Method for obtaining endometrial mesenchymal stem cells from human menstrual blood

Abstract

Provided are a method for obtaining endometrial mesenchymal stem cells from human menstrual blood and endometrial mesenchymal stem cells prepared thereby. The method comprises: 1) grading a menstrual blood sample through a cell sieve to separate tissue fragments, wherein the pore size of the cell sieve decreases step by step, and the cell sieve includes a filtering cell sieve for collecting a filtrate and a retaining cell sieve for collecting a retentate, wherein the pore size of the retaining cell sieve is smaller than that of the filtering cell sieve; 2) collecting the retentate on the retaining cell sieve, namely the tissue fragments; 3) culturing the tissue fragments in a mesenchymal stem cell culture medium; and 4) collecting adherent cells, namely the endometrial mesenchymal stem cells.

Description

Technical Field

[0001] The present application relates to the field of methods for preparing mesenchymal stem cells, and specifically to a method for obtaining endometrial mesenchymal stem cells from human menstrual blood. Background Art

[0002] In recent years, mesenchymal stem cells (MSCs) have attracted widespread attention from researchers due to their advantages such as strong proliferation capacity, low immunogenicity, multidirectional differentiation potential, and directional migration to sites of tissue damage. Bone marrow-derived stem cells (BMSCs) are the earliest discovered mesenchymal stem cells and are currently the most commonly used stem cells in clinical research. However, the clinical application value of BMSCs is limited by the fact that bone marrow must be obtained by puncture under anesthesia and the number of autologous bone marrow stem cells decreases significantly with age. The human endometrium is a highly dynamic tissue that cyclically undergoes a proliferation phase, a secretory phase, and a menstrual phase. It has a remarkable regenerative capacity and can grow from the initial 0.5-1 mm after menstruation to 5-7 mm. The female endometrium undergoes self-renewal, proliferation, differentiation, and shedding more than 400 times throughout a woman's lifetime, and possesses a high regenerative capacity. Mesenchymal stem cells derived from endometrial tissue exist not only in the basal layer but also in menstrual blood, making them easy to obtain. Therefore, human endometrial mesenchymal stem cells (hEMSCs) are gaining increasing attention. Using endometrial mesenchymal stem cells as seed cells has great application prospects in cell therapy, tissue engineering, and regenerative medicine.

[0003] Currently, the main methods for isolating mesenchymal stem cells include tissue digestion and gradient density centrifugation. Tissue digestion can quickly obtain large numbers of viable cells from living organisms, and the cell characteristics are close to those in vivo and reflect growth characteristics, making it ideal for experimental research such as drug testing and cell differentiation. However, its disadvantages are also significant. Digestion is not only cumbersome and prone to contamination, but improper digestion timing can directly affect the purity and biological properties of primary mesenchymal stem cells. It can also damage cell structure, interfere with cell proliferation and differentiation, and even produce heterogeneous cell populations. Furthermore, some digestive enzymes are expensive, inevitably increasing experimental costs. Gradient density centrifugation (mononuclear method) uses the different density and diameter of particles, resulting in different sedimentation rates in solution. Under a certain centrifugal force, each particle settles at a specific rate, allowing cells to be sorted using gravity and centrifugal force. While suitable for purifying mononuclear cells, it is not suitable for isolating and purifying cells from tissues. Therefore, finding an alternative method for isolating and purifying endometrial mesenchymal stem cells from human menstrual blood is a critical issue for the development of endometrial mesenchymal stem cell-based drugs. Summary of the Invention

[0004] The present invention provides a method for obtaining endometrial mesenchymal stem cells from human menstrual blood, the method comprising:

[0005] 1) grading the menstrual blood sample through a cell sieve to separate tissue fragments, wherein the pore size of the cell sieve decreases step by step, including a filtering cell sieve for collecting the filtrate and a retaining cell sieve for collecting the retentate, and the pore size of the retaining cell sieve is smaller than that of the filtering cell sieve;

[0006] 2) collecting the retained material on the retained cell sieve, i.e., tissue fragments;

[0007] 3) culturing tissue fragments in mesenchymal stem cell culture medium;

[0008] 4) Collecting adherent cells is endometrial mesenchymal stem cells.

[0009] In some embodiments, the graded cell sieve comprises at least three levels of cell sieves, preferably at least four levels of cell sieves.

[0010] In some embodiments, the fractionated cell sieve comprises one, two, or more stages of filtration cell sieves, and / or one, two, or more stages of retention cell sieves. Preferably, the fractionated cell sieve comprises two or more stages of retention cell sieves, and step 2) collecting tissue fragments retained on the cell sieves comprises combining tissue fragments from multiple stages of retention cell sieves.

[0011] In some embodiments, the step 2) collecting the tissue fragments retained on the cell sieve includes backwashing the tissue fragments retained on the cell sieve with a cleaning solution.

[0012] In some embodiments, the step 2) collecting the tissue fragments retained on the cell sieve further includes washing the tissue fragments with a washing solution.

[0013] In another aspect, the present invention provides endometrial mesenchymal stem cells obtained by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure shows the comparison of expansion folds of endometrial mesenchymal stem cells prepared by the mononuclear method and the method of the present invention.

[0015] Figure 2 The doubling time of endometrial mesenchymal stem cells prepared by the mononuclear method and the method of the present invention is compared.

[0016] Figure 3A The figure shows the osteogenic differentiation ability staining of endometrial mesenchymal stem cells prepared by the method of the present invention; Figure 3BThe staining diagram shows the chondrogenic differentiation ability of endometrial mesenchymal stem cells prepared by the method of the present invention. Specific implementation plan

[0017] In this specification, unless otherwise specified, the components or preferred components involved can be combined with each other to form a new technical solution.

[0018] In this specification, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution.

[0019] In this specification, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0020] If not specifically stated, the terms "a" and "an" used in this specification mean "at least one".

[0021] "Range" as disclosed herein is in the form of lower limits and upper limits. There can be one or more lower limits, and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower limit and upper limit define the boundaries of the particular range. All ranges that can be defined in this manner are inclusive and combinable, i.e., any lower limit can be combined with any upper limit to form a range.

[0022] Specifically, the present invention first provides a method for obtaining endometrial mesenchymal stem cells from human menstrual blood, the preparation method comprising:

[0023] 1) grading the menstrual blood sample through a cell sieve to separate tissue fragments, wherein the pore size of the cell sieve decreases step by step, including a filtering cell sieve for collecting the filtrate and a retaining cell sieve for collecting the retentate, and the pore size of the retaining cell sieve is smaller than that of the filtering cell sieve;

[0024] 2) collecting the retained material on the retained cell sieve, i.e., tissue fragments;

[0025] 3) culturing tissue fragments in mesenchymal stem cell culture medium;

[0026] 4) Collecting adherent cells is endometrial mesenchymal stem cells.

[0027] In one embodiment, the method comprises

[0028] 1) Tissue separation: The menstrual blood sample is graded and separated through a cell sieve with a pore size that becomes smaller and smaller;

[0029] 2) Tissue backwashing: backwash the tissue fragments trapped on the cell sieve with cleaning solution;

[0030] 3) Tissue washing: resuspend the washed tissue fragments with washing solution and centrifuge to obtain the tissue fragment pellet;

[0031] 4) Tissue culture: Resuspend the tissue fragments in culture medium, inoculate them into culture bottles, add culture medium and culture in an incubator;

[0032] 5) Collect the adherent cells that grow out, which are endometrial mesenchymal stem cells.

[0033] Some embodiments further comprise expanding and culturing the collected endometrial mesenchymal stem cells.

[0034] A cell sieve can be any porous structure that can separate substances by particle size without contaminating tissues and cells. Gradual sieving involves using cell sieves of varying pore sizes to pass particles through the sieves in descending order, thereby obtaining multiple levels of retained products with varying size distributions, or obtaining multiple levels of filtrate containing components with varying size distributions.

[0035] In some embodiments of the present invention, a large-pore filter molecular sieve is first used to remove oversized tissue fragments by interception, and then a small-pore retaining molecular sieve is used to intercept and collect the tissue fragments for culture. In the present invention, "tissue fragments" refers to fragmented tissue and does not specifically refer to products obtained through a pulverization operation.

[0036] In some embodiments, the pore size of the cell sieve can be 70-160 mesh, preferably 80-160 mesh. The present invention includes at least one level of cell sieve, but can and preferably includes two or more levels of cell sieves with decreasing pore size. When multiple levels of cell sieves are included, tissue fragments retained on different levels of cell sieves can be combined for subsequent steps, for example, by backwashing with a cleaning solution and combining them, or scraping and combining them before resuspension, washing, and other treatments.

[0037] In the present invention, oversized tissue fragments are removed by filtering through a cell sieve and the filtrate is collected. Preferably, tissue fragments with a pore size of less than 30 mesh, less than 35 mesh, less than 40 mesh, or even smaller are collected. Accordingly, the cell sieve includes a cell sieve with a pore size of no more than 30 mesh, no more than 35 mesh, no more than 40 mesh, or even smaller. For example, tissue fragments with a pore size of less than 36 mesh can be collected. Accordingly, the cell sieve includes at least one stage of 36 mesh cell sieve. Removal of oversized tissue fragments can be achieved by single-stage screening or by multi-stage screening. Therefore, the present invention includes at least one stage of cell sieve filtering, but can and preferably includes two or more stages of cell sieve filtering with decreasing pore size. The pore size of the cell sieve filtering can be 18-60 mesh, preferably 36-60 mesh, more preferably 36-50 mesh, and even more preferably 36-40 mesh.

[0038] In some embodiments, the step of fractionating and filtering the cells of the present invention comprises at least three stages of cell sieves, preferably at least four stages of cell sieves. For example, the step of fractionating and filtering the cells may comprise at least two stages of filtration cell sieves and at least one stage of retention cell sieve; or at least one stage of filtration cell sieves and at least two stages of retention cell sieves; or at least two stages of filtration cell sieves and at least two stages of retention cell sieves.

[0039] Any suitable method can be used to harvest tissue fragments from the retained cell sieve, such as scraping, picking, backwashing, etc., and those skilled in the art can make their own decisions based on the circumstances. In some embodiments, the present invention uses a backwashing method to flush the tissue fragments retained on the cell sieve with a cleaning solution. Herein, "tissue backwashing" and "backwashing" both refer to the process of washing the retained material with a cleaning solution in the opposite direction of the flow when passing through the molecular sieve. In order to recover as much retained material as possible, the flushing can be stopped when there is no tissue visible to the naked eye on the cell sieve.

[0040] For example, the specific steps of backwashing may include: collecting the cell sieve with retained tissue fragments; placing a certain number of cell sieves with retained tissue fragments in a culture dish, the number of which depends on the size of the culture dish, for example, 10 cm 2 No more than 5 cell sieves should be placed in the culture dish; add cleaning solution to the culture dish containing the cell sieve with retained tissue fragments, and the amount of cleaning solution added should just cover the sieve in the culture dish; use a Pasteur pipette to absorb the cleaning solution in the culture dish to backwash the tissue fragments retained on the cell sieve; stop rinsing when there is no tissue visible to the naked eye on the cell sieve.

[0041] In some embodiments, the method of the present invention further comprises a tissue washing step, i.e., washing the tissue fragments harvested from the trapped cell sieve. For example, the tissue fragments are washed by resuspending with a washing solution. Then, the washed tissue fragment precipitate can be obtained by solid-liquid separation (e.g., filtration or centrifugation). For example, when backwashing is used to collect trapped tissue fragments, the tissue washing step may include: collecting the washing solution, collecting the tissue fragment precipitate by solid-liquid separation (e.g., filtration or centrifugation, discarding the supernatant), adding the washing solution; and repeatedly washing the tissue fragments in this way to obtain a washed tissue fragment precipitate. If centrifugation is used, the conditions for the centrifugal operation may be a centrifugal force of 400g, a temperature of 20-22°C, preferably 20°C, and a centrifugation time of 10 minutes.

[0042] In the present invention, the "cleaning solution" can be a buffer commonly used in cell manipulation, such as a cleaning solution based on a common buffer such as a phosphate buffer, to which other components can be added as needed, for example, antibiotics can be added in the present invention. In some embodiments, the cleaning solution is a phosphate buffer, and in some preferred embodiments, it is a phosphate buffer containing antibiotics. Preferably, the antibiotic is selected from gentamicin, penicillin, streptomycin, amphotericin B or a combination thereof. Preferably, if the cleaning solution contains gentamicin, its concentration can be 50-100 U / mL, preferably 100 U / mL; if the cleaning solution contains penicillin, its concentration can be 180-200 U / mL, preferably 200 U / mL; if the cleaning solution contains streptomycin, its concentration can be 0.15-0.2 mg / mL, preferably 0.2 mg / mL; if the cleaning solution contains amphotericin B, its concentration can be 4-5 μg / mL, preferably 5 μg / mL.

[0043] Step 3) of the present invention is to culture tissue fragments in a mesenchymal stem cell culture medium, also known as "tissue culture", and includes inoculating the tissue fragments in a mesenchymal stem cell culture medium and culturing them under mesenchymal stem cell culture conditions, thereby growing mesenchymal stem cells with adherent properties from the tissue fragments. In some embodiments, the inoculation includes resuspending the tissue fragment pellet with a mesenchymal stem cell culture medium for inoculation, and the culture can be carried out in a culture bottle. Optionally, after ensuring that the endometrial mesenchymal stem cells are firmly adhered to the wall (for example, after 48 hours of tissue culture), non-adherent miscellaneous cells (such as red blood cells) can be removed to prevent miscellaneous cells from contaminating the endometrial mesenchymal stem cell culture, and then the culture is continued until the confluence reaches 90%-100% and then passaged.

[0044] The mesenchymal stem cell culture medium used in the present invention can be any culture medium for mesenchymal stem cell culture known to those skilled in the art, and can be prepared by oneself or obtained through commercial channels. The mesenchymal stem cell culture medium is selected from a serum-free culture medium for primary MSC cells (available from Beijing Youkang Technology Co., Ltd.) and an amniotic fluid cell culture medium (available from Guangzhou Baiyunshan Baidi Biomedicine Co., Ltd.).

[0045] In some embodiments, the tissue culture conditions are conventional mesenchymal stem cell culture conditions, such as 5% CO 2 and 37° C. The culture time is 4-6 days, preferably 5 days.

[0046] In some embodiments, the method further comprises isolating adherent endometrial mesenchymal stem cells after tissue culture and performing expansion culture.

[0047] In some embodiments, a step can be added before step 1) to check the contamination of the collected menstrual blood sample. The step includes mixing the collected menstrual blood sample with a cleaning solution (preferably a phosphate buffered saline solution containing antibiotics, such as phosphate buffered saline solution containing gentamicin and amphotericin B), adding the mixed solution to a culture medium, and culturing the mixture in an incubator to observe the contamination. The menstrual blood sample and cleaning solution are mixed in a 1:1 ratio.

[0048] The present invention also provides endometrial mesenchymal stem cells obtained by the method of the present invention. Compared with endometrial mesenchymal stem cells obtained by existing technologies, such as the mononuclear method, the endometrial mesenchymal stem cells obtained by the present invention have a higher proliferation rate and passage expansion multiple, and have good differentiation potential.

[0049] Example

[0050] The present invention will be described in further detail below in conjunction with examples. However, it should be understood that these examples are only for illustrative purposes and are not intended to limit the scope of the present invention.

[0051] Example 1: Preparation of endometrial mesenchymal stem cells

[0052] Endometrial mesenchymal stem cells were prepared according to the method of the present invention. The specific experimental method is as follows:

[0053] (1) Tissue separation:

[0054] The menstrual blood sample in the 50 mL centrifuge tube was sequentially passed through an 18-mesh stainless steel cell sieve, a 36-mesh stainless steel cell sieve, an 80-mesh stainless steel cell sieve, and a 160-mesh stainless steel cell sieve.

[0055] (2) Organizational backlash:

[0056] 1) Place the 80-mesh and 160-mesh stainless steel cell sieves that were used to filter the menstrual blood sample in step (1) into a 10 cm 2 No more than 5 cell sieves, each 10 cm, are placed in each culture dish. 2 Add 40 mL of washing solution to the culture dish, so that the washing solution just covers the mesh that retains the tissue fragments; the washing solution used is phosphate buffered saline and contains 100 U / mL gentamicin and 5 μg / mL amphotericin B;

[0057] 2) Use a 3 mL Pasteur pipette to draw out the cleaning solution in the culture dish to backwash the tissue fragments trapped on the 80-mesh and 160-mesh cell sieves.

[0058] (3) Tissue cleaning:

[0059] 1) After rinsing, collect the rinsing fluid containing tissue fragments in the above step into 50 mL centrifuge tubes, 40 mL per tube, and centrifuge at 20°C and 400g for 10 minutes, discarding the supernatant;

[0060] 2) Use a pipette to draw up the cleaning solution and add it to the centrifuge tubes. Add 10 mL of cleaning solution to each tube. Resuspend the pellet again and combine into a 50 mL centrifuge tube. Centrifuge at 400 g at 20°C for 10 minutes and discard the supernatant.

[0061] 3) Use a pipette to draw 30 mL of washing solution into the centrifuge tube, resuspend the pellet, and centrifuge at 400 g for 10 minutes at 20°C. Discard the supernatant.

[0062] (IV) Tissue culture:

[0063] 1) Use a pipette to draw up 1 mL of culture medium (amniotic fluid cell culture medium, abbreviated as YS, purchased from Guangzhou Baiyunshan Biopharmaceutical Co., Ltd.) to resuspend the pellet. Inoculate 500 μL of the resuspension into a T25 culture flask and add 10 mL of culture medium to resuspend.

[0064] 2) Mix the cells in a cross or eight shape and culture in a 37°C, 5% CO2 incubator.

[0065] 3) After 48 hours of cell culture, change the medium to wash away non-adherent cells (e.g., red blood cells) and continue culturing. Subculture can be performed when the cell confluence reaches 90%.

[0066] Example 2: Growth detection of endometrial mesenchymal stem cells obtained by different preparation methods

[0067] Endometrial mesenchymal stem cells were prepared according to the mononuclear method described in "Isolation, culture and identification of menstrual blood-derived endometrial mesenchymal stem cells" by Zhou Yunfan, Yang Bo, Hu Xiang, etc. (Chinese Journal of Tissue Engineering Research, 2010, 14(32): 5952-5956).

[0068] The endometrial mesenchymal stem cells prepared in Example 1 and the endometrial mesenchymal stem cells prepared by the above mononuclear method were respectively cultured for passage, with 5.0×10 3 cells / cm 2The density was inoculated into T75 culture flasks, in which the culture medium was amniotic fluid cell culture medium (abbreviated as YS, purchased from Guangzhou Baiyunshan Baidi Biotechnology Co., Ltd.). When the cell fusion rate reached about 90%, the culture medium was discarded, the cells were gently washed with phosphate buffered saline (PBS) and the washing solution was discarded, and 3ml of 0.25% trypsin was added to digest the cells for 4 minutes. 6ml of PBS mixture containing 5% fetal bovine serum was added to terminate cell digestion. The cell suspension was collected into two centrifuge tubes, centrifuged at 1500r / min for 5min. The supernatant was discarded and 1mL of amniotic fluid cell culture medium (abbreviated as YS, purchased from Guangzhou Baiyunshan Baidi Biotechnology Co., Ltd.) was added to resuspend the cells. The AO / PI (AO (Acridine Orange) acridine orange, PI (Propidium Iodide) propidium iodide) double staining cell apoptosis detection kit (DNA probe double staining cell nucleus method, purchased from Shanghai Ruiyu Biotechnology Co., Ltd.) was used to detect the number of cells harvested in each generation, and the expansion times and doubling time of each generation were calculated. The experimental results are shown in Table 1, Appendix Figure 1-2 .

[0069]

[0070] Experimental Conclusion: The mesenchymal stem cells obtained by the preparation method of the present invention exhibited higher expansion times in culture at different passages than endometrial mesenchymal stem cells obtained by the mononuclear method. The doubling time of the obtained cells was also shorter than that obtained by the mononuclear method. While the doubling time of mesenchymal stem cells obtained by the mononuclear method fluctuates significantly depending on the passage number, the doubling time required for passaged mesenchymal stem cells obtained by the preparation method of the present invention exhibited less fluctuation. Therefore, the preparation method of the present invention exhibits advantages over the mononuclear method in terms of both the doubling time and expansion times of the obtained endometrial mesenchymal stem cells.

[0071] Example 3: Test of differentiation ability of endometrial mesenchymal stem cells prepared by the preparation method of the present invention

[0072] Experimental method: The endometrial mesenchymal stem cells 0031 prepared by the preparation method of Example 1 were taken and the cell concentration was 1.0×10 5 Each well was evenly plated with amniotic fluid cell culture medium (abbreviated as YS, purchased from Guangzhou Baiyunshan Baidi Biotechnology Co., Ltd.). Place in a 5% CO2, 37°C incubator and culture for 3-4 days until the cell fusion rate reaches 90%-100%. Aspirate the culture medium in the well in the clean bench and add 1 mL of osteogenic differentiation complete culture medium (purchased from Saiye Biotechnology Co., Ltd.) to each well. The culture medium was replaced every 3 days. On the 21st day of osteogenic differentiation, the osteogenic differentiated cells were stained with Alizarin Red S. The results are shown in the attached figure. Figure 3A .

[0073] The endometrial mesenchymal stem cells 0031 prepared by the preparation method of Example 1 were taken and the cell concentration was 1.0×10 7 / ml amniotic fluid cell culture medium resuspended, take the suspension and drop it evenly to 10cm 2 Cover the culture dish. Incubate in a 5% CO2, 37°C incubator for 24 hours. Pipette the pellet into a 6-well plate in a clean bench and add 2 mL of chondrogenic differentiation medium (purchased from STEMCELL) to each well. Change the medium every 3 days. On day 28 of chondrogenic differentiation, stain the chondrogenic differentiation sections with Alcian blue. See the attached results. Figure 3B .

[0074] Depend on Figure 3A It can be seen that the endometrial mesenchymal stem cells prepared by the method described in Example 1 showed obvious orange-red color after being stained with Alizarin Red S, indicating that they had good osteogenic differentiation ability; Figure 3B It can be seen that the endometrial mesenchymal stem cells produced by the method described in Example 1 showed blue interstitial color after staining with Alcian blue, indicating that they have the ability to form cartilage. In summary, the endometrial mesenchymal stem cells produced by the method described in the present invention have excellent differentiation ability.

[0075] Although the present invention has been described with reference to specific examples, it will be apparent to those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Therefore, the appended claims cover all such variations within the scope of the present invention.

Claims

1. A method for obtaining endometrial mesenchymal stem cells from human menstrual blood, the method comprising: 1) grading the menstrual blood sample through a cell sieve to separate tissue fragments, wherein the pore size of the cell sieve decreases step by step, including a filtering cell sieve for collecting the filtrate and a retaining cell sieve for collecting the retentate, and the pore size of the retaining cell sieve is smaller than that of the filtering cell sieve; 2) collecting the retained material on the retained cell sieve, i.e., tissue fragments; 3) culturing tissue fragments in mesenchymal stem cell culture medium; 4) Collecting adherent cells is endometrial mesenchymal stem cells; The pore size of the retained cell sieve is 70-160 mesh, and the pore size of the filtered cell sieve is 18-60 mesh.

2. The method according to claim 1, wherein the pore size of the retained cell sieve is 80-160 mesh.

3. The method according to claim 1, wherein the pore size of the filtered cell sieve is no more than 30 meshes.

4. The method according to claim 1, wherein the pore size of the filtered cell sieve does not exceed 35 meshes.

5. The method according to claim 1, wherein the pore size of the filtered cell sieve is no more than 40 meshes. The method according to claim 1 , wherein the pore size of the cell filter is 36-60 mesh.

7. The method according to claim 1, wherein the pore size of the cell filter is 36-50 mesh.

8. The method according to claim 1, wherein the pore size of the cell filter is 36-40 mesh.

9. The method according to claim 1, wherein in step 1), the grading through the cell sieve comprises passing through at least three levels of cell sieves.

10. The method according to claim 1, wherein in step 1), the fractionated cell sieve comprises at least four levels of cell sieves.

11. The method according to claim 1, wherein the fractionated cell sieve comprises passing through one or two or more stages of filtration cell sieves and / or passing through one or two or more stages of retention cell sieves.

12. The method according to claim 1, wherein the fractionated cell sieve comprises two or more levels of retained cell sieves, and the step 2) collecting the tissue fragments retained on the cell sieve comprises combining the tissue fragments on multiple levels of retained cell sieves.

13. The method according to claim 1, wherein the step 2) collecting the tissue fragments trapped on the cell sieve comprises backwashing the tissue fragments trapped on the cell sieve with a cleaning solution.

14. The method according to claim 1, wherein the step 2) collecting the tissue fragments retained on the cell sieve further comprises washing the tissue fragments with a washing solution. The method according to claim 13 or 14, wherein the cleaning solution is phosphate buffered saline. The method according to claim 13 or 14, wherein the cleaning solution is a phosphate buffer containing antibiotics.

17. The method according to claim 16, wherein the antibiotic is selected from gentamicin, penicillin, streptomycin, amphotericin B or a combination thereof.

18. The method according to claim 17, wherein in the cleaning solution, the concentration of gentamicin is 50-100 U / mL, the concentration of penicillin is 180-200 U / mL, the concentration of streptomycin is 0.15-0.2 mg / mL, and / or the concentration of amphotericin B is 4-5 μg / mL.

19. The method according to claim 17, wherein in the cleaning solution, the concentration of gentamicin is 100 U / mL, the concentration of penicillin is 200 U / mL, the concentration of streptomycin is 0.2 mg / mL, and / or the concentration of amphotericin B is 5 μg / mL.

20. The method according to claim 1, wherein the mesenchymal stem cell culture medium is selected from a serum-free culture medium for primary MSC cells and an amniotic fluid cell culture medium.

21. The method according to claim 1, wherein the time for step 3) tissue culture is 4-6 days.

22. The method according to claim 1, wherein the time for tissue culture in step 3) is 5 days.

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