Three-dimensional skin model

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

3D skin model

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

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

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

[0004] Here, the dermal fibroblasts contract the collagen hydrogel, causing the epidermal layer to peel off from the cell culture insert during culture, making it difficult to evaluate by directly applying a test substance (such as a cosmetic ingredient) from the stratum corneum side. Furthermore, because the dermal layer contracts during culture of the skin model, prolonged culture periods may affect the morphology and responsiveness of the skin model. Therefore, it is necessary to develop a skin model in which the contraction of the dermal layer can be controlled.

[0005] Therefore, 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 (Non-Patent Document 1).

[0006] Freia F. Schmidt, Sophia Nowakowski and Petra J. Kluger, Frontiers in Bioengineering and Biotechnology, 08 May 2020, Volume 8, Article 388: 1-11.

[0007] 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 contraction of the dermis layer.

[0008] The present invention has been made in consideration of the above 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.

[0009] As a result of extensive research, the present 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).

[0010] (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, wherein the intervening membrane is a porous membrane containing extracellular matrix on at least its surface.

[0011] (2) The three-dimensional skin model described in (1) above, wherein collagen hydrogel is blended in an amount of 0 w / v% or more and less than 0.6 w / v%, and v means the volume of the second layer.

[0012] (3) A skin barrier comprising an epidermis layer, a dermis layer, and an intervening membrane interposed between the epidermis layer and the dermis layer, wherein the epidermis layer has a stratum corneum, a granular layer, a spinous layer, and a basal layer, and the dermis layer has a multi-layer structure and a cell density of 2000 cells / cm. ２ That's it, the three-dimensional skin model.

[0013] (4) A three-dimensional skin model having an epidermal layer, a dermal layer, and an intervening membrane interposed between the epidermal layer and the dermal layer, wherein the epidermal layer has a stratum corneum, a granular layer, a spinous layer, and a basal layer, and the dermal layer has a multilayer structure, and wherein collagen hydrogel is blended in an amount of 0 w / v% or more and less than 0.6 w / v%, where v means the volume of the dermal layer.

[0014] (5) A culture vessel comprising the three-dimensional skin model described in any one of (1) to (4), a plurality of wells containing a culture solution, and a support portion that supports the three-dimensional skin model within the wells and allows the culture solution to communicate with the second layer side or the dermis layer side of the three-dimensional skin model.

[0015] (6) The culture vessel according to (5), having 12, 24, 48 or 96 wells.

[0016] (7) A method for evaluating a sample, comprising: 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 sample administration.

[0017] (8) The evaluation method described in (7) above, wherein the culture period is 6 days or more.

[0018] (9) The evaluation method according to (7) or (8), 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 samples.

[0019] (10) The evaluation method according to any one of (7) to (9), wherein the three-dimensional skin model to which at least no sample is administered has, after the culture period has elapsed, an epidermal layer having a stratum corneum, a granular layer, a spinous layer, and a basal layer, and a dermal layer having a multi-layer structure.

[0020] (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 condition 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 has elapsed is 0.7 times or more the planar area of ​​the dermal layer before the culture period has elapsed.

[0021] (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.

[0022] According to the present invention, a three-dimensional skin model can be provided that can suppress contraction of the dermis layer and maintain the structures of the epidermis and dermis for a long period of time.

[0023] FIG. 1 shows an image of a skin tissue section of a three-dimensional skin model after hematoxylin-eosin staining. FIG. 2(a) is a plan view of a culture vessel having multiple wells, and FIG. 2(b) is a cross-sectional view taken along line A-A in FIG. 2(a). FIG. 3(a) and FIG. 3(b) are images of a skin tissue section of a three-dimensional skin model after culturing for a predetermined period of time. FIG. 4(a) and FIG. 4(b) are images of a skin tissue section of a three-dimensional skin model after immunohistochemical staining. FIG. 5 is a graph showing the results of an analysis of the barrier function of a three-dimensional skin model. FIG. 6 is a graph showing the results of an analysis of the barrier function of a three-dimensional skin model. FIG. 7(a) and FIG. 7(b) are graphs showing the results of an analysis of the responsiveness of a three-dimensional skin model to physiological stimuli. FIG. 8(a) and FIG. 8(b) are graphs showing the results of an analysis of the responsiveness of a three-dimensional skin model to physiological stimuli. FIG. 9(a) and FIG. 9(b) are images of a skin tissue section after an analysis of the responsiveness of a three-dimensional skin model to physiological stimuli. (a) to (d) of Figure 10 are graphs showing the analysis results of the responsiveness of a three-dimensional skin model to cosmetics. (a) to (d) of Figure 11 are graphs showing the analysis results of the responsiveness of a three-dimensional skin model to cosmetics. (a) and (b) of Figure 12 are diagrams showing the analysis results of protein expression. (a) to (f) of Figure 13 are graphs showing the analysis results of the responsiveness to a commercially available cosmetic (serum). (a) to (f) of Figure 14 are graphs showing the analysis results of the responsiveness to a commercially available cosmetic (lotion). (a) to (f) of Figure 15 are graphs showing the analysis results of the responsiveness to a commercially available cosmetic (lotion). (a) to (f) of Figure 16 are graphs showing the analysis results of the responsiveness to a commercially available cosmetic (cream).

[0024] Specific embodiments of the present invention will be described in detail below. Note that the present invention is not limited to the following embodiments, and can be modified as appropriate within the scope of the present invention.

[0025] [Three-dimensional Skin Model] (First Embodiment) As shown in Figure 1, a three-dimensional skin model according to a 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 interposed between the first layer 11 and the second layer 12. The intervening membrane 13 contains an extracellular matrix. The second layer 12 preferably contains a collagen hydrogel in an amount of 0 w / v % or more and less than 0.6 w / v %. Note that the above "v" refers to the volume of the second layer.

[0026] Each component will be described below.

[0027] <<First Layer>> The first layer 11 contains keratinocytes or cells differentiated from keratinocytes. Examples of the cells differentiated from keratinocytes include one or more cells selected from the group consisting of keratinocytes, granular cells, spinous cells, and basal cells, which will be described later.

[0028] Keratinocytes are cells of the epidermis that divide in the basal layer, the lowest layer of the epidermis, and migrate to the skin surface.

[0029] <<Second Layer>> The second layer 12 contains dermal fibroblasts.

[0030] Dermal fibroblasts (fibroblasts) 14 are cells that produce collagen fibers, elastic fibers, mucopolysaccharides, etc., and have a long, spindle-shaped morphology.

[0031] The collagen hydrogel is preferably blended 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, in the second layer 12. When the amount of collagen hydrogel is within the above range, contraction of the dermis layer constituting the three-dimensional skin model during culture can be suppressed.

[0032] Furthermore, the proliferation ability of dermal fibroblasts is less likely to be inhibited by inhibiting cell activity, and the proliferation of dermal fibroblasts is less likely to be inhibited because there is a large space in which the proliferated cells can reside. As a result, the cell density of the dermal layer is likely to be maintained at a high level over a long period of time, which makes it easy to evaluate the effect of a sample on the dermal layer with high sensitivity. Furthermore, proteins involved in crosstalk (cytokines, chemokines, etc.), lipid components (eicosanoids, etc.), etc. are produced and transmitted to the epidermal layer, making it easy to maintain the differentiated structure of the epidermal layer constituting the three-dimensional skin model being cultured at a high level over a long period of time.

[0033] <<Intervening Membrane>> The intervening membrane 13 is a porous membrane that allows substances to pass through, and includes an extracellular matrix at least on its surface. By including an extracellular matrix at least on its surface, the intervening membrane can easily adhere keratinocytes and fibroblasts without relying on a collagen hydrogel.

[0034] The intervening membrane may consist essentially of only an extracellular matrix layer, or may contain extracellular matrix on the surface of the substrate. The extracellular matrix on the surface is not particularly limited, but is preferably a coating membrane due to its high frequency of occurrence.

[0035] Examples of the substrate material include a membrane of polyethylene terephthalate (PET) or polycarbonate (PC), and PET. Examples of the extracellular matrix include type I collagen, type III collagen, type IV collagen, fibronectin, gelatin, etc.

[0036] 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 be promoted satisfactorily. The pore size of the intervening membrane can be determined, for example, by measuring the major axis using an electron microscope image.

[0037] The average thickness of the intervening membrane is preferably 20 μm or less, from the viewpoint of easily obtaining a skin model in which the epidermal and dermal structures are maintained over 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. More preferably, it may be 15 μm or less, 10 μm or less, 7.5 μm or less, or 5 μm or less. However, in cases where specific medium components (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) are used or additional layers are provided, the average thickness of the intervening membrane is not limited thereto, and may be, for example, 20 μm or more and 200 μm or less. When the thickness of the intervening membrane is within the above range, the strength of the three-dimensional skin model is increased, and the crosstalk between the dermis and epidermis is enhanced, making it easier to maintain a state in which the epidermal layer is appropriately differentiated (the spinous layer and granular layer are thick) and the basal layer is highly dense. The thickness of the intervening membrane can be measured, for example, using an optical microscope image of a histochemical section.

[0038] The intervening membrane may be a membrane from a commercially available cell culture insert.

[0039] The three-dimensional skin model according to this embodiment may or may not include other layers in addition to the first layer, second layer, and intervening membrane, including a subcutaneous tissue layer, such as a fat layer, located on the opposite side of the second layer from the intervening membrane.

[0040] When evaluating the additional layer itself, it is preferable to have the additional layer. On the other hand, an embodiment with an additional layer further promotes crosstalk and makes it easier to maintain the differentiation structure, but has less flexibility in culture conditions (e.g., medium composition) suitable for all of the first layer, second layer, and additional layer. In this embodiment, an embodiment without an additional layer is preferred because it makes it easier to maintain the differentiation structure without relying on the additional layer.

[0041] 1, a three-dimensional skin model according to a second embodiment of the present invention has an epidermal layer 11, a dermal layer 12, and an intervening membrane 13 interposed between the epidermal layer 11 and the dermal layer 12. The epidermal layer 11 has a stratum corneum 15, a granular layer 16, a spinous layer 17, and a basal layer 18.

[0042] Each component will be described below.

[0043] <Epidermal Layer> The epidermal layer 11 has a four-layer structure in which cornified cells form the stratum corneum 15 in the uppermost layer, granular cells form the granular layer 16 immediately below the stratum corneum 15, spinous cells (or spinous cells) form the spinous layer 17 immediately below the granular layer 16, and basal cells differentiated from keratinocytes form the basal layer 18 immediately below the spinous layer 17 (lowest layer). The epidermal layer 11 may also contain pigment cells (melanocytes).

[0044] <Stratum corneum> As described above, the stratum corneum 15 is located at the top of the epidermal layer 11 that constitutes the three-dimensional skin model, and is composed of a layered structure constructed by keratinocytes. Furthermore, the keratinocytes have a flat shape and lose their nuclei during the differentiation process. Furthermore, keratinocytes contain aggregates of keratin fibers within their cytoplasm, are acidophilic, and stain light to dark red with hematoxylin-eosin (HE) staining.

[0045] <Granular Layer> As described above, the granular layer 16 is located directly below the stratum corneum 15 and directly above the stratum spinosum 17, which will be described later, and is composed of a layered structure constructed by granular cells. The granular cells have a flat shape and contain granules composed of basophilic components within the cells. The granules within the cells are stained blue-purple to pale 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. Furthermore, 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.

[0046] <Stratum spinosum> As described above, the stratum spinosum 17 is located directly below the stratum granulosum 16 and directly above the stratum basale 18 (described later), and is composed of a layered structure constructed by spinous cells. The spinous cells have a flat shape and spines 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. Furthermore, 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.

[0047] <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. Unlike keratinocytes, granular cells, and spinous cells, the basal cells have a cubic to cylindrical shape and an oval nucleus. Basal cells are basophilic and stain indigo to pale blue with hematoxylin-eosin (HE) staining. The average thickness of the basal layer of the three-dimensional skin model according to this embodiment is preferably 7 μm or more, more preferably 9 μm or more, and even more preferably 14 μm or more. Furthermore, the average thickness of the spinous layer of the three-dimensional skin model is preferably 7 μm or more and 20 μm or less. The thickness can be measured, for example, using an optical microscope image of a histochemical section.

[0048] [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).

[0049] "Multilayer structure" refers to a structure in which fibroblasts form two or more layers in the vertical direction (toward the stratum corneum). Here, the orientation of fibroblasts is not uniform. Therefore, the image of a vertically sliced ​​skin tissue section may appear spindle-shaped due to horizontal orientation of fibroblasts, or circular due to vertical orientation of fibroblasts.

[0050] The cell density of the dermal layer (dermal fibroblasts) is 2000 cells / cm ２ or more, 5000 cells / cm ２ It is preferable that the density is 6000 cells / cm or more. ２ The cell density of the dermis layer is preferably 2000 cells / cm or more. ２ More than 10000cells / cm ２ Preferably, it is 5000 cells / cm or less. ２ More than 8000cells / cm ２ More preferably, it is 6000 cells / cm or less. ２ More than 7700cells / cm ２ It is more preferable that the cell density of the dermis layer is within the above range. 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.

[0051] 1 , a three-dimensional skin model according to a third embodiment of the present invention has an epidermal layer 11, a dermal layer 12, and an intervening membrane 13 interposed between the epidermal layer 11 and the dermal layer 12. The epidermal layer 11 has a stratum corneum 15, a granular layer 16, a spinous layer 17, and a basal layer 18.

[0052] Furthermore, the dermal layer 12 has a multilayer structure and contains collagen hydrogel in an amount of 0 w / v% or more and less than 0.6 w / v%. The "v" above refers to the volume of the dermal layer. It is more preferable that the dermal layer 12 contains collagen hydrogel in an amount of 0 w / v% or more and less than 0.3 w / v%. Alternatively, the dermal layer 12 does not have unevenly dispersed collagen fibers as determined by hematoxylin-eosin (HE) staining. This provides ample space for proliferated cells to reside, making it less likely to inhibit the proliferation ability of dermal fibroblasts. Therefore, contraction of the dermal layer that constitutes the three-dimensional skin model during culture can be suppressed.

[0053] Furthermore, the proliferation ability of dermal fibroblasts is less likely to be inhibited by inhibiting cell activity, and the proliferation of dermal fibroblasts is less likely to be inhibited because there is a large space in which the proliferated cells can reside. As a result, the cell density of the dermal layer is likely to be maintained at a high level over a long period of time, which makes it easy to evaluate the effect of a sample on the dermal layer with high sensitivity. Furthermore, proteins involved in crosstalk (cytokines, chemokines, etc.), lipid components (eicosanoids, etc.), etc. are produced and transmitted to the epidermal layer, making it easy to maintain the differentiated structure of the epidermal layer constituting the three-dimensional skin model being cultured at a high level over a long period of time.

[0054] The three-dimensional skin model according to this embodiment may or may not have other layers in addition to the epidermis, dermis, and intervening membrane, including a subcutaneous tissue layer, such as a fat layer, located on the opposite side of the dermis from the intervening membrane.

[0055] In the embodiment having an additional layer, crosstalk is further promoted and the differentiation structure is easily maintained, but the degree of freedom in culture conditions (e.g., medium composition) suitable for all of the epidermal layer, dermal layer, and additional layer is low. In this embodiment, the differentiation structure is easily maintained without relying on the additional layer, so the embodiment not having the additional layer is preferred.

[0056] 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 22 that supports the three-dimensional skin model 10 within the wells 21. The three-dimensional skin model 10 is positioned so that it can communicate with the culture solution 23 on the second layer 12 side or the dermis layer 12 side.

[0057] The culture vessel 20 may have any number of wells, including but not limited to 12, 24, 48, or 96 wells, allowing for simultaneous evaluation of multiple samples and application conditions.

[0058] [Sample Evaluation Method] The sample evaluation method of the present invention is performed using a culture vessel. Specifically, the sample evaluation method includes: (1) culturing a three-dimensional skin model using a culture vessel 20 shown in Figures 2(a) and 2(b) (culture step), (2) administering a sample to the three-dimensional skin model during culture (sample administration step), and (3) 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 sample administration (evaluation step).

[0059] Each step will be described below.

[0060] (Culturing step) More specifically, the culturing step is a step 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.

[0061] The culture period is preferably 6 days or more, more preferably 12 days or more, and even 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 evaluation period, allowing for satisfactory evaluation results to be obtained.

[0062] (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 using a predetermined method.

[0063] The sample to be administered is not particularly limited, and may be one or more isolated substances, or a mixture of various components (e.g., cosmetics, pharmaceuticals). The form of the sample is not particularly limited, and may be a liquid, gel, cream, powder, etc. The method of administering the sample is also not particularly limited, and may be applied to the surface of the epidermis, injected into the epidermis, injected into the dermis, or added to a culture solution.

[0064] Different samples may be applied to each well, or the same sample may be applied in different ways (doses and / or administration methods), allowing quantitative comparison of the effects of the samples on the skin and estimation of a preferred administration method (dosage and / or administration method) for imparting a desired function to the skin.

[0065] (Evaluation step) More specifically, the evaluation step is a step 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 secretions secreted by the cells after a culture period of several hours or more has elapsed since administration of the sample.

[0066] The analysis preferably includes gene expression, protein expression, histochemical analysis, or skin barrier function analysis, and may include a combination of two or more of these.

[0067] The evaluation process may also include, during the above-mentioned culture period, processing the three-dimensional skin model 10, administering the same or different samples to the three-dimensional skin model 10 two or more times, or removing the samples administered to the three-dimensional skin model 10.

[0068] The process of processing the three-dimensional skin model 10 is a process of creating three-dimensional skin models in different conditions. Processing methods are not particularly limited, but include adding an inflammatory substance to induce oxidative stress, irradiating with light such as UV light to induce oxidative stress, and the like. This processing can take anywhere from several seconds to several days, but the skin model used in this embodiment can maintain the structure of the epidermis and dermis over a long period of time, making it easy to freely perform evaluations using the processed skin model. By creating three-dimensional skin models in different conditions, it is possible to evaluate not only normal skin, but also rough skin, inflamed skin, and the like, thereby obtaining information on the effectiveness of samples for various skin conditions.

[0069] 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, or different samples may be administered by the same method or by different methods. As used herein, "method" refers to dosage and / or administration method.

[0070] Furthermore, for comparison with a three-dimensional skin model to which a sample has been administered, a three-dimensional skin model to which at least 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, or 30 days or less) in the above-described culture step. Here, it is preferable to culture the three-dimensional skin model to which a sample has not been administered under conditions such that, after the culture period has elapsed, the three-dimensional skin model to which a sample has not been administered has an epidermal layer having a stratum corneum, granular layer, spinous layer, and basal layer, and a dermal layer having a multilayer structure.

[0071] 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 preferably satisfies the relation "planar area of ​​the epidermal layer≦planar area of ​​the dermal layer," or the planar area of ​​the dermal layer after the culture period is preferably 0.7 times or more, and is not particularly limited to, more preferably 0.9 times or more, of the planar area of ​​the dermal layer before the culture period. 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, thereby enabling long-term evaluation. Furthermore, the planar area of ​​the epidermal layer is preferably 0.7 to 1.0 times, and more preferably 0.9 to 1.0 times, the planar area of ​​the dermal layer before the culture period.

[0072] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0073] [Construction of a three-dimensional skin model] A cell culture insert with 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 DMEM containing 10% FBS were seeded on one side of the cell culture insert and incubated at 37°C for 1 day in a CO atmosphere. ２ Then, 500,000 normal human epidermal keratinocytes suspended in Humedia KG2 (Kurabo) were seeded on the other side of the cell culture insert, and the cells were cultured at 37°C for 1 day under CO ２ After culturing in an incubator, the medium was replaced with a medium for three-dimensional culture, and the culture was continued for another day. After culturing, the medium on the normal human keratinized epidermal cells side was removed, and gas-phase culture was initiated. Epidermal layer differentiation was induced by gas-phase culture for 12 days, 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 Kurabo Industries, Ltd.

[0074] 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 After 20 days of gas phase culture, the cell density was 5843±349 cells / cm ２ According to the inventors' investigation, the cell density of the dermis layer of the model disclosed in Non-Patent Document 1 was 1300±339 cells / cm ２ In addition, the planar areas of the dermal layer and epidermal layer of the constructed three-dimensional skin model after the culture period had elapsed were each one time the planar areas of the dermal layer and epidermal layer before the culture period had elapsed.

[0075] [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 method described above was fixed with Superfix KY-500 (Kurabo Industries, Ltd.) and then embedded in Paraplast X-tra (Leica Biosystems) to prepare paraffin-embedded sections (skin tissue sections). The skin tissue sections were stained with hematoxylin and eosin (HE) and then analyzed using an all-in-one fluorescence microscope "BZ-X800" (Keyence Corporation). "Paraplast" is a registered trademark of Leica Biosystems Richmond.

[0076] (Results) As shown in the image of the skin tissue section after HE staining in Figure 1, it was confirmed that the three-dimensional skin model, like human skin tissue, has an epidermal layer composed of a stratum corneum, a granular layer (flattened cells containing intracellular granules), a spinous layer (with spines around the cells), and a basal layer (cuboidal). It was also confirmed that fibroblasts are arranged in layers as the dermal layer directly below the basal layer, demonstrating the basic morphological characteristics of a skin model.

[0077] [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 culturing, the skin tissue was stained with hematoxylin and eosin (HE) and analyzed using the all-in-one fluorescence microscope "BZ-X800."

[0078] (Results) Comparing the image of a skin tissue slice of skin tissue (a) cultured in a gas phase for 12 days with the image of a skin tissue slice of skin tissue (b) cultured for 20 days shown in Figure 3, it was confirmed that the four-layer structure of the epidermis layer 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.

[0079] [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 phase for 12 days using the method described above was fixed with Superfix KY-500 and then embedded in Paraplast X-tra to prepare paraffin-embedded sections (skin tissue sections). The prepared skin tissue sections were subjected to immunohistochemical staining using antibodies against proteins known to be localized in the epidermal layer.

[0080] (Results) As shown in (a) of Figure 4, the expression of the differentiation marker cytokeratin 10 (CK10) 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. Furthermore, as shown in (b) of Figure 4, the expression of filaggrin (not shown), a component of keratohyalin granules, was confirmed in the granular layer 16 (granules within granular cells), and the expression of claudin 1 (not shown), a tight junction-constituting adhesion molecule, was confirmed in the spinous layer 16 (periphery of spinous cells). As shown in (a) and (b) of Figure 4, the morphological characteristics of the three-dimensional skin model could be confirmed from the localization of the differentiation marker molecules.

[0081] [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 to 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 method described above, and the model was incubated at 37°C for 1 hour in CO . ２ After the incubation, the SDS solution remaining on the epidermal surface was removed, and the remaining SDS solution was washed off with phosphate-buffered saline (PBS) (-). The cells were then incubated at 37°C for 24 hours in a CO ２ The cells were then cultured in an incubator for recovery. Cytotoxicity after culture was measured using Cytotoxicity LDH Assay Kit-WST (manufactured by Dojindo Laboratories) using the activity of cytoplasmic oxidoreductase (LDH) leaked into the medium as an indicator.

[0082] (Results) 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.

[0083] [Barrier function analysis of three-dimensional cultured skin model 2] (Analysis method) Using the method described above, normal human dermal fibroblasts and normal human epidermal keratinocytes were seeded onto the inserts, and the day on which gas-phase culture was initiated was set as day 0, and the transepithelial electrical resistance (TEER) of the three-dimensional skin model was measured over time.

[0084] (Results) As shown in Figure 6, it was confirmed that the barrier function of the three-dimensional skin model was enhanced as the model grew, and that this barrier function was maintained even after the three-dimensional skin model matured.

[0085] [Analysis 1 of the responsiveness of a three-dimensional skin model to physiological stimuli] (Analysis method) A 10 μM solution of retinoic acid (ATRA) was applied to the epidermis (surface) of a 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 cultured for an additional 5 hours. RNA was then extracted from the skin tissue and gene expression analysis was performed using real-time PCR.

[0086] (Results) As shown in Figures 7(a) and (b), application of the ATRA solution was confirmed to induce the expression of heparin-binding EGF-like growth factor gene (HBEGF) and epidermal hyaluronic acid synthase gene 3 (HAS3).

[0087] [Analysis 2 of the responsiveness of a three-dimensional skin model to physiological stimuli] (Analysis method) The three-dimensional skin model was cultured in the air phase for 12 days using the method described above, and then transformed growth factor β (TGFβ) was added to the medium to a final concentration of 10 nM. The model was then cultured for 48 hours, and gene expression analysis of the reconstructed three-dimensional skin model was performed. Immunohistochemical staining was also performed using antibodies against proteins known to be localized in the dermis layer.

[0088] (Results) 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.

[0089] As shown in Figures 9(a) and (b), immunohistochemical staining was performed to confirm the expression of type I collagen (41) and fibrillin-1 (42) in the dermal fibroblast layer 14 (dermis layer). In the three-dimensional skin model (40) cultured with the addition of TGFβ, increased expression and localization of fibrous proteins were confirmed. This confirmed that the reconstructed skin model was responsive to physiological stimuli.

[0090] [Analysis of the responsiveness of the three-dimensional skin model to cosmetics] (Analysis method) Commercially available cosmetics (skin care products) 1 to 5 were applied to the epidermal surface of the three-dimensional skin model that had been cultured in the gas phase for 12 days using the method described above, and the three-dimensional skin model was incubated at 37°C for 1 hour in CO ２ The tissues were cultured in an incubator. After the culture, cosmetics were removed, and the tissues were cultured for an additional 5 hours. RNA was 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 (hyaluronan synthase 2 (HAS2)) was analyzed using real-time PCR.

[0091] (Results) As shown in Figures 10(a) to 10(d) and 11(a) to 11(d), it was confirmed that different types of epidermal genes showed different responses to cosmetics 1 to 5. This confirmed that the functionality of products (cosmetics, etc.) can be evaluated by analyzing a three-dimensional skin model to which the product has been applied.

[0092] [Protein Expression Analysis of Three-Dimensional Skin Model] (Analysis Method) The epidermal layer of the three-dimensional skin model that had been cultured in an aerated phase for 14 days using the method described above 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.

[0093] (Results) As shown in (a) and (b) of FIG. 12, the synthesis and metabolism of filaggrin and caspase 14 in the granular layer could be confirmed by Western blotting.

[0094] [Analysis of responsiveness of 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) such as serums (S001-S016), lotions (L001-L012), emulsions (E001-E011), or creams (C001-C012) were applied to the epidermal surface of a three-dimensional skin model cultured in an air phase for 12 days, and the models were incubated at 37°C for 1 hour in CO ２ After incubation in an incubator, cosmetics were removed from the epidermal surface, and the tissues were further incubated for 23 or 47 hours. RNA was then extracted from the skin tissues 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.

[0095] 13(a) to 13(f) and 16(a) to 16(f), it was confirmed that different types of epidermal and dermal genes showed different responsiveness to each of the beauty serums (S001 to S016), lotions (L001 to L012), emulsions (E001 to E011), and creams (C001 to C012). This confirmed that it is possible to evaluate the functionality of skin care products by analyzing three-dimensional skin models to which commercially available cosmetics (skin care products) of the same category (formulation) have been applied.

[0096] According to the present invention, contraction of the dermis layer can be suppressed and the structures of the epidermis and dermis can be maintained for a long period of time, and therefore the present invention is effective for efficient sample evaluation of cosmetics and the like using a culture vessel having many wells.

[0097] 10, 30, 40 Three-dimensional skin model 11 First layer, epidermal layer 12 Second layer, dermal layer 13 Intervening membrane 14 Dermal fibroblast layer 15 Stratum corneum 16 Granular layer 17 Spongium layer 18 Basal layer 20 Culture vessel 21 Well 22 Support part 23 Culture medium 41 Type I collagen 42 Fibrin-1 43 Nucleus

Claims

1. 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, the intermediate film is not included within the second layer; The second layer side is arranged in a well having a support part supporting the first layer, the second layer, and the intervening membrane so as to be capable of communicating with a culture medium. Three-dimensional skin model.

2. The three-dimensional skin model of claim 1 , wherein 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. 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 stratum granulosum, the stratum spinosum, and the stratum basale; The dermis layer has a multi-layer structure and a cell density of 2000 cells / cm ２ That's all. the intervening membrane is not contained within the dermis layer; The dermis layer side is arranged in a well having a support part supporting the epidermis layer, the dermis layer, and the intervening membrane so as to be in communication with a culture solution. Three-dimensional skin model.

4. The epidermis layer, The dermis layer, An intervening membrane interposed between the epidermis layer and the dermis layer; having the intervening membrane is not contained within the dermis layer; the dermis layer side is disposed in a well having a support part supporting the epidermis layer, the dermis layer, and the intervening membrane so as to be in communication with a culture solution; The epidermis layers include the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale; 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. 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. The culture vessel according to claim 5 , which has 12, 24, 48 or 96 of the wells.

7. 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. The evaluation method according to claim 7 , wherein the culture period is 6 days or longer.

9. 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. 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. 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 planar area of ​​the epidermal layer≦planar area of ​​the dermal layer, or the planar area of ​​the dermal layer after the culture period has elapsed is 0.7 times or more the planar area of ​​the dermal layer before the culture period has elapsed.

12. The method of claim 7 , wherein the analysis comprises gene expression, protein expression, histochemical analysis, or skin barrier function analysis.