus subtilis CGMCC 1.15792 obtained in step 1 at a volume ratio of 8:0.5-1:0.5-1.
2. The preparation method according to claim 1, characterized in that, The preparation process of the sterile supernatant of durable enterococci CGMCC1.3249 described in step 1 is as follows: Take one loopful of durable enterococci CGMCC1.3249 and inoculate it into MRS solid medium and streak it, then incubate it at 37℃ for 24h; pick colonies from the plate and inoculate them into MRS liquid medium at a 1% inoculum, incubate them at 37℃ for 24h, centrifuge at 5000rpm for 5min, collect the supernatant, adjust the pH to 7.0 with NaOH, and sterilize it through a 0.22μm membrane to obtain the product.
3. The preparation method according to claim 1, characterized in that, The preparation process of the sterile supernatant of Bacillus subtilis CGMCC 1.15792 described in step 1 is as follows: Take one loop of Bacillus subtilis CGMCC 1.15792 and inoculate it into nutrient agar medium and streak it. Incubate at 37℃ for 24h. Pick colonies from the plate and inoculate them into nutrient agar liquid medium at an inoculation rate of 1%. Incubate at 37℃ for 24h. Centrifuge at 5000rpm for 5min and collect the supernatant. Adjust the pH to 7.0 with NaOH and sterilize by passing through a 0.22μm membrane.
4. The preparation method according to claim 1, characterized in that, Step 2, the preparation of mouse endometrial mesenchymal stem cells, includes: (1) Take the weaned mice after multiparous birth, kill them by cervical dislocation, and aseptically remove the uterus. Wash with 1×PBS and cut off the mesentery. Cut the uterus into 1mm×1mm pieces. (2) Add 2 times the volume of 0.25% trypsin-EDTA solution, digest in a constant temperature water bath at 37℃ for 30 min, and blow once every 10 min during the process; (3) Add an equal volume of DMEM / F12 medium containing 10% FBS to terminate digestion. After filtering through a 70μm cell filter, centrifuge at 1000r / min for 10min. Resuspend the precipitate in culture medium and centrifuge again. (4) The resulting precipitate was resuspended in culture medium and the cell density was adjusted to 1×10⁻⁶. 5 -5×10 5 Cells / mL were seeded in 25cm² culture flasks and cultured at 37℃ with 5% CO2. When the cell confluence reached 80%-90%, the cells were passaged at a 1:3 ratio to the 3rd-5th passages. Qualified P3-P5 passage mesenchymal stem cells were selected, added to trypsin-EDTA solution, incubated at 37℃ for 2-3 minutes, and the cells were pipetted to form a suspension. After centrifugation at 1000rpm for 5 minutes, qualified mesenchymal stem cells were obtained. (5) The positive rate of the cell surface markers CD29, CD44 and CD105 is ≥95% to be considered qualified.