Preparation method of human amniotic mesenchymal stem cell and cell patch thereof
By isolating and culturing human amniotic mesenchymal stem cells and preparing decellularized amniotic membranes, human amniotic mesenchymal stem cell sheets were constructed, solving the problems of low mechanical strength and poor operability, and achieving efficient cell utilization and preservation of bioactivity.
Patent Information
- Application Number
- CN202511015838.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-23
- Publication Date
- 2025-11-07
AI Technical Summary
The existing technology has low mechanical strength and poor maneuverability of human amniotic mesenchymal stem cell membranes, which are difficult to effectively solve using traditional preparation methods.
A method for isolating and culturing human amniotic mesenchymal stem cells and preparing decellularized amniotic membrane was adopted. Human amniotic mesenchymal stem cell sheets were constructed by digestion, filtration, centrifugation and seeding culture, and the decellularized amniotic membrane was used as a matrix to enhance mechanical strength.
It enhances the mechanical strength of the cell membrane, improves clinical operability, and ensures cell bioactivity and high cell utilization.
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Figure CN120905135A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of cell biology, and particularly relates to a preparation method of human amniotic mesenchymal stem cells and cell membrane sheets thereof. BACKGROUND
[0002] Human amniotic mesenchymal stem cells (HAMSCs) are obtained from discarded placental amnion, have a wide source and no additional harm to the human body, have high abundance, low immunogenicity, immunomodulation, anti-inflammatory properties and no tumorigenicity. Compared with adipose mesenchymal stem cells, human amniotic mesenchymal stem cells have good proliferation activity and osteogenic activity, and their osteogenic differentiation ability is similar to that of bone marrow mesenchymal stem cells; compared with human umbilical cord mesenchymal stem cells, human amniotic mesenchymal stem cells have excellent immunosuppressive activity.
[0003] Traditional cell application often needs to digest cells into single cells with trypsin, which destroys the ion channels on the cell surface and reduces the cell utilization rate when the single free cells are implanted in vivo. The application of cell membrane sheet technology can avoid the traditional cell utilization method, not only avoid the digestion of cells, but also have high biological activity and can fully retain the biological active factors in the extracellular matrix, and has high cell utilization rate when implanted in vivo. At present, although there are many preparation methods of cell membrane sheets, the preparation method of human amniotic mesenchymal stem cell membrane sheets is less studied, and the cell membrane sheets prepared by the traditional preparation method have the problems of low mechanical strength and poor operability.
[0004] Therefore, the present application provides a preparation method of human amniotic mesenchymal stem cells and cell membrane sheets thereof. SUMMARY
[0005] To solve the above technical problems, the present application provides a preparation method of human amniotic mesenchymal stem cells and cell membrane sheets thereof.
[0006] In order to achieve the above purpose, the present application provides the following technical scheme:
[0007] The present application provides a preparation method of human amniotic mesenchymal stem cell membrane sheets, comprising the following steps:
[0008] S1, isolation and culture of human amniotic mesenchymal stem cells
[0009] The amnion is cut into 1cm*1cm fragments, digested with a first digestion solution, filtered, and the filtrate is discarded; the second digestion solution is used for digestion, filtration, centrifugation of the filtrate, discarding of the supernatant, obtaining of human amniotic mesenchymal stem cell cell precipitate, resuspension, inoculation and cell passage culture of human amniotic mesenchymal stem cells;
[0010] S2, preparation of decellularized amnion
[0011] The amniotic membrane is placed in a 10 cm cell culture dish, and a third digestive solution is used for digestion treatment, and the digestive solution is discarded; the mucus is scraped off with a cell scraper, and then the decellularized amniotic membrane is obtained by cutting.
[0012] S3, preparation of human amniotic membrane mesenchymal stem cell membrane pieces
[0013] The decellularized amniotic membrane obtained in step S2 is placed in a cell culture dish, and the human amniotic membrane mesenchymal stem cells obtained in step S1 are inoculated on the decellularized amniotic membrane and cultured for 3 days, the complete culture medium is removed, and a membrane piece induction culture medium is added for continuous culture for 14 days, to obtain human amniotic membrane mesenchymal stem cell membrane pieces.
[0014] Preferably, the first digestive solution and the third digestive solution are both trypsin / EDTA solutions with a concentration of 0.25%; the second digestive solution is a type II collagenase with a concentration of 0.75%, and the amount of the first digestive solution, the second digestive solution and the third digestive solution is 1-1.5 times the volume of the amniotic membrane.
[0015] Preferably, the digestion treatment specifically involves incubation at 35-38°C in a constant-temperature shaker for 40-50 min, and the rotation speed of the constant-temperature shaker is 160-200 r / min.
[0016] Preferably, the filtration specifically involves filtration with a filter screen with a mesh size of 250-300, and the amniotic membrane in the filter screen is washed with sterile PBS for 2-3 times; before filtration, complete culture medium is added to stop the digestion.
[0017] Preferably, the resuspension involves resuspension of the human amniotic membrane mesenchymal stem cell precipitate with complete culture medium; and the complete culture medium is DMEM-F12 complete culture medium.
[0018] Preferably, the centrifugation specifically involves centrifugation at a rotation speed of 1000-1500 r / min for 4-8 min.
[0019] Preferably, the cutting specifically involves placing the decellularized amniotic membrane with the mucus scraped off on a dry and sterile nitrocellulose membrane so that it completely adheres, and then cutting the decellularized amniotic membrane into a specific size and a specific shape for standby use; the amniotic membrane needs to be washed with sterile PBS for 2-3 times before and after the mucus is scraped off.
[0020] Preferably, the inoculation in step S1 specifically involves inoculating the resuspended human amniotic membrane mesenchymal stem cells into a T75 cell culture bottle at a cell density of 5×10 4 / cm 2 The resuspended human amniotic membrane mesenchymal stem cells are primary cells.
[0021] Preferably, the inoculation in step S3 is specifically inoculating the human amniotic membrane mesenchymal stem cells obtained in step 1 onto the acellular amniotic membrane obtained in step S2 at a density of 5×10 4 / cm 2 , and the human amniotic membrane mesenchymal stem cells obtained in step 1 are the first generation of cells.
[0022] The application also provides a human amniotic membrane mesenchymal stem cell membrane sheet prepared by the preparation method.
[0023] The application has the following beneficial effects:
[0024] The application constructs a cell membrane sheet using an acellular amniotic membrane as a matrix, thereby avoiding the problems of low mechanical strength and poor operability of traditional cell membrane sheets. The experimental results also confirm that the human amniotic membrane mesenchymal stem cells and the acellular amniotic membrane have good biocompatibility, and the construction of a human amniotic membrane mesenchymal stem cell membrane sheet using an acellular amniotic membrane as a matrix enhances the mechanical strength of the cell membrane sheet and the clinical operability. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 is a morphological observation of human amniotic membrane mesenchymal stem cells (A is the P0 generation of hAMSCs, B is the P1 generation of hAMSCs, and C is the P2 generation of hAMSCs, with a magnification of 100X);
[0026] Figure 2 is a flow cytometry detection of the phenotype molecules and purity of the first generation of hAMSCs (A is CD44, B is CD73, C is CD86, D is CD90, E is CD105, and F is CD-34-CD11b-CD19);
[0027] Figure 3 is an immunofluorescence detection of the expression of vimentin and CK-19 of hAMSCs (A is CK-19, B and E are DAPI, D is vimentin, C is a combination of A and B, F is a combination of D and E, with a magnification of 100X);
[0028] Figure 4 is a general observation of human amniotic membranes and acellular amniotic membranes (A is a peeled human amniotic membrane, and B is an acellular amniotic membrane);
[0029] Figure 5 is HE staining, Sirius red staining, and Masson staining of human amniotic membranes and acellular amniotic membranes (with a magnification of 40X);
[0030] Figure 6 is an electron microscope image of human amniotic membranes and acellular amniotic membranes (A is a human amniotic membrane; C is an acellular amniotic membrane; B is a local enlarged view of A, and D is a local enlarged view of C);
[0031] Figure 7Figure 1 is the wave form protein and phalloidin immunofluorescence detection of the hAMSCs membrane sheet (100X magnification).
[0032] Figure 8 Figure 2 is the electron microscope graph of the hAMSCs membrane sheet (A is the 1st day of cell culture; B is the 3rd day of cell culture; C is the 14th day of cell culture). DETAILED DESCRIPTION
[0033] The experimental methods used in the following examples are all conventional methods unless otherwise specified.
[0034] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.
[0035] In order to make the objectives, technical solutions and advantages of the present application more apparent, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.
[0036] Example 1 Isolation and subculture of hAMSCs
[0037] Under sterile conditions, fresh placenta was obtained, the amnion was peeled off, and the amnion surface was repeatedly washed with sterile PBS to remove the blood stains and mucus on the surface of the amnion. The amnion was cut into pieces with a size of about 1 cm 2 , which was placed into a 50 mL centrifuge tube, 20 mL of 0.25% trypsin / EDTA solution was added, and it was placed into a constant temperature water bath shaker for 45-50 min (37°C, 180 r / min) for digestion. DMEM-F12 complete medium was added to terminate the digestion, and the amnion in the filter screen was washed twice with sterile PBS. The amnion from which the human amnion epithelial cells were removed was placed into a new 50 mL centrifuge tube, and an equal volume of 0.75% collagenase type II was added, which was placed into a constant temperature water bath shaker for 40 min (37°C, 180 r / min) for digestion. The filter screen was filtered, and centrifuged at 1500 r / min for 5 min. The supernatant was discarded, and the hAMSCs cell precipitate was obtained. The hAMSCs precipitate was resuspended with DMEM-F12 complete medium, and the obtained primary cells were inoculated into a T75 cell culture bottle at a cell density of 5x10 4 / cm 2 The cell growth was observed and recorded, and the cell subculture was carried out. According to the observation, the hAMSCs adherent growth was in a spindle or rod shape in the primary culture, and after 1-2 passages, the cells were mostly in a long spindle shape and grew in a spiral shape. Figure 1
[0038] Example 2 Identification of hAMSCs
[0039] 1. Phenotype detection of hAMSCs
[0040] The first generation of hAMSCs was taken, the original culture medium was discarded, and the hAMSCs were washed with PBS twice for 2 minutes each time. Then, 0.25% trypsin was added to digest the hAMSCs for 2 minutes. The digestion was terminated by adding a termination solution. The cell suspension was centrifuged, and the supernatant was discarded. The cells were washed with PBS, and the cell suspension was centrifuged again. The cell density was adjusted to 5.0 x 10 6 μL of the cell suspension was added to each flow tube. Mouse anti-human monoclonal antibodies CD44-PE, CD105-PERCP-Cy, CD90-FITC, CD73-APC, negative control mixture CD34-PE, CD19-PE, CD11b-PE, and CD86-PE were added under light shielding conditions. The cells were incubated for 30 minutes under light shielding conditions. The cells were washed with PBS, centrifuged, and the supernatant was discarded. 250 μL of PBS was added to each flow tube to resuspend the cells. The cells were detected on a flow cytometer, and the positive rate of the surface antigens of the hAMSCs was recorded.
[0041] In this embodiment, the phenotype of the first generation of hAMSCs was detected by flow cytometry. The detection results showed that the hAMSCs highly expressed CD44, CD73, CD90, and CD105, and the expression rate was as high as more than 99%. The hAMSCs lowly expressed or did not express CD34, CD11b, CD19, and CD86. Figure 2
[0042] 1.2 Identification of hAMSCs markers
[0043] The first generation of hAMSCs was taken, and 24-well plate inserts were placed in the culture plates. The cells were seeded in the 24-well plates at a concentration of 1.0 x 10 5 μL of the cell suspension was added to each flow tube. Mouse anti-human monoclonal antibodies CD44-PE, CD105-PERCP-Cy, CD90-FITC, CD73-APC, negative control mixture CD34-PE, CD19-PE, CD11b-PE, and CD86-PE were added under light shielding conditions. The cells were incubated for 30 minutes under light shielding conditions. The cells were washed with PBS, centrifuged, and the supernatant was discarded. 250 μL of PBS was added to each flow tube to resuspend the cells. The cells were detected on a flow cytometer, and the positive rate of the surface antigens of the hAMSCs was recorded.
[0044] The hAMSCs were washed with PBS for 3 times for 5 minutes each time. The cells were fixed with 4% paraformaldehyde for 30 minutes. The cells were treated with 0.3% Triton-X100 at room temperature for 30 minutes, washed with PBS for 3 times for 5 minutes each time, and blocked with goat serum for 45 minutes. 100 μL of mouse anti-human vimentin and CK-19 monoclonal antibodies (diluted at 1:200) were added dropwise, and the mixture was incubated at 4°C overnight. PBS was used instead of the primary antibody for the control group. 100 μL of Alexa Fluor 488-labeled rabbit anti-mouse secondary antibody (diluted at 1:500 with an antibody diluent) was added dropwise, and the mixture was incubated at 37°C for 60 minutes. The cells were washed with PBS for 2 times for 5 minutes each time. The cell nuclei were stained with DAPI for 8 minutes, and the cells were washed with PBS for 2 times for 5 minutes each time. The cells were observed and photographed under a fluorescence microscope.
[0045] The present embodiment adopts immunofluorescence to detect markers (vimentin and CK19) in the first generation of hAMSCs, and the results show that hAMSCs highly express MSC marker vimentin and lowly express or even do not express epithelial cell marker CK19.
[0046] Example 3 Construction and identification of decellularized amniotic membrane
[0047] 1.1 Construction of decellularized amniotic membrane:
[0048] Fresh placenta was taken, and the amnion was peeled off. The surface of the amnion was repeatedly washed with sterile PBS to remove blood stains and mucus remaining on the surface of the amnion. The freshly obtained amnion was placed in a cell culture dish with a diameter of 10 cm, 30 mL of 0.25% trypsin / EDTA solution was added to the culture dish, and it was incubated in a constant temperature shaker for 45 min (37°C, 180 r / min). After constant temperature incubation for 45 min, the 0.25% trypsin / EDTA solution was discarded, and the cells were washed twice with sterile PBS. The surface of the amnion was lightly scraped with a cell scraper to thoroughly remove the epithelial cells and mucus remaining on the surface of the amnion. The decellularized amnion was washed again with sterile PBS for 2 times, and then placed on a dry and sterile nitrocellulose membrane to completely adhere. The decellularized amnion was cut into a specific size and shape for standby use.
[0049] In this embodiment, the surface of the amnion (hAM) was peeled off after the freshly obtained placenta was washed clean, and then the fresh amnion was treated by mechanical-enzyme digestion to obtain the decellularized amnion (hAAM). The hAAM was a thin, white or milky white translucent film. Figure 3 ) that could be observed.
[0050] 1.2 Identification of decellularized amniotic membrane
[0051] Fresh hAM and hAAM were randomly selected, and then the hAM and hAAM were fixed with 4% paraformaldehyde for 30 min. After fixation, paraffin embedding was performed, and paraffin sections with a thickness of 5 μm were prepared for HE staining, Masson staining and Sirius red staining, respectively.
[0052] HE staining: The paraffin sections were placed in a 65°C oven for 30 min, and the paraffin sections were routinely dewaxed to water. The sections were stained with hematoxylin for 3 min and washed with running water for 5 min; differentiated with acid differentiation solution for 30 s; washed with distilled water for 15 min; stained with eosin for 25 s, washed with running water for 8 min, and dehydrated with gradient ethanol. The sections were placed in xylene, mounted, and photographed under a microscope.
[0053] Masson staining: paraffin sections were placed in a 65°C oven for 30 min, and the paraffin sections were routinely dewaxed to water. Weigert hematoxylin staining for 5 min, acid ethanol differentiation for 15 s, water washing for 5 min; Masson blue solution back blue for 5 min, water washing for 5 min; ponceau red staining solution for 6 min, weak acid liquid washing for 20 s, phosphomolybdate washing for 3 min; aniline blue staining for 3 min, weak acid liquid washing for 2 min, anhydrous ethanol dehydration twice, 10 s each time. Xylene transparency, mounting, and later microscope photography.
[0054] Sirius red staining: paraffin sections were placed in a 65°C oven for 30 min, and the paraffin sections were routinely dewaxed to water. Iron hematoxylin staining solution was prepared and added dropwise to the paraffin sections for 6 min, water washing for 30 s, and the excess staining solution was washed away, tap water washing for 5 min; Sirius red staining solution for 25 min, tap water washing for 5 min, anhydrous ethanol dehydration. Xylene transparency, mounting, and later microscope photography.
[0055] The results of HE staining, Sirius red staining, and Masson staining showed that the uppermost layer of the normal human amniotic membrane contained rich epithelial cells, and below it was the basement membrane; below the basement membrane was the compact layer and the fibroblast layer. After decellularization treatment, the epithelial layer of the decellularized amniotic membrane was completely removed, the compact layer was retained, and the collagen components in the extracellular matrix were still uniformly distributed, and the collagen was arranged in a crisscross manner. Figure 5
[0056] 1.3 Scanning electron microscope detection of amniotic membrane and decellularized amniotic membrane
[0057] Randomly selected fresh hAM and hAAM were washed with PBS twice, 5 min each time; fixed with electron microscope fixing solution for 2 h, washed with PBS three times, 5 min each time; dehydrated with ethanol at low concentration to high concentration once, 5 min each time; washed with tert-butyl alcohol at low concentration to high concentration twice, 5 min each time; the tissue sample was dried, the sample was sprayed with gold, and scanning electron microscope observation was performed.
[0058] The scanning electron microscope results showed that the human amniotic membrane contained rich collagen, which was arranged in a crisscross manner, and the amniotic membrane surface contained rich epithelial cells. After decellularization treatment, the epithelial cell component was completely removed, and the decellularized amniotic membrane still contained rich collagen components arranged in a crisscross manner, and the collagen was uniformly distributed. Figure 6
[0059] Example 4 Construction of human amniotic membrane mesenchymal stem cell membrane sheet
[0060] 1.1 Construction of human amniotic membrane mesenchymal stem cell membrane sheet
[0061] The decellularized amniotic membrane prepared in Example 3 was placed in a cell culture dish, and the first generation of human amniotic membrane mesenchymal stem cells obtained in Example 1 was inoculated onto the decellularized amniotic membrane at a density of 5x10 4 / cm 2 , and was cultured in DMEM-F12 complete medium. After 3 days, the DMEM-F12 medium was removed, and a film-forming piece induction medium was added for culture. The medium was changed every 2 days, and after continuous culture for 14 days, the human amniotic membrane mesenchymal stem cell film formed on the decellularized amniotic membrane was obtained.
[0062] 1.2 Biocompatibility detection of human amniotic membrane mesenchymal stem cell film
[0063] Randomly selected human amniotic membrane mesenchymal stem cell film constructed on the decellularized amniotic membrane in the cell culture dish, removed the film-forming piece induction medium in the cell culture dish, washed with PBS for 3 times, 5 min each time. 4% paraformaldehyde fixed cells, using PBS washing 3 times, 5 min each time. 0.3% Triton-X100 room temperature for 30 min, PBS wash 2 times, 5 min each time. Goat serum blocking 45 min. Drop mouse anti-human vimentin antibody 700 μL (1:300 dilution), 4℃ incubate overnight, the second day PBS clean 3 times, 5 min each time, then add 1 mL FITC labeled phalloidin working solution per well, gently shake 6 well plate, so that the phalloidin working solution can fully cover the sample, 37℃ incubate for 30 min in the dark, PBS wash 3 times, 5 min each time. Drop 1 mL Alexa Fluor 594 labeled rabbit anti-mouse secondary antibody (1:400, diluted with antibody diluent), 37℃ incubate for 60 min, PBS wash 2 times, 5 min each time. 37℃, using DAPI restain cell nucleus 5 min, PBS rinse 2 times, 5 min each time, observe and take pictures under fluorescence microscope.
[0064] The results show that human amniotic membrane mesenchymal stem cells can adhere and proliferate on the decellularized amniotic membrane, and the human amniotic membrane mesenchymal stem cells can spread well, the cytoskeleton is clear, the cell morphology is normal, and the cells grow in a crisscross manner ( Figure 7 ).
[0065] 1.3 Scanning electron microscope detection of human amniotic membrane mesenchymal stem cell film
[0066] Randomly selected human amniotic membrane mesenchymal stem cell film constructed on the decellularized amniotic membrane in the cell culture dish, removed the film-forming piece induction medium, washed with PBS for 3 times, 5 min each time. Electron microscope fixing fluid fixation 2h, using PBS washing 3 times, 5 min each time; ethanol low concentration to high concentration dehydration each 1 times, 5 min each time; tert-butyl alcohol low concentration to high concentration dehydration 2 times, 5 min each time; dry the sample, then sample spray gold, scanning electron microscope observation.
[0067] The scanning electron microscope results show that on the first day, the human amniotic mesenchymal stem cells can adhere to the acellular amnion, and the cell spreading is not obvious. On the third day, the cells have better adhesion, proliferation and spreading on the acellular amnion, and the cells are spindle-shaped. On the 14th day, the cells form a lamellar structure on the acellular amnion, the cells proliferate in large quantities, the cells are crisscrossed, a large amount of matrix is secreted outside the cells, and the cells are surrounded by the extracellular matrix. Figure 8
[0068] The above-described embodiments only express the relatively optimal implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as the limitation on the scope of the present application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.
Claims
1. A method of preparing a sheet of human amniotic mesenchymal stem cells, characterized by, It comprises the following steps: S1, isolation and culture of human amniotic mesenchymal stem cells The amnion is cut into pieces, digested with a first digestive solution, filtered, and the filtrate is discarded; the amnion is digested with a second digestive solution, filtered, and the filtrate is centrifuged, the supernatant is discarded, and a human amniotic mesenchymal stem cell pellet is obtained, which is resuspended, inoculated, and subcultured to obtain human amniotic mesenchymal stem cells; S2, preparation of decellularized amnion The amnion is placed in a cell culture dish, digested with a third digestive solution, and the digestive solution is discarded; the amnion is cut after the mucus is scraped off with a cell scraper to obtain a decellularized amnion; S3, preparation of human amniotic mesenchymal stem cell membrane pieces The decellularized amnion obtained in step S2 is placed in a cell culture dish, and the human amniotic mesenchymal stem cells obtained in step S1 are inoculated on the decellularized amnion and cultured for 3 days, the complete culture medium is removed, and a membrane piece induction culture medium is added for continuous culture to obtain human amniotic mesenchymal stem cell membrane pieces.
2. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 1, characterized in that, The first digestive solution and the third digestive solution are both trypsin / EDTA solutions with a concentration of 0.25%, and the second digestive solution is a type II collagenase with a concentration of 0.75%, and the amount of the first digestive solution, the second digestive solution, and the third digestive solution is 1-1.5 times the volume of the amnion.
3. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 2, characterized in that, The digestion is specifically incubated at 35-38℃ in a constant temperature shaker for 40-50 min, and the rotation speed of the constant temperature shaker is 160-200 r / min.
4. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 1, characterized in that, The filtration is specifically filtered with a 250-300 mesh filter, and the amnion in the filter is washed with sterile PBS for 2-3 times, and before filtration, complete culture medium is added to stop digestion.
5. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 5, characterized in that, The resuspension is resuspension of the human amniotic mesenchymal stem cell pellet with complete culture medium; the complete culture medium is DMEM-F12 complete culture medium.
6. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 1, characterized in that, The centrifugation is specifically centrifuged at a rotation speed of 1000-1500 r / min for 4-8 min.
7. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 1, characterized in that, The cutting is specifically placing the decellularized amnion after scraping off the mucus on a dry and sterile nitrocellulose membrane so that it completely adheres, and then cutting the decellularized amnion into a specific size and shape for standby; before and after scraping off the mucus, the amnion is washed with sterile PBS for 2-3 times.
8. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 1, characterized in that, The seeding in step S1 is in particular seeding of the resuspended obtained human amniotic mesenchymal stem cells, which are primary cells, at a cell density of 5 x 10 4 / cm 2 in a T75 cell culture flask.
9. The method for preparing a human amniotic mesenchymal stem cell membrane according to claim 1, characterized in that, The inoculation in step S3 is specifically inoculating the human amniotic membrane mesenchymal stem cells obtained in step 1 onto the decellularized amniotic membrane obtained in step S2 at a density of 5 x 10 4 / cm 2 , the human amniotic membrane mesenchymal stem cells obtained in step 1 being the first generation cells.
10. The human amniotic mesenchymal stem cell membrane piece prepared by the preparation method of any one of claims 1-9.