Artificial skin containing basement membrane and preparation method thereof

Abstract

The present invention provides an artificial skin containing a basement membrane and a preparation method. The method uses dermal fibroblasts, keratinocytes, and the structural components of the basement membrane to prepare the artificial skin containing a basement membrane. In this method, the structural components of the basement membrane, extracellular matrix components, and components required to maintain cell growth are added to the dermal fibroblasts. These components can promote the growth and proliferation of the dermal fibroblasts, or can serve as scaffold materials for the dermal fibroblasts, or can enhance intercellular adhesion and cell-matrix adhesion, or can stimulate and promote the secretion of some extracellular matrix components by dermal cells or epidermal cells to form a basement membrane between the dermis and epidermis, thereby strengthening the connection between the dermis and epidermis. The artificial skin comprises an epidermis, a basement membrane, and a dermis, contains living cells, and has a structure similar to normal skin. It can be used to treat skin wounds caused by various traumas and to prepare skin models.

Description

Technical Field

[0001] The invention belongs to the technical field of artificial skin and relates to artificial skin containing a basement membrane and a preparation method thereof. Background Art

[0002] The skin, the largest organ on the human body's surface, is composed of the epidermis and dermis. The basement membrane (EBM) is a crucial connecting structure between the epidermis and dermis. The EBM's primary structural components include laminin, collagen type IV, collagen type VII, entactin, and perlecan. These interact to form a network structure that supports the EBM's diverse biological functions, including epidermal-dermal connectivity, signal transduction, and permeability barrier. A healthy EBM can fulfill these diverse biological functions, ensuring smooth circulation between the epidermis, EBM, and dermis, maintaining the skin's health and integrity.

[0003] In daily life and work, skin damage is unavoidable, with trauma, burns, and scalds being particularly common. To prevent infection, promote and accelerate wound healing, and treat deep burns and scalds, artificial skin is applied to the human body. This artificial skin, developed in vitro using the principles and methods of engineering and cell biology, is used to repair and replace damaged skin tissue.

[0004] Currently, the most ideal construction model in the field of tissue-engineered skin research is a double-layer tissue-engineered skin. However, due to the lack of a complete basement membrane, there is a major drawback that the epidermis and dermis are easily separated, which to a certain extent also restricts its clinical application. Currently, the patent disclosed in CN101954124B uses amniotic membrane to construct the skin basement membrane. This method requires fresh amniotic membrane and the preparation process is complicated, which is not conducive to widespread promotion. In the patent disclosed in CN 102781484 A, matrix metalloproteinase inhibitors and heparanase inhibitors are added to the culture medium containing keratinocytes derived from human epidermis and fibroblasts derived from human dermis to prepare artificial skin. Summary of the Invention

[0005] In order to solve the technical problem, the present invention provides an artificial skin containing a basement membrane and a preparation method.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing artificial skin containing a basement membrane, the method comprising:

[0008] S01: After the dermal fibroblasts are repeatedly resuspended in reagent A, centrifuged, washed with PBS, and centrifuged again, coated dermal fibroblasts are obtained.

[0009] Dermal fibroblasts are cells found in the dermis and are the main cellular component of loose connective tissue. They synthesize and secrete collagen and elastin, forming collagen fibers, reticular fibers, and elastic fibers. They also synthesize and secrete matrices such as glycosaminoglycans and glycoproteins.

[0010] In the present application, an extracellular matrix component is coated on the dermal fibroblasts, and a certain extracellular matrix component is incubated on the dermal layer to form a dermal layer containing a basement membrane. Specifically, the dermal fibroblasts are resuspended using reagent A to form a cell suspension. At this time, a layer of reagent A is coated on the outside of the dermal fibroblasts. Among them, reagent A includes: PBS (English name: phosphate buffered saline; Chinese name: phosphate buffered saline) buffer containing fibronectin and gelatin at a concentration of 0.01-0.1 mg / mL, Tris-HCl (English name: TRIShydrochloride; Chinese name: tris(hydroxymethyl)aminomethane hydrochloride) buffer or other buffer or culture medium. The buffer or culture medium uses the buffer or culture medium commonly used in the preparation of artificial skin. In the present application, the amount of dermal fibroblasts used is 10 5 -6×10 7 cells / cm 2 .

[0011] The cell suspension was centrifuged at 5-500 rpm for 1-5 minutes. The cells were then washed with PBS buffer and centrifuged again to remove any uncoated Reagent A. This process of resuspension, centrifugation, PBS washing, and further centrifugation was repeated 5-10 times to obtain dermal fibroblasts coated with Reagent A.

[0012] Furthermore, before using reagent A to coat the dermal fibroblasts, the process also includes: adding gelatin, collagen or other coating matrices to the support membrane, solidifying at room temperature or 37°C to obtain a coated support membrane. Dermal fibroblasts are added to the coated support membrane, and after repeated resuspension with reagent A, centrifugation, PBS washing, and re-centrifugation, coated dermal fibroblasts are obtained. Preparing a dermis layer on the coated support membrane can form a well-structured basement membrane between the dermis and the epidermis, and the prepared dermis layer is more tightly attached to prevent the dermis from wrinkling or premature detachment from the support membrane.

[0013] S02: Reagent B, reagent C, reagent D and reagent E are mixed to form a mixed solution, and the coated dermal fibroblasts are added to the mixed solution after adjusting the pH value to neutral. After mixing, the mixture is incubated and cultured at room temperature or 37° C. to obtain a dermal cell mixture.

[0014] Reagents B, C, D, and E are mixed to form a mixed solution. After adjusting the pH of the mixed solution to neutral, the encapsulated dermal fibroblasts are added to the pH-adjusted mixed solution. After mixing, the mixture is quickly transferred to a culture device and incubated at room temperature or 37°C for 0.5-3 hours to obtain a dermal cell mixture. Reagent B includes at least one of commonly used culture media such as DMEM (Dulbecco's modified eagle medium), MEM (2-Methoxyethoxymethylchloride), and Ham's F12 nutrient medium (animal cell culture medium). Reagent C includes a 200 mM L-glutamine solution. Reagent D includes serum. Reagent E is a scaffold material comprising at least one of collagen, silk fibroin, hyaluronic acid, gelatin, and chitosan. The amount of reagent B, reagent C, reagent D and reagent E added is determined according to the actual amount of dermal fibroblasts used.

[0015] S03: The dermal cell mixture is cultured at 37° C. and 5% CO 2 to obtain a dermal layer.

[0016] The dermal cell mixture is cultured at a temperature of 37°C and 5% CO2 for 1-5 days to obtain a dermal layer. In the present application, structural components of the basement membrane, extracellular matrix components, and components required to maintain cell growth are added to the dermal fibroblasts, wherein these components include reagent A, reagent B, reagent C, reagent D, and reagent E. These components can promote the growth and proliferation of dermal fibroblasts, or can serve as a scaffold material for dermal fibroblasts, or can enhance intercellular adhesion and cell-matrix adhesion, or can stimulate and promote dermal cells or epidermal cells to secrete some extracellular matrix components, form a basement membrane between the dermis and the epidermis, and make the connection between the dermis and the epidermis tighter.

[0017] S04: adding reagent F to the dermis layer and incubating to form reagent F / dermis layer.

[0018] After the dermis culture is complete, the culture medium is discarded. Reagent F is added to the dermis and incubated for 30 minutes to coat the surface of the dermis, thereby obtaining Reagent F / dermis. Reagent F is at least one of type IV collagen, entactin, and heparan sulfate proteoglycan at a concentration of 0.01-0.9 mg / mL. The amount of Reagent F used is determined based on the size of the dermis.

[0019] S05: Keratinocytes are seeded onto the reagent F / dermis layer and cultured in an artificial skin epidermis culture medium.

[0020] Keratinocytes were seeded onto the reagent F / dermis layer, and an appropriate amount of artificial skin epidermal culture medium was added and cultured for 1-5 days. 5 -6×10 8 cells / cm 2 Artificial skin epidermal culture medium is a culture medium commonly used in the preparation of artificial skin.

[0021] S06: After the culture is completed, the keratinization medium is replaced and immersed in culture for 1-5 days, and then the keratinization medium is replaced again for air-liquid surface culture to form an artificial skin containing a basement membrane.

[0022] After the culture is complete, the artificial skin epidermal culture medium is replaced with a keratinized medium and culture is continued for 1-5 days. This keratinized medium is commonly used in the preparation of artificial skin. After the keratinized medium is replaced again, air-liquid surface culture is performed, and the medium is changed daily for 3-14 days to form an artificial skin containing a basement membrane.

[0023] The present application also provides an artificial skin containing a basement membrane, which is prepared by the above-mentioned preparation method.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides an artificial skin containing a basement membrane and a preparation method thereof. The method uses dermal fibroblasts, keratinocytes, and the structural components of the basement membrane to prepare the artificial skin containing a basement membrane. In this method, the structural components of the basement membrane, extracellular matrix components, and components required to maintain cell growth are added to the dermal fibroblasts. These components include reagent A, reagent B, reagent C, reagent D, and reagent E. These components can promote the growth and proliferation of dermal fibroblasts, or can serve as scaffold materials for dermal fibroblasts, or can enhance intercellular adhesion and cell-matrix adhesion, or can stimulate and promote dermal cells or epidermal cells to secrete some extracellular matrix components, forming a basement membrane between the dermis and epidermis, thereby strengthening the connection between the dermis and epidermis. The prepared artificial skin containing a basement membrane comprises an epidermis, a basement membrane, and a dermis, and contains living cells. It has a structure similar to normal skin and can be used to treat skin wounds caused by various traumas and can also be used to prepare skin models. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 HE staining of artificial skin containing basement membrane provided in the examples of this application;

[0027] Figure 2HE staining images of other artificial skins provided in the examples of this application;

[0028] Figure 3 This is a PAS staining image of the artificial skin containing the basement membrane provided in the examples of the present application;

[0029] Figure 4 This is a PAS staining image of other artificial skins provided in the embodiments of the present application;

[0030] Figure 5 This is a collagen type IV staining image of the artificial skin containing the basement membrane provided in the examples of the present application;

[0031] Figure 6 This is a type IV collagen staining image of other artificial skin provided in the examples of this application;

[0032] Figure 7 This is a laminin staining image of the artificial skin containing the basement membrane provided in the examples of the present application;

[0033] Figure 8 This is a laminin staining image of other artificial skin provided in the examples of this application. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0035] Example 1

[0036] The present invention provides a method for preparing artificial skin containing a basement membrane, the method comprising:

[0037] S101: Add gelatin, collagen or other coating matrix to the support membrane and solidify at 37°C to obtain a coated support membrane. Add dermal fibroblasts to the coated support membrane and resuspend the dermal fibroblasts in PBS buffer containing 0.1 mg / mL fibronectin and gelatin. The amount of dermal fibroblasts used is 6×10 7 cells / cm 2 The resuspended dermal fibroblasts were centrifuged at 500 rpm for 5 minutes. The cells were then washed with PBS buffer and centrifuged again to remove uncoated PBS buffer containing 0.1 mg / mL fibronectin and gelatin. This resuspension, centrifugation, PBS washing, and recentrifugation cycle was repeated 10 times to obtain dermal fibroblasts.

[0038] S102: Mix DMEM, 200 mM L-glutamine solution, serum, and type I collagen to form a mixed solution. Add the coated dermal fibroblasts to the pH-neutralized mixed solution, mix thoroughly, and quickly transfer to a culture device. Incubate at 37°C for 3 hours to obtain a dermal cell mixture.

[0039] S103: The dermal cell mixture was cultured at 37°C and 5% CO2 for 5 days to obtain the dermis layer.

[0040] S104: Add type IV collagen with a concentration of 0.1 mg / mL to the dermis layer and incubate for 30 minutes to form reagent F / dermis layer.

[0041] S105: Press 5×10 5 cells / cm 2 Keratinocytes were seeded onto the reagent F / dermis layer with an inoculum size of 100 μg / mL and cultured in an artificial skin epidermis culture medium for 5 days.

[0042] S106: After the culture is completed, the keratinization medium is replaced and immersed in culture for 5 days, and then the keratinization medium is replaced again and air-liquid surface culture is performed for 14 days to form an artificial skin containing a basement membrane.

[0043] Example 2

[0044] The present invention provides a method for preparing artificial skin containing a basement membrane, the method comprising:

[0045] S201: Add gelatin, collagen or other coating matrix to the support membrane and solidify at room temperature to obtain a coated support membrane. Add dermal fibroblasts to the coated support membrane and resuspend the dermal fibroblasts in Tris-HCl buffer containing fibronectin and gelatin at a concentration of 0.01 mg / mL. The amount of dermal fibroblasts used is 1×10 5 cells / cm 2 The resuspended dermal fibroblasts were centrifuged at 5 rpm for 1 minute. The cells were then washed with PBS and centrifuged again to remove uncoated Tris-HCl buffer containing 0.01 mg / mL fibronectin and gelatin. This resuspension, centrifugation, PBS washing, and re-centrifugation cycle was repeated five times to obtain dermal fibroblasts.

[0046] S202: MEM, 200 mM L-glutamine solution, serum, and chitosan are mixed to form a mixed solution. The coated dermal fibroblasts are added to the pH-adjusted mixed solution, mixed thoroughly, and quickly transferred to a culture device. After mixing thoroughly, the mixture is incubated at room temperature for 0.5 hours to obtain a dermal cell mixture.

[0047] S203: The dermal cell mixture was cultured at 37°C and 5% CO2 for 1 day to obtain the dermis layer.

[0048] S204: Add nestin at a concentration of 0.9 mg / mL to the dermis layer and incubate for 30 minutes to form reagent F / dermis layer.

[0049] S205: will be 6×10 8 cells / cm 2 Keratinocytes were seeded onto the reagent F / dermis layer with an inoculum size of 100 μg / mL and cultured in an artificial skin epidermis culture medium for 1 day.

[0050] S206: After the culture is completed, the keratinization medium is replaced and immersed in culture for 1 day, and then the keratinization medium is replaced again and air-liquid surface culture is performed for 3 days to form an artificial skin containing a basement membrane.

[0051] Example 3

[0052] The present invention provides a method for preparing artificial skin containing a basement membrane, the method comprising:

[0053] S301: Add gelatin, collagen or other coating matrix to the support membrane and solidify at 37°C to obtain a coated support membrane. Add dermal fibroblasts to the coated support membrane and resuspend the dermal fibroblasts in PBS buffer containing fibronectin and gelatin at a concentration of 0.05 mg / mL. The amount of dermal fibroblasts used is 1×10 6 cells / cm 2 The resuspended dermal fibroblasts were centrifuged at 200 rpm for 3 minutes. The cells were then washed with PBS buffer and centrifuged again to remove uncoated fibronectin and gelatin in PBS buffer (0.05 mg / mL). This resuspension, centrifugation, PBS washing, and recentrifugation cycle was repeated eight times to obtain dermal fibroblasts.

[0054] S302: F12, 200 mM L-glutamine solution, serum, type I collagen, and hyaluronic acid are mixed to form a mixed solution. The coated dermal fibroblasts are added to the pH-adjusted mixed solution, mixed thoroughly, and quickly transferred to a culture device. After mixing thoroughly, the mixture is incubated at 37°C for 2.5 hours to obtain a dermal cell mixture.

[0055] S303: The dermal cell mixture was cultured at 37°C and 5% CO2 for 3 days to obtain the dermis layer.

[0056] S304: Add heparan sulfate proteoglycan at a concentration of 0.01 mg / mL to the dermis layer and incubate for 30 minutes to form reagent F / dermis layer.

[0057] S305: will be 6×10 7 cells / cm 2 Keratinocytes were seeded onto the reagent F / dermis layer with an inoculum size of 100 μg / mL and cultured in an artificial skin epidermis culture medium for 4 days.

[0058] S306: After the culture is completed, the keratinization medium is replaced and immersed in culture for 4 days, and then the keratinization medium is replaced again and air-liquid surface culture is performed for 10 days to form an artificial skin containing a basement membrane.

[0059] Example 4

[0060] The present invention provides a method for preparing artificial skin containing a basement membrane, the method comprising:

[0061] S401: Add gelatin, collagen or other coating matrix to the support membrane and solidify at 37°C to obtain a coated support membrane. Add dermal fibroblasts to the coated support membrane and resuspend the dermal fibroblasts in PBS buffer containing 0.05 mg / mL fibronectin and gelatin. The amount of dermal fibroblasts used is 2×10 7 cells / cm 2 The resuspended dermal fibroblasts were centrifuged at 300 rpm for 3 minutes. The cells were then washed with PBS buffer and centrifuged again to remove uncoated fibronectin and gelatin in PBS buffer (0.05 mg / mL). This resuspension, centrifugation, PBS washing, and recentrifugation cycle was repeated eight times to obtain dermal fibroblasts.

[0062] S402: DMEM, MEM, 200 mM L-glutamine solution, serum, gelatin, and chitosan are mixed to form a mixed solution. The coated dermal fibroblasts are added to the pH-adjusted mixed solution. Mix thoroughly and quickly transfer to a culture device. After mixing, incubate at 37°C for 2 hours to obtain a dermal cell mixture.

[0063] S403: The dermal cell mixture was cultured at 37°C and 5% CO2 for 4 days to obtain the dermis layer.

[0064] S404: Add a composition of type IV collagen and entactin at a concentration of 0.5 mg / mL to the dermis layer and incubate for 30 minutes to form reagent F / dermis layer.

[0065] S405: will be 4×10 8 cells / cm2 Keratinocytes were seeded onto the reagent F / dermis layer with an inoculum size of 100 μg / mL and cultured in an artificial skin epidermis culture medium for 4 days.

[0066] S406: After the culture is completed, the keratinization medium is replaced and immersed in culture for 4 days, and then the keratinization medium is replaced again and air-liquid surface culture is performed for 10 days to form an artificial skin containing a basement membrane.

[0067] The examples of this application also subjected the prepared artificial skin containing a basement membrane to HE staining (English name: hematoxylin-eosin staining; Chinese name: hematoxylin-eosin staining), PAS staining (English name: periodic acid-schiff staining), type IV collagen staining, laminin staining, and transmembrane potential testing. HE staining is used to observe the histological structure of the artificial skin. PAS staining, type IV collagen staining, and laminin staining are immunohistochemical staining used to observe the basement membrane components of the artificial skin. Transmembrane potential testing is used to indicate the barrier function of the artificial skin. The following detailed description uses the artificial skin containing a basement membrane prepared in Example 1 as the subject.

[0068] The artificial skin containing a basement membrane prepared in Example 1 and other artificial skins were subjected to conventional paraffin sectioning, with a section thickness of about 4 μm. The sample group was the artificial skin containing a basement membrane prepared in Example 1 of the present application, and the control group was artificial skin prepared by other methods.

[0069] 1. HE staining

[0070] HE staining was performed on the artificial skin sections to observe the tissue structure of the artificial skin. The specific HE staining steps are as follows: xylene staining 3 times, 4 minutes each time; 100% ethanol staining 2 times, 4 minutes each time; 95% ethanol staining 2 times, 4 minutes each time; 95% ethanol staining 4 minutes; 70% ethanol staining 4 minutes. After rinsing with distilled water, hematoxylin staining for 4 minutes, washing with water for 5 minutes, eosin staining for 30 seconds, 70% ethanol staining 2 times, 15 seconds each time; 95% ethanol staining 2 times, 15 seconds each time; 95% ethanol staining 15 seconds, 100% ethanol staining 2 times, 30 seconds each time; xylene staining 2 times, 5 minutes each time. After staining, fix and seal the sections. Observe the staining under a microscope to obtain attached Figure 1 、 2 .

[0071] By the attached Figure 1 、 2As can be seen in the HE staining of the artificial skin of the sample group, a basement membrane is formed between the epidermis and dermis, and the connection is tight, making it difficult to separate when sectioning. In the HE staining of the artificial skin of the control group, a basement membrane is not formed between the epidermis and dermis, and the connection is not tight, so the epidermis and dermis are easy to separate when sectioning.

[0072] 2. PAS staining

[0073] The artificial skin sections were PAS stained to observe the basement membrane of the artificial skin. The specific PAS staining steps are as follows: after dewaxing and washing, stain with 0.5% Periodic acid solution for 10 minutes. Rinse with distilled water and stain with Schiff solution for 10 minutes. Rinse with distilled water and stain twice with 1% sodium bisulfite solution for 3 minutes each time. After washing with water for 5 minutes, dehydrate, fix and seal. Observe the staining under a microscope to obtain the attached Figure 3 、 4 .

[0074] By the attached Figure 3 、 4 As can be seen in the PAS staining of the artificial skin from the sample group, a homogeneous purple-red band between the epidermis and dermis indicates a positive PAS reaction. In the PAS staining of the artificial skin from the control group, a discontinuous purple-red band exists between the epidermis and dermis. This indicates that in the artificial skin provided in the examples of this application, the epidermis and dermis are tightly connected via the basement membrane.

[0075] 3. Type IV collagen staining

[0076] The artificial skin sections were stained for type IV collagen to observe the basement membrane of the artificial skin. The specific steps for type IV collagen staining are as follows: paraffin-embedded specimens were taken, serially sliced, and routine immunohistochemical staining was performed. The primary antibody was mouse anti-human type IV collagen monoclonal antibody (1:150), the secondary antibody was horseradish peroxidase-labeled goat anti-mouse antibody, ABC method, diaminobenzidine color development, and hematoxylin counterstaining. The staining was observed under a microscope to obtain the attached Figure 5 、 6 .

[0077] By the attached Figure 5 、 6 As can be seen, in the collagen IV staining of the artificial skin of the sample group, the epidermis and dermis were uniformly stained with collagen IV, while in the collagen IV staining of the artificial skin of the control group, the epidermis and dermis were completely separated and no collagen IV staining was observed.

[0078] 4. Laminin staining

[0079] The artificial skin sections were stained with laminin to observe the basement membrane of the artificial skin. The specific steps of laminin staining were as follows: paraffin-embedded specimens were taken, serially sliced, and routine immunohistochemical staining was performed. The primary antibody was mouse anti-human Laminin monoclonal antibody (1:150), the secondary antibody was horseradish peroxidase-labeled goat anti-mouse antibody, ABC method, diaminobenzidine color development, and hematoxylin counterstaining. The staining was observed under a microscope to obtain the attached Figure 7 、 8 .

[0080] By the attached Figure 7 、 8 As can be seen, in the laminin staining image of the artificial skin of the sample group, the epidermis and dermis are evenly stained with laminin, while in the laminin staining image of the artificial skin of the control group, the epidermis and dermis are completely separated and no laminin staining is found.

[0081] 5. Transmembrane potential test

[0082] The transmembrane resistance of the artificial skin in the sample group and the control group was tested using a transcutaneous resistance meter under the same test conditions. The test results are shown in Table 1.

[0083] Table 1: Transmembrane resistance test results of the sample group and the control group

[0084]

[0085] As can be seen from Table 1, the transmembrane resistance value of the artificial skin of the sample group is much higher than that of the artificial skin of the control group, which shows that the barrier function of the artificial skin prepared in the embodiment of the present application is better than that of artificial skin prepared by other methods.

[0086] The above tests demonstrate that in the artificial skin containing a basement membrane provided in the examples of this application, the epidermis and dermis are tightly connected via the basement membrane, and the basement membrane exhibits highly uniform staining for PAS, type IV collagen, and laminin. Furthermore, the barrier function of the artificial skin prepared in the examples of this application is superior to that of artificial skin prepared by other methods.

[0087] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing artificial skin containing a basement membrane, characterized in that: include: adding gelatin or collagen to the support membrane to obtain a coated support membrane; Adding dermal fibroblasts to the coating support membrane, and repeatedly resuspending the dermal fibroblasts in reagent A, centrifuging, washing with PBS, and centrifuging again to obtain coated dermal fibroblasts; Reagent B, reagent C, reagent D, and reagent E are mixed to form a mixed solution, the coated dermal fibroblasts are added to the mixed solution after adjusting the pH value to neutral, the mixture is mixed evenly, and then incubated at room temperature or 37° C. to obtain a dermal cell mixture; The dermal cell mixture is cultured at a temperature of 37° C. and 5% CO 2 to obtain a dermal layer; adding reagent F to the dermis layer and incubating to form reagent F / dermis layer; Inoculating keratinocytes onto the reagent F / dermis layer and culturing them in an artificial skin epidermis culture medium; After the culture is completed, the keratinized medium is replaced and immersed in culture for 1-5 days, and then the keratinized medium is replaced again for air-liquid surface culture to form an artificial skin containing a basement membrane; Wherein, the reagent A comprises: a buffer or culture medium containing fibronectin and gelatin; The reagent B comprises: at least one of DMEM, MEM and F12 culture medium; The reagent C includes: L-glutamine solution; The reagent D includes: serum; The reagent E comprises: at least one of collagen, silk fibroin, hyaluronic acid, gelatin and chitosan; The reagent F comprises: at least one of type IV collagen, entactin, and heparan sulfate proteoglycan; The concentration of the fibronectin in the buffer or culture medium is 0.01-0.1 mg / mL, and the concentration of the L-glutamine solution is 200 mM. The amount of dermal fibroblasts used is 10 5 -6×10 7 cells / cm 2 .

2. The method for preparing an artificial skin containing a basement membrane according to claim 1, wherein: Incubate at room temperature or 37°C for 0.5-3 hours.

3. The method for preparing an artificial skin containing a basement membrane according to claim 1, wherein: Culture at 37°C, 5% CO2 for 1-5 days.

4. The method for preparing an artificial skin containing a basement membrane according to claim 1, wherein: The seeding number of the keratinocytes is 10 5 -6×10 8 cells / cm 2 .

5. The method for preparing artificial skin containing basement membrane according to claim 1, wherein: The keratinocytes are cultured on the reagent F / dermis layer for 1-5 days and on the air-liquid surface for 3-14 days.

6. The method for preparing artificial skin containing a basement membrane according to claim 1, wherein: The concentration of the reagent F is 0.01-0.9 mg / mL.

7. The artificial skin containing basement membrane prepared by any one of the preparation methods described in claims 1-6.

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