WO2024090514A1 - Three-dimensional skin model - Google Patents

Three-dimensional skin model Download PDF

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WO2024090514A1
WO2024090514A1 PCT/JP2023/038677 JP2023038677W WO2024090514A1 WO 2024090514 A1 WO2024090514 A1 WO 2024090514A1 JP 2023038677 W JP2023038677 W JP 2023038677W WO 2024090514 A1 WO2024090514 A1 WO 2024090514A1
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layer
skin model
dimensional skin
culture
dermis
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PCT/JP2023/038677
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French (fr)
Japanese (ja)
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克成 手塚
博章 七里
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株式会社アンズコーポレーション
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a three-dimensional skin model.
  • the full-thickness skin model uses collagen hydrogel as the extracellular matrix, and is a skin model in which epidermal keratinocytes are seeded onto the dermis layer in which dermal fibroblasts are dispersed within the collagen hydrogel, and the epidermis layer is reconstructed by gas phase culture.
  • the dermal fibroblasts contract the collagen hydrogel, so the epidermal layer peels off from the cell culture insert during culture, making it difficult to use an evaluation method in which the test substance (cosmetic ingredients, etc.) is applied directly from the stratum corneum side.
  • the dermal layer contracts during culture of the skin model, a long culture period may affect the morphology of the skin model and the skin's responsiveness, so it is necessary to consider a skin model in which the contraction of the dermal layer can be controlled.
  • Non-Patent Document 1 A skin model has been disclosed in which a polyethylene terephthalate membrane is placed between the epidermis and dermis layers to separate the two layers, thereby mitigating the effect of dermal contraction on the epidermis layer.
  • Non-Patent Document 1 uses collagen hydrogel as a matrix in the dermis layer, it is difficult to sufficiently suppress the contraction of the dermis layer.
  • the present invention was made in consideration of these points, and aims to provide a three-dimensional skin model that can suppress contraction of the dermis layer and maintain the structure of the epidermis and dermis for a long period of time.
  • the present invention is configured as follows: (1) to (12).
  • a three-dimensional skin model having a first layer containing keratinocytes or cells differentiated from keratinocytes, a second layer containing dermal fibroblasts, and an intervening membrane interposed between the first layer and the second layer, the intervening membrane being a porous membrane containing an extracellular matrix at least on its surface.
  • a three-dimensional skin model having an epidermis layer, a dermis layer, and an intervening membrane interposed between the epidermis layer and the dermis layer, the epidermis layer having a stratum corneum, a granular layer, a stratum spinosum, and a basal layer, the dermis layer having a multi-layered structure, and a cell density of 2000 cells/ cm2 or more.
  • a three-dimensional skin model having an epidermis layer, a dermis layer, and an intervening membrane interposed between the epidermis layer and the dermis layer, the epidermis layer having a stratum corneum, a granular layer, a stratum spinosum, and a basal layer, the dermis layer having a multi-layer structure, and containing collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%, where v means the volume of the dermis layer.
  • a culture vessel comprising a three-dimensional skin model according to any one of (1) to (4), a plurality of wells for containing a culture medium, and a support section for supporting the three-dimensional skin model within the wells and enabling the culture medium to communicate with the second layer side or the dermis layer side of the three-dimensional skin model.
  • a method for evaluating a sample comprising the steps of culturing a three-dimensional skin model using the culture vessel described in (5) or (6), administering a sample to the three-dimensional skin model during culture, and evaluating the function of the sample on the three-dimensional skin model by analyzing the three-dimensional skin model or the culture supernatant after a predetermined culture period has elapsed since the administration of the sample.
  • evaluation step includes, during the culture period, processing the three-dimensional skin model, administering the same or different samples two or more times, or removing the administered sample.
  • the present invention provides a three-dimensional skin model that can suppress contraction of the dermis layer and maintain the structure of the epidermis and dermis for a long period of time.
  • FIG. 1 is an image of a skin tissue section of a three-dimensional skin model after hematoxylin and eosin staining.
  • FIG. 2(a) is a plan view of a culture vessel having a plurality of wells, and (b) is a cross-sectional view taken along line AA of (a).
  • Figures 3(a) and (b) are images of skin tissue sections of the three-dimensional skin model after culturing for a specified period of time.
  • Figures 4(a) and (b) are images of skin tissue sections of a three-dimensional skin model subjected to immunohistochemical staining.
  • FIG. 5 is a graph showing the analysis results of the barrier function of a three-dimensional skin model.
  • FIGS. 6 is a graph showing the analysis results of the barrier function of a three-dimensional skin model.
  • 7A and 7B are graphs showing the analysis results of the responsiveness of the three-dimensional skin model to physiological stimuli.
  • 8A and 8B are graphs showing the analysis results of the responsiveness of the three-dimensional skin model to physiological stimuli.
  • 9(a) and (b) are images of skin tissue slices after analysis of the responsiveness of the three-dimensional skin model to physiological stimuli.
  • 10A to 10D are graphs showing the analysis results of the responsiveness of a three-dimensional skin model to cosmetics.
  • FIGS. 11A to 11D are graphs showing the analysis results of the responsiveness of a three-dimensional skin model to cosmetics.
  • 12(a) and (b) show the analysis results of protein expression.
  • FIGS. 13A to 13F are graphs showing the analysis results of the responsiveness to commercially available cosmetics (beauty essences).
  • FIG. 14 (a) to (f) are graphs showing the analysis results of the responsiveness to a commercially available cosmetic product (lotion).
  • FIG. 15(a) to (f) are graphs showing the analysis results of the responsiveness to commercially available cosmetics (lotions).
  • FIGS. 16(a) to 16(f) are graphs showing the analysis results of the responsiveness to commercially available cosmetics (creams).
  • the three-dimensional skin model according to the first embodiment of the present invention has a first layer 11 containing keratinocytes or cells differentiated from keratinocytes, a second layer 12 containing dermal fibroblasts, and an intervening membrane 13 intervening between the first layer 11 and the second layer 12.
  • the intervening membrane 13 contains an extracellular matrix.
  • the second layer 12 preferably contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%.
  • the above v means the volume of the second layer.
  • the first layer 11 contains keratinocytes or cells differentiated from keratinocytes.
  • the cells differentiated from keratinocytes include one or more types selected from the group consisting of keratinocytes, granular cells, spinous cells, and basal cells, which will be described later.
  • Keratinocytes are cells of the epidermis that divide in the basal layer, the lowest layer of the epidermis, and migrate to the skin surface.
  • the second layer 12 comprises dermal fibroblasts.
  • Dermal fibroblasts (fibroblasts) 14 are cells that produce collagen fibers, elastic fibers, mucopolysaccharides, etc., and have a long, spindle-shaped morphology.
  • the second layer 12 preferably contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%, and more preferably 0 w/v% or more and 0.3 w/v% or less. If the amount of collagen hydrogel is within the above range, it is possible to suppress contraction of the dermis layer that constitutes the three-dimensional skin model during culture.
  • the proliferation ability of dermal fibroblasts is not easily inhibited by inhibition of cell activity, and since there is a large space in which proliferated cells can exist, the proliferation of dermal fibroblasts is not easily inhibited.
  • the cell density in the dermis layer is easily maintained at a high level for a long period of time, which makes it easy to evaluate the effect of the sample on the dermis layer with high sensitivity.
  • proteins involved in crosstalk cytokines, chemokines, etc.
  • lipid components eicosanoids, etc.
  • the intervening membrane 13 is a porous membrane that allows substances to pass through and contains an extracellular matrix at least on its surface. By containing an extracellular matrix at least on its surface, the intervening membrane can easily adhere keratinocytes and fibroblasts without relying on a collagen hydrogel.
  • 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 frequent presence.
  • substrate materials include PET (polyethylene terephthalate) and PC (polycarbonate) membranes, and PET.
  • extracellular matrices include type I collagen, type III collagen, type IV collagen, fibronectin, gelatin, etc.
  • the average pore size of the intervening membrane is preferably 0.4 ⁇ m or more.
  • the average pore size of the intervening membrane may be 8.0 ⁇ m or less, and is preferably 6.0 ⁇ m or less.
  • the pore size of the intervening membrane can be determined, for example, by measuring the long axis using an image taken with an electron microscope.
  • the average thickness of the intervening membrane is preferably 20 ⁇ m or less, and more preferably 15 ⁇ m or less, 10 ⁇ m or less, 7.5 ⁇ m or less, or 5 ⁇ m or less, from the viewpoint of obtaining a skin model in which the structure of the epidermis and dermis is maintained for a long period of time without relying on medium components (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) or additional layers described below.
  • medium components substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.
  • the average thickness of the intervening membrane may be, for example, 20 ⁇ m or more and 200 ⁇ m or less, in cases where a specific medium component (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) is used or an additional layer is provided.
  • a specific medium component such as KGF, HGF, IGF-1, etc.
  • the thickness of the intervening membrane is within the above range, the strength of the three-dimensional skin model is increased, and since the crosstalk between the dermis layer and the epidermis layer is highly performed, it is easy to maintain a state in which the differentiation of the epidermis layer has progressed appropriately (the spinous layer and granular layer are thick) and a state in which the density of the basal layer is high.
  • the thickness of the intervening membrane can be measured, for example, using an optical microscope image of a histochemical section.
  • the intervening membrane may be a membrane from a commercially available cell culture insert.
  • the three-dimensional skin model according to this embodiment may or may not have other layers in addition to the first layer, second layer, and intervening membrane. Additional layers include a subcutaneous tissue layer, such as an adipose layer, located on the opposite side of the second layer from the intervening membrane.
  • a subcutaneous tissue layer such as an adipose layer
  • an embodiment having an additional layer further promotes crosstalk and makes it easier to maintain the differentiated structure, but there is less freedom in the culture conditions (e.g., medium composition) suitable for all of the first layer, second layer, and additional layer.
  • an embodiment not having an additional layer is preferable, since it is easier to maintain the differentiated structure without relying on the additional layer.
  • a three-dimensional skin model according to a second embodiment of the present invention has an epidermis layer 11, a dermis layer 12, and an intervening membrane 13 interposed between the epidermis layer 11 and the dermis layer 12.
  • the epidermis layer 11 has a stratum corneum 15, a granular layer 16, a spinous layer 17, and a basal layer 18.
  • the epidermal layer 11 has a four-layer structure in which keratinocytes form the stratum corneum 15 in the uppermost layer, granular cells form the granular layer 16 just below the stratum corneum 15, spinous cells (or spinous cells) form the spinous layer 17 just below the granular layer 16, and basal cells differentiated from keratinocytes form the basal layer 18 just below the spinous layer 17 (lowest layer).
  • the epidermal layer 11 may also contain pigment cells (melanocytes).
  • the stratum corneum 15 is located at the top of the epidermal layer 11 constituting the three-dimensional skin model, and is composed of a layered structure constructed by keratinocytes.
  • the keratinocytes have a flat shape and lose their nuclei during the differentiation process.
  • the keratinocytes contain aggregates of keratin fibers in their cytoplasm, are acidophilic, and stain light red to dark red when stained with hematoxylin and eosin (HE).
  • HE hematoxylin and eosin
  • the granular layer 16 is located immediately below the stratum corneum 15 and immediately above the spinous layer 17 described below, and is composed of a layered structure constructed by granular cells.
  • the granular cells have a flat shape and contain granules made of basophilic components within the cells.
  • the granules within the cells are stained blue-purple to light blue by hematoxylin-eosin (HE) staining.
  • the thickness of the granular layer of the three-dimensional skin model according to this embodiment is preferably 3 ⁇ m or more on average, more preferably 6 ⁇ m or more on average, and even more preferably 8 ⁇ m or more on average.
  • the thickness of the granular layer of the three-dimensional skin model is preferably 3 ⁇ m or more and 10 ⁇ m or less on average. The thickness can be measured, for example, using an optical microscope image of a histochemical section.
  • the spinous layer 17 is located directly below the granular layer 16 and directly above the basal layer 18 described later, and is composed of a layered structure constructed by spinous cells.
  • the 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 (HE) staining.
  • the thickness of the spinous layer of the three-dimensional skin model according to this embodiment is preferably 18 ⁇ m or more on average, more preferably 26 ⁇ m or more on average, and even more preferably 39 ⁇ m or more on average.
  • the thickness of the spinous layer of the three-dimensional skin model is preferably 18 ⁇ m or more and 50 ⁇ m or less on average. The thickness can be measured, for example, using an optical microscope image of a histochemical section.
  • the basal layer 18 is located at the bottom of the epidermal layer 11 and is composed of a layer structure constructed by basal cells.
  • the basal cells unlike keratinocytes, granular cells, and spinous cells, have a cubic to cylindrical shape and an elliptical nucleus.
  • the basal cells are basophilic and stained in indigo to light blue by hematoxylin-eosin (HE) staining.
  • the thickness of the basal layer of the three-dimensional skin model according to this embodiment is preferably 7 ⁇ m or more on average, more preferably 9 ⁇ m or more on average, and even more preferably 14 ⁇ m or more on average.
  • the thickness of the spinous layer of the three-dimensional skin model is preferably 7 ⁇ m or more and 20 ⁇ m or less on average. The thickness can be measured, for example, using an optical microscope image of a histochemical section.
  • the dermis layer 12 has a multi-layer structure. Specifically, the dermis layer 12 has a tissue structure composed of fibroblasts, collagen fibers (such as collagen) synthesized and secreted by the fibroblasts, elastic fibers (such as fibrillin and elastin), and other extracellular matrix components (such as hyaluronic acid).
  • tissue structure composed of fibroblasts, collagen fibers (such as collagen) synthesized and secreted by the fibroblasts, elastic fibers (such as fibrillin and elastin), and other extracellular matrix components (such as hyaluronic acid).
  • Multilayer structure refers to a structure in which fibroblasts form two or more layers in the vertical direction (towards the stratum corneum).
  • the orientation of the fibroblasts is not uniform.
  • the image of a vertically sliced skin tissue section may appear spindle-shaped due to the fibroblasts being oriented horizontally, or circular due to the fibroblasts being oriented vertically.
  • the cell density of the dermis layer is 2000 cells/cm 2 or more, preferably 5000 cells/cm 2 or more, and preferably 6000 cells/cm 2 or more.
  • the cell density of the dermis layer is preferably 2000 cells/cm 2 or more and 10000 cells/cm 2 or less, more preferably 5000 cells/cm 2 or more and 8000 cells/cm 2 or less, and even more preferably 6000 cells/cm 2 or more and 7700 cells/cm 2 or less.
  • the layer structure of the three-dimensional skin model can be maintained for a long period of time.
  • a three-dimensional skin model according to a third embodiment of the present invention has an epidermis layer 11, a dermis layer 12, and an intervening membrane 13 interposed between the epidermis layer 11 and the dermis layer 12.
  • the epidermis layer 11 has a stratum corneum 15, a granular layer 16, a spinous layer 17, and a basal layer 18.
  • the dermis layer 12 has a multi-layer structure, and contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%.
  • v refers to the volume of the dermis layer. It is more preferable that the dermis layer 12 contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.3 w/v%.
  • the dermis layer 12 does not have collagen fibers unevenly dispersed as determined by hematoxylin-eosin (HE) staining. This provides a large space in which proliferated cells can exist, making it difficult to inhibit the proliferation ability of dermal fibroblasts. This makes it possible to suppress the contraction of the dermis layer that constitutes the three-dimensional skin model during culture.
  • HE hematoxylin-eosin
  • the proliferation ability of dermal fibroblasts is not easily inhibited by inhibiting cell activity, and since there is a large space in which proliferated cells can exist, the proliferation of dermal fibroblasts is not easily inhibited.
  • the cell density in the dermis layer is easily maintained at a high level for a long period of time, which makes it easy to evaluate the effect of the sample on the dermis layer with high sensitivity.
  • proteins involved in crosstalk cytokines, chemokines, etc.
  • lipid components eicosanoids, etc.
  • the three-dimensional skin model according to this embodiment may or may not have other layers in addition to the epidermis layer, dermis layer, and intervening membrane. Additional layers include a subcutaneous tissue layer, such as an adipose layer, located on the opposite side of the dermis layer from the intervening membrane.
  • a subcutaneous tissue layer such as an adipose layer
  • crosstalk is further promoted and the differentiated structure is easily maintained, but there is less freedom in the culture conditions (e.g., medium composition) suitable for all of the epidermal layer, dermal layer, and additional layer.
  • the differentiated structure is easily maintained without relying on the additional layer, so an embodiment not having an additional layer is preferred.
  • the culture vessel 20 used in the first to third embodiments has a plurality of wells 21 that contain a culture solution 23, and a support portion 22 that supports the three-dimensional skin model 10 in the wells 21.
  • the three-dimensional skin model 10 is arranged so as to be in communication with the culture solution 23 on the second layer 12 side or the dermis layer 12 side.
  • the culture vessel 20 may have any number of wells, including but not limited to 12, 24, 48, or 96 wells. This allows for the evaluation of multiple samples and application conditions simultaneously.
  • sample evaluation method The sample evaluation method of the present invention is carried out using a culture vessel.
  • the sample evaluation method includes the steps of (1) culturing a three-dimensional skin model using a culture vessel 20 shown in (a) and (b) of Figure 2 (culture step), (2) administering a sample to the three-dimensional skin model being cultured (sample administration step), and (3) analyzing the three-dimensional skin model or culture supernatant after a predetermined culture period has elapsed since the administration of the sample, thereby evaluating the function that the sample provides to the three-dimensional skin model (evaluation step).
  • the culture process is a process of placing the second layer 12 side or the dermis layer 12 side of the three-dimensional skin model 10 in the well 21 of the culture container 20 so that it can communicate with the culture solution 23, and culturing the three-dimensional skin model 10 for a predetermined period of time.
  • the culture period is 6 days or more, preferably 12 days or more, and more preferably 20 days or more.
  • the culture period may be 30 days or less.
  • a culture period of 6 days or more provides a long period for evaluation, making it possible to obtain sufficient evaluation results.
  • sample administration step is a step of administering a sample to the three-dimensional skin model 10 placed in the well 21 of the culture container 20 in a predetermined manner.
  • the sample to be administered is not particularly limited, but may be one or more isolated substances, or may be 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.
  • samples may be applied to each well, or the same sample may be applied in different ways (dosages and/or administration methods). This allows for quantitative comparison of the performance of the samples on the skin, and allows for estimation of a preferred administration method (dosage and/or administration method) for imparting a desired function to the skin.
  • the evaluation process is a process of evaluating the function that the sample provides to the three-dimensional skin model 10 by analyzing the three-dimensional skin model 10 or the culture supernatant containing secretory fluids secreted by the cells after a culture period of several hours or more has elapsed since administration of the sample.
  • the above analyses preferably include gene expression, protein expression, histochemical analysis, or skin barrier function analysis, and may include a combination of two or more of these.
  • the evaluation process may also include a process of processing the three-dimensional skin model 10 during the above-mentioned culture period, a process of administering the same or different samples to the three-dimensional skin model 10 two or more times, or a process of removing the samples administered to the three-dimensional skin model 10.
  • the process of processing the three-dimensional skin model 10 is a process of producing three-dimensional skin models in different conditions.
  • the processing method is not particularly limited, but includes the addition of an inflammatory substance to induce oxidative stress, and irradiation with light such as UV light that induces oxidative stress.
  • This processing can take anywhere from a few seconds to a few days, but the skin model used in this embodiment can maintain the structure of the epidermis and dermis layers for a long period of time, making it easy to freely perform evaluations using the processed skin model.
  • the same sample may be administered by the same method or by different methods.
  • different samples may be administered by the same method or by different methods.
  • “method” refers to dosage and/or method of use.
  • At least a three-dimensional skin model to which a sample has not been administered may be cultured for a predetermined period (e.g., 6 days or more, preferably 12 days or more, 20 days or more, and may be 30 days or less) in the above-mentioned culture process.
  • a predetermined period e.g. 6 days or more, preferably 12 days or more, 20 days or more, and may be 30 days or less
  • it is preferable to culture under conditions such that the three-dimensional skin model to which a sample has not been administered has an epidermis layer having a stratum corneum, granular layer, spinous layer, and basal layer, and a dermis layer having a multi-layer structure, after the culture period has elapsed.
  • the relationship between the planar area of the epidermal layer and the planar area of the dermal layer of the three-dimensional skin model is preferably such that the planar area of the epidermal layer is less than or equal to the planar area of the dermal layer, or the planar area of the dermal layer after the culture period is 0.7 times or more of the planar area of the dermal layer before the culture period, and is not particularly limited, but is more preferably 0.9 times or more. Furthermore, the planar area of the dermal layer after the culture period is preferably 1.0 times or less of the planar area of the dermal layer before the culture period.
  • the planar area of the epidermal layer is preferably 0.7 times or more and 1.0 times or less of the planar area of the dermal layer before the culture period, and more preferably 0.9 times or more and 1.0 times or less.
  • a cell culture insert having a collagen membrane (ad-MED Vitrigel 2 08364-96 (Kanto Chemical Co., Ltd.), AteloCell CM-24 (Koken Co., Ltd.) was prepared. 10,000 normal human dermal fibroblasts suspended in 10% FBS-containing DMEM were seeded on one side of the cell culture insert and cultured at 37°C for 1 day in a CO2 incubator.
  • the thicknesses of the granular layer, spinous layer, and basal layer of the constructed three-dimensional skin model were 3-8 ⁇ m, 18-39 ⁇ m, and 7-14 ⁇ m, respectively, and the cell density of the dermis layer was 7114 ⁇ 339 cells/cm 2 , and 5843 ⁇ 349 cells/cm 2 after 20 days of gas phase culture.
  • the cell density of the dermis layer of the model disclosed in Non-Patent Document 1 was 1300 ⁇ 339 cells/cm 2 , which was significantly lower than that of this example.
  • the planar areas of the dermis layer and epidermis layer of the constructed three-dimensional skin model after the culture period were each 1-fold the planar areas of the dermis layer and epidermis layer before the culture period.
  • the three-dimensional skin model like human skin tissue, is composed of the epidermis layer, stratum corneum, stratum granulosum (flattened cells containing intracellular granules), stratum spinosum (having spines around the cells), and stratum basale (cuboidal shape). It was also confirmed that fibroblasts are arranged in layers as the dermis layer just below the stratum basale, showing the basic morphological characteristics of a skin model.
  • cytokeratin 10 As shown in (a) of FIG. 4, the expression of cytokeratin 10 (CK10), a differentiation marker, was confirmed from the stratum corneum 15 to the spinous layer 17 of the three-dimensional skin model 30, and the expression of cytokeratin 14 (CK14) was confirmed in the basal layer 18. As shown in (b) of FIG. 4, the expression of filaggrin (not shown), a component of keratohyalin granules, was confirmed in the granular layer 16 (granules in granular cells), and the expression of claudin 1 (not shown), a tight junction-constituting adhesion molecule, was confirmed in the spinous layer 16 (cell periphery of spinous cells). From (a) and (b) of FIG. 4, the morphological characteristics of the three-dimensional skin model could be confirmed from the localization of the differentiation marker molecules.
  • PBS phosphate buffered saline
  • Cytotoxicity after culture was measured using the Cytotoxicity LDH Assay Kit-WST (manufactured by Dojindo Laboratories, Inc.) using the cytoplasmic oxidoreductase (LDH) activity leaked into the medium as an indicator.
  • LDH cytoplasmic oxidoreductase
  • a 10 ⁇ M solution of retinoic acid (ATRA) was applied to the epidermis (surface) of the three-dimensional skin model that had been gas-phase cultured for 12 days using the method described above, and the model was cultured for 1 hour at 37° C. After culture, the retinoic acid solution was removed and the model was further cultured for 5 hours, after which RNA was extracted from the skin tissue and gene expression analysis was performed using real-time PCR.
  • ATRA retinoic acid
  • TGF ⁇ Transforming growth factor ⁇
  • epidermal genes filamentaggrin (FLG)/caspase 14 (CASP14)/transglutaminase 1 (TGM1)/occludin (OCLN)/heparin-binding EGF-like growth factor (HBEGF)/lysosomal protease cathepsin V (CTSV)/syntaxin (STX3)
  • dermal genes hyaluronic acid synthase 2 (HAS2)
  • GAA glycose
  • SPTLC1 very long-chain fatty acid e
  • the present invention can suppress the contraction of the dermis layer and maintain the structure of the epidermis and dermis for a long period of time, making it effective for efficient sample evaluation of cosmetics, etc., using a culture vessel with many wells.
  • Three-dimensional skin model 11 First layer, epidermis layer 12 Second layer, dermis layer 13 Intervening membrane 14 Dermal fibroblast layer 15 Stratum corneum 16 Granular layer 17 Spongy layer 18 Basal layer 20 Culture vessel 21 Well 22 Support 23 Culture medium 41 Type I collagen 42 Fibrin-1 43 Nuclear

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Abstract

A three-dimensional skin model according to one embodiment of the present invention comprises: a first layer including keratinocytes or cells with differentiated keratinocytes; a second layer including dermal fibroblasts; and an intermediate membrane which is interposed between the first layer and the second layer. The intermediate membrane is a porous membrane including, at least on the surface thereof, an extracellular matrix. Further, a three-dimensional skin model according to another embodiment of the present invention comprises an epidermal layer, a dermal layer, and an intermediate membrane which is interposed between the epidermal layer and the dermal layer. The epidermal layer has a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale. The dermal layer has a multi-layer structure and has a cellular density of no less than 2,000 cells/cm2.

Description

三次元皮膚モデル3D Skin Model
　本発明は、三次元皮膚モデルに関する。 The present invention relates to a three-dimensional skin model.
　世界的な動物愛護の気運の高まりに伴い、動物実験代替法の開発が進められている。皮膚の動物実験代替法として、入手が困難なヒトの皮膚の代わりに、化学繊維の皮膚、三次元皮膚モデル等の人工の皮膚モデルが開発されている。 As interest in animal welfare grows worldwide, efforts are underway to develop alternatives to animal testing. As alternatives 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.
　たとえば、製品（化粧品成分等）の機能性試験では、表皮と真皮の応答性を確認できる全層皮膚モデルが開発されている。全層皮膚モデルは、細胞外マトリクスとしてコラーゲンハイドロゲルを用い、当該コラーゲンハイドロゲル中の真皮線維芽細胞が分散配置された真皮層上に表皮角化細胞を播種し、気相培養により表皮層が再構築された皮膚モデルである。 For example, in functionality tests for products (such as cosmetic ingredients), a full-thickness skin model has been developed that can confirm the responsiveness of the epidermis and dermis. The full-thickness skin model uses collagen hydrogel as the extracellular matrix, and is a skin model in which epidermal keratinocytes are seeded onto the dermis layer in which dermal fibroblasts are dispersed within the collagen hydrogel, and the epidermis layer is reconstructed by gas phase culture.
　ここで、上記真皮線維芽細胞は上記コラーゲンハイドロゲルを収縮させるため、培養中に表皮層がセルカルチャーインサートから剥がれてしまい、角層側から被験物質（化粧品成分等）を直接塗布するような評価方法は困難である。また、皮膚モデルを培養中に真皮層の収縮が生じていることから、培養期間が長くなると、皮膚モデルの形態や皮膚の応答性に影響を与える可能性があるため、真皮層の収縮をコントロール可能な皮膚モデルの検討が必要である Here, the dermal fibroblasts contract the collagen hydrogel, so the epidermal layer peels off from the cell culture insert during culture, making it difficult to use an evaluation method in which the test substance (cosmetic ingredients, etc.) is applied directly from the stratum corneum side. In addition, because the dermal layer contracts during culture of the skin model, a long culture period may affect the morphology of the skin model and the skin's responsiveness, so it is necessary to consider a skin model in which the contraction of the dermal layer can be controlled.
　そこで、表皮層と真皮層との間にポリエチレンテレフタレート膜を配置して、両層を分画することで、真皮層の収縮が表皮層に与える影響を緩和した皮膚モデルが開示されている（非特許文献１）。 A skin model has been disclosed in which a polyethylene terephthalate membrane is placed between the epidermis and dermis layers to separate the two layers, thereby mitigating the effect of dermal contraction on the epidermis layer (Non-Patent Document 1).
　しかし、非特許文献１に開示される皮膚モデルは、真皮層にコラーゲンハイドロゲルをマトリクスとして用いる以上、真皮層の収縮を十分には抑制し難い。 However, since the skin model disclosed in Non-Patent Document 1 uses collagen hydrogel as a matrix in the dermis layer, it is difficult to sufficiently suppress the contraction of the dermis layer.
　本発明は、かかる点に鑑みてなされたものであり、真皮層の収縮を抑制し、表皮及び真皮の構造を長期間にわたって維持することができる三次元皮膚モデルを提供することを目的とする。 The present invention was made in consideration of these points, and aims to provide a three-dimensional skin model that can suppress contraction of the dermis layer and maintain the structure of the epidermis and dermis for a long period of time.
　本発明者は、鋭意検討の結果、上記課題を解決できることを見出し、本発明を完成するに至った。具体的には、本発明は、以下の（１）～（１２）のように構成される。 As a result of extensive research, the inventors have found that the above problems can be solved, and have completed the present invention. Specifically, the present invention is configured as follows: (1) to (12).
　（１）角化細胞又は角化細胞が分化した細胞を含む第１の層と、真皮線維芽細胞を含む第２の層と、前記第１の層と前記第２の層との間に介在する介在膜と、を有し、前記介在膜は細胞外マトリクスを少なくとも表面に含む多孔性の膜である、三次元皮膚モデル。 (1) A three-dimensional skin model having a first layer containing keratinocytes or cells differentiated from keratinocytes, a second layer containing dermal fibroblasts, and an intervening membrane interposed between the first layer and the second layer, the intervening membrane being a porous membrane containing an extracellular matrix at least on its surface.
　（２）コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合され、前記ｖは、前記第２の層の体積を意味する、前記（１）に記載の三次元皮膚モデル。 (2) A three-dimensional skin model as described in (1) above, in which collagen hydrogel is blended in an amount of 0 w/v% or more and less than 0.6 w/v%, where v represents the volume of the second layer.
　（３）表皮層と、真皮層と、表皮層と真皮層との間に介在する介在膜と、を有し、前記表皮層は角層、顆粒層、有棘層、及び基底層を有し、前記真皮層は多層構造を有し、かつ、細胞密度が２０００ｃｅｌｌｓ／ｃｍ２以上である、三次元皮膚モデル。 (3) A three-dimensional skin model having an epidermis layer, a dermis layer, and an intervening membrane interposed between the epidermis layer and the dermis layer, the epidermis layer having a stratum corneum, a granular layer, a stratum spinosum, and a basal layer, the dermis layer having a multi-layered structure, and a cell density of 2000 cells/ cm2 or more.
　（４）表皮層と、真皮層と、表皮層と真皮層との間に介在する介在膜と、を有し、前記表皮層は角層、顆粒層、有棘層、及び基底層を有し、前記真皮層は多層構造を有し、かつ、コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合され、前記ｖは、前記真皮層の体積を意味する、三次元皮膚モデル。 (4) A three-dimensional skin model having an epidermis layer, a dermis layer, and an intervening membrane interposed between the epidermis layer and the dermis layer, the epidermis layer having a stratum corneum, a granular layer, a stratum spinosum, and a basal layer, the dermis layer having a multi-layer structure, and containing collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%, where v means the volume of the dermis layer.
　（５）前記（１）～（４）のいずれかに記載の三次元皮膚モデルと、培養液を収容する複数のウェルと、前記ウェル内で前記三次元皮膚モデルを支持し、かつ、培養液を前記三次元皮膚モデルの前記第２の層側又は真皮層側で連通可能とする支持部と、を備える培養容器。 (5) A culture vessel comprising a three-dimensional skin model according to any one of (1) to (4), a plurality of wells for containing a culture medium, and a support section for supporting the three-dimensional skin model within the wells and enabling the culture medium to communicate with the second layer side or the dermis layer side of the three-dimensional skin model.
　（６）前記ウェルを１２、２４、４８又は９６個有する、前記（５）に記載の培養容器。 (6) The culture vessel according to (5) above, having 12, 24, 48 or 96 wells.
　（７）前記（５）又は（６）に記載の培養容器を用いて、三次元皮膚モデルを培養する工程と、培養中の前記三次元皮膚モデルにサンプルを投与する工程と、サンプル投与から所定の培養期間経過後の前記三次元皮膚モデル又は培養上清を分析することで、前記サンプルが前記三次元皮膚モデルに与える機能を評価する工程と、を含むサンプルの評価方法。 (7) A method for evaluating a sample, comprising the steps of culturing a three-dimensional skin model using the culture vessel described in (5) or (6), administering a sample to the three-dimensional skin model during culture, and evaluating the function of the sample on the three-dimensional skin model by analyzing the three-dimensional skin model or the culture supernatant after a predetermined culture period has elapsed since the administration of the sample.
　（８）前記培養期間は６日以上である、前記（７）に記載の評価方法。 (8) The evaluation method described in (7) above, wherein the culture period is 6 days or longer.
　（９）前記評価する工程は、前記培養期間において、前記三次元皮膚モデルの加工を行う工程、２回以上の同一又は異なるサンプルの投与を行う工程、又は投与したサンプルの除去を行う工程を含む、前記（７）又は（８）に記載の評価方法。 (9) The evaluation method according to (7) or (8), wherein the evaluation step includes, during the culture period, processing the three-dimensional skin model, administering the same or different samples two or more times, or removing the administered sample.
　（１０）少なくともサンプルを投与しない三次元皮膚モデルが、前記培養期間経過後において、角層、顆粒層、有棘層、及び基底層を有する表皮層と、多層構造を有する真皮層と、を有する、前記（７）～（９）のいずれかに記載の評価方法。 (10) The evaluation method according to any one of (7) to (9), wherein the three-dimensional skin model to which at least a sample is not administered has, after the culture period has elapsed, an epidermal layer having a stratum corneum, a granular layer, a stratum spinosum, and a basal layer, and a dermal layer having a multi-layer structure.
　（１１）前記培養期間経過後における、三次元皮膚モデルの表皮層の平面積と真皮層の平面積の大きさとの関係が、表皮層の平面積≦真皮層の平面積を満たすか、又は前記培養期間経過後における真皮層の平面積が、前記培養期間経過前における真皮層の平面積の０．７倍以上である、前記（７）～（１０）のいずれかに記載の評価方法。 (11) The evaluation method according to any one of (7) to (10), wherein the relationship between the planar area of the epidermal layer and the planar area of the dermal layer of the three-dimensional skin model after the culture period has elapsed satisfies the planar area of the epidermal layer ≦ the planar area of the dermal layer, or the planar area of the dermal layer after the culture period is 0.7 times or more the planar area of the dermal layer before the culture period has elapsed.
　（１２）前記分析は、遺伝子発現、タンパク質発現、組織化学分析、又は皮膚バリア機能分析を含む、前記（７）～（１１）のいずれかに記載の評価方法。 (12) The evaluation method according to any one of (7) to (11), wherein the analysis includes gene expression, protein expression, histochemical analysis, or skin barrier function analysis.
　本発明によれば、真皮層の収縮を抑制し、表皮及び真皮の構造を長期間にわたって維持することができる三次元皮膚モデルを提供することができる。 The present invention provides a three-dimensional skin model that can suppress contraction of the dermis layer and maintain the structure of the epidermis and dermis for a long period of time.
図１はヘマトキシリン・エオジン染色後の、三次元皮膚モデルの皮膚組織切片画像である。FIG. 1 is an image of a skin tissue section of a three-dimensional skin model after hematoxylin and eosin staining. 図２の（ａ）は複数のウェルを有する培養容器の平面図であり、（ｂ）は（ａ）のＡ－Ａ線の断面図である。FIG. 2(a) is a plan view of a culture vessel having a plurality of wells, and (b) is a cross-sectional view taken along line AA of (a). 図３の（ａ）及び（ｂ）は所定の期間培養後の三次元皮膚モデルの皮膚組織切片画像である。Figures 3(a) and (b) are images of skin tissue sections of the three-dimensional skin model after culturing for a specified period of time. 図４の（ａ）及び（ｂ）は免疫組織化学染色を行った三次元皮膚モデルの皮膚組織切片画像である。Figures 4(a) and (b) are images of skin tissue sections of a three-dimensional skin model subjected to immunohistochemical staining. 図５は三次元皮膚モデルのバリア機能の解析結果を示すグラフである。FIG. 5 is a graph showing the analysis results of the barrier function of a three-dimensional skin model. 図６は三次元皮膚モデルのバリア機能の解析結果を示すグラフである。FIG. 6 is a graph showing the analysis results of the barrier function of a three-dimensional skin model. 図７の（ａ）及び（ｂ）は三次元皮膚モデルの生理的刺激に対する応答性の解析結果を示すグラフである。7A and 7B are graphs showing the analysis results of the responsiveness of the three-dimensional skin model to physiological stimuli. 図８の（ａ）及び（ｂ）は三次元皮膚モデルの生理的刺激に対する応答性の解析結果を示すグラフである。8A and 8B are graphs showing the analysis results of the responsiveness of the three-dimensional skin model to physiological stimuli. 図９の（ａ）及び（ｂ）は三次元皮膚モデルの生理的刺激に対する応答性の解析後の皮膚組織切片画像である。9(a) and (b) are images of skin tissue slices after analysis of the responsiveness of the three-dimensional skin model to physiological stimuli. 図１０の（ａ）～（ｄ）は三次元皮膚モデルの化粧品に対する応答性の解析結果を示すグラフである。10A to 10D are graphs showing the analysis results of the responsiveness of a three-dimensional skin model to cosmetics. 図１１の（ａ）～（ｄ）は三次元皮膚モデルの化粧品に対する応答性の解析結果を示すグラフである。FIGS. 11A to 11D are graphs showing the analysis results of the responsiveness of a three-dimensional skin model to cosmetics. 図１２の（ａ）及び（ｂ）はタンパク質発現の解析結果を示す図である。12(a) and (b) show the analysis results of protein expression. 図１３の（ａ）～（ｆ）は市販の化粧品（美容液）に対する応答性の解析結果を示すグラフである。FIGS. 13A to 13F are graphs showing the analysis results of the responsiveness to commercially available cosmetics (beauty essences). 図１４の（ａ）～（ｆ）は市販の化粧品（ローション）に対する応答性の解析結果を示すグラフである。FIG. 14 (a) to (f) are graphs showing the analysis results of the responsiveness to a commercially available cosmetic product (lotion). 図１５の（ａ）～（ｆ）は市販の化粧品（乳液）に対する応答性の解析結果を示すグラフである。FIG. 15(a) to (f) are graphs showing the analysis results of the responsiveness to commercially available cosmetics (lotions). 図１６の（ａ）～（ｆ）は市販の化粧品（クリーム）に対する応答性の解析結果を示すグラフである。FIGS. 16(a) to 16(f) are graphs showing the analysis results of the responsiveness to commercially available cosmetics (creams).
　以下、本発明の具体的な実施形態について詳細に説明する。なお、本発明は以下の実施形態に限定されるものではなく、本発明の要旨を変更しない範囲で適宜変更が可能である。 Specific embodiments of the present invention are described in detail below. Note that the present invention is not limited to the following embodiments, and can be modified as appropriate without departing from the spirit of the present invention.
　［三次元皮膚モデル］
　（第１の実施形態）
　本発明の第１の実施形態に係る三次元皮膚モデルは、図１に示されるように、角化細胞又は角化細胞が分化した細胞を含む第１の層１１と、真皮線維芽細胞を含む第２の層１２と、前記第１の層１１と前記第２の層１２との間に介在する介在膜１３と、を有する。介在膜１３は細胞外マトリクスを含む。また、第２の層１２はコラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合されることが好ましい。なお、上記ｖは、第２の層の体積を意味する。 [Three-dimensional skin model]
First Embodiment
As shown in Fig. 1, the three-dimensional skin model according to the first embodiment of the present invention has a first layer 11 containing keratinocytes or cells differentiated from keratinocytes, a second layer 12 containing dermal fibroblasts, and an intervening membrane 13 intervening between the first layer 11 and the second layer 12. The intervening membrane 13 contains an extracellular matrix. The second layer 12 preferably contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%. The above v means the volume of the second layer.
　以下に、各構成要素について説明する。 Each component is explained below.
　≪第１の層≫
　第１の層１１は、角化細胞又は角化細胞が分化した細胞を含む。角化細胞が分化した細胞としては、後述の角質細胞、顆粒細胞、有棘細胞、及び基底細胞からなる群より選ばれる１種以上が挙げられる。 <First Layer>
The first layer 11 contains keratinocytes or cells differentiated from keratinocytes. Examples of the cells differentiated from keratinocytes include one or more types selected from the group consisting of keratinocytes, granular cells, spinous cells, and basal cells, which will be described later.
　角化細胞（ケラチノサイト）は、表皮の細胞であり、上記表皮の最下層の基底層で分裂して皮膚表面へと移行する細胞である。 Keratinocytes are cells of the epidermis that divide in the basal layer, the lowest layer of the epidermis, and migrate to the skin surface.
　≪第２の層≫
　第２の層１２は、真皮線維芽細胞を含む。 <<Second Layer>>
The second layer 12 comprises dermal fibroblasts.
　真皮線維芽細胞（線維芽細胞）１４は、膠原線維や弾性線維、ムコ多糖等を産生する細胞であり、細長い紡錘形の形態を呈する。 Dermal fibroblasts (fibroblasts) 14 are cells that produce collagen fibers, elastic fibers, mucopolysaccharides, etc., and have a long, spindle-shaped morphology.
　第２の層１２は、コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合されることが好ましく、０ｗ／ｖ％以上０．３ｗ／ｖ％以下の量で配合されることがより好ましい。コラーゲンハイドロゲルの量が上記範囲内であると、培養中の三次元皮膚モデルを構成する真皮層の収縮を抑制することができる。 The second layer 12 preferably contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%, and more preferably 0 w/v% or more and 0.3 w/v% or less. If the amount of collagen hydrogel is within the above range, it is possible to suppress contraction of the dermis layer that constitutes the three-dimensional skin model during culture.
　また、細胞活性阻害による真皮線維芽細胞の増殖能の阻害が生じにくく、また、増殖した細胞が存在し得る空間が広大にあるため、真皮線維芽細胞の増殖が阻害されにくい。そのため、真皮層の細胞密度が長期間に亘って高く維持されやすく、これにより、サンプルが真皮層に与える評価を高感度に行いやすく、また、クロストークに関与するタンパク質（サイトカイン、ケモカイン等）、脂質成分（エイコサノイド等）等が産生されて表皮層へと伝達する結果、培養中の三次元皮膚モデルを構成する表皮層の分化構造を長時間に亘って高度に維持しやすい。 In addition, the proliferation ability of dermal fibroblasts is not easily inhibited by inhibition of cell activity, and since there is a large space in which proliferated cells can exist, the proliferation of dermal fibroblasts is not easily inhibited. As a result, the cell density in the dermis layer is easily maintained at a high level for a long period of time, which makes it easy to evaluate the effect of the sample on the dermis layer with high sensitivity. In addition, proteins involved in crosstalk (cytokines, chemokines, etc.) and lipid components (eicosanoids, etc.) are produced and transmitted to the epidermis layer, making it easy to maintain the differentiated structure of the epidermis layer that constitutes the three-dimensional skin model during culture at a high level for a long period of time.
　≪介在膜≫
　介在膜１３は、物質を透過させることを可能とする多孔質の膜であり、細胞外マトリクスを少なくとも表面に含む。介在膜が細胞外マトリクスを少なくとも表面に含むことにより、角化細胞（ケラチノサイト）や線維芽細胞（ファイブロブラスト）をコラーゲンハイドロゲルに依存しなくても接着させることが容易になる。 <Intervening membrane>
The intervening membrane 13 is a porous membrane that allows substances to pass through and contains an extracellular matrix at least on its surface. By containing an extracellular matrix at least on its surface, the intervening membrane can easily adhere keratinocytes and fibroblasts without relying on a collagen hydrogel.
　介在膜は、実質的に細胞外マトリクス層のみからなってもよく、基材の表面に細胞外マトリクスを含んでもよい。表面上の細胞外マトリクスは、特に限定されないが、存在頻度の高さからコーティング膜であることが好ましい。 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 frequent presence.
　基材の材質の例には、ＰＥＴ（ポリエチレンテレフタレート）やＰＣ（ポリカーボネート）の膜、ＰＥＴが挙げられる。細胞外マトリクスの例には、Ｉ型コラーゲン、ＩＩＩ型コラーゲン、ＩＶ型コラーゲン、フィブロネクチン、ゼラチン等が含まれる。 Examples of substrate materials include PET (polyethylene terephthalate) and PC (polycarbonate) membranes, and PET. Examples of extracellular matrices include type I collagen, type III collagen, type IV collagen, fibronectin, gelatin, etc.
　また、介在膜の平均孔径は、０．４μｍ以上であることが好ましい。また、介在膜の平均孔径は、８．０μｍ以下であってよく、６．０μｍ以下であることが好ましい。介在膜の平均孔径が上記範囲内であると、クロストークに関与するタンパク質（サイトカイン、ケモカイン等）、脂質成分（エイコサノイド等）の透過を良好に進行させることができる。介在膜の孔径は、例えば、電子顕微鏡の画像を用いて長径を測定することで求められる。 The average pore size of the intervening membrane is preferably 0.4 μm or more. The average pore size of the intervening membrane may be 8.0 μm or less, and is 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 proceed smoothly. The pore size of the intervening membrane can be determined, for example, by measuring the long axis using an image taken with an electron microscope.
　介在膜の平均厚さは、培地成分（クロストークに関与する物質、例えばＫＧＦ、ＨＧＦ、ＩＧＦ－１等）や後述の追加の層に依存しなくても、表皮及び真皮の構造が長期間にわたって維持される皮膚モデルが得られやすい観点で、２０μｍ以下であることが好ましく、より好ましくは１５μｍ以下、１０μｍ以下、７．５μｍ以下、または５μｍ以下であってよい。ただし、特定の培地成分（クロストークに関与する物質、例えばＫＧＦ、ＨＧＦ、ＩＧＦ－１等）を用いたり、追加の層を設けたりする場合等においては、これに限定されず、介在膜の平均厚さは、例えば２０μｍ以上２００μｍ以下であってよい。介在膜の厚さが上記範囲内であると、三次元皮膚モデルの強度が高まり、また、真皮層と表皮層のクロストークが高度になされるため、表皮層の分化が適切に進んだ状態（有棘層及び顆粒層が厚くなる）、基底層の密度が高い状態を維持しやすい。介在膜の厚さは、例えば、組織化学切片の光学顕微鏡の画像を用いて測定することができる。 The average thickness of the intervening membrane is preferably 20 μm or less, and more preferably 15 μm or less, 10 μm or less, 7.5 μm or less, or 5 μm or less, from the viewpoint of obtaining a skin model in which the structure of the epidermis and dermis is maintained for a long period of time without relying on medium components (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) or additional layers described below. However, this is not limited to the above, and the average thickness of the intervening membrane may be, for example, 20 μm or more and 200 μm or less, in cases where a specific medium component (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) is used or an additional layer is provided. When the thickness of the intervening membrane is within the above range, the strength of the three-dimensional skin model is increased, and since the crosstalk between the dermis layer and the epidermis layer is highly performed, it is easy to maintain a state in which the differentiation of the epidermis layer has progressed appropriately (the spinous layer and granular layer are thick) and a state in which the density of the basal layer is high. The thickness of the intervening membrane can be measured, for example, using an optical microscope image of a histochemical section.
　なお、介在膜は、市販のセルカルチャーインサートの膜を用いてもよい。 The intervening membrane may be a membrane from a commercially available cell culture insert.
　本実施形態に係る三次元皮膚モデルは、第１の層、第２の層、及び介在膜に加え、他の層を有してもよいし、有しなくてもよい。追加の層としては、第２の層の介在膜とは反対側に位置する脂肪層等の皮下組織層が挙げられる。 The three-dimensional skin model according to this embodiment may or may not have other layers in addition to the first layer, second layer, and intervening membrane. Additional layers include a subcutaneous tissue layer, such as an adipose layer, located on the opposite side of the second layer from the intervening membrane.
　追加の層自体の評価を行う場合、当該追加の層を有することが好ましい。他方、追加の層を有する態様は、クロストークが更に促進されて、分化構造を維持しやすい一方、第１の層、第２の層及び追加の層のすべてに適した培養条件（例えば培地組成）の自由度が低い。本実施形態では、追加の層に依存しなくても分化構造を維持しやすいことから、追加の層を有しない態様が好ましい。 When evaluating the additional layer itself, it is preferable to have the additional layer. On the other hand, an embodiment having an additional layer further promotes crosstalk and makes it easier to maintain the differentiated structure, but there is less freedom in the culture conditions (e.g., medium composition) suitable for all of the first layer, second layer, and additional layer. In this embodiment, an embodiment not having an additional layer is preferable, since it is easier to maintain the differentiated structure without relying on the additional layer.
　（第２の実施形態）
　本発明の第２の実施形態に係る三次元皮膚モデルは、図１に示されるように、表皮層１１と、真皮層１２と、表皮層１１と真皮層１２との間に介在する介在膜１３と、を有する。表皮層１１は角層１５、顆粒層１６、有棘層１７、及び基底層１８を有する。 Second Embodiment
1 , a three-dimensional skin model according to a second embodiment of the present invention has an epidermis layer 11, a dermis layer 12, and an intervening membrane 13 interposed between the epidermis layer 11 and the dermis layer 12. The epidermis layer 11 has a stratum corneum 15, a granular layer 16, a spinous layer 17, and a basal layer 18.
　以下に、各構成要素について説明する。 Each component is explained below.
　≪表皮層≫
　表皮層１１は、最上層に角層細胞が角層１５を形成し、角層１５の直下に顆粒細胞が顆粒層１６を形成し、顆粒層１６の直下に有棘細胞（または棘細胞）が有棘層１７を形成し、有棘層１７の直下（最下層）に角化細胞（ケラチノサイト）より分化した基底細胞が基底層１８を形成する４層構造を有する。また、表皮層１１は、色素細胞（メラノサイト）を含んでいてもよい。 <Epidermis layer>
The epidermal layer 11 has a four-layer structure in which keratinocytes form the stratum corneum 15 in the uppermost layer, granular cells form the granular layer 16 just below the stratum corneum 15, spinous cells (or spinous cells) form the spinous layer 17 just below the granular layer 16, and basal cells differentiated from keratinocytes form the basal layer 18 just below the spinous layer 17 (lowest layer). The epidermal layer 11 may also contain pigment cells (melanocytes).
　＜角層＞
　角層１５は、上述したように、三次元皮膚モデルを構成する表皮層１１の最上層に位置し、角質細胞が構築する層状構造で構成されている。また、上記角質細胞は扁平な形状を有しており、分化の過程で核を消失している。また、角質細胞は、その細胞質内にケラチン線維が凝集しており、好酸性でヘマトキシリン・エオジン（ＨＥ）染色により淡赤色～濃赤色に染色される。 <Stratum corneum>
As described above, the stratum corneum 15 is located at the top of the epidermal layer 11 constituting the three-dimensional skin model, and is composed of a layered structure constructed by keratinocytes. The keratinocytes have a flat shape and lose their nuclei during the differentiation process. The keratinocytes contain aggregates of keratin fibers in their cytoplasm, are acidophilic, and stain light red to dark red when stained with hematoxylin and eosin (HE).
　＜顆粒層＞
　顆粒層１６は、上述したように、角層１５の直下であり、後述する有棘層１７の直上に位置し、顆粒細胞が構築する層状構造で構成されている。上記顆粒細胞は扁平な形状を有しており、好塩基性成分からなる顆粒を細胞内に含む。細胞内の顆粒はヘマトキシリン・エオジン（ＨＥ）染色により青紫色～淡青色に染色される。本実施形態に係る三次元皮膚モデルの顆粒層の厚さは、平均３μｍ以上であることが好ましく、より好ましくは平均６μｍ以上であり、さらに好ましくは平均８μｍ以上である。また、三次元皮膚モデルの顆粒層の厚さは平均３μｍ以上１０μｍ以下であることが好ましい。厚さは、例えば、組織化学切片の光学顕微鏡の画像を用いて測定することができる。 <Granular layer>
As described above, the granular layer 16 is located immediately below the stratum corneum 15 and immediately above the spinous layer 17 described below, and is composed of a layered structure constructed by granular cells. The granular cells have a flat shape and contain granules made of basophilic components within the cells. The granules within the cells are stained blue-purple to light blue by hematoxylin-eosin (HE) staining. The thickness of the granular layer of the three-dimensional skin model according to this embodiment is preferably 3 μm or more on average, more preferably 6 μm or more on average, and even more preferably 8 μm or more on average. In addition, the thickness of the granular layer of the three-dimensional skin model is preferably 3 μm or more and 10 μm or less on average. The thickness can be measured, for example, using an optical microscope image of a histochemical section.
　＜有棘層＞
　有棘層１７は、上述したように、顆粒層１６の直下であり、後述する基底層１８の直上に位置し、有棘細胞が構築する層状構造で構成されている。上記有棘細胞は扁平な形状を有し、細胞周囲に棘が配置された構造を有する。上記棘はヘマトキシリン・エオジン（ＨＥ）染色により確認することができる。本実施形態に係る三次元皮膚モデルの有棘層の厚さは、平均１８μｍ以上であることが好ましく、より好ましくは平均２６μｍ以上であり、さらに好ましくは平均３９μｍ以上である。また、三次元皮膚モデルの有棘層の厚さは平均１８μｍ以上５０μｍ以下であることが好ましい。厚さは、例えば、組織化学切片の光学顕微鏡の画像を用いて測定することができる。 <Stratum spinosum>
As described above, the spinous layer 17 is located directly below the granular layer 16 and directly above the basal layer 18 described later, and is composed of a layered structure constructed by spinous cells. The 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 (HE) staining. The thickness of the spinous layer of the three-dimensional skin model according to this embodiment is preferably 18 μm or more on average, more preferably 26 μm or more on average, and even more preferably 39 μm or more on average. In addition, the thickness of the spinous layer of the three-dimensional skin model is preferably 18 μm or more and 50 μm or less on average. The thickness can be measured, for example, using an optical microscope image of a histochemical section.
　＜基底層＞
　基底層１８は、上述したように、表皮層１１の最下層に位置し、基底細胞が構築する層構造で構成される。上記基底細胞は、角層細胞、顆粒細胞、及び有棘細胞と異なり、立方体～円柱状の形状を有し、かつ、楕円形の核を有する。基底細胞は好塩基性であり、ヘマトキシリン・エオジン（ＨＥ）染色により藍色～淡青色に染色される。本実施形態に係る三次元皮膚モデルの基底層の厚さは、平均７μｍ以上であることが好ましく、より好ましくは平均９μｍ以上であり、さらに好ましくは平均１４μｍ以上である。また、三次元皮膚モデルの有棘層の厚さは平均７μｍ以上２０μｍ以下であることが好ましい。厚さは、例えば、組織化学切片の光学顕微鏡の画像を用いて測定することができる。 <Basal layer>
As described above, the basal layer 18 is located at the bottom of the epidermal layer 11 and is composed of a layer structure constructed by basal cells. The basal cells, unlike keratinocytes, granular cells, and spinous cells, have a cubic to cylindrical shape and an elliptical nucleus. The basal cells are basophilic and stained in indigo to light blue by hematoxylin-eosin (HE) staining. The thickness of the basal layer of the three-dimensional skin model according to this embodiment is preferably 7 μm or more on average, more preferably 9 μm or more on average, and even more preferably 14 μm or more on average. In addition, the thickness of the spinous layer of the three-dimensional skin model is preferably 7 μm or more and 20 μm or less on average. The thickness can be measured, for example, using an optical microscope image of a histochemical section.
　［真皮層］
　真皮層１２は、多層構造を有する。具体的には、真皮層１２は、線維芽細胞及び線維芽細胞が合成、分泌した膠原線維（コラーゲンなど）、弾性線維（フィブリリン・エラスチンなど）及びそのほかの細胞外マトリックス成分（ヒアルロン酸など）から構成される組織構造を有する。 [Dermis layer]
The dermis layer 12 has a multi-layer structure. Specifically, the dermis layer 12 has a tissue structure composed of fibroblasts, collagen fibers (such as collagen) synthesized and secreted by the fibroblasts, elastic fibers (such as fibrillin and elastin), and other extracellular matrix components (such as hyaluronic acid).
　「多層構造」とは、線維芽細胞が、垂直方向（角層方向）に２層以上の層状を形成している構造をいう。ここで、線維芽細胞の配向は一定ではない。そのため、線維芽細胞が横方向に配向したため、垂直方向に薄切された皮膚組織切片像が紡錘形を呈したり、縦方向に配向したため、皮膚組織切片像が円形を呈することがある。 "Multilayer structure" refers to a structure in which fibroblasts form two or more layers in the vertical direction (towards the stratum corneum). Here, the orientation of the fibroblasts is not uniform. As a result, the image of a vertically sliced skin tissue section may appear spindle-shaped due to the fibroblasts being oriented horizontally, or circular due to the fibroblasts being oriented vertically.
　真皮層（真皮線維芽細胞）の細胞密度は２０００ｃｅｌｌｓ／ｃｍ２以上であり、５０００ｃｅｌｌｓ／ｃｍ２以上であることが好ましく、６０００ｃｅｌｌｓ／ｃｍ２以上であることが好ましい。また、真皮層の細胞密度は２０００ｃｅｌｌｓ／ｃｍ２以上１００００ｃｅｌｌｓ／ｃｍ２以下であることが好ましく、５０００ｃｅｌｌｓ／ｃｍ２以上８０００ｃｅｌｌｓ／ｃｍ２以下であることがより好ましく、６０００ｃｅｌｌｓ／ｃｍ２以上７７００ｃｅｌｌｓ／ｃｍ２以下であることがさらに好ましい。真皮層の細胞密度が上記範囲内であると、三次元皮膚モデルの層構造を長期間維持することができる。 The cell density of the dermis layer (dermal fibroblasts) is 2000 cells/cm 2 or more, preferably 5000 cells/cm 2 or more, and preferably 6000 cells/cm 2 or more. The cell density of the dermis layer is preferably 2000 cells/cm 2 or more and 10000 cells/cm 2 or less, more preferably 5000 cells/cm 2 or more and 8000 cells/cm 2 or less, and even more preferably 6000 cells/cm 2 or more and 7700 cells/cm 2 or less. When the cell density of the dermis layer is within the above range, the layer structure of the three-dimensional skin model can be maintained for a long period of time.
　（第３の実施形態）
　本発明の第３の実施形態に係る三次元皮膚モデルは、図１に示されるように、表皮層１１と、真皮層１２と、表皮層１１と真皮層１２との間に介在する介在膜１３と、を有する。表皮層１１は角層１５、顆粒層１６、有棘層１７、及び基底層１８を有する。 Third Embodiment
1 , a three-dimensional skin model according to a third embodiment of the present invention has an epidermis layer 11, a dermis layer 12, and an intervening membrane 13 interposed between the epidermis layer 11 and the dermis layer 12. The epidermis layer 11 has a stratum corneum 15, a granular layer 16, a spinous layer 17, and a basal layer 18.
　また、真皮層１２は多層構造を有し、かつ、コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合される。なお、上記ｖは、真皮層の体積を意味する。真皮層１２は、コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．３ｗ／ｖ％以下の量で配合されることがより好ましい。あるいは、真皮層１２が、ヘマトキシリン・エオジン（ＨＥ）染色によりコラーゲン線維が不均等に分散した状態ではない。これにより、増殖した細胞が存在し得る空間が広大にあるため、真皮線維芽細胞の増殖能が阻害にくい。そのため、培養中の三次元皮膚モデルを構成する真皮層の収縮を抑制することができる。 The dermis layer 12 has a multi-layer structure, and contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%. The above "v" refers to the volume of the dermis layer. It is more preferable that the dermis layer 12 contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.3 w/v%. Alternatively, the dermis layer 12 does not have collagen fibers unevenly dispersed as determined by hematoxylin-eosin (HE) staining. This provides a large space in which proliferated cells can exist, making it difficult to inhibit the proliferation ability of dermal fibroblasts. This makes it possible to suppress the contraction of the dermis layer that constitutes the three-dimensional skin model during culture.
　また、細胞活性阻害による真皮線維芽細胞の増殖能の阻害が生じにくく、また、増殖した細胞が存在し得る空間が広大にあるため、真皮線維芽細胞の増殖が阻害されにくい。そのため、真皮層の細胞密度が長期間に亘って高く維持されやすく、これにより、サンプルが真皮層に与える評価を高感度に行いやすく、また、クロストークに関与するタンパク質（サイトカイン、ケモカイン等）、脂質成分（エイコサノイド等）等が産生されて表皮層へと伝達する結果、培養中の三次元皮膚モデルを構成する表皮層の分化構造を長時間に亘って高度に維持しやすい。 In addition, the proliferation ability of dermal fibroblasts is not easily inhibited by inhibiting cell activity, and since there is a large space in which proliferated cells can exist, the proliferation of dermal fibroblasts is not easily inhibited. As a result, the cell density in the dermis layer is easily maintained at a high level for a long period of time, which makes it easy to evaluate the effect of the sample on the dermis layer with high sensitivity. In addition, proteins involved in crosstalk (cytokines, chemokines, etc.) and lipid components (eicosanoids, etc.) are produced and transmitted to the epidermis layer, making it easy to maintain the differentiated structure of the epidermis layer that constitutes the three-dimensional skin model during culture at a high level for a long period of time.
　本実施形態に係る三次元皮膚モデルは、表皮層、真皮層、及び介在膜に加え、他の層を有してもよいし、有しなくてもよい。追加の層としては、真皮層の介在膜とは反対側に位置する脂肪層等の皮下組織層が挙げられる。 The three-dimensional skin model according to this embodiment may or may not have other layers in addition to the epidermis layer, dermis layer, and intervening membrane. Additional layers include a subcutaneous tissue layer, such as an adipose layer, located on the opposite side of the dermis layer from the intervening membrane.
　追加の層を有する態様は、クロストークが更に促進されて、分化構造を維持しやすい一方、表皮層、真皮層及び追加の層のすべてに適した培養条件（例えば培地組成）の自由度が低い。本実施形態では、追加の層に依存しなくても分化構造を維持しやすいことから、追加の層を有しない態様が好ましい。 In the case of an embodiment having an additional layer, crosstalk is further promoted and the differentiated structure is easily maintained, but there is less freedom in the culture conditions (e.g., medium composition) suitable for all of the epidermal layer, dermal layer, and additional layer. In this embodiment, the differentiated structure is easily maintained without relying on the additional layer, so an embodiment not having an additional layer is preferred.
　［培養容器］
　第１の実施形態から第３の実施形態で用いられる培養容器２０は、図２の（ａ）及び（ｂ）に示されるように、培養液２３を収容する複数のウェル２１と、ウェル２１内で三次元皮膚モデル１０を支持する支持部２２を有する。三次元皮膚モデル１０は、培養液２３と第２の層１２側又は真皮層１２側で連通可能となるように配置される。 [Culture vessel]
2(a) and 2(b), the culture vessel 20 used in the first to third embodiments has a plurality of wells 21 that contain a culture solution 23, and a support portion 22 that supports the three-dimensional skin model 10 in the wells 21. The three-dimensional skin model 10 is arranged so as to be in communication with the culture solution 23 on the second layer 12 side or the dermis layer 12 side.
　培養容器２０は、任意の数のウェルを有してよく、特に限定されないが、１２、２４、４８又は９６個のウェルを有することができる。これにより、多数のサンプル及び適用条件に関する評価を同時に実施することができる。 The culture vessel 20 may have any number of wells, including but not limited to 12, 24, 48, or 96 wells. This allows for the evaluation of multiple samples and application conditions simultaneously.
　［サンプルの評価方法］
　本発明のサンプル評価方法は、培養容器を用いて行う。サンプル評価方法は、具体的には、（１）図２の（ａ）及び（ｂ）に示される培養容器２０を用いて、三次元皮膚モデルを培養する工程（培養工程）と、（２）培養中の三次元皮膚モデルにサンプルを投与する工程（サンプル投与工程）と、（３）サンプル投与から所定の培養期間経過後の三次元皮膚モデル又は培養上清を分析することで、上記サンプルが三次元皮膚モデルに与える機能を評価する工程（評価工程）と、を含む。 [Sample evaluation method]
The sample evaluation method of the present invention is carried out using a culture vessel. Specifically, the sample evaluation method includes the steps of (1) culturing a three-dimensional skin model using a culture vessel 20 shown in (a) and (b) of Figure 2 (culture step), (2) administering a sample to the three-dimensional skin model being cultured (sample administration step), and (3) analyzing the three-dimensional skin model or culture supernatant after a predetermined culture period has elapsed since the administration of the sample, thereby evaluating the function that the sample provides to the three-dimensional skin model (evaluation step).
　以下、各工程について説明する。 Each process is explained below.
　（培養工程）
　培養工程は、より具体的には、培養容器２０のウェル２１内に、三次元皮膚モデル１０の第２の層１２側又は真皮層１２側を培養液２３と連通可能となるように配置して、三次元皮膚モデル１０を所定の期間培養する工程である。 (Cultivation process)
More specifically, the culture process is a process of placing the second layer 12 side or the dermis layer 12 side of the three-dimensional skin model 10 in the well 21 of the culture container 20 so that it can communicate with the culture solution 23, and culturing the three-dimensional skin model 10 for a predetermined period of time.
　培養期間は、６日以上であり、１２日以上であり、２０日以上であることがより好ましい。また、培養期間は３０日以下であってよい。培養期間が６日以上であることにより、評価できる期間が長いので、十分な評価結果を得ることができる。 The culture period is 6 days or more, preferably 12 days or more, and more preferably 20 days or more. The culture period may be 30 days or less. A culture period of 6 days or more provides a long period for evaluation, making it possible to obtain sufficient evaluation results.
　（サンプル投与工程）
　サンプル投与工程は、より具体的には、培養容器２０のウェル２１内に配置された三次元皮膚モデル１０に対してサンプルを所定の方法で投与する工程である。 (Sample administration step)
More specifically, the sample administration step is a step of administering a sample to the three-dimensional skin model 10 placed in the well 21 of the culture container 20 in a predetermined manner.
　投与するサンプルは、特に限定されないが、単離された１又は２以上の物質であってもよく、多様な成分の混合物（例えば化粧品、医薬品）であってもよい。サンプルの形態は特に限定されず、液剤、ゲル、クリーム、粉体等であってよい。また、サンプルの投与方法は、特に限定されず、表皮の表面に適用してもよいし、表皮の内部に注入してもよいし、真皮の内部に注入してもよいし、培養液に添加してもよい。 The sample to be administered is not particularly limited, but may be one or more isolated substances, or may be 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.
　また、各ウェルには、互いに異なるサンプルを適用してもよく、また、同じサンプルを異なる方法（用量及び／又は用法）で適用してもよい。これにより、サンプルが皮膚に与える性能を定量的に対比したり、所望の機能を皮膚に与えるために好ましい投与方法（用量及び／又は用法）を推測したりすることができる。 Also, different samples may be applied to each well, or the same sample may be applied in different ways (dosages and/or administration methods). This allows for quantitative comparison of the performance of the samples on the skin, and allows for estimation of a preferred administration method (dosage and/or administration method) for imparting a desired function to the skin.
　（評価工程）
　評価工程は、より具体的には、サンプル投与から、数時間以上の培養期間経過後の三次元皮膚モデル１０又は細胞が分泌する分泌液等を含む培養上清を分析することで、サンプルが三次元皮膚モデル１０に与える機能を評価する工程である。 (Evaluation process)
More specifically, the evaluation process is a process of evaluating the function that the sample provides to the three-dimensional skin model 10 by analyzing the three-dimensional skin model 10 or the culture supernatant containing secretory fluids secreted by the cells after a culture period of several hours or more has elapsed since administration of the sample.
　上記分析は、遺伝子発現、タンパク質発現、組織化学分析、又は皮膚バリア機能分析を含むことが好ましく、これらの２種以上の組み合わせを含んでいてもよい。 The above analyses preferably include gene expression, protein expression, histochemical analysis, or skin barrier function analysis, and may include a combination of two or more of these.
　また、評価工程には、上述の培養期間において、三次元皮膚モデル１０の加工を行う工程、三次元皮膚モデル１０に２回以上の同一又は異なるサンプルの投与を行う工程、又は三次元皮膚モデル１０に投与したサンプルの除去を行う工程を含んでもよい。 The evaluation process may also include a process of processing the three-dimensional skin model 10 during the above-mentioned culture period, a process of administering the same or different samples to the three-dimensional skin model 10 two or more times, or a process of removing the samples administered to the three-dimensional skin model 10.
　三次元皮膚モデル１０の加工を行う工程は、状態の異なる三次元皮膚モデルを作製する工程である。加工方法は、特に限定されないが、酸化ストレス誘導のための起炎性物質の添加、ＵＶ等の酸化ストレスを惹起するような光線照射等が含まれる。この加工には数秒間～数日間の時間を要し得るが、本実施形態で使う皮膚モデルは長時間に亘って表皮層及び真皮層の構造を維持できるため、加工後の皮膚モデルを使った評価を自由に行いやすい。状態の異なる三次元皮膚モデルを作製することにより、正常な状態の皮膚に対する評価だけでなく、肌荒れした状態の皮膚や炎症を起こした皮膚等についても評価を行うことができるので、様々な皮膚状態に対するサンプルの有効性等を取得することができる。 The process of processing the three-dimensional skin model 10 is a process of producing three-dimensional skin models in different conditions. The processing method is not particularly limited, but includes the addition of an inflammatory substance to induce oxidative stress, and irradiation with light such as UV light that induces oxidative stress. This processing can take anywhere from a few seconds to a few days, but the skin model used in this embodiment can maintain the structure of the epidermis and dermis layers for a long period of time, making it easy to freely perform evaluations using the processed skin model. By producing three-dimensional skin models in different conditions, it is possible to evaluate not only normal skin, but also rough and inflamed skin, making it possible to obtain the effectiveness of samples for various skin conditions.
　２回以上の同一又は異なるサンプルの投与を行う工程は、同じサンプルを同じ方法で投与してもよいし、異なる方法で投与してもよい。また、異なるサンプルを同じ方法で投与してもよいし、異なる方法で投与してもよい。ここでいう「方法」とは、用量及び／又は用法を指す。 In the step of administering the same or different samples two or more times, the same sample may be administered by the same method or by different methods. Also, different samples may be administered by the same method or by different methods. Here, "method" refers to dosage and/or method of use.
　また、サンプルを投与した三次元皮膚モデルと比較するために、少なくともサンプルを投与しない三次元皮膚モデルを、上述の培養工程で所定の期間（例えば６日間以上、好ましくは１２日以上であり、２０日以上である。３０日以下であってよい。）培養してもよい。ここで、サンプルが投与されていない三次元皮膚モデルが、培養期間経過後において、角層、顆粒層、有棘層、及び基底層を有する表皮層と、多層構造を有する真皮層と、を有するような条件で、培養を行うことが好ましい。 Furthermore, in order to compare with a three-dimensional skin model to which a sample has been administered, at least a three-dimensional skin model to which a sample has not been administered may be cultured for a predetermined period (e.g., 6 days or more, preferably 12 days or more, 20 days or more, and may be 30 days or less) in the above-mentioned culture process. Here, it is preferable to culture under conditions such that the three-dimensional skin model to which a sample has not been administered has an epidermis layer having a stratum corneum, granular layer, spinous layer, and basal layer, and a dermis layer having a multi-layer structure, after the culture period has elapsed.
　培養期間経過後における、三次元皮膚モデルの表皮層の平面積と真皮層の平面積の大きさとの関係が、表皮層の平面積≦真皮層の平面積を満たすか、又は培養期間経過後における真皮層の平面積が、培養期間経過前における真皮層の平面積の０．７倍以上であることが好ましく、特に限定されないが、０．９倍以上であることがより好ましい。また、培養期間経過後における真皮層の平面積が、培養期間経過前における真皮層の平面積の１．０倍以下であることが好ましい。三次元皮膚モデルの表皮層の平面積と真皮層の平面積の大きさとの関係が、上記範囲内にあることにより、培養中の真皮層の収縮が抑制されるので、長期間の評価が可能となる。また、表皮層の平面積は、培養期間経過前における真皮層の平面積の０．７倍以上１．０倍以下であることが好ましく、より好ましくは０．９倍以上１．０倍以下である。 After the culture period, the relationship between the planar area of the epidermal layer and the planar area of the dermal layer of the three-dimensional skin model is preferably such that the planar area of the epidermal layer is less than or equal to the planar area of the dermal layer, or the planar area of the dermal layer after the culture period is 0.7 times or more of the planar area of the dermal layer before the culture period, and is not particularly limited, but is more preferably 0.9 times or more. Furthermore, the planar area of the dermal layer after the culture period is preferably 1.0 times or less of the planar area of the dermal layer before the culture period. By having the relationship between the planar area of the epidermal layer and the planar area of the dermal layer of the three-dimensional skin model within the above range, contraction of the dermal layer during culture is suppressed, making it possible to perform long-term evaluation. Furthermore, the planar area of the epidermal layer is preferably 0.7 times or more and 1.0 times or less of the planar area of the dermal layer before the culture period, and more preferably 0.9 times or more and 1.0 times or less.
　以下、本発明について実施例を示してより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to the following examples.
　［三次元皮膚モデルの構築］
　コラーゲン膜を有するセルカルチャーインサート（ａｄ－ＭＥＤビトリゲル２ ０８３６４－９６（関東化学株式会社製）、ＡｔｅｌｏＣｅｌｌ ＣＭ－２４（株式会社高研製））を準備した。セルカルチャーインサートの片方の面に１０％ＦＢＳ含有ＤＭＥＭに懸濁された正常ヒト皮膚線維芽細胞１０，０００個を播種し、３７℃で１日間、ＣＯ２インキュベーター内で培養した。次いで、セルカルチャーインサートのもう一方の面にＨｕｍｅｄｉａ ＫＧ２（クラボウ社製）に懸濁された正常ヒト表皮角化細胞５００，０００個を播種し、３７℃で１日間、ＣＯ２インキュベーター内で培養後、三次元培養用の培地に置換し、さらに１日間培養を継続した。培養後、正常ヒト角化表皮細胞側の培地を除去し、気相培養を開始した。１２日間気相培養を行うことにより、表皮層分化を誘導し、三次元皮膚モデルを構築した。なお、「ビトリゲル」は国立研究開発法人農業・食品産業技術総合研究機構の、「ＡｔｅｌｏＣｅｌｌ」は株式会社高研の、「Ｈｕｍｅｄｉａ」は倉敷紡績株式会社の登録商標である。 [Construction of a three-dimensional skin model]
A cell culture insert having a collagen membrane (ad-MED Vitrigel 2 08364-96 (Kanto Chemical Co., Ltd.), AteloCell CM-24 (Koken Co., Ltd.)) was prepared. 10,000 normal human dermal fibroblasts suspended in 10% FBS-containing DMEM were seeded on one side of the cell culture insert and cultured at 37°C for 1 day in a CO2 incubator. Next, 500,000 normal human epidermal keratinocytes suspended in Humedia KG2 (Kurabo) were seeded on the other side of the cell culture insert and cultured at 37°C for 1 day in a CO2 incubator, after which the medium was replaced with a medium for three-dimensional culture and culture was continued for another day. After culture, the medium on the normal human keratinized epidermal cell side was removed and gas phase culture was started. By performing gas phase culture for 12 days, epidermal layer differentiation was induced and a three-dimensional skin model was constructed. "Vitrigel" is a registered trademark of the National Agriculture and Food Research Organization, "AteloCell" is a registered trademark of Koken Co., Ltd., and "Humedia" is a registered trademark of Kuraray Industries, Inc.
　構築した三次元皮膚モデルの顆粒層、有棘層、基底層の厚さは、それぞれ３～８μｍ、１８～３９μｍ、７～１４μｍであり、真皮層の細胞密度は、７１１４±３３９ｃｅｌｌｓ／ｃｍ２であり、気相培養を２０日間行った後において５８４３±３４９ｃｅｌｌｓ／ｃｍ２であった。なお、本発明者が調べたところによると、非特許文献１に開示されるモデルの真皮層の細胞密度は１３００±３３９ｃｅｌｌｓ／ｃｍ２であり、本実施例に比べて顕著に低かった。また、構築した三次元皮膚モデルの培養期間経過後における真皮層及び表皮層の平面積が、培養期間経過前における真皮層及び表皮層の平面積のそれぞれ１倍であった。 The thicknesses of the granular layer, spinous layer, and basal layer of the constructed three-dimensional skin model were 3-8 μm, 18-39 μm, and 7-14 μm, respectively, and the cell density of the dermis layer was 7114±339 cells/cm 2 , and 5843±349 cells/cm 2 after 20 days of gas phase culture. According to the inventor's investigation, the cell density of the dermis layer of the model disclosed in Non-Patent Document 1 was 1300±339 cells/cm 2 , which was significantly lower than that of this example. In addition, the planar areas of the dermis layer and epidermis layer of the constructed three-dimensional skin model after the culture period were each 1-fold the planar areas of the dermis layer and epidermis layer before the culture period.
　［三次元皮膚モデルの組織化学解析１］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの皮膚組織をスーパーフィックス ＫＹ－５００（クラボウ社製）で固定後、Ｐａｒａｐｌａｓｔ Ｘ－ｔｒａ（Ｌｅｉｃａ Ｂｉｏｓｙｓｔｅｍｓ社製）中に包埋し、パラフィン包埋切片（皮膚組織切片）を作製した。皮膚組織切片を、ヘマトキシリン・エオジン（ＨＥ）染色で染色後、オールインワン蛍光顕微鏡「ＢＺ－Ｘ８００」（株式会社キーエンス製）で解析を行った。なお、「Ｐａｒａｐｌａｓｔ」は、Ｌｅｉｃａ Ｂｉｏｓｙｓｔｅｍｓ Ｒｉｃｈｍｏｎｄ社の登録商標である。 [Histochemical analysis of three-dimensional skin model 1]
(analysis method)
The skin tissue of the three-dimensional skin model that had been gas-phase cultured for 12 days using the above method was fixed with Superfix KY-500 (Kurabo Industries, Ltd.), embedded in Paraplast X-tra (Leica Biosystems, Inc.), and paraffin-embedded sections (skin tissue sections) were prepared. The skin tissue sections were stained with hematoxylin-eosin (HE) staining, and then analyzed with an all-in-one fluorescent microscope "BZ-X800" (Keyence Corporation). "Paraplast" is a registered trademark of Leica Biosystems Richmond, Inc.
　（結果）
　図１のＨＥ染色後の皮膚組織切片画像から示されるように、三次元皮膚モデルはヒト皮膚組織と同様に、表皮層は角質層、顆粒層（細胞内顆粒を含む扁平化した細胞）、有棘層（細胞周囲に棘を有する）、基底層（立方体形態）から構成されていることを確認できた。また、基底層の直下に真皮層として線維芽細胞が層状に配置しており皮膚モデルとしての基本的な形態的特徴を示していることも確認することができた。 (result)
As shown in the HE stained skin tissue slice image in Fig. 1, it was confirmed that the three-dimensional skin model, like human skin tissue, is composed of the epidermis layer, stratum corneum, stratum granulosum (flattened cells containing intracellular granules), stratum spinosum (having spines around the cells), and stratum basale (cuboidal shape). It was also confirmed that fibroblasts are arranged in layers as the dermis layer just below the stratum basale, showing the basic morphological characteristics of a skin model.
　［三次元皮膚モデルの組織化学解析２］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの皮膚組織に対して、さらに１週間以上の培養を継続した。培養後の皮膚組織を、ヘマトキシリン・エオジン（ＨＥ）染色で染色後、オールインワン蛍光顕微鏡「ＢＺ－Ｘ８００」で解析を行った。 [Histochemical analysis of three-dimensional skin model 2]
(analysis method)
The skin tissue of the three-dimensional skin model that had been gas-phase cultured for 12 days using the method described above was further cultured for at least one week. After the culture, the skin tissue was stained with hematoxylin and eosin (HE) and analyzed using an all-in-one fluorescence microscope "BZ-X800."
　（結果）
　図３に示される、１２日間気相培養した皮膚組織（ａ）の皮膚組織切片画像と２０日間培養した皮膚組織（ｂ）の皮膚組織切片画像とを比較すると、培養２０日後においても表皮層の４層構造が維持されており、従来技術よりも長い期間、三次元皮膚モデルを維持できることを確認することができた。 (result)
Comparing the image of a skin tissue section of skin tissue (a) cultured in a gas phase for 12 days with the image of a skin tissue section of skin tissue (b) cultured for 20 days shown in Figure 3, it was confirmed that the four-layer structure of the epidermis was maintained even after 20 days of culture, and that the three-dimensional skin model could be maintained for a longer period than with conventional technology.
　［三次元皮膚モデルの組織化学解析２］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの皮膚組織をスーパーフィックス ＫＹ－５００で固定後、Ｐａｒａｐｌａｓｔ Ｘ－ｔｒａ中に包埋し、パラフィン包埋切片（皮膚組織切片）を作製した。作製した皮膚組織切片に、表皮層中の局在が明らかとなっているタンパク質に対する抗体を用いた免疫組織化学染色を行った。 [Histochemical analysis of three-dimensional skin model 2]
(analysis method)
The skin tissue of the three-dimensional skin model that had been cultured in the air for 12 days using the method described above was fixed with Superfix KY-500 and embedded in Paraplast X-tra to prepare paraffin-embedded sections (skin tissue sections). The prepared skin tissue sections were subjected to immunohistochemical staining using an antibody against a protein whose localization in the epidermis layer has been clarified.
　（結果）
　図４の（ａ）で示されるように、三次元皮膚モデル３０の角層１５から有棘層１７にかけて、分化マーカーであるサイトケラチン１０（ＣＫ１０）の発現が確認され、基底層１８では、サイトケラチン１４（ＣＫ１４）の発現が確認された。また、図４の（ｂ）で示されるように、ケラトヒアリン顆粒の成分であるフィラグリン（不図示）は顆粒層１６（顆粒細胞内の顆粒）に発現が確認され、タイトジャンクション構成接着分子であるクローディン１（不図示）は有棘層１６（有棘細胞の細胞周囲）に発現が確認された。図４の（ａ）及び（ｂ）より、三次元皮膚モデルの形態的特徴を分化マーカー分子の局在から確認することができた。 (result)
As shown in (a) of FIG. 4, the expression of cytokeratin 10 (CK10), a differentiation marker, was confirmed from the stratum corneum 15 to the spinous layer 17 of the three-dimensional skin model 30, and the expression of cytokeratin 14 (CK14) was confirmed in the basal layer 18. As shown in (b) of FIG. 4, the expression of filaggrin (not shown), a component of keratohyalin granules, was confirmed in the granular layer 16 (granules in granular cells), and the expression of claudin 1 (not shown), a tight junction-constituting adhesion molecule, was confirmed in the spinous layer 16 (cell periphery of spinous cells). From (a) and (b) of FIG. 4, the morphological characteristics of the three-dimensional skin model could be confirmed from the localization of the differentiation marker molecules.
　［三次元培養皮膚モデルのバリア機能解析１］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの表皮表面に０～０．５％（０．１％刻み）の濃度に調製した界面活性剤であるドデシル硫酸ナトリウム（ＳＤＳ）溶液を塗布し、３７℃で１時間、ＣＯ２インキュベーター内で培養した。培養後、表皮表面に残ったＳＤＳ溶液を除去し、リン酸緩衝食塩水（ＰＢＳ）（－）で残存するＳＤＳ溶液を洗い流し、さらに３７℃で２４時間、ＣＯ２インキュベーター内で回復培養を行った。培養後の細胞障害性（Ｃｙｔｏｔｏｘｉｃｉｔｙ）は、培地中に漏出した細胞質内酸化還元酵素（ＬＤＨ）活性を指標としてＣｙｔｏｔｏｘｉｃｉｔｙ ＬＤＨ Ａｓｓａｙ Ｋｉｔ－ＷＳＴ(株式会社同仁化学研究所製)により測定した。 [Barrier function analysis of three-dimensional cultured skin model 1]
(analysis method)
A surfactant sodium dodecyl sulfate (SDS) solution prepared at a concentration of 0-0.5% (in 0.1% increments) was applied to the epidermal surface of the three-dimensional skin model that had been gas-phase cultured for 12 days using the above-mentioned method, and the model was cultured in a CO 2 incubator for 1 hour at 37°C. After culture, the SDS solution remaining on the epidermal surface was removed, the remaining SDS solution was washed away with phosphate buffered saline (PBS) (-), and the model was further cultured for recovery at 37°C for 24 hours in a CO 2 incubator. Cytotoxicity after culture was measured using the Cytotoxicity LDH Assay Kit-WST (manufactured by Dojindo Laboratories, Inc.) using the cytoplasmic oxidoreductase (LDH) activity leaked into the medium as an indicator.
　（結果）
　図５に示されるように、３次元皮膚モデルの細胞障害性は、ＳＤＳ溶液の濃度が高まるにつれて上昇傾向を示したことから、３次元皮膚モデルは表皮におけるバリア機能を備えていることを確認することができた。 (result)
As shown in Figure 5, the cytotoxicity of the three-dimensional skin model tended to increase as the concentration of the SDS solution increased, confirming that the three-dimensional skin model has a barrier function in the epidermis.
　［三次元培養皮膚モデルのバリア機能解析２］
　（解析方法）
　上述の方法で、正常ヒト皮膚線維芽細胞と正常ヒト表皮角化細胞をインサートに播種し気相培養を開始した日を０日として経時的に三次元皮膚モデルの経上皮電気抵抗（ＴＥＥＲ）を測定した。 [Barrier function analysis of three-dimensional cultured skin model 2]
(analysis method)
Using the method described above, normal human skin fibroblasts and normal human epidermal keratinocytes were seeded onto the insert, and the day when the gas phase culture was started was set as day 0, and the transepithelial electrical resistance (TEER) of the three-dimensional skin model was measured over time.
　（結果）
　図６に示されるように、三次元皮膚モデルの成長に伴いバリア機能が増強し、そのバリア機能は三次元皮膚モデルの成熟後も維持されることを確認することができた。 (result)
As shown in Figure 6, it was confirmed that the barrier function was enhanced as the three-dimensional skin model grew, and that the barrier function was maintained even after the three-dimensional skin model matured.
　［三次元皮膚モデルの生理的刺激に対する応答性解析１］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの表皮（表面）にレチノイン酸（ＡＴＲＡ）の１０μＭ溶液を塗布し、３７℃で、１時間培養した。培養後、レチノイン酸溶液を除去し、さらに５時間培養を行った後、皮膚組織からＲＮＡを抽出しリアルタイムＰＣＲ法を用いて遺伝子発現解析を行った。 [Analysis of responsiveness of three-dimensional skin model to physiological stimuli 1]
(analysis method)
A 10 μM solution of retinoic acid (ATRA) was applied to the epidermis (surface) of the three-dimensional skin model that had been gas-phase cultured for 12 days using the method described above, and the model was cultured for 1 hour at 37° C. After culture, the retinoic acid solution was removed and the model was further cultured for 5 hours, after which RNA was extracted from the skin tissue and gene expression analysis was performed using real-time PCR.
　（結果）
　図７の（ａ）及び（ｂ）に示されるように、ＡＴＲＡ溶液の塗布により、ヘパリン結合性ＥＧＦ様増殖因子遺伝子（ＨＢＥＧＦ）及び表皮ヒアルロン酸合成酵素遺伝子３（ＨＡＳ３）の発現の誘導を確認することができた。 (result)
As shown in Figures 7(a) and (b), it was confirmed that application of the ATRA solution induced the expression of the heparin-binding EGF-like growth factor gene (HBEGF) and epidermal hyaluronan synthase gene 3 (HAS3).
　［三次元皮膚モデルの生理的刺激に対する応答性解析２］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの培地中に、終濃度が１０ｎＭとなるように調製したトランスフォーミング増殖因子β（ＴＧＦβ）を添加し、４８時間培養し、再構築された三次元皮膚モデルの遺伝子発現解析を行った。また、真皮層中の局在が明らかとなっているタンパク質に対する抗体を用いた免疫組織化学染色を行った。 [Analysis of responsiveness of three-dimensional skin model to physiological stimuli 2]
(analysis method)
Transforming growth factor β (TGFβ) was added to the medium of the three-dimensional skin model that had been aerated for 12 days, and the model was cultured for 48 hours. Gene expression analysis was performed on the reconstructed three-dimensional skin model. Immunohistochemical staining was also performed using an antibody against a protein known to be localized in the dermis layer.
　（結果）
　図８の（ａ）及び（ｂ）に示されるように、ＣＯＬ１Ａ１（Ｉ型コラーゲン遺伝子）及びＦＢＮ１（フィブリリン－１遺伝子）等の真皮の膠原線維や弾性線維の遺伝子の発現の誘導を確認することができた。 (result)
As shown in Figures 8(a) and (b), induction of expression of genes for collagen fibers and elastic fibers in the dermis, such as COL1A1 (type I collagen gene) and FBN1 (fibrillin-1 gene), was confirmed.
　図９の（ａ）及び（ｂ）に示されるように、免疫組織化学染色の真皮線維芽細胞層１４（真皮層）内のＩ型コラーゲン（４１）とフィブリリン－１（４２）の発現確認を行ったところ、ＴＧＦβを添加して培養した三次元皮膚モデル（４０）では、線維タンパク質の発現上昇及び局在変化を確認することができた。これにより、再構築された皮膚モデルが生理的刺激に対する応答性を有することも確認することができた。 As shown in Figures 9(a) and (b), the expression of type I collagen (41) and fibrillin-1 (42) in the dermal fibroblast layer 14 (dermis layer) was confirmed by immunohistochemical staining, and an increase in the expression and localization change of fibrous proteins was confirmed in the three-dimensional skin model (40) cultured with the addition of TGFβ. This confirmed that the reconstructed skin model was responsive to physiological stimuli.
　［三次元皮膚モデルの化粧品に対する応答性解析］
　（解析方法）
　上述の方法で、１２日間気相培養した三次元皮膚モデルの表皮表面に、それぞれ市販の化粧品（スキンケア製品）１～５を塗布し、３７℃で１時間、ＣＯ２インキュベーター内で培養した。培養後、化粧品を除去し、さらに５時間培養した後、皮膚組織からＲＮＡを抽出しリアルタイムＰＣＲ法を用いて表皮遺伝子（フィラグリン（ＦＬＧ）／カスパーゼ１４（ＣＡＳＰ１４）／トランスグルタミナーゼ１（ＴＧＭ１）／オクルディン（ＯＣＬＮ）／ヘパリン結合性ＥＧＦ様成長因子（ＨＢＥＧＦ）／リソソームプロテアーゼカテプシンＶ（ＣＴＳＶ）／シンタキシン（ＳＴＸ３））ならびに真皮遺伝子（ヒアルロン酸合成酵素２（ＨＡＳ２））の発現を解析した。 [Analysis of response of three-dimensional skin model to cosmetics]
(analysis method)
The epidermal surface of the three-dimensional skin model that had been gas-phase cultured for 12 days using the above-mentioned method was coated with commercially available cosmetics (skin care products) 1 to 5, and the model was cultured in a CO 2 incubator at 37°C for 1 hour. After culturing, the cosmetics were removed and the model was further cultured for 5 hours. RNA was then extracted from the skin tissue and the expression of epidermal genes (filaggrin (FLG)/caspase 14 (CASP14)/transglutaminase 1 (TGM1)/occludin (OCLN)/heparin-binding EGF-like growth factor (HBEGF)/lysosomal protease cathepsin V (CTSV)/syntaxin (STX3)) and dermal genes (hyaluronic acid synthase 2 (HAS2)) was analyzed using real-time PCR.
　（結果）
　図１０の（ａ）～（ｄ）及び図１１（ａ）～（ｄ）に示されるように、表皮遺伝子の種類により化粧品１～５に対して異なる応答性を示すことが確認できた。これにより、製品（化粧品等）が塗布された三次元皮膚モデルを解析することにより、製品の機能性評価が可能であることが確認できた。 (result)
As shown in Figures 10(a) to (d) and 11(a) to (d), it was confirmed that different types of epidermal genes showed different responsiveness to cosmetics 1 to 5. This confirmed that it is possible to evaluate the functionality of a product (such as cosmetics) by analyzing a three-dimensional skin model to which the product has been applied.
　［三次元皮膚モデルのタンパク質発現解析］
　（解析方法）
　上述の方法で、１４日間気相培養した三次元皮膚モデルの表皮層のみを摘出し一般的な細胞溶解液を用いて抽出タンパク質溶液を調製した。調製した抽出タンパク質溶液を用いてウェスタンブロッティング（Western Blotting）法により皮膚組織のタンパク質発現解析を行った。 [Protein expression analysis of three-dimensional skin model]
(analysis method)
The epidermis layer of the three-dimensional skin model that had been cultured in an aerated phase for 14 days was extracted and an extracted protein solution was prepared using a general cell lysis solution. Protein expression analysis of the skin tissue was performed by Western blotting using the extracted protein solution.
　（結果）
　図１２の（ａ）及び（ｂ）に示されるように、ウェスタンブロッティング法により、顆粒層からフィラグリン及びカスパーゼ１４の合成・代謝を確認することができた。 (result)
As shown in (a) and (b) of Figure 12, the synthesis and metabolism of filaggrin and caspase 14 in the granular layer was confirmed by Western blotting.
　［同一剤形の化粧品に対する三次元培養皮膚モデルの応答性解析］
　（解析方法）
　スキンケア製品の同じカテゴリ（剤形）内での機能性評価を行うため、１２日間気相培養した三次元皮膚モデルの表皮表面に、市販の化粧品（スキンケア製品）の美容液（Ｓ００１～Ｓ０１６）、ローション（Ｌ００１～Ｌ０１２）、乳液（Ｅ００１～Ｅ０１１）若しくはクリーム（Ｃ００１～Ｃ０１２）をそれぞれ塗布し、３７℃で１時間、ＣＯ２インキュベーター内で培養した。培養後、表皮表面の化粧品を除去し、さらに２３時間または４７時間培養した後、皮膚組織からＲＮＡを抽出しリアルタイムＰＣＲ法を用いて様々な表皮遺伝子（グルコシルセラミダーゼ（ＧＢＡ）／セリンパルミトイルトランスフェラーゼ長鎖塩基サブユニット1（ＳＰＴＬＣ１）／超長鎖脂肪酸伸長酵素４（ＥＬＯＶＬ４）／トランスグルタミナーゼ１（ＴＧＭ１）／オクルディン（ＯＣＬＮ）／ヘパリン結合性ＥＧＦ様成長因子（ＨＢＥＧＦ）／リソソームプロテアーゼカテプシンＶ（ＣＴＳＶ）／シンタキシン３（ＳＴＸ３）等）、並びに真皮遺伝子（Ｉ型コラーゲンα鎖１（ＣＯＬ１Ａ１）／フィブリリン（ＦＢＮ）／フィブリン５（ＦＢＬＮ５）／潜在型ＴＧＦβ結合タンパク質４（ＬＴＢＰ４）／ヒアルロン酸合成酵素２（ＨＡＳ２）等）の発現を解析した。 [Analysis of response of a three-dimensional cultured skin model to cosmetics of the same formulation]
(analysis method)
To evaluate the functionality of skin care products within the same category (formulation), commercially available cosmetics (skin care products) - serums (S001-S016), lotions (L001-L012), emulsions (E001-E011) or creams (C001-C012) - were applied to the epidermal surface of the three-dimensional skin model that had been cultured in the gas phase for 12 days, and the model was cultured in a CO2 incubator at 37°C for 1 hour. After the culture, the cosmetics on the epidermal surface were removed, and the tissue was further cultured for 23 or 47 hours. RNA was then extracted from the skin tissue, and the expression of various epidermal genes (glucosylceramidase (GBA)/serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/very long-chain fatty acid elongase 4 (ELOVL4)/transglutaminase 1 (TGM1)/occludin (OCLN)/heparin-binding EGF-like growth factor (HBEGF)/lysosomal protease cathepsin V (CTSV)/syntaxin 3 (STX3) etc.) and dermal genes (type I collagen α chain 1 (COL1A1)/fibrillin (FBN)/fibulin 5 (FBLN5)/latent TGF-β binding protein 4 (LTBP4)/hyaluronan synthase 2 (HAS2) etc.) was analyzed using real-time PCR.
　（結果）
　図１３の（ａ）～（ｆ）～図１６の（ａ）～（ｆ）に示されるように、表皮遺伝子、真皮遺伝子の種類により美容液（Ｓ００１～Ｓ０１６）、ローション（Ｌ００１～Ｌ０１２）、乳液（Ｅ００１～Ｅ０１１）若しくはクリーム（Ｃ００１～Ｃ０１２）のそれぞれの製品に対して異なる応答性を示すことが確認できた。これにより、同一カテゴリ（剤形）の市販の化粧品（スキンケア製品）が塗布された三次元皮膚モデルを解析することにより、スキンケア製品の機能性評価が可能であることが確認できた。 (result)
As shown in Figures 13(a) to 13(f) to Figure 16(a) to 16(f), it was confirmed that different types of epidermal and dermal genes showed different responsiveness to each of the products, serums (S001 to S016), lotions (L001 to L012), milky lotions (E001 to E011), and creams (C001 to C012). This confirmed that it is possible to evaluate the functionality of skin care products by analyzing a three-dimensional skin model to which commercially available cosmetics (skin care products) of the same category (formulation) have been applied.
　本発明によれば、真皮層の収縮を抑制し、表皮及び真皮の構造を長期間にわたって維持することができるので、多くのウェルを有する培養容器を用いて行う、化粧料等の効率的なサンプル評価に有効である。 The present invention can suppress the contraction of the dermis layer and maintain the structure of the epidermis and dermis for a long period of time, making it effective for efficient sample evaluation of cosmetics, etc., using a culture vessel with many wells.
　１０、３０、４０　三次元皮膚モデル
　１１　第１の層、表皮層
　１２　第２の層、真皮層
　１３　介在膜
　１４　真皮線維芽細胞層
　１５　角層
　１６　顆粒層
　１７　有棘層
　１８　基底層
　２０　培養容器
　２１　ウェル
　２２　支持部
　２３　培養液
　４１　Ｉ型コラーゲン
　４２　フィブリン－１
　４３　核 10, 30, 40 Three-dimensional skin model 11 First layer, epidermis layer 12 Second layer, dermis layer 13 Intervening membrane 14 Dermal fibroblast layer 15 Stratum corneum 16 Granular layer 17 Spongy layer 18 Basal layer 20 Culture vessel 21 Well 22 Support 23 Culture medium 41 Type I collagen 42 Fibrin-1
43 Nuclear

Claims (12)

  1. 　角化細胞又は角化細胞が分化した細胞を含む第１の層と、
    　真皮線維芽細胞を含む第２の層と、
    　前記第１の層と前記第２の層との間に介在する介在膜と、
    　を有し、
    　前記介在膜は細胞外マトリクスを少なくとも表面に含む多孔性の膜である、
    　三次元皮膚モデル。     a first layer comprising keratinocytes or cells differentiated from keratinocytes;
    a second layer comprising dermal fibroblasts;
    an intervening film interposed between the first layer and the second layer;
    having
    The intermediate membrane is a porous membrane including an extracellular matrix at least on its surface.
    Three-dimensional skin model.
  2. 　コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合され、前記ｖは、前記第２の層の体積を意味する、請求項１に記載の三次元皮膚モデル。 The three-dimensional skin model of claim 1, in which collagen hydrogel is blended in an amount of 0 w/v% or more and less than 0.6 w/v%, where v represents the volume of the second layer.
  3. 　表皮層と、
    　真皮層と、
    　表皮層と真皮層との間に介在する介在膜と、
    　を有し、
    　前記表皮層は角層、顆粒層、有棘層、及び基底層を有し、
    　前記真皮層は多層構造を有し、かつ、細胞密度が２０００ｃｅｌｌｓ／ｃｍ２以上である、
    　三次元皮膚モデル。     The epidermis layer,
    The dermis layer,
    An intervening membrane interposed between the epidermis layer and the dermis layer;
    having
    The epidermis layers include the stratum corneum, the granular layer, the spinous layer, and the basal layer;
    The dermis layer has a multi-layer structure and a cell density of 2000 cells/ cm2 or more.
    Three-dimensional skin model.
  4. 　表皮層と、
    　真皮層と、
    　表皮層と真皮層との間に介在する介在膜と、
    　を有し、
    　前記表皮層は角層、顆粒層、有棘層、及び基底層を有し、
    　前記真皮層は多層構造を有し、かつ、コラーゲンハイドロゲルを、０ｗ／ｖ％以上０．６ｗ／ｖ％未満の量で配合され、
    　前記ｖは、前記真皮層の体積を意味する、
    　三次元皮膚モデル。     The epidermis layer,
    The dermis layer,
    An intervening membrane interposed between the epidermis layer and the dermis layer;
    having
    The epidermis layers include the stratum corneum, the granular layer, the spinous layer, and the basal layer;
    The dermis layer has a multi-layer structure and contains collagen hydrogel in an amount of 0 w/v% or more and less than 0.6 w/v%,
    The v means the volume of the dermis layer.
    Three-dimensional skin model.
  5. 　請求項１～４のいずれか一項に記載の三次元皮膚モデルと、
    　培養液を収容する複数のウェルと、
    　前記ウェル内で前記三次元皮膚モデルを支持し、かつ、培養液を前記三次元皮膚モデルの前記第２の層側又は真皮層側で連通可能とする支持部と、
    　を備える培養容器。     A three-dimensional skin model according to any one of claims 1 to 4,
    A plurality of wells for containing a culture medium;
    a support section that supports the three-dimensional skin model in the well and allows a culture medium to communicate with the second layer side or the dermis layer side of the three-dimensional skin model;
    A culture vessel comprising:
  6. 　前記ウェルを１２、２４、４８又は９６個有する、請求項５に記載の培養容器。 The culture vessel according to claim 5, having 12, 24, 48 or 96 wells.
  7. 　請求項５に記載の培養容器を用いて、三次元皮膚モデルを培養する工程と、
    　培養中の前記三次元皮膚モデルにサンプルを投与する工程と、
    　サンプル投与から所定の培養期間経過後の前記三次元皮膚モデル又は培養上清を分析することで、前記サンプルが前記三次元皮膚モデルに与える機能を評価する工程と、
    　を含むサンプルの評価方法。     Culturing a three-dimensional skin model using the culture vessel according to claim 5;
    administering a sample to the three-dimensional skin model in culture;
    A step of evaluating the function of the sample on the three-dimensional skin model by analyzing the three-dimensional skin model or the culture supernatant after a predetermined culture period has elapsed since administration of the sample;
    How to evaluate the sample, including:
  8. 　前記培養期間は６日以上である、請求項７に記載の評価方法。 The evaluation method according to claim 7, wherein the culture period is 6 days or longer.
  9. 　前記評価する工程は、前記培養期間において、前記三次元皮膚モデルの加工を行う工程、２回以上の同一又は異なるサンプルの投与を行う工程、又は投与したサンプルの除去を行う工程を含む、請求項７に記載の評価方法。 The evaluation method according to claim 7, wherein the evaluation step includes a step of processing the three-dimensional skin model during the culture period, a step of administering the same or different samples two or more times, or a step of removing the administered sample.
  10. 　少なくともサンプルを投与しない三次元皮膚モデルが、前記培養期間経過後において、角層、顆粒層、有棘層、及び基底層を有する表皮層と、多層構造を有する真皮層と、を有する、請求項７に記載の評価方法。 The evaluation method according to claim 7, wherein the three-dimensional skin model to which at least a sample is not administered has, after the culture period has elapsed, an epidermis layer having a stratum corneum, a granular layer, a stratum spinosum, and a basal layer, and a dermis layer having a multi-layer structure.
  11. 　前記培養期間経過後における、三次元皮膚モデルの表皮層の平面積と真皮層の平面積の大きさとの関係が、表皮層の平面積≦真皮層の平面積を満たすか、又は前記培養期間経過後における真皮層の平面積が、前記培養期間経過前における真皮層の平面積の０．７倍以上である、請求項７に記載の評価方法。 The evaluation method according to claim 7, wherein the relationship between the planar area of the epidermal layer and the planar area of the dermal layer of the three-dimensional skin model after the culture period has elapsed satisfies the planar area of the epidermal layer ≦ the planar area of the dermal layer, or the planar area of the dermal layer after the culture period is 0.7 times or more the planar area of the dermal layer before the culture period has elapsed.
  12. 　前記分析は、遺伝子発現、タンパク質発現、組織化学分析、又は皮膚バリア機能分析を含む、請求項７に記載の評価方法。 The evaluation method according to claim 7, wherein the analysis includes gene expression, protein expression, histochemical analysis, or skin barrier function analysis.
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