Cellular membrane biological dressing for treating skin burns and method for preparing the same

Abstract

The present application relates to the technical field of biological medicine, in particular to a cell membrane biological dressing for treating skin injury and a preparation method thereof. The present application provides a cell membrane biological dressing, which comprises a fibroblast cell membrane, an epidermal cell membrane, a hydrogel nutrition layer and a breathable dressing. The fibroblast cells and the epidermal cells are prepared into cell membranes, and the cell membranes are combined with the hydrogel nutrition layer to form a novel biological dressing. The cell membranes can be directly applied to the burn skin wound to promote wound healing, and are good skin substitutes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular, the present application relates to a cell membrane biological dressing for treating skin burns and a preparation method thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, but due to pathological or external reasons, it can cause skin defects, such as burns and scalds, chronic diseases, trauma, etc. Although the skin tissue has strong regenerative ability, the regenerative ability of the skin will be significantly weakened in the case of large area full-thickness skin defect, so the large area skin damage, severe trauma, cosmetic and plastic surgery and postoperative wound covering are currently very important problems in clinical practice. At present, autologous skin grafting is still the most effective method for treating full-thickness skin defects in clinical practice, but this method has the disadvantage of insufficient autologous skin source. Allogeneic or xenogeneic skin grafting is limited by immune rejection and has the potential risk of infection of donor pathogens. Artificial tissue skin replacement refers to culturing tissue substitutes in vitro by tissue engineering methods and then applying them to skin defects. Through the multiplication of cultured cells to form tissue substitutes with a certain cell density, the problem of skin source shortage is solved in principle, which is the future trend of treating skin defects.

[0003] However, in vitro cultured tissue substitutes also face the problem of immune rejection, therefore, it is urgent to develop a drug that can promote the rapid healing of skin burn wounds without rejection. SUMMARY

[0004] The present application aims to at least solve one of the technical problems in the related art. To this end, one object of the present application is to provide a cell membrane biological dressing, which is a good skin substitute by preparing fibroblast cells and epidermal cells into a cell membrane, combining with a hydrogel nutrient layer to form a new biological dressing, and directly applying the cell membrane to the skin wound of the burn to promote wound healing.

[0005] To this end, the first aspect of the present application provides a cell membrane biological dressing. According to the embodiment of the present application, the cell membrane biological dressing comprises a fibroblast cell membrane, an epidermal cell membrane, a hydrogel nutrient layer, and a breathable dressing,

[0006] wherein the fibroblast cell membrane or the epidermal cell membrane is formed on one side surface of the hydrogel nutrient layer, and the breathable dressing is formed on the other side surface of the hydrogel nutrient layer,

[0007] The fibroblast cell membrane and the epidermal cell membrane are stacked between each other.

[0008] Cell sheets as a kind of cell material without exogenous substances have attracted wide attention in cell transplantation applications. Cell sheets refer to the complete monolayer cell sheets obtained by culturing isolated cells on a specific material under in vitro culture conditions, and separating the cells from the culture substrate without destroying the cell connection by using a specific means. 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 required site during the transplantation operation; (2) cell sheets can be directly applied to these sites to play a key therapeutic role; (3) cell sheets completely retain the extracellular matrix, which is beneficial to cell regeneration and in vivo utilization. Therefore, the new type of biological dressing based on cell sheets is an ideal means for treating skin burns.

[0009] The present application provides a cell sheet biological dressing, which comprises a fibroblast cell sheet, an epidermal cell sheet, a hydrogel nutrient layer and a breathable dressing. The cell sheet biological dressing is transplanted to the hydrogel nutrient layer suitable for cell survival after processing different types of cell sheets. The nutrient layer has a microenvironment suitable for cell interaction, can promote fibroblasts and keratinocytes to exert their differentiation potential, rapidly proliferate and divide in a short time, and promote the repair of burn tissue epidermis by secreting growth factors. Fibroblasts and epidermal cells have strong division and migration ability, are seed cells for treating burns, scalds and chronic ulcer treatment, can promote tissue regeneration and accelerate wound healing. In the hydrogel nutrient layer of the present application, gelatin hydrogel is used as the substrate, and nutrients and growth factors necessary for cell activity are contained, which can effectively improve the cell survival rate and prolong the cell survival time.

[0010] The present application can directly apply cell sheets to skin wounds by preparing fibroblasts and epidermal cells into cell sheets and combining them with the hydrogel nutrient layer to form a new type of biological dressing, which can promote wound healing and is a good skin substitute, and is expected to develop into a main means for treating skin defects in the future.

[0011] The cell sheet biological dressing according to the above embodiments of the present application can also have at least one of the following additional technical features:

[0012] According to the embodiments of the present application, the fibroblast cell sheet comprises 1-3 single-layer fibroblast cell sheets.

[0013] According to the embodiments of the present application, the epidermal cell sheet comprises 1-2 single-layer epidermal cell sheets.

[0014] According to an embodiment of the present application, the fibroblasts in the fibroblast thin film are derived from at least one of foreskin, dermis, tendon, lung, umbilical cord of a newborn mammal.

[0015] According to an embodiment of the present application, the epidermal cells in the epidermal cell thin film are derived from at least one of foreskin, dermis, tendon, lung, umbilical cord of a newborn mammal.

[0016] According to an embodiment of the present application, the monolayer fibroblast thin film and the monolayer epidermal cell thin film are obtained by:

[0017] (1) culturing the fibroblasts and the epidermal cells respectively in a culture dish using a fibroblast culture medium or an epidermal cell culture medium, so as to form a monolayer fibroblast thin film and a monolayer epidermal cell thin film;

[0018] (2) removing the fibroblast culture medium and the epidermal cell culture medium respectively, and placing the monolayer fibroblast thin film and the monolayer epidermal cell thin film respectively in a stripping solution, so as to obtain a free monolayer fibroblast thin film and a free monolayer epidermal cell thin film;

[0019] (3) collecting the free monolayer fibroblast thin film and the free monolayer epidermal cell thin film from the stripping solution respectively.

[0020] According to an embodiment of the present application, a plurality of the free monolayer fibroblast thin films are stacked, so as to obtain a multi-layer stacked fibroblast thin film.

[0021] According to an embodiment of the present application, a plurality of the free monolayer epidermal cell thin films are stacked, so as to obtain a multi-layer stacked epidermal cell thin film.

[0022] According to an embodiment of the present application, in step (1), the number of passages of the fibroblasts and the epidermal cells is not more than 7. Using fibroblasts and epidermal cells with a number of passages not more than 7 can further improve the integrity of the obtained fibroblast thin film and epidermal cell thin film, and improve the state of the cell thin film. However, using cells with 8 passages or more to prepare the cell thin film, the cell thin film may occasionally be broken, the cell thin film may automatically fall off, and it may be difficult to form a complete cell thin film. According to an embodiment of the present application, the inner surface of the bottom of the culture dish is coated with a coating layer, and the surface free energy of the coating layer is not more than 90 mJ / m 2 . In this way, the cells can more easily adhere and grow, and the cell thin film can be more easily detached from the culture dish under the stimulation of specific external conditions.

[0023] According to an embodiment of the present application, the surface free energy of the coating layer is not more than 60 mJ / m 2Therefore, the effect of the cell membrane from the culture dish is further improved.

[0024] The surface free energy of the culture dish affects the hydrophilicity / hydrophobicity of the culture dish surface. If the hydrophilicity is too strong, the adhesion protein on the cell surface is too strong to the culture dish, which can cause the cell to be unable to fall off into a single layer of cell membrane, or cause the cell to partially fall off and cause the cell membrane to break; if the hydrophobicity is too strong, the cell can not grow adherently.

[0025] According to an embodiment of the present application, the thickness of the coating is 2-100 nm, preferably 2-50 nm, and further preferably 2-30 nm. Therefore, the effect of the cell membrane from the culture dish is better. In addition, the inventors found that if the thickness of the coating is too thick, the growth of the cell will be adversely affected.

[0026] According to an embodiment of the present application, the coating comprises a high polymer and / or a block copolymer.

[0027] According to an embodiment of the present application, the coating is formed by poly(2-vinylpyridine-co-styrene) block copolymer. The coating can effectively adjust the surface free energy of the culture dish, so that the cell adheres and is easily detached under specific external conditions.

[0028] According to an embodiment of the present application, the coating is formed by solid phase deposition of 2-vinylpyridine and styrene. Therefore, the effect of the cell membrane from the culture dish is better.

[0029] According to an embodiment of the present application, the stripping solution is DPBS buffer.

[0030] According to an embodiment of the present application, the free single layer of fibroblast cell membrane or the free single layer of epidermal cell membrane is collected by using a solid phase support.

[0031] According to an embodiment of the present application, the solid phase support is a high polymer support.

[0032] According to an embodiment of the present application, the solid phase support is a PVDF membrane.

[0033] According to an embodiment of the present application, the PVDF membrane is a star-polydimethylaminoethyl acrylate polymer hydrophilic modified PVDF membrane.

[0034] The second aspect of the present application provides a preparation method of the cell membrane biological dressing of the first aspect. According to an embodiment of the present application, the preparation method comprises:

[0035] 1) attaching the breathable dressing to one side surface of the hydrogel nutrient layer to obtain a breathable dressing-hydrogel nutrient layer;

[0036] 2) stacking the fibroblast cell membrane and epidermal cell membrane in the air-permeable dressing-hydrogel nutrient layer on the other side surface of the hydrogel nutrient layer to obtain the cell membrane biological dressing.

[0037] According to an embodiment of the present application, the hydrogel nutrient layer comprises 10-30% wt gelatin, 0-15% wt serum substitute, 1.5-4.5 g / L glucose, 0-5% wt L-glutamine.

[0038] According to an embodiment of the present application, the thickness of the hydrogel nutrient layer is 0.5-3 mm.

[0039] The third aspect of the present application provides the use of the cell membrane biological dressing of the first aspect or the cell membrane biological dressing prepared by the preparation method of the second aspect in the preparation of a medicament for treating skin burn.

[0040] The fourth aspect of the present application provides a medicament. According to an embodiment of the present application, the medicament contains the cell membrane biological dressing of the first aspect and / or the cell membrane biological dressing prepared by the preparation method of the second aspect.

[0041] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0042] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0043] Figure 1 A nuclear magnetic resonance hydrogen spectrum of a star-shaped-poly (dimethylamino ethyl acrylate) polymer according to an embodiment of the present application is shown;

[0044] Figure 2 A SEM spectrum of a star-shaped-poly (dimethylamino ethyl acrylate) polymer according to an embodiment of the present application is shown;

[0045] Figure 3 A case of treating skin burn of a mouse by each group of cell membrane biological dressings according to Example 3 of the present application is shown. DETAILED DESCRIPTION

[0046] Embodiments of the present application are described below by way of example with reference to the accompanying drawings, in which like or similar elements and / or features are identified by the same reference signs, and in which the drawings are not to scale. The embodiments described below are examples for explaining the present application and are not to be understood as limiting the present application. Unless specifically indicated otherwise, the technical or structural features described in the embodiments are to be understood as being described in the same way as described in the literature or as described in the product specifications. Unless specifically indicated otherwise, the reagents or instruments used are commercially available products.

[0047] In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0048] In addition, the terms "first", "second", and the like are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined with "first", "second", and the like can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, and the like, unless otherwise explicitly specified and limited.

[0049] In the present application, unless otherwise explicitly specified and limited, the "on" or "under" of the first feature to the second feature can be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the "over", "above" and "on" of the first feature to the second feature can be that the first feature is directly above or obliquely above the second feature, or only indicates that the horizontal height of the first feature is higher than that of the second feature. The "under", "below" and "under" of the first feature to the second feature can be that the first feature is directly below or obliquely below the second feature, or only indicates that the horizontal height of the first feature is less than that of the second feature.

[0050] According to one specific embodiment of the present application, the present application provides a cell sheet biological dressing, comprising a fibroblast cell sheet, an epidermal cell sheet, a hydrogel nutrient layer, and a breathable dressing,

[0051] Wherein the fibroblast cell sheet or the epidermal cell sheet is formed on one side surface of the hydrogel nutrient layer, and the breathable dressing is formed on the other side surface of the hydrogel nutrient layer,

[0052] The fibroblast cell sheet and the epidermal cell sheet are stacked between each other.

[0053] According to the embodiment of the present application, the specific type and material of the adhesive dressing is not particularly limited, and those skilled in the art can select a commonly used suitable adhesive dressing according to actual needs, preferably an air-permeable adhesive dressing. The size of the cell membrane biological dressing can also be selected according to the size of the cell membrane contained therein.

[0054] According to the embodiment of the present application, the fibroblast cell membrane includes 1-3 single-layer fibroblast cell membranes. In this way, the toughness and firmness of the cell membrane can be further improved.

[0055] According to the embodiment of the present application, the epidermal cell membrane includes 1-2 single-layer epidermal cell membranes. In this way, the toughness and firmness of the cell membrane can be further improved.

[0056] According to the embodiment of the present application, the fibroblast cells in the fibroblast cell membrane are derived from at least one of the foreskin, dermis, tendon, lung, and umbilical cord of a newborn mammal, and the fibroblast cells in the fibroblast cell membrane are derived from the foreskin of a healthy newborn.

[0057] According to the embodiment of the present application, the epidermal cells in the epidermal cell membrane are derived from at least one of the foreskin, dermis, tendon, lung, and umbilical cord of a newborn mammal.

[0058] According to the embodiment of the present application, the single-layer fibroblast cell membrane and the single-layer epidermal cell membrane are obtained by:

[0059] (1) culturing the fibroblast cells and the epidermal cells in a culture dish using a fibroblast cell culture medium and an epidermal cell culture medium, respectively, so as to form a single-layer fibroblast cell membrane and a single-layer epidermal cell membrane;

[0060] (2) removing the fibroblast cell culture medium and the epidermal cell culture medium, respectively, and placing the single-layer fibroblast cell membrane and the single-layer epidermal cell membrane in a stripping solution, so as to obtain a free single-layer fibroblast cell membrane and a free single-layer epidermal cell membrane;

[0061] (3) collecting the free single-layer fibroblast cell membrane and the free single-layer epidermal cell membrane from the stripping solution, respectively.

[0062] According to the embodiment of the present application, a plurality of the above-mentioned free single-layer fibroblast cell membranes are stacked to obtain a multi-layer stacked fibroblast cell membrane.

[0063] According to the embodiment of the present application, a plurality of the above-mentioned free single-layer epidermal cell membranes are stacked to obtain a multi-layer stacked epidermal cell membrane.

[0064] According to an embodiment of the present invention, in step (1), the number of passages for fibroblasts and epidermal cells does not exceed 7. There is no particular limitation on the number of passages for fibroblasts and epidermal cells, but it is preferred that the number of passages for fibroblasts and epidermal cells does not exceed 7, because fibroblasts and epidermal cells that meet this requirement can further improve the integrity of the obtained fibroblast membrane and epidermal cell membrane, improve the state of the cell membrane, and are less likely to detach prematurely.

[0065] According to embodiments of the present invention, the inoculation density of fibroblasts in culture dishes used for culturing fibroblasts and epidermal cells in culture dishes used for culturing epidermal cells is not particularly limited and can be controlled according to the time required to harvest the cell membrane. For example, if a faster cell membrane is required, the inoculation density of fibroblasts or epidermal cells can be increased. For example, an inoculation density of 24,000-30,000 cells / cm² can be maintained.

[0066] According to embodiments of the present invention, the fibroblast culture medium and epidermal cell culture medium can be conventional culture media suitable for culturing fibroblasts and epidermal cells. Additionally, extra components, such as an appropriate concentration of ascorbic acid, can be added to the culture medium to facilitate the synthesis of extracellular matrix and enhance cell membrane strength.

[0067] 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 cell membrane detaches more effectively from the culture dish.

[0068] 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.

[0069] 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.

[0070] According to an embodiment of the present invention, in step (1), after culturing fibroblasts or epidermal cells in a culture dish to near 100% confluence, the cell membrane should be processed within 48 hours. If the process exceeds 48 hours, there is a chance that the cell membrane will detach or break.

[0071] According to an embodiment of the present application, the stripping solution is DPBS buffer. This stripping solution has a better effect of reducing the binding force between the cell sheet and the culture dish.

[0072] According to an embodiment of the present application, the free cell sheet can be collected by using a PVDF membrane. Specifically, after the monolayer fibroblast cell sheet or the monolayer foreskin cell sheet is detached from the culture dish, the monolayer cell sheet can be adsorbed to the surface of the PVDF membrane, and then washed several times with normal saline according to actual needs. Preferably, the PVDF membrane is a star-shaped PDMAEA hydrophilic modified PVDF membrane. Thus, the adsorption effect of the monolayer cell sheet is better.

[0073] For the convenience of understanding, the star-shaped PDMAEA hydrophilic modified PVDF membrane is described in detail as follows.

[0074] The star-shaped PDMAEA has a structure as shown in Formula I

[0075]

[0076] In Formula I, R is n is a positive integer of 15-105. The degree of polymerization of the star-shaped PDMAEA is between 100 and 400, and the molecular weight is between 10,000 and 60,000 Dalton. The inventors have found that the long alkyl chain in the star-shaped PDMAEA has hydrophobicity and has good compatibility with PVDF; and the PDMAEA chain segment has high hydrophilicity, which improves the hydrophilicity and increases the stability of the modifier combined with the PVDF membrane. Thus, the star-shaped PDMAEA hydrophilic modified PVDF membrane has higher hydrophilicity, permeability and anti-fouling property, and still has high recovery performance after multiple and long-time use.

[0077] According to a specific embodiment of the present application, the preparation method of the star-shaped PDMAEA is as follows:

[0078] A certain amount of initiator azobisisobutyronitrile and dimethylaminoethyl acrylate with a molar ratio of (60-420): 1 and star chain transfer agent four-branched 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (structure as formula II) were dissolved in 50 mL 2-butanone, the oxygen in the reaction vessel was removed by freeze-degassing-freezing method, and nitrogen or argon was injected as protective gas, and was placed in a metal bath at 70-120℃ for stirring reaction for 4-20h; the obtained polymer solution was dropped into hexane, and the product was collected by precipitation-sedimentation method, and the obtained product was impurity-removed and dried to constant weight through steps of rotary evaporation, vacuum drying and the like, to obtain star-polydimethylaminoethyl acrylate polymer, whose nuclear magnetic resonance hydrogen spectrum is as Figure 1 , SEM spectrum is as Figure 2 .

[0079] wherein, R' is

[0080] Subsequently, the prepared star-polydimethylaminoethyl acrylate polymer was added as an additive into a PVDF casting solution, and a star-polydimethylaminoethyl acrylate polymer hydrophilic modified PVDF membrane was prepared by using a doctor blade method. The specific method comprises: taking 50g PVDF, 2g PVP, 2.7g star-polydimethylaminoethyl acrylate polymer and a certain amount of DMF into a reaction vessel, stirring at 70℃ for 10-15h in a metal bath, and then deaerating in a vacuum oven at 60℃ for 8h to obtain a casting solution. A PVDF flat membrane was prepared by using a solvent-induced phase separation method, water was used as a coagulation bath, the temperature was room temperature, the casting solution was poured on a clean glass plate, and an automatic film doctor was used for doctoring, with a doctor blade thickness of 150μm. After the doctoring film was exposed in air for 30s, it was placed in the coagulation bath until it fell off the glass plate, and the prepared membrane (M-0, M-1, M-2, M-3 and M-4) was immersed in distilled water, and the water was changed every 12h to remove the residual solvent and pore-forming agent in the membrane, to obtain a star-polydimethylaminoethyl acrylate polymer hydrophilic modified PVDF membrane product.

[0081] The following will describe the embodiments of the present application in detail. The following described embodiments are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0082] If a specific technology or condition is not specified in the embodiments, the technology or condition described in the literature in the art or according to the product instruction is used. If the used reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by market purchase.

[0083] Example 1: preparation of fibroblast thin film-epidermal cell thin film-hydrogel nutrient layer-air permeable dressing

[0084] 1. Culture dish treatment

[0085] The culture dish bottom used for preparing the fibroblast membrane has a coating of poly(2-vinylpyridine-co-styrene) block copolymer having a thickness of 8 nm. After the culture dish is sterilized by irradiation, it is used to culture fibroblasts to prepare the fibroblast membrane. The surface free energy of the coated culture dish bottom is 49 ± 2.5 mJ / m 2 .

[0086] 2. Preparation of human fibroblast membrane

[0087] Culture medium for culturing fibroblasts: basal medium DMEM, 100 U / ml of penicillin-streptomycin double antibiotic, 10% serum substitute, 16.8 μg / ml ascorbic acid.

[0088] Preparation of human fibroblasts: The prepuce of a male newborn is taken, subcutaneous adipose tissue, fascia and capillary blood vessels are removed, and the prepuce is rinsed twice with PBS (1X) and cut into small pieces. The prepuce is then digested in 0.1% trypsin at 4°C for 2-12 hours, and the degradation of the skin is observed. After the degradation of the skin is observed, the epidermis and dermis are peeled off with tweezers and the front and back of the dermis are scraped with a blade. The dermis is then cut into small pieces and transferred to a sterile culture dish containing 0.1% collagenase type IV, and digested at 37°C for 2-12 hours. After the digestion is completed, the cells are collected by centrifugation and primary cultured. The cells are suspended in a small amount of cell culture medium and transferred to a cell culture dish, and cultured in a 37°C, 5% CO2, 100% relative humidity incubator. After one week, the presence of free cells is observed. If fibroblasts are present, the medium is changed after 3-4 days to allow the cells to reach a certain density. When the cell culture density reaches 70-80%, the cells are rinsed with PBS and digested with 0.25% trypsin at 37°C for 1-5 minutes. The digestion is terminated by adding cell culture medium, and the cells are transferred to a centrifuge tube and centrifuged. The supernatant is discarded, 10 ml of cell culture medium is added to resuspend the cells, and the cells are seeded at a density of 5 x 10 3 ~ 5 x 10 5 / cm 2 in a cell culture dish. The medium is changed after 3-4 days until the cell culture density reaches 70-80%.

[0089] After the primary cells are cultured to 90% confluence, they are digested with trypsin and centrifuged at 300 x g for five minutes. The cells are resuspended in culture medium and seeded at a density of 30,000 cells / cm in a 35 mm diameter low surface free energy culture dish, and the culture dish is placed in a 37°C, 5% carbon dioxide, humidity saturated cell culture incubator. The medium is changed every two days, and the cell growth state is observed. When the cells grow to a supersaturated state (100% confluence), a complete monolayer cell membrane is obtained.

[0090] After the complete removal of the culture medium in the culture dish, DPBS was added to completely cover the cell monolayer, and the culture dish was left at room temperature until the cell monolayer spontaneously detached. After the cell monolayer was washed with DPBS for three times, the cell monolayer completely detached from the culture dish and floated in the DPBS solution in the form of a complete monolayer. The fibroblast cell monolayer was collected using the PVDF membrane product obtained by the method described above, and about 2 ml of DPBS was added to the cell monolayer and incubated at room temperature for 20 minutes. After the incubation, the three-layer fibroblast cell monolayer was transferred to the hydrogel nutrient layer (2 mm thick) with a breathable dressing on the outside, or to the epidermal cell monolayer in the epidermal cell monolayer-hydrogel nutrient layer-breathable dressing, and then the PVDF membrane product was removed, and a cell monolayer biological dressing: fibroblast cell monolayer-epidermal cell monolayer-hydrogel nutrient layer-breathable dressing was successfully prepared.

[0091] Example 2: Preparation of an epidermal cell monolayer-fibroblast cell monolayer-hydrogel nutrient layer-breathable dressing

[0092] 1. Culture dish treatment

[0093] The bottom of the culture dish used to prepare the epidermal cell monolayer was coated with a poly(2-vinylpyridine-co-styrene) block copolymer coating with a thickness of 50 nm. After the culture dish was sterilized by irradiation, it was used to culture epidermal cells to prepare an epidermal cell monolayer. The surface free energy of the coated bottom surface of the culture dish was 43±3.7 mJ / m 2 .

[0094] 2. Preparation of an epidermal cell monolayer

[0095] The culture medium used to culture epidermal cells was prepared by adding the following components to the basal medium DMEM: penicillin and streptomycin double-antibiotic 100 U / ml; amphotericin B 5 ng / ml; adenine 34.3 μg / ml; insulin 10 μg / ml; hydrocortisone 0.4 μg / ml; triiodothyronine 1.3 μg / ml; transferrin 5 μg / ml; and ascorbic acid 21.6 μg / ml to prepare a cell culture medium.

[0096] Healthy male neonatal foreskin tissue was used to prepare human epidermal cells. The foreskin tissue was removed from subcutaneous adipose tissue, fascia and capillary blood vessels, rinsed twice with PBS (1X), cut into small pieces, and immersed in 0.1 trypsin for 2-12 hours at 4°C for digestion. The skin degradation was observed. After the skin tissue was observed to degrade, the epidermis and dermis were peeled off with tweezers and scraped with a blade on the front and back of the dermis. The epidermis was repeatedly kneaded with tweezers and filtered through a 200-mesh nylon filter. The filtrate was mixed with 10 ml of cell culture solution, placed in a cell culture dish, and cultured in a 37°C, 5% CO2, 100% relative humidity incubator. After one week, whether there were cells free was observed. If there were epidermal cells, the liquid was changed after 3-4 days to allow the cells to reach a certain density. When the cell culture density reached 70-80%, after rinsing with PBS, the cells were digested with 0.25% trypsin at 37°C for 1-5 minutes, then cell culture solution was added to stop the digestion, transferred to a centrifuge tube, centrifuged, the supernatant was discarded, 10 ml of cell culture solution was added to resuspend, and inoculated in a cell culture dish at 5x103-5x105 / cm2. The liquid was changed every 3-4 days until the cell culture density reached 70-80%.

[0097] After the primary cells were cultured to 90% confluence, they were trypsinized and centrifuged at 300x g for five minutes. The cells were resuspended in culture medium, inoculated in 35mm diameter low surface free energy culture dishes at 30,000 cells / cm2, and placed in a 37°C, 5% carbon dioxide, humidity saturated cell culture incubator. The culture medium was changed every two days, and the cell growth state was observed. When the cells grew to a supersaturated state (100% confluence), a complete monolayer cell membrane was obtained.

[0098] After the culture medium in the culture dish was completely removed, DPBS was added to completely cover the cell membrane, and the cell membrane was allowed to fall off at room temperature. After the cell membrane was washed three times with DPBS, the cell membrane completely detached from the cell culture dish and floated in the DPBS solution in the form of a complete monolayer membrane. The monolayer cell membrane prepared was collected using the star-poly (dimethylamino ethyl acrylate) polymer hydrophilic modified PVDF membrane product obtained by the method described above, and about 2 ml of DPBS was added and incubated at room temperature for 20 minutes. After the incubation was completed, three layers of monolayer epidermal cell membranes were stacked with tweezers and transferred to a hydrogel nutrient layer (thickness 2 mm) with a breathable dressing on the outside, or to a fibroblast cell membrane-hydrogel nutrient layer-breathable dressing fibroblast cell membrane, and then the PVDF membrane product was removed, successfully preparing a cell membrane biological dressing: epidermal cell membrane-fibroblast cell membrane-hydrogel nutrient layer-breathable dressing.

[0099] Example 3: Cell membrane biological dressing for treating skin burns

[0100] The experiment adopts the self-control method, 36 C57BL / 6 mice (half male and half female) aged 4-8 weeks are selected and divided into three groups, group 1 is the fibroblast cell membrane alone, group 2 is the epidermal cell membrane alone, and group 3 is the composite membrane composed of the fibroblast cell membrane and the epidermal cell membrane. The cell membrane biological adjuvant of group 1, group 2 and group 3 is obtained by the method in examples 1 and 2. After the mice are anesthetized, the back of the mice is shaved, and a 10mm diameter round brass flat head mold heated to 85℃ is pressed on the back of the mice at a constant pressure for 20 seconds to create two burn wounds. After the mice are cultured for three days, the burn wounds are completely formed. After the burn wounds are completely formed, the cell membrane biological adjuvant is applied to the wounds on one side according to the grouping, and physiological saline is added to the wounds on the other side as a control, and the wounds are covered and bandaged with air-permeable gauze. The wounds are continuously observed for 21 days, and the healing of the wounds on both sides is observed at the designated time points, and the wound area is measured. The healing rate is calculated by comparing with the initial wound area, and the results are plotted as shown in the following figure. Figure 3 .

[0101] According to the results in Figure 3 , it is shown that the composite membrane biological dressing containing the fibroblast cell membrane and the epidermal cell membrane can more effectively promote the rapid healing of the mouse skin burn wounds compared with the cell membrane biological dressing using the fibroblast cell or the epidermal cell alone, and the wound healing speed is faster than that of the control group, which indicates that the composite membrane biological dressing containing the fibroblast cell membrane and the epidermal cell membrane provided by the present application has a promoting effect on the burn wound healing and can shorten the course of the disease.

[0102] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0103] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A cell membrane bio-dressing, characterized in that, This includes fibroblast membranes, epidermal cell membranes, hydrogel nutrient layers, and breathable dressings. The fibroblast membrane or epidermal cell membrane is formed on one side of the hydrogel nutrient layer, and the breathable dressing is formed on the other side of the hydrogel nutrient layer. The fibroblast membrane and the epidermal cell membrane are stacked together. The fibroblast membrane comprises 1 to 3 monolayer fibroblast membranes; The epidermal cell membrane comprises 1-2 monolayer epidermal cell membranes. The monolayer fibroblast membrane and the monolayer epidermal cell membrane are obtained through the following methods: (1) Fibroblasts and epidermal cells are cultured in a culture dish using fibroblast culture medium or epidermal cell culture medium, respectively, in order to form a monolayer fibroblast membrane and a monolayer epidermal cell membrane. (2) Remove the fibroblast culture medium and the epidermal cell culture medium respectively, and place the monolayer fibroblast membrane and the monolayer epidermal cell membrane in the peeling solution respectively to obtain the free monolayer fibroblast membrane and the free monolayer epidermal cell membrane. (3) Collect the free monolayer fibroblast membrane and the free monolayer epidermal cell membrane from the stripping solution, respectively. 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 cell membrane bio-dressing according to claim 1, characterized in that, The fibroblasts in the fibroblast membrane are derived from at least one of the foreskin, dermis, tendon, lung, and umbilical cord of a newborn mammal; Optionally, the epidermal cells in the epidermal cell membrane are derived from at least one of the foreskin, dermis, tendon, lung, and umbilical cord of a newborn mammal.

3. The cell membrane bio-dressing according to claim 1, characterized in that, Multiple free monolayer fibroblast membranes are stacked to obtain multilayer stacked fibroblast membranes. Optionally, multiple free monolayer epidermal cell films can be stacked to obtain multilayered epidermal cell films.

4. The cell membrane bio-dressing according to claim 3, characterized in that, In step (1), the fibroblasts and epidermal cells are passaged no more than 7 times; Optionally, the surface free energy of the coating does not exceed 60 mJ / m 2 ; Optionally, the thickness of the coating is 2 to 50 nm; Optionally, the coating is formed by solid-phase deposition of 2-vinylpyridine and styrene.

5. The cell membrane bio-dressing according to claim 4, characterized in that, The thickness of the coating is 2~30 nm.

6. The cell membrane bio-dressing according to claim 3, characterized in that, The free monolayer fibroblast membrane or free monolayer epidermal cell membrane is collected using a solid support.

7. The cell membrane bio-dressing according to claim 6, characterized in that, The solid support is a polymer support.

8. The cell membrane bio-dressing according to claim 6, characterized in that, The solid support is a PVDF membrane.

9. The cell membrane bio-dressing according to claim 8, characterized in that, The PVDF membrane is a star-shaped hydrophilically modified PVDF membrane made of dimethylaminoethyl acrylate polymer.

10. A method for preparing a cell film bio-dressing according to any one of claims 1 to 9, characterized in that, include: 1) The breathable dressing is attached to one side of the hydrogel nutrient layer to obtain a breathable dressing-hydrogel nutrient layer; 2) The fibroblast membrane and the epidermal cell membrane are stacked on the other side of the hydrogel nutrient layer in the breathable dressing-hydrogel nutrient layer to obtain the cell membrane bio-dressing. Monolayer fibroblast membranes and monolayer epidermal cell membranes are obtained through the following methods: (1) Fibroblasts and epidermal cells are cultured in a culture dish using fibroblast culture medium or epidermal cell culture medium, respectively, in order to form a monolayer fibroblast membrane and a monolayer epidermal cell membrane. (2) Remove the fibroblast culture medium and the epidermal cell culture medium respectively, and place the monolayer fibroblast membrane and the monolayer epidermal cell membrane in the peeling solution respectively to obtain the free monolayer fibroblast membrane and the free monolayer epidermal cell membrane. (3) Collect the free monolayer fibroblast membrane and the free monolayer epidermal cell membrane from the stripping solution, respectively. 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.

11. The preparation method according to claim 10, characterized in that, The hydrogel nutrient layer comprises 10-30%wt gelatin, 0-15%wt serum substitute, 1.5-4.5g / L glucose, and 0-5%wt L-glutamine. Optionally, the thickness of the hydrogel nutrient layer is 0.5~3mm.

12. Use of the cell film bio-dressing according to any one of claims 1 to 9, or the cell film bio-dressing prepared by the preparation method according to claim 10 or 11, in the preparation of a medicament for treating skin burns.

13. A drug, characterized in that, The cell film bio-dressing contains any one of claims 1 to 9 and / or the cell film bio-dressing prepared by the preparation method of claim 10 or 11.

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