Method for producing three-dimensionally cultured skin model comprising hair follicle structure using fibroblasts and three-dimensionally cultured skin model produced thereby

Abstract

The present invention relates to a method for producing a three-dimensionally cultured skin model comprising a hair follicle structure using fibroblasts, and a three-dimensionally cultured skin model produced thereby. Specifically, to reproduce a hair follicle structure similar to a three-dimensionally cultured skin comprising a hair follicle structure using dermal papilla cells, unique fibroblasts that exhibit gene expression similar to dermal papilla cells when the dermal papilla cells are treated with a hair growth promoting material, were discovered. Therefore, by replacing dermal papilla cells which are difficult to separate and culture, it is possible to produce a three-dimensionally cultured skin model comprising a hair follicle structure using fibroblasts that can be easily mass-cultured.

Description

Method for manufacturing a three-dimensional cultured skin model including a hair follicle structure using fibroblasts and a three-dimensional cultured skin model manufactured thereby

[0001] The present invention relates to a method for producing a three-dimensional cultured skin model including a hair follicle structure using fibroblasts and a three-dimensional cultured skin model produced thereby.

[0002]

[0003] The skin is the largest organ covering the entire surface of the human body and has various functions such as regulating body temperature and acting as a barrier against the external environment. In adults, its area is approximately 1.2 to 2.3 m 2 It reaches. The skin is composed of the epidermis and the dermis, and the dermis layer is composed of collagen and fibroblasts, and the epidermis layer is composed of keratinocytes and melanin-forming cells. Compared to the epidermis layer, which has a relatively uniform thickness, the thickness of the dermis layer is known to vary greatly depending on the area, but is known to be about three times thicker than the epidermis.

[0004] The epidermis is stratified into the basement membrane, stratum spinosum, stratum granulosum, and stratum corneum, and multiple layers of terminally differentiated dead cells accumulate on the skin surface to perform a protective barrier function. The basement membrane is a thin membrane in which laminin and various extracellular matrices are deposited on type IV collagen lattices, and cells of the basal layer adhere to it. When a wound occurs, the basement membrane decomposes, bringing the cells of the basal layer into contact with type I collagen of the dermis. At this time, epidermal cells migrate laterally, inducing re-epithelialization.

[0005] The dermis is composed of the papillary dermis, which is rich in fibroblasts and microvessels, and the reticular dermis, which is rich in thick collagen fibers. Hair follicles and various skin appendages are located deep within the dermis. Therefore, even in cases where the epidermis is lost, such as in a partial thickness wound, epidermal cells grow from the hair follicles, inducing reepithelialization.

[0006] Hair follicles primarily serve a cosmetic function in social life rather than a vital function. A normal hair follicle grows and is maintained through a repetitive hair cycle: anagen (3-5 years), during which hair matrix cells actively divide and grow; catagen (3 weeks), during which hair growth ceases, the hair papilla separates from the hair within the follicle, and the follicle structure begins to degenerate; and telogen (3 months), during which the hair papilla separates from the hair and the hair falls out.

[0007] However, in modern life, hair loss (alopecia) progresses due to factors such as abnormal hormonal secretions, excessive stress, and environmental pollution, causing significant psychological distress in people's social lives. In the past, passive methods were used, such as simply ignoring hair loss or masking the symptoms with wigs. However, recent research on the biology and molecular biology of hair follicles and hair loss is revealing new facts. With the discovery of stem cells, hair follicles are emerging as a key new area of ​​research.

[0008] To assess the overall progression and status of hair loss and to test the efficacy of appropriate treatments and effective substances, a deeper understanding of the complex structure and regulatory mechanisms of hair follicles is necessary. Furthermore, an in vitro system capable of reproducible, quantitative observations, and artificially controlling various factors is required. To date, research on hair follicles has primarily utilized mouse vibrissae, with very few studies using human hair follicles. Unlike rodents, which shed hair in a regular cycle, human hair follicles have independent cycles, resulting in different trends in growth factors and numerous biological and functional differences. Therefore, to fully understand and study the characteristics of human hair follicles, it is desirable to conduct research using human hair follicle cells.

[0009] In particular, the production of artificial skin using hair papilla cells is being studied, but hair papilla cells, which are involved in hair production and hair follicle formation, require a different separation method from general cells, and when cultured in vitro, they easily lose their unique characteristics and have low proliferation ability, making cell culture difficult.

[0010] Additionally, there are limitations in testing to evaluate the efficacy and safety of hair loss treatments, cosmetics, and pharmaceuticals using breast cancer cells.

[0011] Therefore, there is an urgent need to create a three-dimensional cultured skin model that can replace mammary papilla cells, which are difficult to isolate and culture.

[0012] [Prior Art Literature]

[0013] [Patent Document]

[0014] Domestic Publication Patent No. 10-2016-0007901

[0015] [Non-patent literature]

[0016] Hasan Erbil Abaci et al. Tissue engineering of human hair follicles using a biomimetic developmental approach, NATURE COMMUNICATIONS | (2018) 9:5301 |

[0017]

[0018] Accordingly, as a result of research efforts to solve the above problems, the inventors of the present invention discovered a unique fibroblast that shows gene expression similar to that of dermal papilla cells when dermal papilla cells are treated with a hair growth promoting substance in order to reproduce a hair follicle structure similar to a three-dimensional cultured skin including a hair follicle structure using dermal papilla cells, and thus completed the present invention by developing a three-dimensional cultured skin model including a hair follicle structure using fibroblasts that can be easily mass-cultured as a substitute for dermal papilla cells, which are difficult to isolate and culture.

[0019]

[0020] Accordingly, the purpose of the present invention is to provide a method for manufacturing a three-dimensional cultured skin model including a hair follicle structure using fibroblasts that exhibit gene expression similar to that of hair papilla cells treated with a hair growth promoting substance, thereby enabling easy mass cultivation by replacing hair papilla cells, which are difficult to isolate and culture.

[0021] In addition, another purpose of the present invention is to provide a three-dimensional cultured skin model including a hair follicle structure using fibroblasts that show gene expression similar to that of hair papilla cells treated with a hair growth promoting substance.

[0022]

[0023] The present invention selects specific fibroblasts (HF1) that exhibit gene expression similar to that of the obtained hair papilla cells by treating hair growth promoting substances to hair papilla cells isolated from human scalp tissue, and when using these to manufacture three-dimensional cultured skin including a hair follicle structure, a hair follicle structure similar to that of three-dimensional cultured skin including a hair follicle structure using hair papilla cells is reproduced.

[0024] Accordingly, a three-dimensional cultured skin model including a hair follicle structure can be manufactured using fibroblasts that can be easily mass-cultured to replace hair papilla cells, which are difficult to isolate and culture.

[0025]

[0026] Figure 1 shows the gene expression results of two types of fibroblasts HF1 and HF2, one type of hair papilla cells, and hair papilla cells treated with a hair growth promoting substance through heatmap analysis.

[0027] Figure 2 shows the expression of eight genes that showed high expression in HF1 among two types of fibroblasts, HF1 and HF2, treated with a hair growth promoting substance, through heatmap analysis.

[0028] Figure 3 shows the results of polymerase chain reaction (Real time RT-PCR) performed on HF1, HF2, and DP cells treated with minoxidil for 24 hours.

[0029] Figure 4 shows the results of a western blot analysis of the expression of ITGA2, TBX3, and ITGA6 genes after 24 hours of treatment with minoxidil in HF1, HF2, and DP cells.

[0030] Figure 5 is a schematic representation of the dermal structure in which cylindrical holes exist.

[0031] Figure 6 shows three-dimensional cultured skin with dermal and epidermal layers including hair follicle structures formed using fibroblasts, stained with H&E (Hematoxylin & Eosin) tissue.

[0032] Figure 7 shows three-dimensional cultured skin, including the dermal layer and epidermal layer including the hair follicle structure, manufactured using HF1, HF2 cells and DP cells, confirmed by H&E (Hematoxylin & Eosin) tissue staining.

[0033] Figure 8 shows that a three-dimensional cultured skin model including the dermal layer, epidermal layer, and hair follicle structure was recreated using HF1, which shows high expression of eight genes similar to hair papilla cells treated with a hair growth promoting substance, as confirmed by H&E (Hematoxylin & Eosin) tissue staining.

[0034]

[0035] The present invention relates to a method for producing a three-dimensional cultured skin model including a hair follicle structure using fibroblasts that exhibit gene expression similar to that of hair papilla cells treated with a hair growth promoting substance.

[0036] Specifically, the method for manufacturing a three-dimensional cultured skin model including a hair follicle structure according to the present invention is

[0037] 1) A step of selecting fibroblasts that show gene expression similar to that of hair papilla cells treated with a hair growth promoting substance;

[0038] 2) A step of inoculating a cell mixture containing fibroblasts and collagen into a cell culture device, fixing a cylindrical hole-forming inducing tool, and solidifying the mixture to produce a dermal structure;

[0039] 3) A step of forming a cylindrical hole by removing a cylindrical hole forming induction tool from the structure formed in step 2);

[0040] 4) A step of manufacturing a dermal layer by inoculating fibroblasts or dermal papilla cells selected in step 1) into the cylindrical hole formed in step 3);

[0041] 5) A step of inoculating keratinocytes into the upper layer of the dermis and culturing them until a cell sheet is formed; and

[0042] 6) Step of culturing the cell sheet by exposing it to the air to form an epidermal layer.

[0043] Includes.

[0044] Hereinafter, the manufacturing method of the present invention will be described in detail as follows.

[0045] The term "hair follicle" in the present invention refers to a skin appendage found only in mammals, which is formed from epithelial-mesenchymal interactions during embryogenesis. During embryogenesis, hair follicle development is initiated by a first dermal signal, which results in the thickening of the epithelial layer to form a placode. This epithelial signal from the thickened epithelial plate induces the formation of a dermal condensate of mesenchymal-derived dermal cells, and this condensate then generates a second dermal signal. This signal promotes the proliferation of epithelial cells and simultaneously induces their infiltration into the dermis, surrounding the condensate, resulting in the formation of the dermal papilla. This initial hair follicle structure, upon which epithelial cell proliferation and differentiation continue, develops into a mature hair follicle that forms hair. In mature hair follicles, specialized differentiation of matrix cells occurs through the interaction between the matrix cells and the hair papilla cells, resulting in hair production and growth.

[0046] The term “hair growth promoting substance” of the present invention refers to a substance that prevents hair loss or promotes hair growth, and may specifically be minoxidil and finasteride, which are hair growth promoting drugs approved by the FDA, and CHIR99021 and CHIR98014, which are Wnt signaling activating compounds generally involved in hair growth.

[0047] The term "dermal papilla cell" of the present invention refers to the core cell responsible for hair growth, located at the very bottom of the hair root and surrounded by dermal papilla cells like a wall. Dermal papilla cells are cells that produce hair within the tissue, and exist beneath the hair follicle. They absorb necessary nutrients from the capillaries flowing to the dermal papilla cells and produce hair through division and proliferation, and determine the number, thickness, and regrowth cycle of the hair.

[0048] The term "fibroblast" in the present invention refers to a cell that constitutes an important component of fibrous connective tissue, and particularly, in the case of skin related to the present invention, refers to a major cell constituting the dermal layer. In the present invention, fibroblasts may be derived from humans, mice, rats, etc., but are not limited thereto. For the purposes of the present invention, the fibroblasts used in the preparation of the dermal layer in the process of preparing the three-dimensional cultured skin model of the present invention may be, in particular, of human origin.

[0049] The term "collagen" of the present invention refers to a light protein that exists in both plants and animals, and is mainly present in the bones and skin of animals, and is also distributed in cartilage, organ membranes, hair, etc., and is also called collagen. Collagen is a protein composed of a triple helix structure, and has a non-helical telopeptide at both ends of the triple helix structure. In the present invention, collagen may be derived from humans, cows, pigs, mice, rats, etc., but is not limited thereto. In the present invention, collagen may be not only natural collagen, but also bioengineered variants or bioengineered or chemically treated modified collagen, but is not limited in origin or modification.

[0050] The term "keratinocyte" of the present invention refers to a cell that has keratinization ability among epidermal cells and accounts for the majority of epidermal cells, and generally refers to the same cell as keratinocyte. In the present invention, keratinocytes may be derived from humans, mice, rats, etc., but are not limited thereto. For the purpose of the present invention, the keratinocytes used in the preparation of the epidermal layer in the process of preparing the three-dimensional cultured skin model of the present invention may be, in particular, human-derived keratinocytes, and may be keratinocytes derived from normal tissues or keratinocytes derived from diseased tissues such as chemicals, burns, wounds, scars, and ulcers, but are not limited thereto.

[0051] The term "three-dimensional cultured skin model" of the present invention refers to a skin layer model similar to a natural skin layer, manufactured through an artificial culture process rather than collecting or acquiring cells from animals or the like. The origin of the cells constituting the model is not limited.

[0052]

[0053] According to one embodiment of the present invention,

[0054] First, to reproduce the artificial skin with a hair follicle structure, fibroblasts that show gene expression similar to hair papilla cells treated with a hair growth promoting substance are selected.

[0055] The gene similar to the hair papilla cells treated with the hair growth promoting substance is a gene that shows similar expression between the hair papilla cells and fibroblasts when the hair papilla cells are treated with the hair growth promoting substance as a result of heatmap analysis. The gene similar to the hair papilla cells treated with the hair growth promoting substance may be at least one selected from the group consisting of AFF1, KCTD12, KANK1, NFAT5, RAPGEF6, ITGA2, TBX3, and ITGA6, and preferably may be at least one selected from the group consisting of ITGA2, TBX3, and ITGA6.

[0056] 2) In step 2, a cell mixture containing fibroblasts and collagen is inoculated into a cell culture device, and a cylindrical hole formation induction tool is fixed and solidified to manufacture a dermal structure.

[0057] The above cell culture device may be a container capable of cell culture, and may be specifically a transwell insert, but is not limited thereto.

[0058] The above cylindrical hole formation inducing tool may be, but is not limited to, a needle, syringe, pipette tip, aluminum mold, stainless steel mold, etc. that can induce the creation of a circular hole in a dermal structure to mimic a hair follicle structure.

[0059] At this time, 1 × 10 mouse-derived or human-derived fibroblasts per 1 mL were added to the dermal structure containing fibroblasts and collagen. 4 1 × 10 7 It may contain a number of cells, and may additionally contain one or more cells selected from the group consisting of endothelial cells, adipocytes, and mesenchymal stem cells.

[0060] In step 3), a number of cylindrical holes are formed in the above-mentioned formed dermal structure.

[0061] The above cylindrical holes are intended to mimic the structure of hair follicles within the skin, and are preferably manufactured to be as similar to the size of the hair follicles as possible. Specifically, the diameter of the circular holes is 100 μm to 1000 μm, and the length of the cylinder is 200 μm to 500 μm. The spacing between the holes may be manufactured to be 100 μm to 1000 μm, but is not limited thereto.

[0062] The step of inoculating a composition containing fibroblasts and collagen corresponding to step 2) above into a culture dish and solidifying it is performed for 10 to 48 hours (10-24 hours).

[0063] In step 4), fibroblasts or hair papilla cells selected in step 1) are inoculated into the cylindrical hole to produce a dermal layer including a hair follicle structure.

[0064] At this time, selected fibroblasts or dermal papilla cells are seeded at 1 x 10 to 1 x 10 per cylindrical hole. 6 The number of cells can be inoculated.

[0065] Step 4) above may be performed for 1 to 48 hours.

[0066] In step 5, keratinocytes are seeded into the upper layer of the dermis and cultured until a cell sheet is formed.

[0067] At this time, the dermal layer cm manufactured above 2 1 x 10 keratinocytes per 4 1 x 10 7 It is desirable to inoculate the cell count.

[0068] In addition, the keratinocytes may be, but are not limited to, keratinocytes derived from skin, mucous membranes, cornea, hair follicles, or epithelial tissues. When inoculating the keratinocytes, one or more cells selected from the group consisting of melanocytes, Merkel cells, and Langerhans cells may be additionally included.

[0069] In addition, the above step 5) may be performed for about 2 to 21 days, but this may vary depending on the area of ​​the keratin cells and dermal layer to be inoculated, and is not particularly limited.

[0070] In step 6), the cell sheet is exposed to the air and cultured to form an epidermal layer, thereby manufacturing a three-dimensional cultured skin model.

[0071] Step 6) above may be performed for 7 to 21 days.

[0072] The present invention also includes a three-dimensional cultured skin model manufactured according to the above method.

[0073] The present invention also includes a three-dimensional cultured skin model comprising a dermal layer and an epidermal layer formed by keratinocytes, prepared from a composition comprising fibroblasts and collagen that exhibit gene expression similar to that of hair papilla cells treated with a hair growth promoting substance.

[0074]

[0075] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0076]

[0077] Example 1: Selection of HF1 fibroblasts showing gene expression similar to that of breast papilla cells.

[0078] Dermal papilla cells were isolated from human scalp tissue obtained from donors by microdissection, seeded on culture dishes, and cultured in DMEM culture medium containing 10% FBS to isolate the dermal papilla cells (DP).

[0079] Fibroblasts were isolated from skin tissue obtained from donors and cultured in F12 medium containing 10% FBS.

[0080] 2.2X10 breast papilla cells were seeded in a 60 mm diameter culture dish. 5 After inoculating and culturing for 24 hours, the hair papilla cells were treated with 50 μM Minoxidil, 100 μM Finasteride, and 5 μM CHIR99021, which are hair growth promoting substances, for 24 hours each and cultured, and fibroblasts were seeded at 1.2X10 in a 60 mm diameter culture dish. 5 was inoculated and cultured for 48 hours.

[0081] After completion of culture, RNA was extracted from each obtained cell using TriZol and mRNA-sequencing analysis was performed. Gene expression was analyzed through mRNA-sequencing results between groups treated with hair growth promoting substances for two types of fibroblasts (HF1, HF2), one type of dermal papilla cells (DP), and one type of dermal papilla cells. It was confirmed that gene expression in one type of HF among the two types of fibroblasts was similar to gene expression in the dermal papilla cells treated with the hair growth promoting substance. This fibroblast was named HF1, the other type of fibroblast was named HF2, and the dermal papilla cells were named DP. The results of gene expression were presented using heatmap analysis (Fig. 1).

[0082] Compared to the hair papilla cells, eight genes (AFF1, KCTD12, KANK1, NFAT5, RAPGEF6, ITGA2, TBX3, ITGA6) that commonly showed an increase in expression of more than twofold in the hair papilla cells and HF1 treated with hair growth promoting substances were selected, and the results of intercellular gene expression for the eight genes were presented using heatmap analysis (Fig. 2).

[0083] It was determined that genes whose expression level increases when hair growth promoting substances are treated in mammary papilla cells are likely to be genes related to hair growth.

[0084] To analyze the correlation between hair growth promotion and eight genes selected at the RNA level, RNA was extracted from DP cells, DP cells treated with minoxidil for 24 hours, HF1, and HF2 cells using TriZol, and then polymerase chain reaction (Real-time RT-PCR) was performed using the synthesized cDNA. The polymerase chain reaction was performed using a QuantStudio5 Real-Time PCR system using a mixture of TB Green Premix Ex Taq, ROX dye, and primers designed for each of the eight genes. As a result, it was confirmed that the expression of all eight genes increased in DP cells treated with hair growth promoting substances compared to DP cells, and that the expression was higher in HF1 cells than in HF2 cells. Accordingly, it was confirmed that the expression of the eight genes increased in DP cells by hair growth promoting substances, and that the expression of the eight genes was high specifically in HF1 cells at the RNA level (Fig. 3).

[0085] To analyze the correlation between hair growth promotion and eight genes selected at the protein level, cell lysates were extracted using RIPA buffer from DP cells, DP cells treated with minoxidil for 24 hours, HF1, and HF2 cells, and western blot was performed to observe the expression changes of ITGA2, TBX3, and ITGA6 genes. As a result, it was confirmed that the expression of all three genes increased in DP cells treated with hair growth promoting substances compared to DP cells, and that the expression was higher in HF1 cells than in HF2 cells. Accordingly, it was confirmed that the expression of the three genes increased in DP cells by hair growth promoting substances, and that the expression of the three genes was high specifically in HF1 cells (Fig. 4).

[0086] Therefore, HF1 fibroblasts, which have a similar gene expression pattern to hair follicle cells treated with hair growth promoting substances, were used to produce three-dimensional cultured skin including hair follicle structures.

[0087]

[0088] Example 2: Manufacturing a three-dimensional cultured skin model containing hair follicle structures

[0089] 1) Dermal layer manufacturing

[0090] 1X10 per mL of mouse-derived fibroblasts or human-derived fibroblasts 5 A cell mixture solution was prepared by mixing 2.0 mg / mL collagen solution and 10X Reconstruction buffer (0.2 M sodium bicarbonate, 0.2 M HEPES, 0.05 N NaOH) to include the number of cells. 4 mL of the cell mixture solution was added to each 6-well type transwell insert, and needles with a diameter of 500 μm were fixed at 500 μm intervals within the transwell insert and solidified in a 37°C, 10% CO2 incubator for 24 hours to create a dermal structure. After 2 hours, the needles were removed from the dermal structure to form cylindrical holes with a diameter of 500 μm and a height of 3 mm in the dermal structure (Fig. 5).

[0091] 100 μL of DP cells and HF1, HF2 cells at a number of 3,000 cells per cylindrical structure were inoculated into the dermal layer manufactured above to form a dermal layer containing a hair follicle structure.

[0092] 2) Preparation of the epidermal layer

[0093] 2.5X10 human skin-derived keratinocytes were placed on the dermal layer prepared in 1) above. 5 The cells were inoculated and cultured. The culture medium containing 10 ng / mL EGF was replaced every two days, and the cells were cultured in a 37°C, 10% CO2 incubator for 6 days until keratin sheets were formed.

[0094] Once keratinocytes formed sheets on the dermal layer, they were transferred to a 6-well three-dimensional culture-dedicated vessel for air exposure, filled with a culture medium containing 10 ng / ml of EGF, and the prepared dermal-epidermal layer was exposed to air. Thereafter, the culture medium containing 10 ng / ml of EGF was replaced every 4 days, and the cells were cultured in a 37°C, 10% CO2 incubator for a total of 14 days.

[0095] 3) Tissue staining (H&E staining)

[0096] The structure of a three-dimensional cultured skin model including a hair follicle structure was confirmed by H&E (Hematoxylin & Eosin) tissue staining by manufacturing a dermal layer using HF1 cells in 1) above and manufacturing an epidermal layer as in 2) above and culturing it (Fig. 6). In the case of manufacturing using HF1 cells, it was observed that the hair follicle structure including the hair papilla, hair bulb, and hair root was reproduced.

[0097] As a result of confirming the structure of the three-dimensional cultured skin model including the hair follicle structure by manufacturing the dermal layer with DP cells and HF1, HF2 cells in 1) above and manufacturing the epidermal layer as in 2) above and culturing it, H&E staining was performed, and the hair follicle structure was observed only in the cultured skin using DP cells and HF1 cells (Fig. 7).

[0098] H&E tissue staining was performed using experimental procedures well known in the art.

[0099] When manufactured with fibroblasts (HF1) that showed gene expression similar to that of hair papilla cells, it was possible to reproduce three-dimensional cultured skin including hair papilla, hair bulb, and hair root structures, but when manufactured with fibroblasts (HF2) that did not show specific gene expression, it was impossible to reproduce three-dimensional cultured skin (Fig. 7).

[0100] As a result of confirming the structure of the three-dimensional cultured skin model (dermal layer + epidermal layer formation) containing the hair follicle structure manufactured in the above Example 2) by H&E tissue staining, it was confirmed that a three-dimensional skin model containing the hair follicle structure can be manufactured by forming a basal layer, a granular layer, a spinous layer, and a stratum corneum similar to the human epidermis (Fig. 8).

[0101]

[0102] The present invention is a technology developed through the "Development of 3D cultured skin including hair follicles and animal alternative testing method" of the 2021 Bio-Medical Technology Commercialization Support Project (BT210069) of the Seoul Business Agency of Seoul Metropolitan City.

Claims

1. 1) A step of selecting fibroblasts that show gene expression similar to that of hair papilla cells treated with a hair growth promoting substance; 2) A step of inoculating a cell mixture containing fibroblasts and collagen into a cell culture device, fixing a cylindrical hole-forming inducing tool, and solidifying the mixture to produce a dermal structure; 3) A step of forming a cylindrical hole by removing a cylindrical hole forming induction tool from the structure formed in step 2); 4) A step of producing a dermal layer by inoculating fibroblasts or dermal papilla cells selected in step 1) into the cylindrical hole; 5) A step of inoculating keratinocytes into the upper layer of the dermis and culturing them until a cell sheet is formed; and 6) A method for manufacturing a three-dimensional cultured skin model, comprising a step of culturing a cell sheet by exposing it to the air to form an epidermal layer.

2. In paragraph 1, A manufacturing method wherein genes similar to those of hair papilla cells treated with the above hair growth promoting substance are expressed similarly to those of hair papilla cells when hair papilla cells are treated with the hair growth promoting substance as shown in the heatmap analysis results.

3. In paragraph 1, A manufacturing method wherein the above hair growth promoting substance is at least one selected from the group consisting of minoxidil, finasteride, CHIR99021, and CHIR98014.

4. In paragraph 1, A manufacturing method wherein the gene similar to a hair follicle cell treated with a hair growth promoting substance is at least one selected from the group consisting of AFF1, KCTD12, KANK1, NFAT5, RAPGEF6, ITGA2, TBX3, and ITGA6.

5. In paragraph 1, A manufacturing method wherein the gene similar to a hair follicle cell treated with a hair growth promoting substance is at least one selected from the group consisting of ITGA2, TBX3, and ITGA6.

6. A three-dimensional cultured skin model manufactured according to paragraph 1.

7. A three-dimensional cultured skin model comprising a dermal layer and an epidermal layer formed by keratinocytes, prepared from a composition containing fibroblasts and collagen that exhibit gene expression similar to that of hair papilla cells treated with a hair growth promoting substance.

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