Stem cell thin film biological dressing for treating skin injury and preparation method thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a stem cell film biological dressing for treating skin injury and a preparation method thereof. The present application provides a stem cell film biological dressing, which comprises a composite stem cell film, a hydrogel nutrition layer and an adhesive dressing, wherein the composite stem cell film is formed on one side surface of the hydrogel nutrition layer, the adhesive dressing is formed on the other side of the hydrogel nutrition layer, and the composite stem cell film comprises a plurality of single-layer stem cell films stacked. The stem cell film biological dressing can promote the growth of wound epidermal cells, shorten the wound healing time and shorten the course of disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a stem cell film bio-dressing for treating skin lesions and its preparation method. Background Technology

[0002] Extensive skin damage, severe trauma, cosmetic surgery, and postoperative wound coverage are currently critical clinical issues. Existing treatments, such as autologous skin grafts, allogeneic skin grafts, and skin grafts using synthetic repair materials, all have limitations, increasing the difficulty and cost of their clinical application. Therefore, there is an urgent need to find a treatment method that does not cause rejection and promotes skin repair.

[0003] With the development of biology and tissue engineering technology, mesenchymal stem cells have attracted attention due to their potential repair function. However, current methods of cell suspension infusion often result in low cell regeneration efficiency and utilization after reinfusion because the extracellular matrix (ECM) is damaged by hydrolytic enzymes.

[0004] Therefore, it is urgent to develop a drug that can induce rejection and promote rapid wound healing. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a stem cell film bio-dressing that promotes the growth of epidermal cells in wounds, shortens wound healing time, and reduces the course of disease.

[0006] Therefore, a first aspect of the present invention provides a stem cell film bio-dressing. According to an embodiment of the present invention, the stem cell film bio-dressing includes a composite stem cell film, a hydrogel nutrient layer, and an adhesive dressing.

[0007] The composite stem cell film is formed on one side of the hydrogel nutrient layer, and the adhesive dressing is formed on the other side of the hydrogel nutrient layer.

[0008] The composite stem cell membrane comprises multiple stacked monolayer stem cell membranes.

[0009] Cell sheets refer to a complete monolayer of cells obtained by culturing isolated cells on a specific material under in vitro culture conditions and separating the cells from the culture material using specific methods without disrupting cell connections. Compared with cell injection and polymer-cell chimeric implants, cell sheets have the following advantages in clinical applications: (1) Cell sheets can be directly transplanted to the desired site during transplantation surgery; (2) Cell sheets can be directly applied to these sites to play a key therapeutic role; (3) Cell sheets completely preserve the extracellular matrix, which is conducive to cell regeneration and in vivo utilization. Therefore, novel biological dressings based on stem cell membranes are an ideal means of treating skin injuries.

[0010] This invention provides a stem cell film bio-dressing, comprising a multilayer stem cell film and a hydrogel nutrient layer that provides essential nutrients and growth factors for cell viability. This stem cell film bio-dressing can promote the growth of wound epidermal cells through cell growth factors and extracellular matrix produced by the paracrine function of multiple stem cells, thus shortening wound healing time. Furthermore, the hydrogel nutrient layer in this invention uses gelatin hydrogel as a base and contains various nutrients and growth factors necessary for cell viability, effectively improving cell survival rate and prolonging cell survival time.

[0011] The stem cell film bio-dressing according to the above embodiments of the present invention may further have at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the composite stem cell membrane comprises 3 to 5 stacked monolayer stem cell membranes. This further improves the toughness and robustness of the cell membrane.

[0013] According to an embodiment of the present invention, the composite stem cell membrane is a mesenchymal stem cell membrane.

[0014] According to an embodiment of the present invention, the mesenchymal stem cell membrane is formed from at least one selected from umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

[0015] According to an embodiment of the present invention, the composite stem cell film is obtained by the following means:

[0016] (1) Stem cells are cultured in a culture dish using stem cell culture medium to form a monolayer stem cell film;

[0017] (2) Remove the stem cell culture medium and place the monolayer stem cell membrane in a peeling solution to obtain a free monolayer stem cell membrane;

[0018] (3) Collect the free monolayer stem cell film from the stripping solution and stack multiple free monolayer stem cell films to obtain a composite stem cell film.

[0019] According to an embodiment of the present invention, in step (1), the mesenchymal stem cells are passaged no more than 7 times. Using mesenchymal stem cells with a passage count of no more than 7 times can further improve the integrity of the obtained mesenchymal stem cell membrane and enhance the state of the stem cell membrane. However, when using cells from passage 8 or higher to prepare stem cell membranes, cell membrane breakage and automatic detachment may occasionally occur, making it difficult to form an intact cell membrane.

[0020] According to an embodiment of the present invention, the inner surface of the bottom of the petri dish is coated with a coating, the surface free energy of which does not exceed 90 mJ / m 2 This makes it easier for cells to adhere and grow, and the cell membrane detaches more effectively from the culture dish under specific external stimuli.

[0021] According to an embodiment of the present invention, the surface free energy of the coating does not exceed 60 mJ / m 2 This further improves the efficiency of cell membrane detachment from the culture dish.

[0022] The surface free energy of a culture dish affects its hydrophilicity / hydrophobicity. If the hydrophilicity is too high, the adhesion proteins on the cell surface will have an excessively strong effect on the culture dish, which may prevent cells from detaching to form a monolayer cell membrane, or cause partial cell detachment and cell membrane breakage. If the hydrophobicity is too high, cells may not be able to adhere to the culture dish for growth.

[0023] According to an embodiment of the present invention, the thickness of the coating is 2–100 nm, preferably 2–50 nm, and more preferably 2–30 nm. This improves the detachment of the cell membrane from the culture dish. Furthermore, the inventors have found that if the coating is too thick, cell growth can be adversely affected.

[0024] According to an embodiment of the invention, the coating comprises a polymer and / or a block copolymer.

[0025] According to an embodiment of the invention, the coating is formed from a poly(2-vinylpyridine-co-styrene) block copolymer.

[0026] According to an embodiment of the invention, the coating is formed by solid-phase deposition of 2-vinylpyridine and styrene. This results in better detachment of the cell film from the culture dish.

[0027] According to an embodiment of the present invention, the stripping solution is DPBS buffer.

[0028] According to an embodiment of the present invention, the free monolayer stem cell membrane is collected using a solid support.

[0029] According to an embodiment of the present invention, the solid support is a polymer support.

[0030] According to an embodiment of the present invention, the solid support is a PVDF membrane.

[0031] According to an embodiment of the present invention, the PVDF membrane is a star-shaped hydrophilically modified PVDF membrane of dimethylaminoethyl acrylate polymer.

[0032] A second aspect of the present invention provides a method for preparing the stem cell film bio-dressing described in the first aspect. According to an embodiment of the present invention, the preparation method includes:

[0033] 1) The adhesive dressing is attached to one side of the hydrogel nutrient layer to obtain an adhesive dressing-hydrogel nutrient layer;

[0034] 2) Transfer the composite stem cell film to the other side of the hydrogel nutrient layer in the adhesive dressing-hydrogel nutrient layer to obtain the stem cell film bio-dressing.

[0035] According to an embodiment of the present invention, the hydrogel nutrient layer comprises mesenchymal stem cell culture medium and 10-30% wt gelatin.

[0036] According to an embodiment of the present invention, the thickness of the hydrogel nutrient layer is 0.5–3 mm.

[0037] The third aspect of this invention provides the use of the stem cell film bio-dressing described in the first aspect and the stem cell film bio-dressing prepared by the preparation method described in the second aspect in the preparation of a medicament for treating skin injuries.

[0038] A fourth aspect of the present invention provides a medicament. According to an embodiment of the present invention, the medicament comprises the stem cell film bio-dressing described in the first aspect and / or the stem cell film bio-dressing prepared by the preparation method described in the second aspect.

[0039] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0041] Figure 1The atomic nucleus magnetic resonance (NMR) spectrum of a star-shaped poly(dimethylaminoethyl acrylate) polymer according to an embodiment of the present invention is shown.

[0042] Figure 2 The SEM image of a star-shaped poly(dimethylaminoethyl acrylate) polymer according to an embodiment of the present invention is shown. Detailed Implementation

[0043] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0044] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0045] Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0046] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0047] According to a specific embodiment of the present invention, the present invention provides a stem cell film bio-dressing, comprising a composite stem cell film, a hydrogel nutrient layer, and an adhesive dressing.

[0048] In this structure, a composite stem cell film is formed on one side of the hydrogel nutrient layer, while an adhesive dressing is formed on the other side of the hydrogel nutrient layer.

[0049] The composite stem cell membrane consists of multiple stacked monolayer stem cell membranes.

[0050] According to embodiments of the present invention, there are no particular limitations on the specific type or material of the adhesive dressing. Those skilled in the art can select commonly used and suitable adhesive dressings according to actual needs, preferably breathable adhesive dressings. The size of the stem cell film bio-dress can also be selected according to the size of the stem cell film contained therein.

[0051] According to embodiments of the present invention, the composite stem cell membrane comprises 3 to 5 stacked monolayer stem cell membranes. This further improves the toughness and robustness of the cell membrane.

[0052] The appropriate number of single-layer stem cell membranes in the composite stem cell membrane can be selected based on different applications and indications. For example, the composite stem cell membrane used in mouse animal models contains three stem cell membrane layers.

[0053] According to an embodiment of the present invention, the composite stem cell membrane is a mesenchymal stem cell membrane, wherein the mesenchymal stem cell membrane is formed from at least one selected from bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and placental-derived mesenchymal stem cells. This further enhances the skin damage repair capability of the mesenchymal stem cell membrane.

[0054] According to an embodiment of the present invention, the composite stem cell film is obtained by the following means:

[0055] (1) Mesenchymal stem cells are cultured in a culture dish using stem cell culture medium in order to form a monolayer stem cell membrane;

[0056] (2) Remove the stem cell culture medium and place the monolayer stem cell membrane in the peeling solution to obtain the free monolayer stem cell membrane.

[0057] (3) Collect the free monolayer stem cell membrane from the stripping solution and stack multiple free monolayer stem cell membranes to obtain a composite stem cell membrane.

[0058] According to an embodiment of the present invention, in step (1), the mesenchymal stem cells are passaged no more than 7 times. There is no particular limitation on the number of passages of mesenchymal stem cells, but it is preferred that the number of passages not exceed 7, because mesenchymal stem cells meeting this requirement can further improve the integrity of the obtained mesenchymal stem cell membrane, improve the state of the stem cell membrane, and are less likely to detach prematurely.

[0059] According to embodiments of the present invention, the seeding density of mesenchymal stem cells into the culture medium contained in the culture dish is not particularly limited and can be controlled according to the desired time for harvesting the stem cell membrane. For example, if a faster stem cell membrane is required, the seeding density of mesenchymal stem cells can be increased. A seeding density of 10,000 cells / cm² to 50,000 cells / cm² can achieve a near 100% over-fusion state after culturing in a 35 mm diameter culture dish for 72 hours, which is suitable for preparing stem cell membranes.

[0060] According to embodiments of the present invention, the stem cell culture medium can be a conventional culture medium suitable for culturing mesenchymal stem cells, such as serum-free stem cell culture medium (Cellartis, Takara) or DMEM medium. In addition, additional components can be added to the culture medium, such as 16.8 μg / ml ascorbic acid, to facilitate the synthesis of extracellular matrix by stem cells and enhance cell membrane strength.

[0061] According to an embodiment of the present invention, the inner surface of the bottom of the petri dish is coated with a coating, the surface free energy of which does not exceed 90 mJ / m 2 Preferably not exceeding 60mJ / m 2 Therefore, the stem cell membrane detaches more effectively from the culture dish.

[0062] According to an embodiment of the present invention, the coating thickness is 2–100 nm, preferably 2–50 nm, and more preferably 2–30 nm. This results in better detachment of the cell membrane from the culture dish.

[0063] According to an embodiment of the present invention, the coating comprises a polymer and / or a block copolymer. There are no particular limitations on the type of polymer and / or block copolymer in the coating material, as long as the surface free energy of the coating does not exceed 90 mJ / m². 2 That is, the coating can be formed from a poly(2-vinylpyridine-co-styrene) block copolymer, or the coating can be formed by solid-phase deposition of 2-vinylpyridine and styrene.

[0064] According to an embodiment of the present invention, in step (1), after culturing mesenchymal stem cells in a culture dish to near 100% confluence, the stem cell membrane should be processed within 48 hours. If the process exceeds 48 hours, there is a chance that the stem cell membrane will detach or break.

[0065] According to an embodiment of the present invention, the stripping solution is DPBS buffer. This stripping solution is effective in reducing the adhesion between the cell membrane and the culture dish.

[0066] According to embodiments of the present invention, a PVDF membrane can be used to collect free monolayer stem cell membranes. Specifically, after the monolayer stem cell membrane detaches from the culture dish, a PVDF membrane can be used to adsorb the monolayer stem cell membrane onto the surface, and the membrane can be rinsed several times with physiological saline as needed. Preferably, the PVDF membrane is a star-shaped hydrophilically modified PVDF membrane with dimethylaminoethyl acrylate polymer. Therefore, its adsorption effect on the monolayer stem cell membrane is better.

[0067] For ease of understanding, the following is a detailed description of the star-shaped PVDF membrane modified with dimethylaminoethyl acrylate polymer.

[0068] The star-shaped poly(dimethylaminoethyl acrylate) polymer (PDMAEA) has the structure shown in Formula I.

[0069]

[0070] In Equation I, R is n is a positive integer from 15 to 105. The degree of polymerization of this star-shaped dimethylaminoethyl acrylate polymer is between 100 and 400, and the molecular weight is between 10,000 and 60,000 Daltons. The inventors discovered that the long alkyl chains in the star-shaped dimethylaminoethyl acrylate polymer are hydrophobic and have good compatibility with PVDF; while the dimethylaminoethyl acrylate segments have high hydrophilicity, which improves hydrophilicity and increases the stability of the bond between the modifier and the PVDF membrane. Therefore, the PVDF membrane modified by the star-shaped dimethylaminoethyl acrylate polymer has higher hydrophilicity, permeability, and antifouling properties, and still has high recovery performance after multiple and long-term uses.

[0071] According to a specific embodiment of the present invention, the preparation method of the star-shaped dimethylaminoethyl acrylate polymer is as follows:

[0072] A suitable amount of initiator azobisisobutyronitrile (AIBN), dimethylaminoethyl acrylate (DMCA) in a molar ratio of (60–420):1, and a star-shaped chain transfer reagent, tetra-branched 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (structure as shown in Formula II), were dissolved in 50 mL of 2-butanone. Oxygen in the reaction vessel was removed using a freeze-degassing-thawing method, and nitrogen or argon was injected as a protective gas. The reaction was carried out in a metal bath at 70–120 °C with stirring for 4–20 h. The resulting polymer solution was added dropwise to hexane, and the product was collected by a precipitation-sedimentation method. The obtained product was purified by rotary evaporation and vacuum drying, and dried to constant weight to obtain a star-shaped poly(dimethylaminoethyl acrylate) polymer. Its 1H NMR spectrum is shown in Figure 1. Figure 1 SEM images such as Figure 2 .

[0073] Where R' is

[0074] Subsequently, the prepared star-shaped dimethylaminoethyl acrylate polymer was added as an additive to the PVDF casting solution, and a star-shaped dimethylaminoethyl acrylate polymer hydrophilically modified PVDF membrane was prepared by a blade coating method. The specific method included: adding 50g PVDF, 2g PVP, 2.7g star-shaped dimethylaminoethyl acrylate polymer, and an appropriate amount of DMF to a reaction vessel, stirring and reacting in a metal bath at 70℃ for 10–15 h, and then degassing in a vacuum oven at 60℃ for 8 h to obtain the casting solution. A PVDF flat sheet membrane was prepared using a solvent-induced phase separation method, with water as the coagulation bath at room temperature. The casting solution was poured onto a clean glass plate, and an automatic blade coating machine was used to coat the membrane, with a blade thickness of 150 μm. After exposing the scraped membrane to air for 30 seconds, place it in a coagulation bath until it detaches from the glass plate. Immerse the prepared membranes (M-0, M-1, M-2, M-3, and M-4) in distilled water, changing the water every 12 hours to remove residual solvents and pore-forming agents from the membrane, thus obtaining a star-shaped PVDF membrane product modified with dimethylaminoethyl acrylate polymer.

[0075] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0076] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0077] Example 1: Preparation of mesenchymal stem cell film bio-dressing

[0078] 1. Petri dish treatment

[0079] The culture dish used to prepare mesenchymal stem cell films had an 8 nm thick poly(2-vinylpyridine-co-styrene) block copolymer coating on its bottom. After thorough sterilization by irradiation, the culture dish was used to culture mesenchymal stem cells for stem cell film preparation. The surface free energy of the bottom of the coated culture dish was 49.0 ± 2.5 mJ / m². 2 .

[0080] 2. Preparation of monolayer mesenchymal stem cell membranes

[0081] Human umbilical cord-derived mesenchymal stem cells (fourth generation) were harvested and resuscitated. After resuspending in serum-free stem cell culture medium (Cellartis, Takara), the cells were centrifuged at 300 x g for five minutes. Serum-free stem cell culture medium was added to the cells, and they were seeded at 30,000 cells / cm² in 35 mm diameter low surface energy culture dishes coated with block copolymers. The culture dishes were placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. The culture medium was changed every two days, and cell growth was observed. Once the cells reached supersaturation (100% confluence), a complete monolayer stem cell membrane was obtained. The culture medium was completely removed from the culture dish, and DPBS was added to completely cover the cell membrane. The membrane was then left at room temperature to allow it to detach spontaneously. After washing the cell membrane three times with DPBS, the cell membrane completely detached from the culture dish and floated in the DPBS solution as a complete monolayer. The star-shaped PVDF membrane product modified by the above method was used to prepare a monolayer stem cell film. The three monolayer stem cell films were stacked and transferred onto a hydrogel nutrient layer (2 mm thick) with an outer breathable adhesive dressing. The PVDF membrane product was then removed, and the mesenchymal stem cell membrane biological dressing was successfully prepared.

[0082] The hydrogel nutrient layer consists of: Cellartis DEF-CS 500Xeno-Free Culture Medium (Takara) + 25% wt gelatin + 10% V / V serum substitute.

[0083] Example 2: Preparation of mesenchymal stem cell film bio-dressing

[0084] 1. Petri dish treatment

[0085] The culture dish used to prepare mesenchymal stem cell films had a 50 nm thick poly(2-vinylpyridine-co-styrene) block copolymer coating on its bottom, prepared by the aforementioned vacuum evaporation method. After thorough sterilization by irradiation, the culture dish was used to culture mesenchymal stem cells for stem cell film preparation. The surface free energy of the bottom of the coated culture dish was 43.0 ± 3.7 mJ / m². 2 .

[0086] 2. Preparation of monolayer mesenchymal stem cell membranes

[0087] Human umbilical cord-derived mesenchymal stem cells (fourth generation) were harvested and resuscitated, resuspended in DMEM medium, and centrifuged at 300 x g for five minutes. DMEM medium containing 10% serum substitute was added to the cells, and the cells were seeded at 30,000 cells / cm² in 35 mm diameter low surface energy culture dishes coated with block copolymers. The culture dishes were placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. The medium was changed every three days, and cell growth was observed. Once the cells reached supersaturation (100% confluence), a complete monolayer of stem cell membrane was obtained. After completely removing the medium from the culture dish, DPBS was added to completely cover the cell membrane, and the membrane was left at room temperature to detach spontaneously. After washing the cell membrane three times with DPBS, the cell membrane completely detached from the culture dish and floated in the DPBS solution as a complete monolayer. The star-shaped PVDF membrane product modified by the above method was used to prepare a monolayer stem cell film. The three monolayer stem cell films were stacked and transferred onto a hydrogel nutrient layer (2 mm thick) with an outer breathable adhesive dressing. The PVDF membrane product was then removed, and the mesenchymal stem cell membrane biological dressing was successfully prepared.

[0088] The hydrogel nutrient layer consists of: Cellartis DEF-CS 500Xeno-Free Culture Medium (Takara) + 25% wt gelatin + 10% V / V serum substitute.

[0089] Example 3: Mesenchymal stem cell membrane bio-dressing promotes wound repair

[0090] Using an in vivo control method, two circular incisions with a diameter of 10 mm were created on the backs of 10 C57BL / 6 mice. The left wound was treated with the mesenchymal stem cell film biological dressing (dressing group) prepared in Example 1 of this invention, containing a total of 3,000,000 cells (3.82 million cells per square centimeter). The dressing from Example 1 was applied to the wound. The right wound was treated with a common mesenchymal stem cell suspension (control group), containing a total of 3,000,000 cells (with an average concentration of 3.82 million cells per square centimeter), and covered with gauze.

[0091] Table 1 shows the remaining wound area on the backs of mice in the dressing group and the control group after different numbers of days of drug administration.

[0092] Table 1 Summary of wound area in dressing group and control group

[0093]

[0094]

[0095] The results in Table 1 above show that from day 3 to day 14, the wound area in the dressing group was significantly reduced compared to the control group. This indicates that the mesenchymal stem cell film biological dressing provided by the present invention can promote rapid wound healing in mice compared to mesenchymal stem cell suspension, and its wound healing speed is significantly faster than that of the same amount of mesenchymal stem cell suspension. This suggests that the mesenchymal stem cell film biological dressing of the present invention has a promoting effect on wound healing and can shorten the course of the disease.

[0096] In the description of this specification, the 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 present 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. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A stem cell film bio-dressing, characterized in that, This includes a composite stem cell membrane, a hydrogel nutrient layer, and an adhesive dressing. The composite stem cell film is formed on one side of the hydrogel nutrient layer, and the adhesive dressing is formed on the other side of the hydrogel nutrient layer. The composite stem cell membrane comprises multiple stacked monolayer stem cell membranes. The composite stem cell membrane is obtained through the following method: (1) Culture stem cells in a culture dish using stem cell culture medium to form a monolayer stem cell film; (2) Remove the stem cell culture medium and place the monolayer stem cell membrane in a peeling solution to obtain a free monolayer stem cell membrane; (3) Collect the free monolayer stem cell membrane from the stripping solution, and stack multiple free monolayer stem cell membranes to obtain a composite stem cell membrane. The inner surface of the bottom of the culture dish is coated with a coating, the surface free energy of which does not exceed 90 mJ / m 2 , The thickness of the coating is 2~100 nm. The stripping solution is DPBS buffer; The coating is formed from a poly(2-vinylpyridine-co-styrene) block copolymer.

2. The stem cell film bio-dressing according to claim 1, characterized in that, The composite stem cell membrane comprises 3 to 5 stacked monolayer stem cell membranes.

3. The stem cell film bio-dressing according to claim 1 or 2, characterized in that, The composite stem cell membrane is a mesenchymal stem cell membrane.

4. The stem cell film bio-dressing according to claim 3, characterized in that, The mesenchymal stem cell membrane is formed from at least one of the following: mesenchymal stem cells derived from umbilical cord, mesenchymal stem cells derived from bone marrow, mesenchymal stem cells derived from placenta, mesenchymal stem cells derived from dental pulp, and mesenchymal stem cells derived from adipose tissue.

5. The stem cell film bio-dressing according to claim 3, characterized in that, In step (1), the mesenchymal stem cells are passaged no more than 7 times.

6. The stem cell film bio-dressing according to claim 3, characterized in that, The surface free energy of the coating does not exceed 60 mJ / m 2 .

7. The stem cell film bio-dressing according to claim 3, characterized in that, The thickness of the coating is 2~50nm.

8. The stem cell film bio-dressing according to claim 3, characterized in that, The thickness of the coating is 2~30nm.

9. The stem cell film bio-dressing according to claim 3, characterized in that, The coating is formed by solid-phase deposition of 2-vinylpyridine and styrene.

10. The stem cell film bio-dressing according to claim 3, characterized in that, The free monolayer stem cell membrane was collected using a solid support.

11. The stem cell film bio-dressing according to claim 10, characterized in that, The solid support is a polymer support.

12. The stem cell film bio-dressing according to claim 10, characterized in that, The solid support is a PVDF membrane.

13. The stem cell film bio-dressing according to claim 12, characterized in that, The PVDF membrane is a star-shaped hydrophilically modified PVDF membrane made of dimethylaminoethyl acrylate polymer.

14. A method for preparing a stem cell film bio-dressing according to any one of claims 1 to 13, characterized in that, include: 1) The adhesive dressing is attached to one side of the hydrogel nutrient layer to obtain an adhesive dressing-hydrogel nutrient layer; 2) Transfer the composite stem cell film to the other side of the hydrogel nutrient layer in the adhesive dressing-hydrogel nutrient layer to obtain the stem cell film bio-dressing.

15. The preparation method according to claim 14, characterized in that, The hydrogel nutrient layer comprises mesenchymal stem cell culture medium and 10-30%wt gelatin.

16. The preparation method according to claim 14, characterized in that, The thickness of the hydrogel nutrient layer is 0.5~3mm.

17. Use of the stem cell film bio-dressing according to any one of claims 1 to 13, and the stem cell film bio-dressing prepared by the preparation method according to any one of claims 14 to 16, in the preparation of a medicament for treating skin lesions.

18. A drug, characterized in that, The stem cell film bio-dressing contains any one of claims 1 to 13 and / or the stem cell film bio-dressing prepared by any one of claims 14 to 16.

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