Three-dimensional skin model

Abstract

A three-dimensional skin model according to one embodiment of the present invention has an epidermal layer, a melanocyte layer, and an intervening membrane. The intervening membrane is disposed between the epidermal layer and the melanocyte layer, and the epidermal layer has a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale in this order from the side furthest from the intervening membrane.

Description

3D skin model

[0001] The present invention relates to a three-dimensional skin model.

[0002] As animal welfare becomes more prevalent worldwide, the development of alternatives to animal testing is progressing. As an alternative to animal testing on skin, artificial skin models such as synthetic fiber skin and three-dimensional skin models are being developed to replace human skin, which is difficult to obtain.

[0003] For example, a full-thickness skin model has been developed that can confirm the responsiveness of the epidermis and dermis in functionality tests of products (such as cosmetic ingredients). This skin model uses a collagen hydrogel as the extracellular matrix, and epidermal keratinocytes are seeded on the dermis layer where dermal fibroblasts are dispersed within the collagen hydrogel, and the epidermis layer is reconstructed by gas-phase culture.

[0004] Additionally, skin models containing pigment cells (melanocytes) as constituent cells have been developed for use in skin whitening research. Melanin in the skin is produced in melanocytes in the basal layer, and the produced melanin is taken up by surrounding keratinocytes. The melanin migrates to the stratum corneum along with the keratinocyte metabolism (turnover) and is excreted from the body over approximately 40 days. Skin pigmentation, such as age spots and freckles, is thought to be caused by excessive melanin production in melanocytes due to UV exposure, local inflammation, etc., which results in melanin deposition within keratinocytes in the basal layer. One method for assessing melanin production involves using a skin model containing melanocytes localized in the basal layer to assess the amount of melanin produced by melanocytes upon UV exposure (see, for example, Patent Documents 1 and 2).

[0005] JP 2009-219491 A JP 2022-184025 A

[0006] In conventional 3D skin models, melanocytes can migrate from the basal layer to the upper layers as the epidermis differentiates during culture, resulting in variations in the location of melanocytes between production lots. This leads to variations in the amount of UV exposure to melanocytes, making it difficult to consistently evaluate melanin production. Furthermore, in skin models in which keratinocytes, melanocytes, and fibroblasts are co-cultured in a completely separate state, melanocytes do not migrate. This allows for uniform UV exposure and stabilizes melanin production, but the lack of 3D organization makes it difficult to evaluate these models as skin tissue substitutes. Therefore, an optimal 3D skin model that exhibits good responsiveness to UV exposure is needed.

[0007] The present invention has been made in view of the above points, and has an object to provide a three-dimensional skin model that has stable responsiveness to stimuli such as exposure to ultraviolet rays.

[0008] As a result of extensive research, the present inventors have found that the above-mentioned problems can be solved, and have completed the present invention. Specifically, the present invention is configured as follows [1] to [8].

[0009] [1] A three-dimensional skin model having an epidermal layer, a melanocyte layer, and an intervening membrane, the intervening membrane being interposed between the epidermal layer and the melanocyte layer, and the epidermal layer having, in this order from the side furthest from the intervening membrane, the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale.

[0010] [2] A three-dimensional skin model having an epidermal layer, a melanocyte layer, an intervening membrane, and a dermal layer, wherein the melanocyte layer is located between the epidermal layer and the intervening membrane, and the intervening membrane is interposed between the melanocyte layer and the dermal layer, and the epidermal layer has, in this order from the side furthest from the intervening membrane, the stratum corneum, the granular layer, the spinous layer, and the basal layer.

[0011] [3] A three-dimensional skin model having an epidermal layer, a melanocyte layer, an intervening membrane, and a dermal layer, wherein the intervening membrane is interposed between the epidermal layer and the melanocyte layer, the melanocyte layer is located between the intervening membrane and the dermal layer, and the epidermal layer has, in this order from the side furthest from the intervening membrane, the stratum corneum, the granular layer, the spinous layer, and the basal layer.

[0012] [4] A three-dimensional skin model described in [2] or [3], wherein the epidermal layer is co-cultured with at least one of the melanocyte layer and the dermal layer.

[0013] [5] The three-dimensional skin model according to any one of [1] to [3], wherein the intervening membrane is a porous membrane containing an extracellular matrix at least on its surface.

[0014] [6] A method for evaluating the responsiveness of a melanocyte layer, comprising the steps of culturing the three-dimensional skin model according to any one of [1] to [5] in a culture vessel, adding a melanin synthesis inhibitor to the cultured three-dimensional skin model, applying a physical stimulus and / or a melanin synthesis promoter to the three-dimensional skin model to which the melanin synthesis inhibitor has been added, and observing the production of melanin synthesized in the three-dimensional skin model or measuring the amount of melanin produced after applying the physical stimulus and / or the melanin synthesis promoter.

[0015] [7] The evaluation method described in [6], wherein the step of adding the melanin synthesis inhibitor is carried out at least once.

[0016] [8] The evaluation method according to [6] or [7], wherein the amount of the melanin synthesis inhibitor added is 1 μM to 100 mM.

[0017] According to the present invention, it is possible to provide a three-dimensional skin model that has stable responsiveness to stimuli such as exposure to ultraviolet rays.

[0018] FIG. 1 is a schematic diagram showing the configuration of a three-dimensional skin model according to a first embodiment of the present invention. FIG. 2 is a schematic diagram showing the configuration of a three-dimensional skin model according to a second embodiment of the present invention. FIG. 3 is a schematic diagram showing the configuration of a three-dimensional skin model according to a third embodiment of the present invention. FIG. 4(a) is a plan view of a culture vessel having a plurality of wells, and FIG. 4(b) is a cross-sectional view taken along line A-A in FIG. 5(a). FIG. 5(A) is an image of a skin tissue section of the three-dimensional skin model according to the first embodiment after hematoxylin-eosin staining. FIG. 5(B) is an image of a skin tissue section of the three-dimensional skin model according to the first embodiment after Fontana-Masson staining, and FIG. 5(C) is a partial enlarged image of FIG. 6(A) is an image of a skin tissue section of the three-dimensional skin model according to the second embodiment after hematoxylin-eosin staining. FIG. 6(B) is an image of a skin tissue section of the three-dimensional skin model according to the second embodiment after Fontana-Masson staining, and FIG. 5(C) is a partial enlarged image of FIG. 6(B). FIG. 7A is an image of a skin tissue section of a three-dimensional skin model according to the third embodiment after hematoxylin-eosin staining. FIG. 7B is an image of a skin tissue section of a three-dimensional skin model according to the third embodiment after Fontana-Masson staining. FIG. 8 is a graph showing the results of genetic analysis of a three-dimensional skin model. FIG. 9 is a graph showing the results of genetic analysis of a three-dimensional skin model. FIG. 10A is an image of a three-dimensional skin model, and (A)' is a partially enlarged image of (A). FIG. 10B is an image of a three-dimensional skin model after melanin production promotion, and (B)' is a partially enlarged image of (B). FIG. 11 is a graph showing the results of quantitative analysis of produced melanin. FIG. 12A is an image of a three-dimensional skin model to which only a melanin synthesis promoter has been added, and (B) and (C) are images of three-dimensional skin models to which a melanin synthesis inhibitor and a melanin synthesis promoter have been added. In Fig. 13, (A) is an image showing a three-dimensional skin model to which only a melanin synthesis promoter has been added, and (B) and (C) are images showing three-dimensional skin models to which a melanin synthesis inhibitor and a melanin synthesis promoter have been added. In Fig. 14, (A) is an image showing a three-dimensional skin model to which only a melanin synthesis promoter has been added, and (B) is an image showing a three-dimensional skin model to which a melanin synthesis inhibitor and a melanin synthesis promoter have been added.(A) of Figure 15 is an image showing a three-dimensional skin model to which only a melanin synthesis promoter has been added, and (B) is an image showing a three-dimensional skin model to which a melanin synthesis inhibitor and a melanin synthesis promoter have been added. (A) of Figure 16 is an image showing a three-dimensional skin model to which only a melanin synthesis promoter has been added, and (B) and (C) are images showing three-dimensional skin models to which a melanin synthesis inhibitor and a melanin synthesis promoter have been added. Figure 17 is a graph showing the degree of melanocyte blackening. Figure 18 is a graph showing the results of quantitative analysis of the melanin produced.

[0019] Specific embodiments of the present invention will be described in detail below. Note that the present invention is not limited to the following embodiments, and can be modified as appropriate within the scope of the present invention.

[0020] [Three-dimensional Skin Model] (First Embodiment) As shown in Figure 1, a three-dimensional skin model 100 according to a first embodiment of the present invention has an epidermal layer 10, a melanocyte layer 20, and an intervening membrane 30. The intervening membrane 30 is interposed between the basal layer 14 and the melanocyte layer 20. The epidermal layer 10 has, in this order from the side furthest from the intervening membrane 30, a stratum corneum 11, a stratum granulosum 12, a stratum spinosum 13, and a basal layer 14.

[0021] [Epidermal Layer] The epidermal layer contains keratinocytes or cells differentiated from keratinocytes. Cells differentiated from keratinocytes include keratinocytes, granular cells, spinous cells, and basal cells. Keratinocytes are cells of the epidermis that divide in the basal layer, the lowest layer of the epidermis, and migrate to the skin surface. As shown in Figure 1, the epidermal layer is composed of multiple layers, including the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale.

[0022] <Stratum corneum> The stratum corneum is located at the top of the epidermal layer that makes up the three-dimensional skin model and is composed of a layered structure made up of keratinocytes. Furthermore, keratinocytes have a flat shape and lose their nuclei during the differentiation process. Furthermore, keratinocytes contain aggregates of keratin fibers within their cytoplasm. They are acidophilic and stain light to dark red with hematoxylin-eosin (HE) staining.

[0023] <Granular layer> The granular layer is located directly below the stratum corneum and directly above the stratum spinosum, which will be described later, and is composed of a layered structure constructed by granular cells. Granular cells have a flat shape and contain granules made of basophilic components within the cells. The granules within the cells are stained bluish purple to pale blue with hematoxylin-eosin staining. The thickness of the granular layer of the three-dimensional skin model is preferably 3 μm or more on average, more preferably 6 μm or more on average, and particularly preferably 8 μm or more on average.

[0024] <Stratum spinosum> The stratum spinosum is located directly below the stratum granulosum and directly above the stratum basale (described later), and is composed of a layered structure constructed by spinous cells. Spinous cells have a flat shape and have a structure in which spines are arranged around the cells. The spines can be confirmed by hematoxylin-eosin staining. The thickness of the spinous layer of the three-dimensional skin model is preferably 18 μm or more on average, preferably 26 μm or more on average, and particularly preferably 39 μm or more on average.

[0025] <Basal Layer> The basal layer is located at the bottom of the epidermis and is composed of a layer structure constructed by basal cells. Unlike keratinocytes, granular cells, and spinous cells, basal cells have a cubic to cylindrical shape and an oval nucleus. Basal cells are basophilic and stain indigo to pale blue with hematoxylin-eosin staining. The thickness of the basal layer of the three-dimensional skin model is preferably 7 μm or more on average, more preferably 9 μm or more on average, and particularly preferably 14 μm or more on average.

[0026] The thickness of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale mentioned above can be measured, for example, using light microscope images of histochemical sections.

[0027] [Melanocyte Layer] The melanocyte layer is a layer composed of melanocytes (cultured normal human epidermal melanocytes). Melanocytes are pigment cells that produce melanin. Melanocytes are usually found in the basal layer and have a shape with multiple dendritic processes extended. When stained with hematoxylin and eosin, the cytoplasm of melanocytes appears to be pale. Melanin is produced in melanosomes (organelles containing melanin) within melanocytes, and the produced melanin is passed to nearby keratinocytes while still enclosed in the melanosomes. Melanocytes also produce melanin from tyrosine through the action of a series of enzymes, including tyrosinase.

[0028] [Intervening Membrane] The intervening membrane is a porous membrane that allows substances to pass through and contains an extracellular matrix at least on its surface. The inclusion of an extracellular matrix at least on the surface of the intervening membrane makes it easy to attach keratinocytes and fibroblasts without relying on a collagen hydrogel.

[0029] The intervening membrane may consist essentially of only an extracellular matrix layer, or may contain extracellular matrix on the surface of the substrate. The extracellular matrix on the surface is not particularly limited, but is preferably a coating membrane due to its high frequency of occurrence.

[0030] Examples of the substrate material include PET (polyethylene terephthalate) and PC (polycarbonate) membranes, etc. Examples of the extracellular matrix include type I collagen, type III collagen, type IV collagen, fibronectin, gelatin, etc.

[0031] The average pore size of the intervening membrane is preferably 0.4 μm or more. The average pore size of the intervening membrane is preferably 8.0 μm or less, more preferably 6.0 μm or less. When the average pore size of the intervening membrane is within the above range, the permeation of proteins involved in crosstalk (cytokines, chemokines, etc.) and lipid components (eicosanoids, etc.) can be promoted satisfactorily. The pore size of the intervening membrane can be determined, for example, by measuring the major axis using an electron microscope image.

[0032] The average thickness of the intervening membrane is preferably 20 μm or less, more preferably 15 μm or less, particularly preferably 10 μm or less, and even more preferably 5 μm or less, from the viewpoint of easily obtaining a skin model in which the structure of the epidermal layer is maintained over a long period of time. However, in cases where specific medium components (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) are used or additional layers are provided, the average thickness of the intervening membrane is not limited thereto, and may be, for example, 20 μm or more and 200 μm or less. When the thickness of the intervening membrane is within this range, the strength of the three-dimensional skin model is increased and crosstalk between the dermis and epidermis is enhanced, making it easier to maintain a state in which the epidermal layer is appropriately differentiated (the spinous layer and granular layer are thickened) and the basal layer is highly dense. The thickness of the intervening membrane can be measured, for example, using an optical microscope image of a histochemical section.

[0033] In one embodiment of the present invention, the intervening membrane can be a membrane from a commercially available cell culture insert.

[0034] The three-dimensional skin model according to the first embodiment is partitioned by inserting an intervening membrane between the basal layer and the melanocyte layer, thereby preventing the melanocyte layer from migrating to the epidermis. This reduces the variation in the location of melanocytes, allowing melanin production to occur at the desired location. Furthermore, even with partitioning, intercellular interactions between the epidermis and melanocyte layers can be maintained, allowing melanin produced by exposure to ultraviolet light, etc., to migrate from the melanocyte layer to the epidermis. This allows the three-dimensional skin model according to the first embodiment to be used in research on skin whitening agents.

[0035] 2, a three-dimensional skin model 200 according to a second embodiment of the present invention has an epidermal layer 10, a melanocyte layer 20, an intervening membrane 30, and a dermal layer 40. The melanocyte layer 20 is located between the epidermal layer 10 and the intervening membrane 30. The intervening membrane 30 is interposed between the melanocyte layer 20 and the dermal layer 40. The epidermal layer 10 has, in this order from the side furthest from the intervening membrane 30, a stratum corneum 11, a stratum granulosum 12, a stratum spinosum 13, and a stratum basale 14.

[0036] [Dermis Layer] The dermis layer contains slender, spindle-shaped dermal fibroblasts (fibroblasts) that produce collagen fibers, elastic fibers, mucopolysaccharides, etc. The dermis layer also has a multi-layered structure. Specifically, the dermis layer has a tissue structure composed of fibroblasts and collagen fibers (e.g., collagen), elastic fibers (e.g., fibrillin, elastin), and other extracellular matrix components (e.g., hyaluronic acid) synthesized and secreted by the fibroblasts.

[0037] "Multilayer structure" refers to a structure in which fibroblasts form two or more layers in the vertical direction (toward the stratum corneum). Here, the orientation of fibroblasts is not uniform. Therefore, the image of a vertically sliced ​​skin tissue section may appear spindle-shaped due to horizontal orientation of fibroblasts, or circular due to vertical orientation of fibroblasts.

[0038] The cell density of the dermal layer (dermal fibroblasts) is 2000 cells / cm ２ or more, 5000 cells / cm ２ More than 6000 cells / cm is preferred. ２ More preferably, the cell density of the dermis layer is 2000 cells / cm. ２ More than 10000cells / cm ２ Preferably, 5000 cells / cm or less ２ More than 8000cells / cm ２ More preferably, 6000 cells / cm or less ２ More than 7700cells / cm ２ The following is particularly preferred: When the cell density of the dermis layer is within this range, the layer structure of the three-dimensional skin model can be maintained for a long period of time.

[0039] Furthermore, the proliferation ability of dermal fibroblasts is less likely to be inhibited by inhibiting cell activity, and the space in which proliferated cells can exist is vast, so the proliferation of dermal fibroblasts is less likely to be inhibited. Therefore, the cell density of the dermal layer is likely to be maintained at a high level over a long period of time, making it easy to evaluate the effect of a sample on the dermal layer with high sensitivity. Furthermore, proteins involved in crosstalk (cytokines, chemokines, etc.) and lipid components (eicosanoids, etc.) are produced and transmitted to the epidermal layer, making it easy to maintain a high level of differentiation structure of the epidermal layer that constitutes the three-dimensional skin model during culture over a long period of time.

[0040] The sample is not particularly limited, and may be one or more isolated substances, or a mixture of various components (e.g., cosmetics, pharmaceuticals). The form of the sample is not particularly limited, and may be a liquid, gel, cream, powder, etc. The method of administering the sample is also not particularly limited, and may be applied to the surface of the epidermis, injected into the epidermis, injected into the dermis, or added to a culture solution.

[0041] The three-dimensional skin model according to the second embodiment has a melanocyte layer on one side of the intervening membrane and a dermis layer (dermal fibroblasts) on the other side, thereby preventing the melanocyte layer from migrating toward the dermis. This reduces variation in the location of melanocytes, allowing melanin production to occur at the desired location. Furthermore, because the basal layer and melanocyte layer are adjacent to each other, intercellular interactions can be maintained, allowing melanin produced by exposure to ultraviolet light, etc., to migrate from the melanocyte layer to the epidermis. This allows the three-dimensional skin model according to the second embodiment to be used in research on skin whitening agents.

[0042] 3, a three-dimensional skin model 300 according to a third embodiment of the present invention has an epidermal layer 10, a melanocyte layer 20, an intervening membrane 30, and a dermal layer 40. The intervening membrane 30 is interposed between the epidermal layer 10 and the melanocyte layer 20. The melanocyte layer 20 is located between the intervening membrane 30 and the dermal layer 40. The epidermal layer 10 has, in this order from the side furthest from the intervening membrane 30, a stratum corneum 11, a stratum granulosum 12, a stratum spinosum 13, and a stratum basale 14.

[0043] The three-dimensional skin model according to the third embodiment is able to inhibit the migration of the melanocyte layer to the epidermis layer by separating the basal layer and melanocyte layer through the intervening membrane. This reduces the variation in the location of melanocytes, allowing melanin production to occur at the desired location. Furthermore, the basal layer and melanocyte layer are adjacent to each other, allowing intercellular interactions to be maintained. Furthermore, interactions between the epidermis layer and the melanocyte layer, and between the epidermis layer and the dermis layer, can be maintained. This allows melanin produced by exposure to ultraviolet light, etc., to migrate from the melanocyte layer to the epidermis layer. This allows the three-dimensional skin model according to the third embodiment to be used in research on skin whitening agents.

[0044] Furthermore, the three-dimensional skin models of the first to third embodiments can be used for gene expression and histochemical analysis in addition to research on skin whitening agents.

[0045] [Culture vessel] As shown in Figures 4(a) and (b) , the culture vessel 50 used in the first to third embodiments has a plurality of wells 51 that contain a culture solution, a support portion 53 that supports the three-dimensional skin model in the wells 51, and an intervening membrane 30.

[0046] The culture vessel 50 may have, but is not limited to, 12, 24, 48, or 96 wells, which allows for simultaneous evaluation of a large number of samples and application conditions.

[0047] [Method for Evaluating Responsiveness of a Three-Dimensional Skin Model] A method for evaluating the responsiveness of a three-dimensional skin model according to one embodiment of the present invention is carried out using a culture vessel. Specifically, the method for evaluating responsiveness includes the steps of culturing a three-dimensional skin model (culture step), adding a melanin synthesis inhibitor to the cultured three-dimensional skin model (synthesis inhibitor addition step), applying a physical stimulus and / or a melanin synthesis promoter to the three-dimensional skin model to which the melanin synthesis inhibitor has been added (synthesis promoter addition step), and observing the production of melanin synthesized in the three-dimensional skin model or measuring the amount of melanin produced after the application of the physical stimulus and / or the application of the melanin synthesis promoter (observation or measurement step).

[0048] (Culturing Step) The culturing step is a step of culturing each layer (cell) that constitutes the three-dimensional skin model. Specifically, the culturing step includes a step of culturing normal human epidermal melanocytes on one side of an intervening membrane in a well of a culture vessel and a step of culturing normal human epidermal keratinocytes or normal human dermal fibroblasts on the other side, or a step of culturing normal human epidermal keratinocytes on one side and normal human epidermal melanocytes and normal human dermal fibroblasts on the other side. The culturing period for normal human epidermal melanocytes is preferably 1 to 3 days, the culturing period for normal human epidermal keratinocytes is preferably 1 to 3 days, and the culturing period for normal human dermal fibroblasts is preferably 1 to 3 days.

[0049] (Synthesis inhibitor addition step) The synthesis inhibitor addition step is a step of adding a melanin synthesis inhibitor to the three-dimensional skin model cultured in the above-mentioned culture step by a predetermined method. This step may be performed multiple times at predetermined intervals.

[0050] In one embodiment of the present invention, the melanin synthesis inhibitor refers to a tyrosinase inhibitor. Examples of tyrosinase inhibitors include kojic acid, rucinol, arbutin, magnesium L-ascorbyl phosphate, sodium L-ascorbyl phosphate, L-ascorbic acid, 2-glucoside, ellagic acid, potassium 4-methoxysalicylate (4MSK), 3-O-ethyl ascorbic acid, ascorbyl tetra-2-hexyldecanoate EX, Linole S, 5,5'-dipropyl-biphenyl-2,2'-diol, and the like. The amount of the melanin synthesis inhibitor added is preferably an amount that results in a concentration of 1 μM to 100 mM in the medium used to culture the three-dimensional skin model. When the melanin synthesis inhibitor is kojic acid, the amount added is preferably an amount that results in a concentration of 1 μM to 100 mM in the medium, more preferably an amount that results in a concentration of 10 to 50 μM. When the melanin synthesis inhibitor is rucinol, the amount added is preferably an amount that gives a concentration in the medium of 1 to 20 μM, more preferably an amount that gives a concentration of 1 to 10 μM.

[0051] In the synthesis inhibitor addition step described above, a tyrosinase inhibitor is used as a melanin synthesis inhibitor, but this is not limited thereto. For example, in the synthesis inhibitor addition step, a melanin transfer inhibitor that inhibits the transfer of melanocytes or melanosomes from melanocytes to epidermal cells can be used. Examples of melanin transfer inhibitors include adenosine monophosphate disodium, nicotinamide, dexpanthenol W, placenta extract, etc.

[0052] (Synthesis Accelerator Application Step) The synthesis accelerator application step is a step of applying a physical stimulus (e.g., ultraviolet irradiation) and / or a melanin synthesis accelerator to the three-dimensional skin model to which the synthesis inhibitor has been added, by a predetermined method. Examples of melanin synthesis accelerators include L-dopa. The amount of melanin synthesis accelerator added is preferably an amount that results in a concentration of 1 to 5 mM, and more preferably an amount that results in a concentration of 3 to 4 mM in the culture medium used for the three-dimensional skin model.

[0053] (Observation or measurement step) In the observation step, the melanocyte layer of the three-dimensional skin model that has undergone the above-described synthesis accelerator addition step can be observed and photographed using, for example, a Greenough stereomicroscope "Stemi 508" (manufactured by Carl Zeiss K.K.).

[0054] The measurement step is a step of measuring the degree of melanocyte blackening using image analysis software, for example, "ImageJ" (National Institutes of Health (NIH)), using tissue images of a portion of the skin tissue of the three-dimensional skin model that has undergone the above-mentioned synthesis accelerator addition step, taken with a stereomicroscope.

[0055] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0056] [Construction of a Three-Dimensional Skin Model] A three-dimensional skin model for use in histochemical analysis, genetic analysis, and functionality evaluation was constructed as follows.

[0057] <Construction of a three-dimensional skin model 1> A cell culture insert "ad-MED Vitrigel 2" (Kanto Chemical Co., Ltd., product number 08364-96) was prepared as an intervening membrane. 100,000 normal human epidermal melanocytes suspended in DermaLife (Lifeline Cell Technology) were seeded on one side of the cell culture insert and incubated at 37°C for 1 day under CO ２ Then, 500,000 normal human epidermal keratinocytes suspended in HuMedia-KG2 (manufactured by Kurabo Industries, Ltd.) were seeded on the other side of the cell culture insert, and the cells were cultured at 37°C for 1 day under CO ２ After culturing in an incubator, the medium was replaced with a medium for culturing a three-dimensional skin model, and culturing was continued for another day. After culturing, the medium for normal human epidermal keratinocytes was removed, and gas-phase culture was initiated. By carrying out gas-phase culture for 12 days, differentiation of the epidermal layer was induced, and a three-dimensional skin model 1 was constructed. "DermaLife" is a registered trademark of Lifeline Cell Technology, Inc., and "Humedia" is a registered trademark of Kurabo Industries, Ltd.

[0058] <Construction of a three-dimensional skin model 2> A cell culture insert "ad-MED Vitrigel 2" was prepared. 10,000 normal human dermal fibroblasts suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) were seeded on one side (also referred to as side A) of the cell culture insert, and the insert was incubated at 37°C for 1 day in a CO atmosphere. ２ Then, 100,000 normal human epidermal melanocytes suspended in DermaLife were seeded on the other side (also referred to as side B), and the plate was incubated at 37°C for 1 day under CO ２ Then, 500,000 normal human epidermal keratinocytes suspended in HuMedia-KG2 were seeded on the B side, and the plate was incubated at 37°C for 1 day under CO ２ After culturing in an incubator, the medium was replaced with a medium for culturing a three-dimensional skin model, and culturing was continued for another day. After culturing, the medium for normal human epidermal keratinocytes was removed, and gas-phase culture was initiated. By continuing the gas-phase culture for 12 days, differentiation of the epidermal layer was induced, and three-dimensional skin model 2 was constructed.

[0059] <Construction of a three-dimensional skin model 3> A cell culture insert "ad-MED Vitrigel 2" was prepared. 100,000 normal human epidermal melanocytes suspended in DermaLife were seeded on one side (also referred to as side A) of the cell culture insert, and the insert was incubated at 37°C for 1 day in CO ２ Then, 10,000 normal human dermal fibroblasts suspended in DMEM containing 10% FBS were seeded on the A-side of the plate, and the plate was incubated at 37°C for 1 day in a CO ２ Then, 500,000 normal human epidermal keratinocytes suspended in HuMedia-KG2 were seeded on the other side (also referred to as side B) of the cell culture insert, and the cells were cultured at 37°C for 1 day under CO ２ After culturing in an incubator, the medium was replaced with a medium for three-dimensional culture and continued for another day. After culturing, the medium for normal human epidermal keratinocytes was removed and gas-phase culture was initiated. After 12 days of gas-phase culture, differentiation of the epidermal layer was induced, and three-dimensional skin model 3 was constructed.

[0060] [Histochemical Analysis of Three-Dimensional Skin Model] Using the constructed three-dimensional skin models 1 to 3, histochemical analysis of the three-dimensional skin model was carried out.

[0061] (Analysis Method) The skin tissues of three-dimensional skin models 1 to 3, which had been cultured in the air phase for 12 days, were fixed with Superfix (KY-500), a rapid tissue culture fixative, and then embedded in Paraplast X-TRA (Leica Biosystems) to prepare paraffin-embedded sections (skin tissue sections). The localization of melanin in the prepared skin tissue sections was confirmed using a Fontana-Masson staining kit (ScyTek Laboratories).

[0062] (Results) As shown in Figures 5 to 7(B), melanin accumulation in the melanocyte layer was confirmed. Furthermore, as shown in Figures 5 and 6(C), melanin granules were confirmed in the epidermal layer, confirming the transfer of melanin to the epidermal layer.

[0063] [Genetic Analysis of Three-Dimensional Skin Model 1] Genetic analysis of the three-dimensional skin model was carried out using the constructed three-dimensional skin model 1.

[0064] (Analysis Method) Three-dimensional skin model 1 constructed by adding the melanocyte activating factors α-melanocyte-stimulating hormone (αMSH) (final concentration: 10 nM) and endothelin-1 (ET-1) (final concentration: 10 nM) to the medium, and two groups of culture medium (hereinafter also referred to as control) were subjected to vapor phase culture, and RNA was collected 12 and 19 days after the start of vapor phase culture. The expression levels of melanocyte-specific genes (tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), microphthalmia-associated transcription factor (MITF)) and melanin cascade-related genes (macrophage migration inhibitory factor (MIF), protease-activated receptor 2 (PAR2), and cytaxin (STX3)) from the collected RNA were analyzed by real-time PCR.

[0065] (Results) As shown in Figure 8, the expression levels of melanocyte-specific genes (TYR, TYRP1, MITF) were confirmed to be higher in the three-dimensional skin model with melanocyte activating factor added to the medium than in the control group on both days 12 and 19 after the start of gas-phase culture. This confirmed that the three-dimensional skin model responds to physiological stimuli. Furthermore, as shown in Figure 8, no significant difference was observed between the expression levels of melanin cascade-related genes (MIF, PAR2, STX3), but it was confirmed that a constant expression level was maintained.

[0066] [Genetic analysis of three-dimensional skin model 2] The addition of melanocyte activating factors α-melanocyte stimulating hormone (αMSH) and endothelin-1 (ET-1) to a final concentration of 10 nM was initiated to three-dimensional skin model 1, and UVB (302 nm) was irradiated onto the stratum corneum surface at 10, 25, or 50 mJ / cm every day. ２ The mice were irradiated with a dose of 1000mg / kg. Eight days after the start of UVB irradiation, RNA was collected from the skin tissue. The expression levels of melanocyte-specific genes (tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF)) were analyzed from the collected RNA using real-time PCR.

[0067] (Results) As shown in FIG. 9, it was confirmed that the expression of melanocyte-specific genes (TYR, MITF) was induced by irradiating the three-dimensional skin model 1 with UVB.

[0068] [Functionality Evaluation of Three-Dimensional Skin Model 1-1] Using the constructed three-dimensional skin model 1, the promotion of melanin production was evaluated.

[0069] When living skin is stimulated by exposure to ultraviolet rays or other factors, melanin-stimulating factors are produced, which stimulate melanocytes. Stimulated melanocytes then produce oxidative enzymes such as tyrosinase. Melanin is produced by a multi-step oxidation reaction of tyrosine by these oxidative enzymes, and the reaction rate of the first step, in which L-dopa (L-Dopa) is produced from tyrosine, is known to be slow. Therefore, in functional evaluation, we constructed an evaluation system that efficiently produces melanin by adding L-Dopa to a three-dimensional skin model, making it easy to evaluate the melanin synthesis inhibitory ability.

[0070] (Evaluation Method) The three-dimensional skin model 1 was cultured with L-Dopa, a melanin synthesis promoter, adjusted to a final concentration of 4 mM. ２ The cells were cultured in an incubator for 48 hours.

[0071] The skin tissue of the cultured three-dimensional skin model 1 was fixed with Superfix (KY-500), and then tissue images were taken using a stereomicroscope "Stemi508" (manufactured by Carl Zeiss K.K.). The tissue images are shown in Figure 10 (B) and (B)'. Note that Figure 10 (A) is a tissue image of the three-dimensional skin model to which L-Dopa was not added.

[0072] (Results) As shown in (A) and (A)' of Figure 10, the three-dimensional skin model to which L-Dopa had not been added showed very weak darkening (melanin production), whereas as shown in (B) and (B)' of Figure 10, the three-dimensional skin model to which L-Dopa had been added showed clearly visible darkening (melanin production). This confirmed that the three-dimensional skin model of the present invention can be used for functional evaluation of cosmetic ingredients and whitening cosmetics that have a whitening effect.

[0073] [Functionality Evaluation of Three-Dimensional Skin Model 1-2] Using the constructed three-dimensional skin model 1, the promotion of melanin production was evaluated.

[0074] Forskolin, which has adenyl cyclase activating properties, was added to the three-dimensional culture medium for three-dimensional skin model 1 at a final concentration of 1 μM or 10 μM. Culture was continued for 8 days to promote darkening, resulting in a three-dimensional melanocyte model. Next, melanocytes were detached from the three-dimensional melanocyte model using 0.025% trypsin-EDTA, and a melanocyte pellet was obtained by centrifugation. The supernatant was removed, and 1N NaOH was added to the pellet. The pellet was then treated at 95°C for 10 minutes to dissolve the melanin, and the absorbance was measured at a wavelength of 405 nm. A medium group without forskolin served as a control.

[0075] As shown in Figure 11, it was confirmed that the addition of forskolin to the three-dimensional culture medium increased the amount of melanin in a concentration-dependent manner. This confirmed that the three-dimensional skin model of the present invention can be used for functional evaluation of cosmetic ingredients and whitening cosmetics that have whitening effects.

[0076] [Functionality Evaluation of Three-Dimensional Skin Model 2-1] Using the constructed three-dimensional skin model 1, a study was carried out using cosmetic ingredients that have the effect of suppressing melanin production (tyrosinase inhibitory action).

[0077] (Evaluation 1 using cosmetic raw materials) A cosmetic raw material (10 μM kojic acid) was added to the medium of the three-dimensional skin model 1, and CO ２ The cells were cultured in an incubator for 24 hours. After the culture, the medium was replaced with a whitening cosmetic and L-Dopa adjusted to 4 mM. ２ The cells were cultured in an incubator for 48 hours.

[0078] (Evaluation 2 using cosmetic raw materials) A cosmetic raw material (10 μM kojic acid) was added to the medium of the three-dimensional skin model 1, and CO ２ The medium was changed once every 24 hours in the incubator, and the cells were cultured for 72 hours. After the culture, the medium was changed to one containing a whitening cosmetic and L-Dopa adjusted to 4 mM. ２ The cells were cultured in an incubator for 48 hours.

[0079] (Evaluation 3 Using Cosmetic Raw Material) Culture was carried out under the same conditions as in Evaluation 1, except that the cosmetic raw material (10 μM kojic acid) was changed to a cosmetic raw material (50 μM kojic acid).

[0080] (Evaluation 4 Using Cosmetic Raw Material) Cultivation was carried out under the same conditions as in Evaluation 2, except that the cosmetic raw material (10 μM kojic acid) was changed to a cosmetic raw material (50 μM kojic acid).

[0081] (Evaluation 5 Using Cosmetic Raw Material) Cultivation was carried out under the same conditions as in Evaluation 1, except that the cosmetic raw material (kojic acid) was changed to a cosmetic raw material (1 μM rucinol).

[0082] (Evaluation 6 Using Cosmetic Raw Material) Cultivation was carried out under the same conditions as in Evaluation 2, except that the cosmetic raw material (kojic acid) was changed to a cosmetic raw material (1 μM rucinol).

[0083] (Evaluation 7 Using Cosmetic Raw Material) Cultivation was carried out under the same conditions as in Evaluation 2, except that the cosmetic raw material (1 μM rucinol) was changed to a cosmetic raw material (10 μM rucinol).

[0084] The skin tissues of the three-dimensional skin models of evaluations 2, 4, 6, and 7 were fixed with Superfix, and then tissue images were taken using a stereomicroscope. The tissue images are shown in Figures 12(A) to 12(C) and 13(A) to 13(C). Note that Figures 12(A) and 13(A) are tissue images of three-dimensional skin models to which only L-Dopa was added.

[0085] (Results) As shown in Figure 12 (A), melanin production was confirmed in the three-dimensional skin model to which no cosmetic raw materials were added, and as shown in (B) and (C), melanin production was confirmed to be suppressed in the three-dimensional skin model to which cosmetic raw materials were added. Furthermore, it was found that the three-dimensional skin model to which a higher concentration of cosmetic raw materials was added (Figure 12 (C)) had a greater effect of suppressing melanin production than the three-dimensional skin model to which a lower concentration of cosmetic raw materials was added (Figure 12 (B)).

[0086] As shown in Figure 13 (A), melanin production was confirmed in the three-dimensional skin model to which no cosmetic raw materials were added, and as shown in (B) and (C), melanin production was confirmed to be suppressed in the three-dimensional skin model to which cosmetic raw materials were added. Furthermore, it was found that the three-dimensional skin model to which a higher concentration of cosmetic raw materials was added (Figure 13 (C)) had a greater effect of suppressing melanin production than the three-dimensional skin model to which a lower concentration of cosmetic raw materials was added (Figure 13 (B)).

[0087] [Functionality Evaluation of Three-Dimensional Skin Model 2-2] Using the constructed three-dimensional skin model 1, a study was carried out using whitening cosmetics that have the effect of suppressing melanin production (tyrosinase inhibitory action).

[0088] (Evaluation 1 using whitening cosmetics) A whitening cosmetic containing kojic acid was applied to the surface of the stratum corneum of the three-dimensional skin model 1, and CO ２ The cells were cultured in an incubator for 1 hour. After the culture, the applied cosmetics were removed and the cells were further incubated in a CO ２ The cells were cultured in an incubator for 23 hours. After the culture, L-Dopa was added to the culture medium to a final concentration of 4 mM. ２ The cells were cultured in an incubator for 48 hours.

[0089] (Evaluation 2 using whitening cosmetics) A whitening cosmetic containing kojic acid was applied to the surface of the stratum corneum of the three-dimensional skin model 1, and CO ２ The cells were cultured in an incubator for 1 hour. After the culture, the applied cosmetics were removed. The procedure of applying and removing the whitening cosmetics was repeated once every 24 hours, and the cells were cultured for 72 hours. After the culture, the medium was replaced with one containing L-Dopa adjusted to 4 mM, and the cells were incubated in CO ２ The cells were cultured in an incubator for 48 hours.

[0090] (Evaluation 3 Using Whitening Cosmetics) The same conditions as in Evaluation 2 were used, except that the whitening cosmetics containing kojic acid were replaced with whitening cosmetics containing rucinol.

[0091] The skin tissues of the three-dimensional skin models of evaluations 2 and 3 were fixed with Superfix, and then tissue images were taken using a stereomicroscope. The tissue images are shown in Figures 14(A) and 14(B) and Figures 15(A) and 15(B). Note that Figures 14(A) and 15(A) are tissue images of three-dimensional skin models to which only L-Dopa was added.

[0092] (Results) As shown in Figure 14 (A), melanin production was confirmed in the three-dimensional skin model to which no commercially available cosmetics containing whitening ingredients were applied, and as shown in Figure 14 (B), melanin production was confirmed to be suppressed in the three-dimensional skin model to which commercially available cosmetics containing whitening ingredients were added. Furthermore, as shown in Figure 15 (A), melanin production was confirmed in the three-dimensional skin model to which no commercially available cosmetics containing whitening ingredients were applied, and as shown in Figure 15 (B), melanin production was confirmed to be suppressed in the three-dimensional skin model to which commercially available cosmetics containing whitening ingredients were added.

[0093] [Functionality Evaluation of Three-Dimensional Skin Model 2-3] The culture conditions were changed, and a study was carried out using cosmetic ingredients that have the effect of suppressing melanin production (tyrosinase inhibitory action) and commercially available whitening cosmetics.

[0094] (Evaluation using cosmetic raw materials) A cosmetic raw material (10 μM rucinol) was added to the culture medium of the three-dimensional skin model 1, and CO ２ The cells were cultured in an incubator for 24 hours. After further culture, the medium was replaced with a whitening cosmetic and L-Dopa adjusted to 4 mM. ２ The cells were cultured in an incubator for 48 hours.

[0095] (Evaluation using commercially available whitening cosmetics) A whitening cosmetic containing rucinol was applied to the stratum corneum surface of the three-dimensional skin model 1, and CO ２ The cells were cultured in an incubator for 1 hour. After the culture, the applied cosmetics were removed and the cells were further incubated in a CO ２ The cells were cultured in an incubator for 23 hours. After the culture, the medium was replaced with one containing L-Dopa adjusted to a final concentration of 4 mM, and the cells were incubated in a CO ２ The cells were cultured in an incubator for 48 hours.

[0096] The skin tissue of each evaluation three-dimensional skin model was fixed with Superfix, and then tissue images were taken using a stereomicroscope. The tissue images are shown in Figure 14 (A) to (C). Note that Figure 16 (A) is a tissue image of a three-dimensional skin model to which only L-Dopa was added, Figure 16 (B) is a tissue image of a three-dimensional skin model to which a cosmetic ingredient was added, and Figure 16 (C) is a tissue image of a three-dimensional skin model to which a whitening cosmetic containing rucinol was applied.

[0097] (Results) As shown in Figure 16 (A), melanin production was confirmed in the three-dimensional skin model to which no cosmetic ingredients were added or commercially available cosmetics containing whitening ingredients were applied, and as shown in Figure 16 (B), melanin production was confirmed to be suppressed in the three-dimensional skin model to which cosmetic ingredients were added. Furthermore, as shown in Figure 16 (C), melanin production was confirmed to be suppressed in the three-dimensional skin model to which whitening cosmetics were added.

[0098] [Histochemical analysis of three-dimensional skin models] Three-dimensional skin models (evaluations 2, 4, 6, and 7 using cosmetic ingredients) containing cosmetic ingredients with melanin production inhibitory effects (tyrosinase inhibitory activity) were fixed with Superfix, and tissue images were taken using a stereomicroscope. Using these images, the degree of melanocyte blackening was measured using the image analysis software "ImageJ" (National Institutes of Health (NIH)). Ten areas where melanocytes were not present were randomly selected from the tissue images, and the calculated mean gray value for each was used as the background value. Next, 30 areas where melanocytes were present were randomly selected, and the mean gray value was calculated in the same manner. The value obtained by subtracting the background gray value (shade value) from the mean gray value of the areas where melanocytes were present was used as the degree of melanocyte blackening.

[0099] (Results) As shown in Figure 17, the group to which the cosmetic raw materials were added before adding L-Dopa had a lower degree of blackening than the group to which only L-Dopa was added (control), and it was confirmed that there was no difference from the visual evaluation.

[0100] [Quantitative Analysis of Melanin] A control group was treated with L-Dopa alone. A cosmetic raw material (10 μM rucinol) was added to the medium of the three-dimensional skin model 1, and the medium was then replaced with L-Dopa to promote darkening. Alternatively, a commercially available whitening cosmetic containing rucinol was applied to the stratum corneum surface of the three-dimensional skin model 1, and the medium was then replaced with L-Dopa to promote darkening. From each group, the melanocyte layer was scraped off with tweezers, dispersed in PBS(-), and centrifuged to obtain a melanocyte pellet. The supernatant was removed, and the pellet was added with 1N NaOH and incubated at 95°C for 10 minutes to dissolve the melanin. The absorbance was measured at a wavelength of 405 nm.

[0101] As shown in FIG. 18, when comparing rucinol and commercially available whitening cosmetics containing rucinol with the control, it was confirmed that darkening was suppressed.

[0102] According to the present invention, a three-dimensional skin model capable of producing melanin at a desired position can be provided, which is effective for efficient sample evaluation of whitening cosmetics.

[0103] 10 Epidermal layer 11 Stratum corneum 12 Granular layer 13 Sponge layer 14 Basal layer 20 Melanocyte layer 30 Intercalated membrane 40 Dermal layer 50 Incubator 51 Well 52 Culture medium 53 Support 60 Melanin, melanin granules 100, 200, 300 Three-dimensional skin model

Claims

1. A three-dimensional skin model having an epidermal layer, a melanocyte layer, and an intervening membrane, the intervening membrane being interposed between the epidermal layer and the melanocyte layer, the epidermal layer having, in this order from the side furthest from the intervening membrane, a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale.

2. A three-dimensional skin model having an epidermal layer, a melanocyte layer, an intervening membrane, and a dermal layer, wherein the melanocyte layer is located between the epidermal layer and the intervening membrane, and the intervening membrane is interposed between the melanocyte layer and the dermal layer, and the epidermal layer has, in this order from the side furthest from the intervening membrane, a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale.

3. A three-dimensional skin model having an epidermal layer, a melanocyte layer, an intervening membrane, and a dermal layer, wherein the intervening membrane is interposed between the epidermal layer and the melanocyte layer, the melanocyte layer being located between the intervening membrane and the dermal layer, and the epidermal layer having, in this order from the side furthest from the intervening membrane, a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale.

4. The three-dimensional skin model according to claim 2 or 3, wherein the epidermal layer is co-cultured with at least one of the melanocyte layer and the dermal layer.

5. The three-dimensional skin model according to claim 1, 2 or 3, wherein the intervening membrane is a porous membrane that contains an extracellular matrix at least on its surface.

6. A method for evaluating the responsiveness of a melanocyte layer, comprising the steps of: culturing a three-dimensional skin model according to any one of claims 1 to 3 in a culture vessel; adding a melanin synthesis inhibitor to the cultured three-dimensional skin model; applying a physical stimulus and / or a melanin synthesis promoter to the three-dimensional skin model to which the melanin synthesis inhibitor has been added; and observing the production of melanin synthesized in the three-dimensional skin model or measuring the amount of melanin produced after the application of the physical stimulus and / or the application of the melanin synthesis promoter.

7. The evaluation method according to claim 6, wherein the step of adding the melanin synthesis inhibitor is carried out at least once.

8. The evaluation method according to claim 6, wherein the amount of the melanin synthesis inhibitor added is 1 μM to 100 mM.