Artificial cultured skin and method for producing the same

Abstract

To provide an artificial cultured skin with excellent skin tissue (for example, properly formed horny layers and appropriately expressed genes).SOLUTION: An artificial cultured skin comprises: a basal tissue layer, comprising adipose-derived stem cells and a gel; a dermal tissue layer, disposed on the basal tissue layer and comprising fibroblasts and a gel; and an epidermal layer, disposed on the dermal tissue layer and comprising epidermal cells. Preferably, the gel is a collagen gel or a fibrin gel. Preferably, the adipose-derived stem cells are human adipose-derived stem cells. Preferably, the fibroblasts are normal human dermal fibroblasts. Preferably, the epidermal cells are normal human epidermal keratinocytes.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an artificial cultured skin that can be used in dermatology-related research (e.g., screening of cosmetics or topical skin agents, etc.) and a method for producing the same. [Background technology]

[0002] In recent years, artificial cultured skin formed by culturing cells three-dimensionally has been developed. Artificial cultured skin is useful for screening tests of cosmetics and topical skin agents. Patent Document 1 discloses a method for producing artificial skin tissue including a dermal tissue layer and an epidermal layer. Patent Document 2 discloses a method for producing regenerated human skin tissue by culturing human adipose tissue stem cells together with human fibroblasts and human keratinocytes.

[0003] However, in the development of artificial cultured skin, there is a demand for artificial cultured skin that is closer to human skin, and in particular, there is a demand for artificial cultured skin that has good skin tissue in which the state of each skin layer (e.g., good formation of the stratum corneum, gene expression, etc.) is closer to that of real skin. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6933855 [Patent Document 2] Patent Publication No. 2022-018946 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide artificial cultured skin having good skin tissue (e.g., good formation of the stratum corneum, good gene expression, etc.) and a method for producing artificial cultured skin having good skin tissue. [Means for solving the problem]

[0006] The present invention includes, but is not limited to, the embodiments listed below. [1] A base tissue layer comprising adipose-derived stem cells and a gel; a dermal tissue layer disposed over the base tissue layer, the dermal tissue layer comprising fibroblasts and a gel; and an epidermis layer disposed above the dermis tissue layer and including epidermal cells; The artificial cultured skin comprises [2] The artificial cultured skin described in item [1], wherein the gel of the base tissue layer is a collagen gel or a fibrin gel, and the gel of the dermis tissue layer is a collagen gel or a fibrin gel. [3] The artificial cultured skin described in item [1] or [2], wherein the adipose-derived stem cells are human adipose-derived stem cells, the fibroblasts are normal human skin fibroblasts, and the epidermal cells are normal human epidermal keratinocytes. [4] The artificial cultured skin according to any one of items [1] to [3], wherein the epidermal layer has a stratum corneum formed by differentiation of epidermal cells by air-liquid interface culture. [5] The artificial cultured skin according to any one of items [1] to [4], wherein the epidermal layer is an epidermal layer in which expression of one or more genes selected from basement membrane-related factors, basal cell-related factors, barrier function-related factors, and moisturizing-related factors is activated. [6] The artificial cultured skin described in any one of items [1] to [5], in which expression of one or more genes selected from COL17A1, KRT15, ​​COL4A1, COL7A1, OCLN, HAS1, COL1A1, FGF10, and VEGF is activated, or expression of the MMP-1 gene is suppressed. [7] A method for producing an artificial cultured skin according to any one of items [1] to [6], (a) providing a mixture comprising adipose-derived stem cells and a gel to form a base tissue layer; (b) providing a mixture containing fibroblasts and a gel on the base tissue layer to form a dermis tissue layer; (c) providing epidermal cells onto the dermal tissue layer to form an epidermal layer; and (d) culturing a laminate comprising a basal tissue layer, a dermal tissue layer and an epidermal layer; A method for producing artificial cultured skin comprising: [8] The method according to item [7], further comprising the step of culturing the laminate comprising the base tissue layer and the dermis tissue layer after step (b). [9] The method according to item [7] or [8], wherein step (d) includes a step of immersing the laminate in a culture medium for undifferentiation and culturing it, a step of immersing the laminate in a culture medium for differentiation and culturing it, and a step of culturing the laminate at an air-liquid interface in the culture medium for differentiation.

[10] The method according to any one of items [7] to [9], wherein step (d) uses a tubular device having protrusions on its inner wall, and engages the protrusions with the base tissue layer or the dermal tissue layer to perform the culture.

[11] The method according to item

[10] , wherein in step (d), a base having a plurality of holes extending vertically therethrough is used, and the device is placed on the base to perform the culture.

[12] Contacting the artificial cultured skin according to any one of items [1] to [6] with a drug; and Analyzing the artificial cultured skin after contact with the drug to examine at least one selected from a change in the state of the artificial cultured skin and a state of the drug in the artificial cultured skin; A method for evaluating a drug using artificial cultured skin, comprising: Effect of the Invention

[0007] According to the present invention, it is possible to provide artificial cultured skin having good skin tissue (e.g., good formation of the stratum corneum, good gene expression, etc.). It is also possible to provide a method for producing artificial cultured skin having good skin tissue. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a perspective cross-sectional view that shows a schematic diagram of artificial cultured skin 1. [Diagram 2] A device 40 for producing artificial cultured skin 1 is shown, in which FIG. 2A is a schematic cross-sectional view and FIG. 2B is a plan view. [Diagram 3]FIG. 1 is a schematic cross-sectional view showing the production of an artificial cultured skin 1, in particular showing the formation of a base tissue layer 10. [Figure 4] 1 is a schematic cross-sectional view showing the production of artificial cultured skin 1, in particular the formation of a dermal tissue layer 20. FIG. [Diagram 5] FIG. 1 is a schematic cross-sectional view showing the production of an artificial cultured skin 1, in particular the culture of a laminate comprising a base tissue layer 10 and a dermal tissue layer 20. [Figure 6] FIG. 2 is a schematic cross-sectional view showing the production of artificial cultured skin 1, in particular showing the state of a laminate including a base tissue layer 10 and a dermal tissue layer 20 after culture. [Figure 7] FIG. 1 is a schematic cross-sectional view showing the production of an artificial cultured skin 1, in particular the preparation for the formation of an epidermal layer 30. [Figure 8] FIG. 1 is a schematic cross-sectional view showing the manufacture of an artificial cultured skin 1, in particular the preparation of the laminate for culture. [Figure 9] FIG. 2 is a plan view of a base used in manufacturing artificial cultured skin 1. [Figure 10] FIG. 1 is a schematic cross-sectional view showing the production of an artificial cultured skin 1, in particular showing the culture of a laminate. [Figure 11] FIG. 2 is a schematic cross-sectional view showing the production of artificial cultured skin 1, in particular showing the air-liquid interface culture of the laminate. [Figure 12] FIG. 2 is a schematic cross-sectional view showing artificial cultured skin 1A of a comparative example. [Figure 13] 1 shows images of skin sections of artificial cultured skin obtained in Example 1 and Comparative Example 1. [Figure 14] 1 shows images of skin sections of artificial cultured skin obtained in Example 1 and Comparative Example 1. [Figure 15] 1 is an image of a skin section of artificial cultured skin obtained in Example 2. [Figure 16] 1 shows images of skin sections of artificial cultured skin obtained in Example 2 and Comparative Examples 2 and 3. [Figure 17] 1 shows images of skin sections of artificial cultured skin obtained in Example 2 and Comparative Examples 2 and 3. [Figure 18]These are images showing the results of immunostaining (Collagen 4, Collagen 17). [Figure 19] These are images showing the results of immunostaining (Collagen7, Occludin). [Figure 20] 13 shows images showing the results of immunostaining (CK15, Loricrin). [Figure 21] 1 shows images showing the results of immunostaining (FLG, HAS1). [Figure 22] 1 is a graph showing the results of PCR measurement (COL4A1). [Figure 23] 1 is a graph showing the results of PCR measurement (COL7A1). [Figure 24] 1 is a graph showing the results of PCR measurement (COL17A1). [Diagram 25] 1 is a graph showing the results of PCR measurement (CK15). [Figure 26] 1 is a graph showing the results of PCR measurement (OCLN). [Figure 27] 1 is a graph showing the results of PCR measurement (HAS1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, embodiments of the artificial cultured skin and the method for producing the same of the present invention will be described with reference to Figs. 1 to 11. The following embodiment shows one aspect of the present invention, does not limit the present invention, and can be modified as desired within the scope of the technical idea of ​​the present invention. In addition, in the following drawings, the scale and number of each structure are different from the actual structure in order to make each configuration easier to understand.

[0010] artificial cultured skin First, the artificial cultured skin according to the present invention (hereinafter also referred to as "artificial cultured skin of the present invention") will be described with reference to Fig. 1. Fig. 1 is a perspective cross-sectional view that shows a schematic diagram of artificial cultured skin 1, which is one embodiment of the artificial cultured skin.

[0011] The artificial cultured skin 1 includes a base tissue layer 10, a dermal tissue layer 20, and an epidermal layer 30. The base tissue layer 10, the dermal tissue layer 20, and the epidermal layer 30 are laminated in this order. The dermal tissue layer 20 is disposed in contact with the base tissue layer 10, and the epidermal layer 30 is disposed in contact with the dermal tissue layer 20. The artificial cultured skin 1 is formed by extending three layers, the base tissue layer 10, the dermal tissue layer 20, and the epidermal layer 30, in a predetermined direction along a plane perpendicular to the direction in which they are laminated (the vertical direction in FIG. 1, hereinafter referred to as the lamination direction). Here, for convenience, with regard to the predetermined direction along the plane, the left-right direction in FIG. 1 is hereinafter referred to as the first direction, and the direction perpendicular to both the first direction and the vertical direction (the diagonal direction in FIG. 1) is hereinafter referred to as the second direction.

[0012] The base tissue layer 10 includes adipose-derived stem cells 11 (also referred to as first cells) and a gel 12 (also referred to as first gel). The base tissue layer 10 is formed by culturing the adipose-derived stem cells 11 in the gel 12. The base tissue layer 10 is disposed at the bottom of the artificial cultured skin 1. The gel 12 functions as a tissue surrounding the adipose-derived stem cells. The gel 12 is not particularly limited, but examples thereof include collagen (type I, type II, type III, type V, type XI, etc.), a basement membrane component (product name: Matrigel) reconstituted from mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, etc. From the viewpoint of obtaining good skin tissue (particularly the epidermis layer and dermis tissue layer), the gel of the base tissue layer is preferably a collagen gel or a fibrin gel, and more preferably a fibrin gel.

[0013] In the artificial cultured skin of the present invention, a layer containing adipose-derived stem cells (ADSC: Adipose-Derived Stem Cells) is placed under a layer containing fibroblasts. Adipose-derived stem cells are mesenchymal stem cells that exist in subcutaneous adipose tissue and can differentiate into other things besides fat. When differentiating into fat, they differentiate from mesenchymal stem cells (adipose-derived stem cells) to preadipocytes and then to mature adipocytes. They have the advantage of being multipotent and being able to collect a large amount of cells relatively easily. Development is also being carried out in the field of regenerative medicine. In the present invention, it was found that the condition of the skin tissue was improved by providing a layer of adipose-derived stem cells. Specifically, as shown in the examples below, it was found that the condition of the epidermis layer was significantly improved (e.g., good formation of the stratum corneum, gene expression, etc.) by providing a layer of adipose-derived stem cells. In addition, as shown in the examples below, it was found that the condition of the dermis tissue layer and the entire skin tissue including it was improved (gene expression, etc.) by providing a layer of adipose-derived stem cells. Without being bound by theory, it is speculated that the improvement in the condition of the epidermal and dermal tissue layers is related to the stabilization of the upper epidermal and dermal tissue layers by the basal tissue layer due to the influence of substances secreted from adipose-derived stem cells (extracellular vesicles such as exosomes, various cytokines, etc.).

[0014] Examples of the adipose-derived stem cells include cells derived from mammals such as humans, mice, and rats, and human adipose-derived stem cells are preferred. Examples of the human adipose-derived stem cells are human subcutaneous adipose-derived stem cells. The adipose-derived stem cells are preferably those that have not been passaged many times (e.g., passaged 3 or less, 2 or less, or 1 or less) and have high stemness without differentiation induction.

[0015] The base tissue layer can mimic subcutaneous tissue. That is, in the actual subcutaneous tissue of animals such as humans, fat cells, loose connective tissue, Pacinian corpuscles, etc. are present around the adipose-derived stem cells. The subcutaneous tissue supports the epidermis and dermis. The base tissue layer supports the epidermis layer and dermis tissue layer, just like the subcutaneous tissue. In the artificial cultured skin of the present invention, the base tissue layer functions like subcutaneous tissue, making the artificial cultured skin closer to actual skin.

[0016] Here, the base tissue layer 10 is formed by culturing adipose-derived stem cells 11 in the gel 12, and the cells contained in the base tissue layer 10 may be cells differentiated from the adipose-derived stem cells 11. This is because the artificial cultured skin 1 is obtained through a culture process, and the adipose-derived stem cells 11 may have differentiation ability as described above. Therefore, the base tissue layer 10 may be a layer containing only the adipose-derived stem cells 11, or may be a layer containing the adipose-derived stem cells 11 and cells differentiated from the adipose-derived stem cells 11, or may be a layer containing only cells differentiated from the adipose-derived stem cells 11.

[0017] The dermal tissue layer 20 includes fibroblasts 21 (also referred to as second cells) and a gel 22 (also referred to as a second gel). The dermal tissue layer 20 is provided on the base tissue layer 10. The dermal tissue layer 20 is formed by culturing the fibroblasts 21 in the gel 22. The dermal tissue layer 20 is disposed in the middle of the artificial cultured skin 1 (between the base tissue layer 10 and the epidermis layer 30). The gel 22 functions as an extracellular matrix. The gel 22 is not particularly limited, but examples thereof include collagen (type I, type II, type III, type V, type XI, etc.), a basement membrane component (product name: Matrigel) reconstituted from mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.), gelatin, agar, agarose, fibrin, glycosaminoglycan, hyaluronic acid, proteoglycan, and the like. From the viewpoint of obtaining good skin tissue (particularly the epidermis layer and dermis tissue layer), the gel for the dermis tissue layer is preferably a collagen gel or a fibrin gel, and more preferably a fibrin gel.

[0018] Examples of fibroblasts include cells derived from mammals such as humans, mice, and rats, and human-derived fibroblasts are preferred. Examples of human-derived fibroblasts include normal human dermal fibroblasts (NHDF), human pulmonary fibroblasts (HPF), human cardiac fibroblasts (HCF), human aortic adventitial fibroblasts (HAoAF), human uterine fibroblasts (HUF), and human villous mesenchymal fibroblasts (HVMF), and normal human dermal fibroblasts (NHDF) are preferred.

[0019] The dermal tissue layer can mimic the actual dermis. That is, in the actual dermis of animals such as humans, cells such as fibroblasts are present, and extracellular matrices such as collagen are present around the cells. The dermal tissue layer contains cells and extracellular matrices, similar to the actual dermis. In the artificial cultured skin of the present invention, the dermal tissue layer performs a dermal function, making the artificial cultured skin closer to the actual skin.

[0020] Here, with regard to the gels of each layer, it is preferable that the gel 12 (first gel) of the base tissue layer 10 is a collagen gel or a fibrin gel, and the gel 22 (second gel) of the dermal tissue layer 20 is a collagen gel or a fibrin gel. It is also preferable that the first gel and the second gel are the same gel. For example, the first gel and the second gel may both be collagen gels, or the first gel and the second gel may both be fibrin gels. It is more preferable that the first gel and the second gel are both fibrin gels.

[0021] The gel of each layer can be obtained by solidifying the liquid gel component. A collagen gel can be obtained, for example, by heating (incubating) a collagen solution to 37°C under neutral conditions. A fibrin gel can be obtained, for example, by adding thrombin, which functions as a coagulant, to fibrinogen. A gel containing cells can be formed for each layer (base tissue layer 10 and dermal tissue layer 20) by mixing a cell suspension with a liquid gel component and solidifying the mixture.

[0022] The epidermal layer 30 includes epidermal cells 31 (also referred to as third cells). The epidermal layer 30 is provided on the dermal tissue layer 20. The epidermal layer 30 is formed by seeding and culturing the epidermal cells 31 on the dermal tissue layer 20. As the epidermal cells 31, for example, epidermal keratinocytes can be used.

[0023] Examples of epidermal cells include cells derived from mammals such as humans, mice, and rats, and human-derived epidermal cells are preferred. Examples of human-derived epidermal cells include epidermal keratinocytes and epidermal melanocytes, and epidermal keratinocytes are preferred. Examples of epidermal keratinocytes include normal human epidermal keratinocytes (NHEK). Examples of epidermal melanocytes include normal human epidermal melanocytes (NHEM). From the viewpoint of obtaining a suitable human skin model, it is preferred that the epidermal cells are normal human epidermal keratinocytes (NHEK). In the artificial cultured skin of the present invention, it is preferred that the adipose-derived stem cells are human adipose-derived stem cells, the fibroblasts are normal human skin fibroblasts, and the epidermal cells are normal human epidermal keratinocytes.

[0024] Here, the epidermal cells 31 can be differentiated by culture, and the epidermal layer 30 of the artificial cultured skin 1 can be composed of the entire epidermal cells 31 including the differentiated epidermal cells 31. The epidermal cells 31 can differentiate to form each layer of the epidermis including the basal layer, the spinous layer, the granular layer, and the stratum corneum. In this way, the epidermal layer of the artificial cultured skin can mimic the actual epidermis. That is, in the actual epidermis of animals such as humans, epidermal cells exist, and the epidermal cells can differentiate to form each layer of the epidermis. In the artificial cultured skin of the present invention, the epidermal layer performs the same function as the actual epidermis, making the artificial cultured skin closer to the actual skin. And, the artificial cultured skin of the present invention has a basal tissue layer corresponding to the subcutaneous tissue, a dermal tissue layer corresponding to the dermis, and an epidermal layer corresponding to the epidermis, so that an artificial cultured skin close to the actual skin (especially human skin) can be obtained.

[0025] As described below, the epidermal layer 30 of the artificial cultured skin 1 can have a stratum corneum formed by differentiation of epidermal cells by air-liquid interface culture. Therefore, the artificial cultured skin 1 can provide a skin model close to the condition of actual skin.

[0026] In addition, the epidermis layer 30 may be an epidermis layer in which the expression of one or more genes selected from basement membrane-related factors, basal cell-related factors, barrier function-related factors, and moisturizing-related factors is activated, as shown in the examples below. Therefore, the artificial cultured skin 1 can provide a skin model having a good epidermis layer. In addition, the artificial cultured skin 1 may be an artificial cultured skin in which the expression of one or more genes selected from COL17A1, KRT15, ​​COL4A1, COL7A1, OCLN, HAS1, COL1A1, FGF10, and VEGF is activated, or the expression of the MMP-1 gene is suppressed. Therefore, the artificial cultured skin 1 can provide a skin model having a good epidermis layer and dermis tissue layer.

[0027] Here, COL17A1 is a gene related to Collagen 17. KRT15 is a gene related to Keratin 15. COL4A1 is a gene related to Collagen 4. COL7A1 is a gene related to Collagen 7. OCLN is a gene related to Occludin. HAS1 is a gene related to Hyaluronan Synthase 1. COL1A1 is a gene related to Collagen 1. FGF10 is a gene related to Fibroblast Growth Factor 10. VEGF is a gene related to Vascular Endothelial Growth Factor. MMP-1 is a gene related to Matrix Metalloproteinase-1. These are known genes and are known to be genes related to the skin. For information regarding gene expression in the skin, publicly known literature can be referred to as appropriate, for example, literature such as Journal of Histochemistry & Cytochemistry 2015, Vol. 63(2) 129-141 can be referred to, and information such as the human protein expression information database The Human Protein Atlas (https: / / www.proteinatlas.org / ) can be referred to.

[0028] Method for producing artificial cultured skin As an example of a method for producing artificial cultured skin, a method for producing artificial cultured skin 1 will be described below with reference to Figures 2 to 11. Note that the following description also describes a preferred embodiment of the above-mentioned artificial cultured skin.

[0029] The method for producing artificial cultured skin according to the present invention comprises the steps of: (a) providing a mixture comprising adipose-derived stem cells and a gel to form a base tissue layer; (b) providing a mixture containing fibroblasts and a gel on the base tissue layer to form a dermis tissue layer; (c) providing epidermal cells onto the dermal tissue layer to form an epidermal layer; and (d) culturing a laminate comprising a basal tissue layer, a dermal tissue layer and an epidermal layer; Includes.

[0030] In the method for producing artificial cultured skin, preferably, after step (b), a step of culturing the laminate including the base tissue layer and the dermal tissue layer may be further included. Also, preferably, step (d) may include a step of immersing and culturing the laminate in a culture medium for undifferentiation, a step of immersing and culturing the laminate in a culture medium for differentiation, and a step of culturing the laminate at an air-liquid interface in the culture medium for differentiation.

[0031] In producing the artificial cultured skin 1, a device for producing artificial cultured skin is used. FIG. 2 shows a schematic diagram of a device 40 for producing artificial cultured skin, FIG. 2A is a cross-sectional view, and FIG. 2B is a plan view. The device 40 is placed on a culture dish 50 for use. A culture tank is formed by the device 40 and the culture dish 50. The device 40 is made of, for example, plastic, and can be produced by, for example, molding with a 3D printer using a resin. The device 40 can preferably be subjected to a coating treatment (e.g., parylene coating) to enhance biocompatibility. A commercially available cell culture dish 50 can be used. The culture tank can be formed by adhering the device 40 to the culture dish 50 with an adhesive (e.g., PDMS; polydimethylsiloxane). Note that in FIG. 2A, only the bottom of the culture dish 50 is shown, and the side wall is not shown.

[0032] The device 40 is formed in a cylindrical shape having a culture space 43 that is surrounded by side walls 41 and penetrates in the vertical direction. The shape of the culture space 43 may be a rectangular cylinder, or a polygonal cylinder such as a circular cylinder, an elliptical cylinder, or a hexagonal cylinder. In this embodiment, the four side walls 41 are arranged in a rectangular shape, and the culture space 43 is formed in a rectangular cylinder. The device 40 may have a structure in which cylindrical structures are connected. One cylindrical structure can be one section for cell culture in the device 40. One section can have a size of, for example, 0.1 to 5 cm x 0.1 to 5 cm.

[0033] The device 40 is a cylindrical device having protrusions on the inner wall. Specifically, the opposing side walls 41 and 41 of the device 40 are each provided with a protrusion 42. A plurality of protrusions 42 are provided on each side wall 41. The protrusions 42 protrude toward the inside of the device 40. The protrusions 42 are composed of a protrusion base 42a and a protrusion tip 42b formed with a cross-sectional diameter larger than that of the protrusion base 42a. As described later, when the gel contracts during culture, the gel is caught by the protrusion tip 42b, so that the gel contraction is suppressed. The protrusions 42 are provided on each of the four side walls 41. Note that, as the hook (anchor) structure, the protrusion 42 with a larger tip is shown, but the hook (anchor) structure is not limited to this structure.

[0034] Formation of base tissue layer 10 and dermis tissue layer 20 The above-mentioned device 40 is used to form the base tissue layer 10 and the dermal tissue layer 20. The base tissue layer 10 and the dermal tissue layer 20 can be formed by step (a) of supplying a mixture containing adipose-derived stem cells 11 and a gel 12 to form the base tissue layer 10, and step (b) of supplying a mixture containing fibroblasts 21 and a gel 22 to form the dermal tissue layer 20.

[0035] The base tissue layer 10 can be formed by pouring a mixture of adipose-derived stem cells and a first gel (gel material before solidification) into the culture space 43 of the culture tank, as shown in FIG. 3. Collagen or fibrinogen is preferably used as the component of the first gel. Here, it is preferable to shift the position of the upper end of the poured gel from the position of the protrusion 42. Specifically, as shown in FIG. 3, the position of this upper end is made higher or lower than the protrusion 42. This allows the protrusion 42 to be positioned offset from the boundary position of the base tissue layer 10 and the dermal tissue layer 20, which are the joining portion of the two layers. Therefore, as described later, the protrusion 42 can be firmly hooked onto the gel, and gel contraction can be reliably suppressed.

[0036] After pouring the mixture containing the adipose-derived stem cells and the first gel, the gel is solidified. In the case of collagen, the gel can be solidified by, for example, incubating at 37°C for about 5 to 10 minutes under neutral conditions. In the case of fibrinogen, a mixture containing fibrinogen and thrombin can be prepared under cooling, and this mixture can be poured into the device 40 at room temperature to immediately solidify. The solidification of the gel forms the base tissue layer 10. After the formation of the base tissue layer 10, the dermis tissue layer 20 is formed.

[0037] The dermal tissue layer 20 can be formed by pouring a mixture of fibroblasts and the second gel into the culture space 43 (see FIG. 3) of the culture vessel, as shown in FIG. 4. Collagen or fibrinogen is preferably used as a component of the second gel.

[0038] After pouring the mixture containing fibroblasts and the second gel, the gel is solidified. In the case of collagen, the gel can be solidified by, for example, incubating at 37°C for about 5 to 10 minutes under neutral conditions. In the case of fibrinogen, a mixture containing fibrinogen and thrombin can be prepared under cooling, and the mixture can be poured into the device 40 at room temperature to immediately solidify it. The dermis tissue layer 20 is formed by the solidification of the gel.

[0039] Next, the laminate of the base tissue layer 10 and the dermal tissue layer 20 is cultured. The culture can be performed by immersing the laminate in a culture medium. For example, as shown in FIG. 5, the culture can be performed by placing a culture medium 60 in a culture dish 50 so that the laminate is completely immersed in the culture medium. As the culture medium, a mixture of a medium for adipose-derived stem cells and a medium for fibroblasts (for example, a mixture ratio of 1:2 to 2:1, specifically 1:1) can be used. The culture conditions are, for example, at a temperature of 37°C for 1 to 14 days, preferably 3 to 10 days, and more preferably 6 to 8 days.

[0040] The gels of the base tissue layer 10 and the dermal tissue layer 20 contract due to the culture. Here, since the gel is caught and engaged with the protrusions 42, the contraction in the stacking direction is not restricted, but the contraction in the horizontal directions (first and second directions) perpendicular to the stacking direction is restricted. Furthermore, the gel is pressed against the outer periphery of the protrusions 42 due to the contraction in the stacking direction. Therefore, the gel is caught more strongly with the protrusions 42, and the resistance to the contraction in the first and second directions is increased. In this way, the protrusions 42 can be engaged with the base tissue layer 10 or the dermal tissue layer 20 to perform the culture. This suppresses the contraction of the gel, and an artificial cultured skin 1 with a good shape can be obtained.

[0041] Upon completion of the culture of the laminate including the base tissue layer 10 and the dermal tissue layer 20, the laminate assumes a gel-shrunk shape as shown in Fig. 6. As described above, the shrinkage of the laminate in the first and second directions is suppressed more than the shrinkage in the lamination direction.

[0042] Formation of epidermal layer 30 The epidermal layer 30 can be formed by a step (c) of supplying epidermal cells to form the epidermal layer. Specifically, the epidermal layer 30 can be formed by removing the culture medium, exposing the laminate of the base tissue layer 10 and the dermal tissue layer 20, and seeding the epidermal cells 31 on the laminate (i.e., on the dermal tissue layer 20), as shown in FIG. 7. At this time, a cylindrical body 44 (e.g., a plastic ring) or the like can be placed to prevent the epidermal cells 31 from flowing or falling, and a cell suspension containing the epidermal cells 31 can be poured into the cylindrical body 44. After the seeding of the epidermal cells 31, if it is left for several hours (e.g., 1 to 24 hours), the epidermal cells 31 will adhere to the dermal tissue layer 20. After the adhesion, the cylindrical body 44 is removed. As a result, a laminate including the base tissue layer 10, the dermal tissue layer 20, and the epidermal layer 30 is formed. Note that when the cylindrical body 44 is removed, the epidermal layer 30 may slightly expand.

[0043] 8, in preparation for culturing (including air-liquid interface culturing) the laminate (epidermal layer 30 / dermal tissue layer 20 / base tissue layer 10), a pedestal 51 is inserted between the device 40 and a culture dish 50. Specifically, the device 40 including the laminate is temporarily removed from the culture dish 50, a coarse mesh pedestal 51 is placed on the culture dish 50, and the device 40 including the laminate is placed on the pedestal 51. Then, a culture medium is poured into the culture dish 50 so that the laminate is immersed.

[0044] FIG. 9 is a plan view (viewed in the vertical direction) of the base 51. The base 51 is made of, for example, plastic, like the device 40, and can be manufactured by, for example, molding with a 3D printer using resin. The base 51 is in a coarse mesh shape and has a plurality of holes 52 penetrating vertically. The holes 52 are smaller than the culture tank (cylindrical structure) of the device 40. The culture medium 60 can pass under the base 51. Therefore, the culture medium can pass under the base 51 and contact the lower surface of the laminate (i.e., the bottom of the base tissue layer 10). In this way, it is preferable to use the base 51 having a plurality of holes 52 penetrating vertically, and place the device 40 on the base 51 to perform culture. As a result, the culture medium can pass through the holes 52, so that the culture medium can contact the bottom surface of the culture medium base tissue layer 10, and it becomes easy to supply the culture medium components to the adipose-derived stem cells. Furthermore, by providing the pedestal 51, the position of the upper surface of the dermal tissue layer 20 is raised, so that the air-liquid interface culture can be performed more stably.

[0045] Next, as shown in FIG. 10, the laminate (epidermal layer 30 / dermal tissue layer 20 / base tissue layer 10) is cultured. The culture is preferably performed by immersing the laminate in a culture medium for undifferentiation, then immersing the laminate in a culture medium for differentiation, and then performing air-liquid interface culture of the laminate in the culture medium for differentiation. As the culture medium, a mixture of a culture medium for adipose-derived stem cells, a culture medium for fibroblasts, and a culture medium for epidermal cells (for example, a mixing ratio of 1-2:1-2:1-2, specifically a mixing ratio of 1:1:1) can be used. As the culture medium, for example, a culture medium not containing calcium chloride (CaCl2) can be used as the culture medium for undifferentiation, and a culture medium for differentiation can be used in which calcium chloride (CaCl2) is added to this culture medium for undifferentiation. Calcium chloride is a component that can promote differentiation of cells, particularly epidermal cells. In the culture, first, the cells are cultured in a culture medium for undifferentiation (a culture medium not containing a component that promotes differentiation). This allows each cell to be stabilized. Then, the medium is replaced and the cells are cultured in a differentiation medium (a medium containing components that promote differentiation). This allows differentiation of cells with differentiation potential (particularly epidermal cells) to begin. Culture in the undifferentiated medium can be performed for, for example, 1 to 14 days (7 days as a specific example), and culture in the differentiation medium can be performed for, for example, 1 to 7 days (3 days as a specific example).

[0046] Then, as shown in FIG. 11, the laminate is cultured at the gas-liquid interface. In the gas-liquid interface culture, the culture medium is first removed, and then the culture medium is placed in the culture dish 50 again. At that time, the position of the upper end of the culture medium is set to be the same as or slightly lower than the upper surface of the dermal tissue layer 20. In FIG. 11, the culture medium 60 is placed slightly lower than the upper surface of the dermal tissue layer 20. This allows the epidermal layer 30 to be exposed to gas, and the base tissue layer 10 and the dermal tissue layer 20 to be cultured (cultured at the interface between gas and liquid) while being immersed in liquid. The gas-liquid interface culture can be performed for, for example, 3 days to 4 weeks. The gas-liquid interface culture can promote the differentiation of epidermal cells and can also advance the keratinization of epidermal cells. Thus, in this example, the step (d) includes a step of immersing the laminate in a culture medium for undifferentiation and culturing it, a step of immersing the laminate in a culture medium for differentiation and culturing it, and a step of gas-liquid interface culturing the laminate in a culture medium for differentiation.

[0047] The air-liquid interface culture induces differentiation of the epidermal cells 31. This allows the epidermal cells 31 to form the epidermal layer 30 containing keratin. The actual human epidermis has, from the bottom up, a basal layer, a spinous layer, a granular layer, and a stratum corneum, and a layer structure resembling this can be formed.

[0048] By the above operations, it is possible to produce artificial cultured skin 1 as shown in Fig. 1. As shown in the examples described later, the artificial cultured skin 1 produced as described above has a good skin condition, particularly the condition of the epidermis layer (e.g., good formation of the stratum corneum, gene expression, etc.), and also has a good overall skin tissue condition (e.g., gene expression, etc.), which is closer to real skin.

[0049] How to evaluate drugs As a further aspect, the present invention relates to a method for evaluating a drug using the above artificial cultured skin. The method for evaluating a drug includes contacting the above artificial cultured skin with a drug, and analyzing the artificial cultured skin after contact with the drug to examine at least one selected from a change in the state of the artificial cultured skin and the state of the drug in the artificial cultured skin. This method makes it possible to evaluate the effectiveness, toxicity, or irritation of a drug on skin. Here, the drug means a substance to be evaluated. Drugs include drugs such as medicines, cosmetics, and quasi-drugs. The drug may be a single component, or a composition or formulation containing two or more components. The components of the drug may include organic compounds, inorganic compounds, and the like. Changes in the state of the artificial cultured skin include changes in the appearance of skin tissue (stratum corneum, epidermis layer, dermis tissue layer, etc.), changes in gene expression, and responses to stimuli caused by contact with a drug. The state of the drug in the artificial cultured skin includes the permeability and retention of the drug into the skin tissue.

[0050] According to the above evaluation method, for example, a drug can be evaluated in a skin model that is closer to actual skin than conventional methods. Moreover, the above evaluation method is extremely useful, for example, in the kinetic evaluation of drugs with various molecular weights in the creation (screening) of new drugs, and in the evaluation in the development of cosmetics, quasi-drugs, etc. EXAMPLES

[0051] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0052] 1. Preparation of Devices and Culture Chambers A device (6 compartments per dish, plastic, 6 mm high) as described in Figure 2 was printed using a 3D printer (Keyence, Agilista-3100). The printed object was then coated with parylene using a parylene coater (Japan Parylene, PDS2010), placed in a culture dish (diameter 6 cm), and fixed with PDMS polymer (Dow-Toray, SILPOT 184 Silicone Elastmer). A few drops of 100% ethanol solution containing 0.5% Lipidure (NOF, Lipidure-CM5206) were placed on the bottom of the culture dish and coated by heat treatment.

[0053] ADSC (adipose derived stem cells) were obtained from PromoCell. NHDF (normal human dermal fibroblasts) were obtained from CellResearchCorp. NHEK (normal human epidermal keratinocytes) were obtained from PromoCell.

[0054] Example 1. Production of artificial cultured skin using collagen gel ADSC cell suspension MSCGM2 (C-28009, PromoCell) supplemented with penicillin and streptomycin was used as the culture medium for ADSC culture (hereinafter referred to as "ADSC culture medium"). ADSCs (adipose-derived stem cells) were suspended in the ADSC culture medium, and the cell suspension of ADSCs (0.7 to 2 × 10 5 A concentration of 100 mM NaCl (10 mM / mL) was prepared.

[0055] NHDF cell suspension As the medium for NHDF culture (hereafter referred to as "NHDF medium"), DMEM (08459-64, Nacalai Tesque) containing 10% FBS and supplemented with ascorbic acid, penicillin, and streptomycin was used. NHDF (normal human dermal fibroblasts) were suspended in the NHDF medium, and the NHDF cell suspension (0.7 to 2 × 10 5 A concentration of 100 mM NaCl (10 mM / mL) was prepared.

[0056] NHEK cell suspension KGM2 (C-20111, PromoCell) was used as the medium for culturing NHEK (hereinafter referred to as "NHEK medium"). NHEK (normal human epidermal keratinocytes) were suspended in the NHEK medium, and the NHEK cell suspension (0.7 to 4 × 10 5 A concentration of 100 mM NaCl (10 mM / mL) was prepared.

[0057] Collagen Gel Material A collagen acidic solution (IAC-50, Atelocollagen, Koken) was used as the collagen gel material.

[0058] ADSC / collagen mixture The collagen acidic solution, 10x PBS (D1408, Sigma) solution, and ADSC cell suspension were mixed in a ratio of 9:1:5 on ice to prepare an ADSC / collagen mixture.

[0059] NHDF / collagen mixture An acidic collagen solution, a 10x PBS (D1408, Sigma) solution, and a cell suspension of NHDF were mixed in a ratio of 9:1:5 on ice to prepare an NHDF / collagen mixture.

[0060] Cell seeding and incubation The ADSC / collagen mixture was added to the device, which was then left to stand in an incubator at 37°C for 5-10 minutes to solidify the collagen, yielding an ADSC gel layer (base tissue layer) (see Figure 3). The NHDF / collagen mixture was added on top of the ADSC gel layer, which was then left to stand in an incubator at 37°C for 5-10 minutes to solidify the collagen, yielding an NHDF gel layer (dermal tissue layer) (see Figure 4). A medium made by mixing ADSC medium and NHDF medium in a 1:1 ratio was added so that the laminate of the ADSC gel layer and the NHDF gel layer was immersed (see Figure 5). The device containing the obtained cell-containing gel was incubated for 5 days. The gel contracted during incubation, but the protrusions on the device suppressed gel contraction compared to when there were no protrusions (see Figure 6). The medium outside the gel was removed until the surface of the NHDF gel layer was exposed. A ring was placed on the NHDF gel layer, and a cell suspension of NHEK was added inside the ring (see FIG. 7). After leaving it for 3 hours, the NHEK adhered to the NHDF gel layer. The ring was removed. The medium outside the gel was removed, and a medium made by mixing ADSC medium, NHDF medium, and NHEK medium in a 1:1:1 ratio was added, and the obtained cell-containing gel laminate was immersed in the medium and incubated for 1 day. The device containing the laminate was removed from the culture dish, a coarse mesh base was placed on the culture dish, and the device containing the laminate was placed on the base (see Figure 8). A medium made by mixing ADSC medium, NHDF medium, and NHEK medium in a ratio of 1:1:1 was added, and the laminate was immersed in the medium and incubated for 7 days (see Figure 10). Calcium chloride (CaCl2) was added to a 1:1:1 mixture of ADSC medium, NHDF medium, and NHEK medium to prepare a medium containing 1.8 mM CaCl2 (cell differentiation medium). Note that the medium before calcium chloride was added was a medium for maintaining cells in an undifferentiated state (cell undifferentiation medium), and the medium with calcium chloride added was a cell differentiation medium. The medium was replaced with a cell differentiation medium, and the laminate was immersed in the medium and incubated for 3 days. Next, the medium was removed, and cell differentiation medium was added so that the surface of the medium was positioned slightly below the upper surface of the NHDF gel layer. This caused the collagen gel portion to be immersed in the medium, and the NHEKs were positioned outside the medium, making it possible to culture them at the air-liquid interface (see Figure 11). The laminate was incubated for 1 to 3 weeks. Through incubation, the NHEKs differentiated, and a structure corresponding to the epidermal layer was formed. This resulted in artificial cultured skin 1 being obtained. The obtained artificial cultured skin 1 was removed from the device and subjected to the tests described below.

[0061] Comparative Example 1. Production of artificial cultured skin (not containing ADSCs) using collagen gel As an artificial cultured skin of a comparative example, an artificial cultured skin without an ADSC gel layer was produced. Fig. 12 shows a schematic cross-sectional view of the artificial cultured skin 1A produced in the comparative example. In Fig. 12, the artificial cultured skin 1A is composed of a dermis tissue layer 20 (the thickness corresponds to the combined thickness of the base tissue layer 10 and the dermis tissue layer 20 in Example 1) and an epidermis layer 30.

[0062] Cell suspension and collagen material The cell suspension and collagen material used were the same as those used in Example 1.

[0063] Cell seeding and incubation The NHDF / collagen mixture was added to the device, which was then placed in an incubator at 37°C for 5-10 minutes to allow the collagen to solidify. NHDF medium was added so that the NHDF gel layer was submerged. The resulting device containing the cell-containing gel was incubated for 5 days. The medium outside the gel was removed until the surface of the NHDF gel layer was exposed. A ring was placed on top of the NHDF gel layer, and a cell suspension of NHEK was added inside the ring. After leaving it for 3 hours, the NHEK adhered to the NHDF gel layer. The ring was removed. The medium outside the gel was removed, and a medium made by mixing NHDF medium and NHEK medium in a 1:1 ratio was added. The resulting laminate was immersed in the medium and incubated for 1 day. The device containing the laminate was removed from the culture dish, a coarse mesh base was placed on the culture dish, and the device containing the laminate was placed on the base. A medium made by mixing NHDF medium and NHEK medium in a 1:1 ratio was added, and the laminate was immersed in the medium and incubated for 7 days. Calcium chloride was added to a 1:1 mixture of NHDF medium and NHEK medium to prepare a medium containing 1.8 mM CaCl2 (cell differentiation medium). The medium was replaced with a cell differentiation medium, and the laminate was immersed in the medium and incubated for 3 days. Next, the medium was removed, and the cell differentiation medium was added so that the surface of the medium was positioned slightly below the upper surface of the NHDF gel layer. This caused the collagen gel portion to be immersed in the medium, and the NHEKs were positioned outside the medium, enabling air-liquid interface culture. The laminate was incubated for 1 to 3 weeks. Through incubation, the NHEKs differentiated and a structure corresponding to the epidermal layer was formed. This resulted in the artificial cultured skin 1A (see FIG. 12).

[0064] Example 2. Preparation of artificial cultured skin using fibrin gel 6-Aminocaproic acid in PBS A PBS solution of 6-aminocaproic acid (A2504, Merck) was prepared at a concentration of 150 mg / mL.

[0065] ADSC cell suspension (for fibrin gel) The medium for culturing ADSCs (hereinafter referred to as "ADSC medium") was prepared by adding the above-mentioned 6-aminocaproic acid PBS solution to MSCGM2 (C-28009, PromoCell) (100-fold dilution of the 6-aminocaproic acid PBS solution). ADSCs (adipose-derived stem cells) were suspended in the ADSC medium, and the cell suspension of ADSCs (0.7 to 2 × 10 5 A concentration of 100 mM NaCl (10 mM / mL) was prepared.

[0066] NHDF cell suspension (for fibrin gel) The medium for culturing NHDF (hereafter referred to as "NHDF medium") was prepared by adding the above-mentioned 6-aminocaproic acid PBS solution to DMEM containing 10% FBS and supplemented with ascorbic acid, penicillin, and streptomycin (100-fold dilution of 6-aminocaproic acid PBS solution). NHDF (normal human dermal fibroblasts) were suspended in the NHDF medium, and the NHDF cell suspension (0.7-2 × 10 5 A concentration of 100 mM NaCl (10 mM / mL) was prepared.

[0067] NHEK cell suspension (for fibrin gel) The medium for culturing NHEK (hereafter referred to as "NHEK medium") was prepared by adding the above 6-aminocaproic acid PBS solution to KGM2 (C-20111, Takara Bio) (100-fold dilution of the 6-aminocaproic acid PBS solution). NHEK (normal human epidermal keratinocytes) were suspended in the NHEK medium, and the NHEK cell suspension (0.7-2 × 10 5 A concentration of 100 mM NaCl (10 mM / mL) was prepared.

[0068] Fibrin gel material Fibrinogen (F8630-1G, Merck, powder) was used as a material for the fibrin gel, and was dissolved in PBS to a concentration of 7.5 to 20 mg / mL (fibrinogen solution).

[0069] ADSC / fibrinogen mixture On ice, fibrinogen solution, a solution of thrombin (T4648-10KU, Merck) diluted with PBS to 50-100 UN, and the ADSC cell suspension were mixed in a ratio of 10:1:14 to prepare an ADSC / fibrinogen mixture.

[0070] NHDF / fibrinogen mixture A fibrinogen solution, a solution of thrombin (T4648-10KU, Merck) diluted with PBS to 50-100 UN, and a NHDF cell suspension were mixed in a ratio of 10:1:14 on ice to prepare an NHDF / fibrinogen mixture.

[0071] Cell seeding and incubation The ADSC / fibrinogen mixture was added to the device (see Figure 3). Immediately after addition, the fibrinogen solidified and became a fibrin gel. This resulted in an ADSC gel layer (base tissue layer). The NHDF / fibrinogen mixture was added on top of the ADSC gel layer (see Figure 4). Immediately after addition, the fibrinogen solidified and became a fibrin gel. This resulted in an NHDF gel layer (dermal tissue layer). A medium made by mixing ADSC medium and NHDF medium in a 1:1 ratio was added so that the laminate of the ADSC gel layer and the NHDF gel layer was immersed (see Figure 5). The device containing the obtained cell-containing gel was incubated for 7 days. The gel contracted during incubation, but the protrusions on the device suppressed gel contraction compared to when there were no protrusions (see Figure 6). The medium outside the gel was removed until the surface of the NHDF gel layer was exposed. A ring was placed on the NHDF gel layer, and a cell suspension of NHEK was added inside the ring (see Figure 7). After leaving it for 3 hours, the NHEK adhered to the NHDF gel layer. The ring was removed. A medium made by mixing ADSC medium, NHDF medium, and NHEK medium in a 1:1:1 ratio was added, and the resulting laminate was immersed in the medium and incubated for 1 day. The device containing the laminate was removed from the culture dish, a coarse mesh base was placed on the culture dish, and the device containing the laminate was placed on the base (see Figure 8). A medium (medium for undifferentiated cells) made by mixing ADSC medium, NHDF medium, and NHEK medium in a ratio of 1:1:1 was added, and the laminate was immersed in the medium and incubated for 7 days (see Figure 10). Calcium chloride (CaCl2) was added to a medium obtained by mixing ADSC medium, NHDF medium, and NHEK medium in a 1:1:1 ratio to prepare a medium containing 1.8 mM CaCl2 (cell differentiation medium). The medium was replaced with a cell differentiation medium, and the laminate was immersed in the medium and incubated for 3 days. Next, the medium was removed, and cell differentiation medium was added so that the surface of the medium was positioned slightly below the upper surface of the NHDF gel layer. This caused the fibrin gel portion to be immersed in the medium, and the NHEKs were positioned outside the medium, making it possible to culture them at the air-liquid interface (see Figure 11). The laminate was incubated for 1 to 3 weeks. Through incubation, the NHEKs differentiated, and a structure corresponding to the epidermal layer was formed. This resulted in the production of an artificial cultured skin. The resulting artificial cultured skin was removed from the device and subjected to the tests described below.

[0072] Comparative Example 2. Production of artificial cultured skin (not containing ADSCs) using fibrin gel As a comparative example, artificial cultured skin without an ADSC gel layer was produced (see FIG. 12).

[0073] Cell suspension and fibrin material The cell suspension and fibrin material used were the same as those used in Example 2.

[0074] Cell seeding and incubation The NHDF / fibrinogen mixture was added to the device. The fibrinogen quickly solidified to form a fibrin gel. NHDF medium was added so that the NHDF gel layer was submerged. The resulting device containing the cell-laden gel was incubated for 7 days. The medium outside the gel was removed until the surface of the NHDF gel layer was exposed. A ring was placed on top of the NHDF gel layer, and a cell suspension of NHEK was added inside the ring. After leaving it for 3 hours, the NHEK adhered to the NHDF gel layer. The ring was removed. The medium outside the gel was removed, and a medium made by mixing NHDF medium and NHEK medium in a 1:1 ratio was added. The resulting laminate was immersed in the medium and incubated for 1 day. The device containing the laminate was removed from the culture dish, a coarse mesh base was placed on the culture dish, and the device containing the laminate was placed on the base. A medium made by mixing NHDF medium and NHEK medium in a 1:1 ratio was added, and the laminate was immersed in the medium and incubated for 7 days. Calcium chloride was added to a 1:1 mixture of NHDF medium and NHEK medium to prepare a medium containing 1.8 mM CaCl2 (cell differentiation medium). The medium was replaced with a cell differentiation medium, and the laminate was immersed in the medium and incubated for 3 days. Next, the medium was removed, and the cell differentiation medium was added so that the surface of the medium was positioned slightly below the upper surface of the NHDF gel layer. This caused the fibrin gel portion to be immersed in the medium, and the NHEKs were positioned outside the medium, enabling air-liquid interface culture. The laminate was incubated for 1 to 3 weeks. Through incubation, the NHEKs differentiated and a structure corresponding to the epidermal layer was formed. This resulted in an artificial cultured skin (see Figure 12).

[0075] Comparative Example 3. Production of artificial cultured skin using fibrin gel (ADSC culture medium supernatant added) In Comparative Example 2, a medium in which the supernatant of culture fluid obtained by separately culturing ADSCs was added to the above-mentioned NHDF medium (the ratio of NHDF medium:ADSC culture supernatant was 2:1 by volume) was used as the NHDF medium, and a medium in which the supernatant of culture fluid obtained by separately culturing ADSCs was added to the above-mentioned NHEK medium (the ratio of NHEK medium:ADSC culture supernatant was 2:1 by volume) was used as the NHEK medium. A laminate in which an NHDF gel layer and an NHEK layer were layered was formed, and the laminate was cultured (immersion culture and air-liquid interface culture) in the same manner as in Comparative Example 2.

[0076] Test Example 1. Evaluation of skin sections by HE staining Preparation of skin sections The artificial cultured skin produced in the examples and comparative examples was taken out, immersed in 4% paraformaldehyde, and fixed at 4°C for 3 hours. The liquid was replaced with PBS, and the tissue was left at 4°C for 30 minutes. The tissue was immersed in a 30% sucrose solution in PBS, and kept at 4°C for 24 hours. For example 1 and comparative example 1 (in the case of the test in Figure 13 and Figure 14), and example 2 (in the case of the test in Figure 15), the tissue was embedded in OCT Compound (Tissue-Tek) and frozen, and stored in a freezer at -80°C. For example 2 and comparative examples 2 and 3 (in the case of the test in Figure 16 and Figure 17), OCT Compound (Tissue-Tek) was added, and the tissue was left at 4°C for 24 hours, and then the tissue was embedded in new OCT Compound and frozen, and stored in a freezer at -80°C.

[0077] HE staining The skin sections prepared as described above were stained with hematoxylin and eosin (HE) in the usual manner and observed under a microscope.

[0078] FIG. 13 shows the cell suspension concentration (NHEK: 2×10 5 Pieces / model, NHDF and ADSC: 2×10 5 13A shows an image of a skin slice obtained from an artificial cultured skin produced under the conditions of a concentration of 1000 cells / mL and one week of air-liquid interface culture. FIG. 13A shows a skin slice obtained in Comparative Example 1, and FIG. 13B shows a skin slice obtained in Example 1. For ease of understanding, the approximate ranges of the dermis layer, epidermis layer (excluding the stratum corneum), and stratum corneum are shown on the right side of FIG. 13B. The dermis layer (dermal tissue layer) is formed from the NHDF gel layer, and the epidermis layer (including the stratum corneum) is formed from the NHEK layer (by cell differentiation). Note that "cells / model" refers to the number of cells in one skin model (one section).

[0079] FIG. 14 shows the cell suspension concentration (NHEK: 0.7×10 5 Pieces / model, NHDF and ADSC: 2×10 514A shows an image of a skin section obtained from an artificial cultured skin produced under the conditions of a concentration of 100 μg / mL of 100 μg / mL and one week of air-liquid interface culture. Fig. 14A shows a skin section obtained in Comparative Example 1, and Fig. 14B shows a skin section obtained in Example 1.

[0080] FIG. 15 shows the cell suspension concentration (NHEK: 2×10 5 Pieces / model, NHDF and ADSC: 2×10 5 The figures show images of skin sections obtained from artificial cultured skin produced under the conditions of (1) 0.01% cell / mL (cells / mL) and two weeks of air-liquid interface culture.

[0081] FIG. 16 shows the cell suspension concentration (NHEK: 0.7×10 5 Pieces / model, NHDF and ADSC: 2×10 5 16A shows an image of a skin section obtained from an artificial cultured skin produced under the conditions of a concentration of 100 μg / mL of 100 μg / mL and one week of air-liquid interface culture. Fig. 16A shows a skin section obtained in Comparative Example 2, Fig. 16B shows a skin section obtained in Example 2, and Fig. 16C shows a skin section obtained in Comparative Example 3.

[0082] FIG. 17 shows the cell suspension concentration (NHEK: 0.7×10 5 Pieces / model, NHDF and ADSC: 2×10 5 17A shows images of skin sections obtained from artificial cultured skin produced under the conditions of a concentration of 100 μg / mL of 100 μg / mL and cultured at an air-liquid interface for 3 weeks. Fig. 17A shows a skin section obtained in Comparative Example 2, Fig. 17B shows a skin section obtained in Example 2, and Fig. 17C shows a skin section obtained in Comparative Example 3.

[0083] evaluation In artificial cultured skin, the state of epidermal differentiation is important, and in particular, the quality of the formation of the basal layer that serves as the base for cell differentiation is key. As shown in Figs. 13-14, in the artificial cultured skin obtained in Example 1, the basal layer was formed neatly compared to Comparative Example 1, and specifically, it was confirmed that the basal cells were crushed and formed neatly in layers. Also, as shown in Figs. 15-17, in the artificial cultured skin obtained in Example 2, the basal layer was formed neatly compared to Comparative Examples 2 and 3, and specifically, it was confirmed that the basal cells were crushed and formed neatly in layers. From Figs. 16-17, it was confirmed that the state of the basal layer was improved by using the culture supernatant of ADSC (Comparative Example 3) compared to the case where no ADSC was used at all (Comparative Example 2), but the state of the basal layer was even better in the artificial cultured skin using ADSC cells (Example 2).

[0084] Test Example 2. Evaluation of skin sections by immunostaining Skin slices were prepared from artificially cultured skin that had been cultured at the air-liquid interface for one week in the same manner as in Test Example 1. The skin slices were immunostained using a commercially available immunostaining kit to examine gene expression. The genes examined were Collagen4, Collagen17, Collagen7, Occludin, CK15 (also called KRT15), Loricrin, FLG (also called Filaggrin), and HAS1. Each of these genes encodes a corresponding protein.

[0085] 18 to 21 show images obtained by immunostaining of skin sections according to the examples and comparative examples. FIG. 18 shows Collagen 4 and Collagen 17, and their merge, FIG. 19 shows Collagen 7 and Occludin, and their merge, FIG. 20 shows CK15 and Loricrin, and their merge, and FIG. 21 shows FLG and HAS1, and their merge. For FLG and HAS1, Example 1 (collagen gel K+F+A) and Comparative Example 1 (collagen gel K+F) were not performed (FIG. 21). The merge was performed to confirm the state of gene expression, and was performed on skin with slightly different expression sites. Note that "K+F+A" means a (three-layer) skin model containing NHEK, NHDF, and ADSC, and "K+F" means a (two-layer) skin model containing NHEK and NHDF.

[0086] From each of the immunostaining images, a comparison between Example 1 and Comparative Example 1, and a comparison between Example 2 and Comparative Example 2, it can be said that the case with a base tissue layer "K+F+A" (Examples 1 and 2) has better gene expression than the case without a base tissue layer "K+F" (Comparative Examples 1 and 2). Furthermore, a comparison between Example 2 and Comparative Example 3 shows that the case with a base tissue layer "K+F+A" (Example 2) has better gene expression than the case with the addition of ADSC culture supernatant "K+F(+A supernatant)" (Comparative Example 3).

[0087] The results of the comprehensive evaluation of gene expression by immunostaining are summarized in Table 1 below. [Table 1]

[0088] The above results suggest that the presence of ADSCs may activate the expression of factors involved in the epidermal basement membrane, basal cells, barrier function, and moisturizing function, and may homogenize their localization, thereby stabilizing the epidermal structure.

[0089] Test Example 3. Analysis of epidermal gene expression by PCR The artificial cultured skin of the examples and comparative examples obtained by the same method as above was homogenized, and PCR was performed according to a known PCR method to examine gene expression. The genes examined were COL17A1, KRT15, ​​COL4A1, COL7A1, OCLN, HAS1, COL1A1, FGF10, VEGF, and MMP-1. These genes each encode the corresponding protein. The measurement results were normalized by GAPDH. GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase.

[0090] Figures 22 to 27 are graphs showing the results of PCR measurements (representative results). Figure 22 shows COL4A1, Figure 23 shows COL7A1, Figure 24 shows COL17A1, Figure 25 shows CK15 (another name for KRT15), Figure 26 shows OCLN, and Figure 27 shows HAS1.

[0091] The results of gene expression levels (%) by PCR (relative levels with "K+F" being 100%) are summarized in Table 2 below. Table 2 shows the average values ​​of two tests. [Table 2]

[0092] 22 to 27 and Table 2 show that the expression of many genes related to skin conditions is well controlled (activated or inhibited). From the results of gene expression by PCR, a comparison between Example 1 and Comparative Example 1, and a comparison between Example 2 and Comparative Example 2, it can be said that the gene expression is better in the case of "K+F+A" with a basal tissue layer (Examples 1 and 2) than in the case of "K+F" without a basal tissue layer (Comparative Examples 1 and 2).

[0093] Among the above genes, COL17A1, KRT15, ​​COL4A1, COL7A1, OCLN, and HAS1 are genes that can particularly affect the epidermis, and activation of these gene expressions (increased expression) is thought to be able to improve the epidermal layer. Also, COL1A1, FGF10, VEGF, and MMP-1 are genes that can particularly affect the dermis. And activation of COL1A1, FGF10, and VEGF (increased expression) and inhibition of MMP-1 (collagen decomposition enzyme) (decreased expression) are thought to be able to improve the dermal tissue.

[0094] The above results suggest that the presence of ADSCs may stabilize the epidermal structure by activating the expression of factors related to the basement membrane, basal cells, barrier function, and moisturizing function of the epidermis and by homogenizing their localization. It is also suggested that the presence of ADSCs leads to favorable formation of the epidermal layer as well as the dermal tissue layer and the entire skin tissue including it. [Industrial Applicability]

[0095] According to the present invention, it is possible to provide artificial cultured skin that can be used for drug evaluation, etc. [Explanation of symbols]

[0096] 1 Artificial cultured skin 10 Base tissue layer 11 Adipose-derived stem cells 12 Gel (First Gel) 20 Dermal tissue layer 21 Fibroblasts 22 Gel (Second Gel) 30 Epidermal layer 31 Epidermal cells 40 Devices 41 Side wall 42 Protrusion 42a Protrusion base 42b Tip of protrusion 43 Culture space 44 Cylindrical body 50 culture dishes 51 Pedestal 52 holes 60 Culture medium

Claims

1. a base tissue layer comprising adipose-derived stem cells and a gel; a dermal tissue layer disposed on the base tissue layer and including fibroblasts and a gel; and an epidermal layer disposed above the dermal tissue layer and including epidermal cells; Artificial cultured skin, including

2. The artificial cultured skin of claim 1, wherein the gel of the base tissue layer is a collagen gel or a fibrin gel, and the gel of the dermal tissue layer is a collagen gel or a fibrin gel.

3. The artificial cultured skin of claim 1, wherein the adipose-derived stem cells are human adipose-derived stem cells, the fibroblasts are normal human skin fibroblasts, and the epidermal cells are normal human epidermal keratinocytes.

4. The artificial cultured skin of claim 1, wherein the epidermal layer has a stratum corneum formed by differentiation of epidermal cells through air-liquid interface culture.

5. The artificial cultured skin of claim 4, wherein the epidermal layer is an epidermal layer in which expression of one or more genes selected from basement membrane-related factors, basal cell-related factors, barrier function-related factors, and moisturizing-related factors is activated.

6. The artificial cultured skin according to claim 1, wherein expression of one or more genes selected from COL17A1, KRT15, ​​COL4A1, COL7A1, OCLN, HAS1, COL1A1, FGF10, and VEGF is activated, or expression of the MMP-1 gene is suppressed.

7. A method for producing artificial cultured skin according to any one of claims 1 to 6, (a) providing a mixture comprising adipose-derived stem cells and a gel to form a base tissue layer; (b) providing a mixture containing fibroblasts and a gel on the base tissue layer to form a dermal tissue layer; (c) supplying epidermal cells onto the dermal tissue layer to form an epidermal layer; and (d) culturing the laminate comprising the basal tissue layer, the dermal tissue layer and the epidermal layer; A method for producing artificial cultured skin, comprising:

8. The method of claim 7, further comprising the step of culturing the laminate comprising the basal tissue layer and the dermal tissue layer after step (b).

9. The method according to claim 7, wherein step (d) comprises a step of immersing the laminate in a culture medium for undifferentiation and culturing it, a step of immersing the laminate in a culture medium for differentiation and culturing it, and a step of culturing the laminate at an air-liquid interface in a culture medium for differentiation.

10. The method according to claim 7, wherein step (d) uses a cylindrical device having protrusions on its inner wall, and the protrusions are engaged with the base tissue layer or the dermal tissue layer to perform the culture.

11. The method according to claim 10, wherein step (d) comprises culturing the device by placing the device on a base having a plurality of holes penetrating vertically.

12. Contacting the artificial cultured skin according to any one of claims 1 to 6 with a drug; and Analyzing the artificial cultured skin after contact with the drug to examine at least one selected from a change in the state of the artificial cultured skin and the state of the drug in the artificial cultured skin; A method for evaluating a drug using artificial cultured skin, comprising:

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