Preparation method of mesenchymal stem cell membrane and application of mesenchymal stem cell membrane in promoting cell proliferation

Abstract

The invention relates to the technical field of preparation of cell membranes, in particular to a preparation method of a mesenchymal stem cell membrane and application of the mesenchymal stem cell membrane in promotion of cell proliferation, and the preparation method comprises the following steps: step 1, taking mesenchymal stem cells, putting the mesenchymal stem cells in a culture dish, and culturing primary mesenchymal stem cells; 2, when the mesenchymal stem cells in the culture dish are arranged in order and the culture dish is full of the mesenchymal stem cells, performing passage treatment on the primary mesenchymal stem cells; and 3, taking the mesenchymal stem cells in the logarithmic phase for cell patch induction to obtain the mesenchymal stem cell patch. The mesenchymal stem cell membrane prepared by the method can promote cell proliferation and angiogenesis, and is used for treating healing of diabetic wounds. The umbilical cord-derived mesenchymal stem cells used in the preparation method have low immunity, so that the prepared mesenchymal stem cell membrane reduces immunological rejection in allograft.

Description

Technical Field

[0001] The invention relates to the technical field of cell membrane preparation, and in particular to a preparation method of a mesenchymal stem cell membrane and an application of the membrane in promoting cell proliferation. Background Art

[0002] In recent years, with the continuous exploration in the field of regenerative medicine, stem cell therapy has gradually emerged and become an emerging treatment option that has attracted much attention. Among them, mesenchymal stem cells (MSCs), as a multipotent stem cell derived from the mesoderm, are widely distributed in the human body and exist in most tissues. Compared with other treatment methods and means, MSCs have the advantages of self-replication, self-renewal, multidirectional differentiation, and low immunogenicity. They have been widely used in the treatment of diseases such as cancer, diabetes, neurological diseases and cardiovascular diseases. They are ideal "seeds" for repairing cell damage. MSCs also have great potential in the treatment of wound healing disorders. The presence of mesenchymal stem cells in skin tissue, coupled with their indispensable role in normal wound healing and skin homeostasis, means that mesenchymal stem cells may be beneficial to the healing of chronic wounds.

[0003] As a new type of tissue regeneration therapy, cell sheet technology has been widely used to regenerate various types of tissues and organs, including the repair of skin, oral cavity, urethra, cartilage, heart, cornea, etc. The composition of cell sheets is similar to that of natural tissues, avoiding inflammation and immune response caused by scaffolds. In addition, since cell sheets do not need to be treated with proteolytic enzymes, the number of cells, intercellular connections and structures are well preserved, thereby improving the regeneration capacity.

[0004] All cell sheets currently used in clinical practice are derived from autologous cells, which eliminates the risk of immune response after transplantation. The use of allogeneic cell sheets is a promising treatment strategy that solves the high production and time costs required to manufacture cell sheets from each patient. Therefore, cell sheet technology makes it possible for MSCs to continuously and precisely focus on the surface of the infarct area to function. Summary of the invention

[0005] In order to overcome the problems of high production cost and time cost of cell membrane sheets derived from autologous cells in the prior art, the present invention provides a method for preparing a mesenchymal stem cell membrane sheet using mesenchymal stem cells and its application in promoting cell proliferation.

[0006] The present invention adopts the following technical solutions:

[0007] A method for preparing a mesenchymal stem cell membrane comprises the following steps:

[0008] Step 1: Take mesenchymal stem cells and place them in a culture dish to culture primary mesenchymal stem cells;

[0009] Step 2: When the mesenchymal stem cells are neatly arranged in the culture dish and cover the entire culture dish, the primary mesenchymal stem cells are passaged;

[0010] Step 3: Take mesenchymal stem cells in the logarithmic growth phase to induce cell membrane sheets to obtain mesenchymal stem cell membrane sheets.

[0011] Furthermore, the mesenchymal stem cells in step 1 are obtained from the umbilical cord using a tissue adhesion method, and the specific steps for obtaining the mesenchymal stem cells derived from the umbilical cord are: the umbilical cord is soaked in physiological saline containing 1% double antibody for 30 minutes and then placed in a 150mm cell culture dish, washed with sterile physiological saline 2-4 times to wash away the umbilical cord blood attached to the surface of the umbilical cord, and stored in 20ml of sterile physiological saline, one vein and two arteries of the umbilical cord are peeled off, and the remaining tissue is flattened and cut into small sections with a length of 0.5-1cm with scissors, and evenly spread in a 60mm cell culture dish, 2ml of mesenchymal stem cell complete culture medium containing 15% fetal bovine serum is added to the culture dish, and the dish is placed in a constant temperature 37°C cell culture incubator for culture, and the cell adhesion is observed.

[0012] Furthermore, the specific operation of step 2 is as follows: the mesenchymal stem cells are first digested with 0.25% trypsin solution in the passaging treatment, and then the culture medium containing the mesenchymal stem cells is transferred to a centrifuge tube, centrifuged at 1200 rpm for 5 minutes at room temperature, the supernatant is discarded with a pipette, and the precipitate is retained, and then the stem cell complete culture medium is added, and the cells are passaged at a ratio of 1:2 to 1:3, and the cells are placed in a constant temperature incubator for culture.

[0013] Furthermore, the specific operation of the digestion treatment in step 2 is: discard the original culture medium with a pipette, add 1 ml of trypsin solution along the wall of the culture dish to cover the cell layer, discard the trypsin solution and then add 1 ml of trypsin solution, place it in a 37°C incubator for digestion for 3 minutes, and after the digestion is completed, observe the cell morphology under a microscope and change to a round shape. When the cells can be seen floating and falling off from the bottom of the culture dish by tapping the culture dish, 1 ml of culture medium containing fetal bovine serum is added thereto to terminate the digestion process.

[0014] Furthermore, the specific operation of step three is: taking a mesenchymal stem cell suspension of the logarithmic growth phase of generation P3-P5, fully discretizing it into single cells, and placing 5×10 6 Mesenchymal stem cells were inoculated into a cell culture dish with a diameter of 100 mm and shaken in an "8" shape to ensure that the cells were evenly distributed in the dish. After the cells adhered to the wall, the culture medium was replaced with a cell membrane induction medium. The culture medium was replaced every other day. When a curled white film appeared on the edge of the cell culture dish, a mesenchymal stem cell membrane was obtained.

[0015] Furthermore, the cell sheet induction medium in step three is 120 μg / ml ascorbic acid and stem cell culture medium. During the process of inducing the cell sheet with ascorbic acid, the thickness of the cell sheet increases with the increase of the induction days.

[0016] Furthermore, the mesenchymal stem cell membrane is stacked by 3 or more layers of mesenchymal stem cells.

[0017] The present invention also provides another technical solution:

[0018] A mesenchymal stem cell membrane is used in promoting cell proliferation. The mesenchymal stem cell membrane prepared by the above technical scheme is used to treat the healing of diabetic wounds. The specific operation is: applying the prepared mesenchymal stem cell membrane to the diabetic wound surface, using a transparent semi-closed adhesive dressing to cover the wound, and protecting the transplanted cell membrane.

[0019] The present invention has at least the following beneficial effects:

[0020] 1. The present invention provides a method for preparing a mesenchymal stem cell membrane. Umbilical cord-derived mesenchymal stem cells have low immunity, so that the prepared mesenchymal stem cell membrane reduces immune rejection in allogeneic transplantation. The cell membrane prepared from umbilical cord-derived mesenchymal stem cells has low production cost, simple operation steps, and can promote cell proliferation and angiogenesis;

[0021] 2. The use of the mesenchymal stem cell membrane provided by the present invention in promoting cell proliferation. The mesenchymal stem cell membrane is used to treat diabetic wounds, which can promote wound healing, enable the epidermal cells at the wound to self-repair, and effectively prevent infection caused by external pathogens;

[0022] 3. The use of the mesenchymal stem cell membrane provided by the present invention in promoting cell proliferation. After culturing the mesenchymal stem cell membrane, exosomes are obtained by ultrahigh-speed centrifugation. The exosomes of the mesenchymal stem cell membrane can accelerate wound hemostasis, promote cell proliferation, and promote angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A flow chart of a method for preparing a mesenchymal stem cell membrane provided by the present invention;

[0024] Figure 2 This is a culture identification diagram of a mesenchymal stem cell membrane prepared by the preparation method provided by the present invention;

[0025] Figure 3 This is a CCK-8 test diagram of the mesenchymal stem cell membrane prepared by the preparation method provided by the present invention;

[0026] Figure 4 This is a scratch test diagram of the mesenchymal stem cell membrane prepared by the preparation method provided by the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Embodiment 1

[0029] Combination Figure 1 As shown, the present invention discloses a method for preparing a mesenchymal stem cell membrane, and the specific operation steps are as follows:

[0030] Step 1: Take mesenchymal stem cells and culture them in a culture dish:

[0031] Mesenchymal stem cells are obtained from the umbilical cord using the tissue adhesion method. After delivery, the umbilical cord is transferred to a sterile tissue sampling bottle and transported to the laboratory at 4°C. Within 4-6 hours of tissue ex vivo, it is transferred to a UV-sterilized biosafety cabinet or clean bench in the laboratory for subsequent operations.

[0032] The umbilical cord was taken out from the sampling bottle and transferred to a 50ml centrifuge tube in a clean bench. The umbilical cord was soaked in saline containing 1% double antibody for 30 minutes and then placed in a 150mm cell culture dish. The cord blood attached to the surface of the umbilical cord was washed 2-4 times with sterile saline to remove the cord blood. The cord without cord blood was stored in 20ml of sterile saline. A vein and two arteries were stripped from the cleaned umbilical cord using autoclaved hemostatic forceps and tweezers. The surface of the umbilical cord was kept moist during the stripping process. A small amount of saline was added in time. The remaining tissue was flattened and cut into small sections with a length of 0.5-1cm with scissors. The sections were evenly spread in a 60mm cell culture dish. 2ml of complete medium for mesenchymal stem cells containing 15% fetal bovine serum was added to the culture dish. It was appropriate to make sure that the tissue blocks were in contact with the liquid and that the tissue did not float. The culture was placed in a constant temperature 37℃ cell culture incubator for culture. To reduce residual red blood cells, the culture medium in the culture dish was replaced the next day, and the medium was changed every 2 days thereafter. Observe the cells under a microscope to see whether they are attached to the wall and their density. If necessary, adjust the position of the tissue in the dish to ensure that the cells are evenly distributed in the dish.

[0033] Step 2: When the mesenchymal stem cells are neatly arranged and cover the entire culture dish, the primary mesenchymal stem cells are passaged:

[0034] Preheat 0.25% trypsin solution and complete stem cell culture medium in a 37°C constant temperature water bath for 10-20 minutes.

[0035] Passaging treatment: First, use 0.25% trypsin solution to digest the mesenchymal stem cells, discard the original culture medium with a pipette, add 1 ml of trypsin solution along the wall of the culture dish to cover the cell layer, immediately discard the trypsin solution to avoid excessive digestion and damage to the cells, add 1 ml of trypsin solution, place in a 37°C incubator for digestion for 3 minutes, and after digestion, observe the cell morphology under a microscope and see that the cells float and fall off from the bottom of the culture dish by tapping the culture dish, then add 1 ml of culture medium containing fetal bovine serum to terminate the digestion process;

[0036] Then, gently blow the cells that have not fallen off in the culture dish to ensure that they can fall off from the surface of the culture dish smoothly, transfer the culture medium containing mesenchymal stem cells to a centrifuge tube, centrifuge at 1200 rpm for 5 minutes at room temperature, discard the supernatant with a pipette, retain the precipitate, add stem cell complete culture medium, perform cell subculture operation at a ratio of 1:2 to 1:3, and culture in a constant temperature incubator;

[0037] Step 3: Take mesenchymal stem cells in the logarithmic growth phase to induce cell membranes to obtain mesenchymal stem cell membranes:

[0038] Take the mesenchymal stem cell suspension at the generation number of P3-P5 in the logarithmic growth phase, fully disperse it into single cells, and place 5×10 6 Mesenchymal stem cells were inoculated into a cell culture dish with a diameter of 100 mm and shaken in an "8" shape to ensure that the cells were evenly distributed in the culture dish. After the cells adhered to the wall, the culture medium was replaced with a cell membrane induction medium containing 120 μg / ml ascorbic acid and stem cell culture medium. The culture medium was replaced every other day. After about 10 days, the edge of the cell culture dish curled up and a white film appeared, and a mesenchymal stem cell membrane sheet was obtained. Figure 2 -A.

[0039] After obtaining the mesenchymal stem cell membrane, it was immersed in 4% paraformaldehyde fixative, embedded in paraffin, and sliced ​​at 5 μm for HE staining. Figure 2 -B shows that the mesenchymal stem cell membrane is composed of 3 or more layers of mesenchymal stem cells. The cells are arranged tightly and neatly, and there is a large amount of cell matrix between the cells. The morphology and structure of the cell membrane are observed under an optical microscope. The cells are stacked layer by layer. Figure 2 -C.

[0040] In order to verify that mesenchymal stem cell membranes have the effect of promoting cell proliferation and angiogenesis, the effects of mesenchymal stem cells and mesenchymal stem cell membranes on endothelial cells will be compared as follows:

[0041] Take 1×10 endothelial cells 6 The number of cells / ml was inoculated into two 100 mm culture dishes, and the culture medium in the culture dishes was fresh stem cell complete medium, which were recorded as culture dishes A and culture dishes B.

[0042] When the cells almost fill the entire culture dish A and the degree of confluence reaches 90%, the culture medium is replaced with serum-free DMEM medium containing 1% double antibody. After culturing for 48 hours, the supernatant is collected, centrifuged at 1500 rpm for 10 minutes, filtered using a 0.22 μm filter membrane, and stored in a 4°C refrigerator awaiting the next step. When used, 5% fetal bovine serum is added to prepare a mesenchymal stem cell conditioned medium.

[0043] When the cells almost fill the entire culture dish B and the degree of confluence reaches 90%, the culture medium is replaced with a cell membrane induction medium containing ascorbic acid. On the 7th day of culture, the cells are washed with PBS and replaced with a serum-free DMEM medium containing 1% double antibody. After 48 hours, the supernatant is collected, centrifuged, filtered, and stored at 4°C for later use. When used, 5% fetal bovine serum is added to prepare a mesenchymal stem cell membrane conditioned medium.

[0044] At the same time, a blank culture medium (1% double antibody serum-free DMEM + 5% fetal bovine serum) was prepared as a control group, and the CCK-8 method was used to detect the proliferation of endothelial cells under the three culture medium conditions. The cell proliferation was detected at 12h, 24h, 48h, 72h, and 96h. At the same time, the three culture media were subjected to scratch experiments to detect the migration of endothelial cells, and cell scratch images were collected on days 0, 1, and 2.

[0045] CCK-8 reagent is converted into an orange-yellow substance with excellent water solubility through the reduction reaction of dehydrogenase inside mitochondria, thus effectively indicating the activity status of cells. It is worth noting that the amount of this orange-yellow product produced is directly proportional to the number of living cells. The more active the dehydrogenase, the more cells proliferate and the darker the color. The final three groups of results are as follows Figure 3 As shown in the figure, it can be clearly seen that both mesenchymal stem cells and mesenchymal stem cell membrane sheets can promote the proliferation of endothelial cells, and the promoting effect of mesenchymal stem cell membrane sheets is better.

[0046] The scratch test is a quick and convenient method to determine the migration ability of cells. By comparing the changes in the width and size of the scratch before and after, the migration and growth ability of cells are compared, the initial and final wound widths of migration are measured, and the migration rate of endothelial cells is calculated. The image collection and scratch area calculation of the scratch healing after different treatments are performed on the 0th day, the 1st day, and the 2nd day. The red line is the scratch edge. Figure 4 -A shows that the width of the scratches was similar at the beginning, but as time went on, the width of the scratches in each group shortened to varying degrees. The cells treated with the supernatant of the conditioned culture of the mesenchymal stem cell membrane were significantly faster than those in the other two groups, which was consistent with the healing rate of the scars on the second day. Figure 4 -B, the healing rate of the mesenchymal stem cell group was higher than that of the control group, the healing rate of the mesenchymal stem cell membrane group was higher than that of the mesenchymal stem cell group, and the migration speed of the mesenchymal stem cell membrane group was the fastest, significantly higher than that of the other two groups.

[0047] In summary, the mesenchymal stem cell membrane sheet prepared by the preparation method of the mesenchymal stem cell membrane sheet disclosed in the present invention has a promoting effect on the proliferation of endothelial cells, and can promote cell healing and migration.

[0048] Embodiment 2

[0049] The present invention also discloses an application of a mesenchymal stem cell membrane in promoting cell proliferation. The mesenchymal stem cell membrane prepared by the technical solution of Example 1 is used to treat the healing of diabetic wounds. The specific operation is: applying the prepared mesenchymal stem cell membrane to the wound surface of diabetic wounds, and using a transparent semi-closed adhesive dressing to cover the wound to protect the transplanted cell membrane. After the mesenchymal stem cell membrane is treated, the total wound healing time is shortened, and the wound healing quality is better, the collagen deposition is richer, and the number of new blood vessels is also greater.

[0050] Embodiment 3

[0051] In order to further study the effect of mesenchymal stem cell membrane on cell proliferation, the present invention also discloses that the mesenchymal stem cell membrane prepared by the technical solution of Example 1 is further cultured, and exosomes are obtained by ultrahigh-speed centrifugation. Experiments show that the cell proliferation activity is positively correlated with the exosome concentration.

[0052] Take the mesenchymal stem cell suspension of generation P3-P5 in step 3 of Example 1 for subculture, and add 5×10 6 The cells were inoculated in a 150 mm cell culture dish, and the culture medium was replaced with serum-free medium after the cells adhered to the wall. The culture medium was collected after 24 hours of culture and stored at 4°C for later use. After the supernatant was collected to a certain volume, it was filtered with a 0.45 μm filter to remove cell debris, and then filtered with a 30 kD membrane bag and concentrated to 5 times the original concentration.

[0053] Transfer the concentrated supernatant to a sterilized 50ml centrifuge tube and prepare for centrifugation. Before centrifugation, lower the temperature of the centrifuge to 4°C to prevent high-temperature degradation of exosomes. First, centrifuge the sample at 2000g for 20 minutes. After centrifugation, retain the supernatant and discard the precipitate, adjust the speed of the desktop centrifuge to 10000g, and centrifuge the supernatant sample again for 30 minutes.

[0054] Transfer the supernatant to a centrifuge tube dedicated to the ultracentrifuge, and accurately balance it before centrifugation to ensure that the error between the two corresponding tubes is less than 0.01g. Then, centrifuge at a high speed of 100,000g for 90 minutes. After centrifugation, quickly discard the supernatant to prevent re-dissolution, and retain the required white or brown precipitate. Add 1-2ml of sterile PBS solution to each ultracentrifuge tube and resuspend until the precipitate is no longer visible. Enrich all the sterile PBS solution that has dissolved the precipitate into the ultracentrifuge tube and balance it again. After balancing, centrifuge at a speed of 100,000g for 70 minutes.

[0055] After the centrifugation operation is completed, the supernatant is discarded and the precipitate with a darker color than that in the previous step and easier to be observed by the naked eye is retained. 1-2 ml of sterile PBS is used to dissolve all the precipitates and transfer them to an EP tube. The EP tube is sealed with a sealing film to obtain the exosomes of the mesenchymal stem cell membrane.

[0056] At the same time, as a control group, ultra-high-speed centrifugation was used to obtain exosomes from mesenchymal stem cells. It was found that the content of exosomes obtained from mesenchymal stem cell membranes was higher than that obtained from mesenchymal stem cells.

[0057] The exosome samples of the mesenchymal stem cell membrane were taken, and the CCK-8 test method and the scratch test were also used to detect the effect of exosomes on the proliferation and migration of endothelial cells. The final results showed that the exosomes of the mesenchymal stem cell membrane can promote the proliferation of endothelial cells and the migration of endothelial cells, and are positively correlated with the concentration of the exosomes. As the concentration increases, the cell proliferation activity increases, which corresponds to the result that the mesenchymal stem cell membrane of Example 1 is more conducive to cell proliferation.

[0058] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell membrane, characterized in that: The following steps are involved: Step 1: Take mesenchymal stem cells and place them in a culture dish to culture primary mesenchymal stem cells; Step 2: When the mesenchymal stem cells are neatly arranged in the culture dish and cover the entire culture dish, the primary mesenchymal stem cells are passaged; Step 3: Take mesenchymal stem cells in the logarithmic growth phase to induce cell membrane sheets to obtain mesenchymal stem cell membrane sheets.

2. The method for preparing a mesenchymal stem cell membrane according to claim 1, characterized in that: The mesenchymal stem cells are obtained from the umbilical cord using a tissue adhesion method, and the culture dish in step 1 contains 2 ml of a complete mesenchymal stem cell culture medium containing 15% fetal bovine serum.

3. The method for preparing a mesenchymal stem cell membrane according to claim 2, characterized in that: The specific steps of obtaining the mesenchymal stem cells from the umbilical cord are as follows: the umbilical cord is soaked in physiological saline containing 1% double antibody for 30 minutes and then placed in a 150mm cell culture dish, washed with sterile physiological saline for 2-4 times to wash away the umbilical cord blood attached to the surface of the umbilical cord, and stored in 20ml of sterile physiological saline, one vein and two arteries of the umbilical cord are peeled off, the remaining tissue is flattened and cut into small sections with a length of 0.5-1cm with scissors, and evenly spread in a 60mm cell culture dish, 2ml of mesenchymal stem cell complete culture medium containing 15% fetal bovine serum is added to the culture dish, and the dish is placed in a constant temperature 37°C cell culture incubator for culture, and the cell adhesion is observed.

4. The method for preparing a mesenchymal stem cell membrane according to claim 1, characterized in that: The specific operation of step 2 is as follows: the mesenchymal stem cells are first digested with 0.25% trypsin solution in the passaging treatment, and then the culture medium containing the mesenchymal stem cells is transferred to a centrifuge tube, centrifuged at 1200 rpm for 5 minutes at room temperature, the supernatant is discarded with a pipette, and the precipitate is retained, and then the stem cell complete culture medium is added, and the cells are passaged at a ratio of 1:2 to 1:3, and the cells are placed in a constant temperature incubator for culture.

5. The method for preparing a mesenchymal stem cell membrane according to claim 4, characterized in that: The specific operation of the digestion treatment in step 2 is: discard the original culture medium with a pipette, add 1 ml of trypsin solution along the wall of the culture dish to cover the cell layer, discard the trypsin solution and then add 1 ml of trypsin solution, place it in a 37°C incubator for digestion for 3 minutes, and after the digestion is completed, observe the cell morphology under a microscope and see that the cells have changed into a round shape and can be seen floating and falling off from the bottom of the culture dish, then add 1 ml of culture medium containing fetal bovine serum to terminate the digestion process.

6. The method for preparing a mesenchymal stem cell membrane according to claim 5, characterized in that: The specific operation of step 3 is: take a mesenchymal stem cell suspension of the logarithmic growth phase of generation P3-P5, fully disperse it into single cells, and place 5×10 6 Mesenchymal stem cells were inoculated into a cell culture dish with a diameter of 100 mm. After the cells adhered to the wall, the cell membrane induction medium was replaced. The medium was replaced every other day. When the edge of the cell culture dish curled and a white film appeared, the mesenchymal stem cell membrane was obtained.

7. The method for preparing a mesenchymal stem cell membrane according to claim 6, characterized in that: The cell sheet induction medium in step 3 is 120 μg / ml ascorbic acid and stem cell culture medium.

8. The method for preparing a mesenchymal stem cell membrane according to claim 7, characterized in that: The mesenchymal stem cell membrane sheet is stacked by 3 or more layers of mesenchymal stem cells.

9. Use of a mesenchymal stem cell membrane in promoting cell proliferation, characterized in that: The mesenchymal stem cell membrane sheet described in any one of claims 1 to 8 is used to treat the healing of diabetic wounds, and the specific operation is: applying the prepared mesenchymal stem cell membrane sheet to the diabetic wound surface, and using a transparent semi-occlusive adhesive dressing to cover the wound to protect the transplanted cell membrane sheet.

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