Three-dimensional skin model

Abstract

To provide a three-dimensional skin model capable of suppressing contraction of a dermal layer and maintaining the structure of an epidermis and a dermis over the long term.SOLUTION: 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, wherein 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, wherein the epidermal layer has a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale, and the dermal layer has a multi-layer structure and has a cellular density of 2000 cells / cm2 or more.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] With the growing global trend of animal welfare, the development of alternative methods to animal experiments is underway. As alternatives to animal experiments on the skin, artificial skin models such as chemical fiber skin and three-dimensional skin models have been developed to replace human skin, which is difficult to obtain.

[0003] For example, in the functional test of products (such as cosmetic ingredients), a full-thickness skin model capable of confirming the responsiveness of the epidermis and dermis has been developed. The full-thickness skin model uses collagen hydrogel as an extracellular matrix, and epidermal keratinocytes are seeded on the dermis layer in which dermal fibroblasts are dispersed in the collagen hydrogel, and the epidermal layer is reconstructed by gas-phase culture.

[0004] Here, since the above dermal fibroblasts contract the above collagen hydrogel, the epidermal layer peels off from the cell culture insert during culture, and an evaluation method such as directly applying a test substance (such as a cosmetic ingredient) from the stratum corneum side is difficult. In addition, since the contraction of the dermis layer occurs during the culture of the skin model, if the culture period is prolonged, it may affect the morphology of the skin model and the skin responsiveness. Therefore, it is necessary to study a skin model capable of controlling the contraction of the dermis layer.

[0005] Therefore, a skin model has been disclosed in which a polyethylene terephthalate film is disposed between the epidermis layer and the dermis layer to fractionate the two layers, thereby alleviating the influence of the contraction of the dermis layer on the epidermis layer (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, the skin model disclosed in Non-Patent Document 1 is difficult to sufficiently suppress the shrinkage of the dermis layer as long as it uses a collagen hydrogel as a matrix in the dermis layer.

[0008] In view of this point, the present invention has been made, and an object thereof is to provide a three-dimensional skin model that can suppress the shrinkage of the dermis layer and maintain the structures of the epidermis and dermis over a long period of time.

Means for Solving the Problems

[0009] As a result of intensive studies, the present inventor has found that the above problems can be solved, and has completed the present invention. Specifically, the present invention is configured as follows in (1) to (12) below.

[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 an extracellular matrix at least on the surface.

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

[0012] (3) It has an epidermis, a dermis, and an intervening membrane intervening between the epidermis and the dermis. The epidermis has a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale. The dermis has a multilayer structure and a cell density of 2000 cells / cm 2 or more, a three-dimensional skin model.

[0013] (4) It has an epidermis, a dermis, and an intervening membrane intervening between the epidermis and the dermis. The epidermis has a stratum corneum, a stratum granulosum, a stratum spinosum, and a stratum basale. The dermis 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 means the volume of the dermis, a three-dimensional skin model.

[0014] (5) A culture vessel comprising the three-dimensional skin model according to any one of (1) to (4) above, a plurality of wells for accommodating a culture solution, and a support portion for supporting the three-dimensional skin model in the well and enabling 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) above, having 12, 24, 48, or 96 of the wells.

[0016] (7) A method for evaluating a sample, comprising the steps of culturing a three-dimensional skin model using the culture vessel according to (5) or (6) above, administering a sample to the three-dimensional skin model during the 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 sample administration.

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

[0018] (9) The evaluation method according to (7) or (8) above, wherein the step of evaluating 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.

[0019] (10) The evaluation method according to any one of (7) to (9) above, wherein at least a three-dimensional skin model not administered with a sample has, after the lapse of the culture period, an epidermal layer having a stratum corneum, a granular layer, a spinous layer, and a basal layer, and a dermal layer having a multilayer structure.

[0020] (11) The evaluation method according to any one of (7) to (10) above, 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 lapse of the culture period 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 lapse of the culture period is 0.7 times or more the planar area of the dermal layer before the lapse of the culture period.

[0021] (12) The analysis includes gene expression, protein expression, histochemical analysis, or skin barrier function analysis, and the evaluation method according to any one of (7) to (11) above. [Advantages of the Invention]

[0022] According to the present invention, it is possible to provide a three-dimensional skin model that suppresses the shrinkage of the dermal layer and can maintain the structures of the epidermis and dermis over a long period of time. [Brief Description of the Drawings]

[0023]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0024] Hereinafter, specific embodiments of the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and can be appropriately changed without changing the gist of the present invention.

[0025] [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 includes 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. Further, it is preferable that the second layer 12 is formulated with a collagen hydrogel in an amount of 0 w / v% or more and less than 0.6 w / v%. Here, v means 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 cells differentiated from keratinocytes include one or more selected from the group consisting of keratinocytes, granular cells, spinous cells, and basal cells, which will be described later.

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

[0029] ≪Second layer≫ The second layer 12 contains dermal fibroblasts.

[0030] Dermal fibroblasts 14 are cells that produce collagen fibers, elastic fibers, mucopolysaccharides, etc., and exhibit an elongated spindle shape.

[0031] The second layer 12 is preferably formulated with a collagen hydrogel in an amount of 0 w / v% or more and less than 0.6 w / v%, and more preferably in an amount of 0 w / v% or more and 0.3 w / v% or less. When the amount of the collagen hydrogel is within the above range, shrinkage of the dermal layer constituting the three-dimensional skin model during culture can be suppressed.

[0032] In addition, since the inhibition of the proliferation ability of dermal fibroblasts due to cell activity inhibition is less likely to occur and there is a vast space where proliferated cells can exist, the proliferation of dermal fibroblasts is less likely to be inhibited. Therefore, the cell density in the dermis layer is likely to be maintained high over a long period, which enables the evaluation of the sample on the dermis layer to be performed with high sensitivity. As a result of the production of proteins (cytokines, chemokines, etc.) and lipid components (eicosanoids, etc.) involved in crosstalk and their transmission to the epidermis layer, the differentiated structure of the epidermis layer constituting the three-dimensional skin model during culture can be easily maintained at a high level for a long time.

[0033] ≪Intervening membrane≫ The intervening membrane 13 is a porous membrane that allows substances to permeate and contains at least the surface with the extracellular matrix. Since the intervening membrane contains at least the surface with the extracellular matrix, it becomes easier to adhere keratinocytes and fibroblasts without depending on the collagen hydrogel.

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

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

[0036] In addition, the average pore diameter of the intervening membrane is preferably 0.4 μm or more. Also, the average pore diameter of the intervening membrane may be 8.0 μm or less, and is preferably 6.0 μm or less. When the average pore diameter of the intervening membrane is within the above range, the permeation of proteins (cytokines, chemokines, etc.) and lipid components (eicosanoids, etc.) involved in crosstalk can proceed well. The pore diameter 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, more preferably 15 μm or less, 10 μm or less, 7.5 μm or less, or 5 μm or less, from the viewpoint that a skin model in which the structures of the epidermis and dermis are maintained over a long period can be easily obtained without depending on the culture medium components (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) or the additional layers described below. However, when using specific culture medium components (substances involved in crosstalk, such as KGF, HGF, IGF-1, etc.) or providing additional layers, etc., it is not limited thereto, and the average thickness of the intervening membrane 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 increases, and the crosstalk between the dermis layer and the epidermis layer becomes advanced, so that the differentiation of the epidermis layer appropriately proceeds (the spinous layer and the granular layer become thick), and a state with a high density of the basal layer is easily maintained. The thickness of the intervening membrane can be measured, for example, using an image of an optical microscope of a histochemical section.

[0038] Note that a commercially available membrane of a cell culture insert may be used as the intervening membrane.

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

[0040] When evaluating the additional layer itself, it is preferable to have the additional layer. On the other hand, the aspect of having an additional layer promotes crosstalk more and easily maintains the differentiated structure, while the degree of freedom of the culture conditions (for example, the medium composition) suitable for all of the first layer, the second layer, and the additional layer is low. In the present embodiment, since the differentiated structure can be easily maintained without depending on the additional layer, the aspect of not having an additional layer is preferable.

[0041] (Second Embodiment) As shown in FIG. 1, the three-dimensional skin model according to the second embodiment of the present invention includes an epidermis layer 11, a dermis layer 12, and an intervening film 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.

[0042] Each component will be described below.

[0043] ≪Epidermis layer≫ In the epidermis layer 11, corneocytes form the stratum corneum 15 at the uppermost layer, granular cells form the granular layer 16 immediately below the stratum corneum 15, spinous cells (or prickle 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 (the lowermost layer). The epidermis layer 11 may also contain pigment cells (melanocytes).

[0044] <Stratum corneum> As described above, the stratum corneum 15 is located at the uppermost layer of the epidermis layer 11 that constitutes the three-dimensional skin model, and is composed of a layered structure constructed by corneocytes. The corneocytes have a flat shape and have lost their nuclei during the differentiation process. Also, the corneocytes have keratin fibers aggregated in their cytoplasm and are eosinophilic and stained light red to dark red by hematoxylin and eosin (HE) staining.

[0045] <Granular layer> As described above, the granular layer 16 is immediately below the stratum corneum 15 and is located immediately above the spinous layer 17 to 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 inside the cells. The intracellular granules are stained blue-violet to light blue by hematoxylin and eosin (HE) staining. The thickness of the granular layer of the three-dimensional skin model according to the present embodiment is preferably 3 μm or more on average, more preferably 6 μm or more on average, and still more preferably 8 μm or more on average. Also, 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 image of an optical microscope of a histochemical section.

[0046] <Stratum spinosum> As described above, the stratum spinosum 17 is directly below the stratum granulosum 16 and directly above the stratum basale 18 to be described later, and is composed of a layered structure constructed by spinous cells. The spinous cells have a flat shape and a structure in which spines are arranged around the cells. The spines can be confirmed by hematoxylin-eosin (HE) staining. The thickness of the stratum spinosum of the three-dimensional skin model according to the present embodiment is preferably 18 μm or more on average, more preferably 26 μm or more on average, and still more preferably 39 μm or more on average. Further, the thickness of the stratum spinosum 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 image of an optical microscope of a histochemical section.

[0047] <Stratum basale> As described above, the stratum basale 18 is located at the lowermost layer of the epidermis 11 and is composed of a layer structure constructed by basal cells. Unlike the stratum corneum cells, stratum granulosum cells, and stratum spinosum cells, the basal cells have a cubic to columnar shape and an oval nucleus. The basal cells are basophilic and are stained blue to light blue by hematoxylin-eosin (HE) staining. The thickness of the stratum basale of the three-dimensional skin model according to the present embodiment is preferably 7 μm or more on average, more preferably 9 μm or more on average, and still more preferably 14 μm or more on average. Further, the thickness of the stratum spinosum 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 image of an optical microscope of a histochemical section.

[0048] [Dermis] The dermis 12 has a multilayer structure. Specifically, the dermis 12 has a tissue structure composed of fibroblasts and collagen fibers (such as collagen), elastic fibers (such as fibrillin elastin), and other extracellular matrix components (such as hyaluronic acid) synthesized and secreted by the fibroblasts.

[0049] The "multi-layer structure" refers to a structure in which fibroblasts form two or more layers in the vertical direction (stratum corneum direction). Here, the orientation of fibroblasts is not constant. Therefore, due to the horizontal orientation of fibroblasts, the skin tissue section image thinly sliced in the vertical direction may exhibit a spindle shape, or due to the vertical orientation, the skin tissue section image may exhibit a circular shape.

[0050] The cell density of the dermis layer (dermal fibroblasts) is 2000 cells / cm 2 or more, preferably 5000 cells / cm 2 or more, and more preferably 6000 cells / cm 2 or more. Also, 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, 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.

[0051] (Third Embodiment) As shown in FIG. 1, the three-dimensional skin model according to the third embodiment of the present invention includes 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.

[0052] In addition, the dermis layer 12 has a multilayer structure, and a collagen hydrogel is incorporated in an amount of 0 w / v% or more and less than 0.6 w / v%. Here, the above v means the volume of the dermis layer. It is more preferable that the dermis layer 12 incorporates the collagen hydrogel in an amount of 0 w / v% or more and 0.3 w / v% or less. Alternatively, the dermis layer 12 is not in a state where collagen fibers are unevenly dispersed by hematoxylin and eosin (HE) staining. As a result, since there is a large space where proliferated cells can exist, the proliferative ability of dermal fibroblasts is less likely to be inhibited. Therefore, shrinkage of the dermis layer constituting the three-dimensional skin model during culture can be suppressed.

[0053] In addition, the inhibitory effect on the proliferative ability of dermal fibroblasts due to cell activity inhibition is less likely to occur, and since there is a large space where proliferated cells can exist, the proliferation of dermal fibroblasts is less likely to be inhibited. Therefore, the cell density of the dermis layer is likely to be maintained high over a long period of time. As a result, it is easy to highly sensitively evaluate the sample on the dermis layer, and proteins (cytokines, chemokines, etc.), lipid components (eicosanoids, etc.), etc. involved in crosstalk are produced and transmitted to the epidermis layer, so that the differentiated structure of the epidermis layer constituting the three-dimensional skin model during culture can be highly maintained over a long time.

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

[0055] In the aspect having an additional layer, crosstalk is further promoted and it is easy to maintain the differentiated structure, while the degree of freedom of culture conditions (for example, medium composition) suitable for all of the epidermis layer, the dermis layer, and the additional layer is low. In the present embodiment, since it is easy to maintain the differentiated structure without depending on the additional layer, an aspect without an additional layer is preferable.

[0056] [Culture vessel] The culture vessel 20 used in the first to third embodiments has, as shown in FIGS. 2(a) and 2(b), a plurality of wells 21 for accommodating a culture solution 23, and a support portion 22 for supporting the three-dimensional skin model 10 within the wells 21. The three-dimensional skin model 10 is arranged so as to be communicable 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 and is not particularly limited, but can have 12, 24, 48, or 96 wells. Thereby, evaluations regarding a large number of samples and application conditions can be carried out simultaneously.

[0058] [Method for evaluating samples] The sample evaluation method of the present invention is carried out using a culture vessel. Specifically, the sample evaluation method includes: (1) a step of culturing a three-dimensional skin model using the culture vessel 20 shown in FIGS. 2(a) and 2(b) (culture step); (2) a step of administering a sample to the three-dimensional skin model during culture (sample administration step); and (3) a step of evaluating the function exerted by the above 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 from the sample administration (evaluation step).

[0059] Hereinafter, each step will be described.

[0060] [Culture step] More specifically, the culture step is a step of arranging 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 vessel 20 so as to be communicable with the culture solution 23, and culturing the three-dimensional skin model 10 for a predetermined period.

[0061] The culture period is 6 days or more, more preferably 12 days or more, and even more preferably 20 days or more. Also, the culture period may be 30 days or less. Since the evaluation period is long when the culture period is 6 days or more, sufficient evaluation results can be obtained.

[0062] [Sample administration step] The sample administration step is, more specifically, a step of administering a sample to the three-dimensional skin model 10 disposed in the well 21 of the culture vessel 20 by a predetermined method.

[0063] The sample to be administered is not particularly limited and 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. Also, the method of administering the sample is not particularly limited and may be applied to the surface of the epidermis, injected into the epidermis, injected into the dermis, or added to the culture solution.

[0064] Also, different samples may be applied to each well, or the same sample may be applied in different ways (dosage and / or usage). Thereby, the performance of the sample on the skin can be quantitatively compared, and the preferable administration method (dosage and / or usage) for imparting the desired function to the skin can be inferred.

[0065] (Evaluation step) The evaluation step is, more specifically, a step of evaluating the function of the sample on the three-dimensional skin model 10 by analyzing the culture supernatant containing the secretion secreted by the three-dimensional skin model 10 or cells after a culture period of several hours or more from sample administration.

[0066] The above 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] Also, the evaluation step may include a step of processing the three-dimensional skin model 10, a step of administering the same or different samples to the three-dimensional skin model 10 two or more times, or a step of removing the sample administered to the three-dimensional skin model 10 during the above-mentioned culture period.

[0068] The process of processing the three-dimensional skin model 10 is a process of producing three-dimensional skin models in different states. The processing method is not particularly limited, and includes addition of inflammatory substances for inducing oxidative stress, light irradiation that causes oxidative stress such as UV, etc. This processing may take several seconds to several days, but since the skin model used in this embodiment can maintain the structures of the epidermal layer and the dermal layer over a long period of time, it is easy to freely perform evaluations using the processed skin model. By producing three-dimensional skin models in different states, it is possible to evaluate not only normal skin but also rough skin, inflamed skin, etc., so that the effectiveness of samples for various skin states can be obtained.

[0069] The step of administering the same or different samples two or more times may involve administering the same sample in the same way or in different ways. Also, different samples may be administered in the same way or in different ways. The "way" referred to here refers to dosage and / or usage.

[0070] In addition, in order to compare with the three-dimensional skin model to which the sample has been administered, at least a three-dimensional skin model to which no sample has been administered may be cultured in the above-described culturing step for a predetermined period (for example, 6 days or more, preferably 12 days or more, and 20 days or more. It may be 30 days or less). Here, it is preferable to perform the culturing under conditions such that the three-dimensional skin model to which no sample has been administered has an epidermal layer having a stratum corneum, a granular layer, a spinous layer, and a basal layer, and a dermal layer having a multilayer structure after the lapse of the culturing period.

[0071] After the cultivation 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 condition that the planar area of the epidermal layer ≤ the planar area of the dermal layer, or the planar area of the dermal layer after the cultivation period is preferably 0.7 times or more the planar area of the dermal layer before the cultivation period. Although not particularly limited, it is more preferably 0.9 times or more. Also, the planar area of the dermal layer after the cultivation period is preferably 1.0 times or less the planar area of the dermal layer before the cultivation period. When 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 within the above range, shrinkage of the dermal layer during cultivation is suppressed, enabling long-term evaluation. Also, the planar area of the epidermal layer is preferably 0.7 times or more and 1.0 times or less the planar area of the dermal layer before the cultivation period, more preferably 0.9 times or more and 1.0 times or less.

Examples

[0072] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

[0073] [Construction of three-dimensional skin model] A cell culture insert having a collagen membrane (ad-MED Vitrigel 208364-96 (manufactured by Kanto Chemical Co., Inc.), AteloCell CM-24 (manufactured by Takara Bio Inc.)) 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 cultured in a CO2 incubator at 37°C for 1 day. Next, 500,000 normal human epidermal keratinocytes suspended in Humedia KG2 (manufactured by Kurabo Industries Ltd.) were seeded on the other side of the cell culture insert, cultured in a CO2 incubator at 37°C for 1 day, then replaced with a medium for three-dimensional culture, and the culture was continued for another 1 day. After the culture, the medium on the normal human keratinocyte side was removed, and air-phase culture was started. By performing air-phase culture for 12 days, epidermal layer differentiation was induced to construct a three-dimensional skin model. Note that "Vitrigel" is a registered trademark of the National Agriculture and Food Research Organization, "AteloCell" is a registered trademark of Takara Bio Inc., 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 dermal layer was 7114 ± 339 cells / cm 2 and was 5843 ± 349 cells / cm 2 after 20 days of air-phase culture. According to the investigation by the present inventor, the cell density of the dermal layer of the model disclosed in Non-Patent Document 1 was 1300 ± 339 cells / cm 2 and was significantly lower than that of this example. In addition, the planar areas of the dermal layer and epidermal layer of the constructed three-dimensional skin model after the culture period were 1 times those of the dermal layer and epidermal layer before the culture period, respectively.

[0075] [Histochemical Analysis of Three-Dimensional Skin Model 1] (Analysis Method) Using the above method, after fixing the skin tissue of the three-dimensional skin model that had been cultured in the gas phase for 12 days with Superfix KY-500 (manufactured by Kurabo Industries Ltd.), it was embedded in Paraplast X-tra (manufactured by Leica Biosystems), and paraffin-embedded sections (skin tissue sections) were prepared. After staining the skin tissue sections with hematoxylin and eosin (HE), analysis was performed using an all-in-one fluorescence microscope "BZ-X800" (manufactured by KEYENCE CORPORATION). Note that "Paraplast" is a registered trademark of Leica Biosystems Richmond.

[0076] (Results) As shown from the image of the skin tissue section after HE staining in Fig. 1, it was confirmed that, similar to human skin tissue, the three-dimensional skin model was composed of an epidermis layer consisting of a stratum corneum, a granular layer (flattened cells containing intracellular granules), a spinous layer (cells with spines around them), and a basal layer (cuboidal morphology). Also, it was confirmed that fibroblasts were arranged in layers as the dermis layer directly below the basal layer, showing the basic morphological characteristics as a skin model.

[0077] [Histochemical Analysis 2 of Three-Dimensional Skin Model] (Analysis Method) Using the above method, the skin tissue of the three-dimensional skin model that had been cultured in the gas phase for 12 days was further cultured for more than one week. After staining the cultured skin tissue with hematoxylin and eosin (HE), analysis was performed using an all-in-one fluorescence microscope "BZ-X800".

[0078] (Results) Comparing the skin tissue section image of the skin tissue (a) cultured in the gas phase for 12 days and the skin tissue section image of the skin tissue (b) cultured for 20 days, as shown in Fig. 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 in the prior art.

[0079] [Histochemical Analysis 2 of Three-Dimensional Skin Model] (Analysis Method) Using the above method, the skin tissue of the three-dimensional skin model cultured in the gas phase for 12 days was fixed with Superfix KY-500, embedded in Paraplast X-tra, and paraffin-embedded sections (skin tissue sections) were prepared. Immunohistochemical staining was performed on the prepared skin tissue sections using an antibody against a protein whose localization in the epidermal layer was clear.

[0080] (Results) As shown in Fig. 4(a), 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. Also, as shown in Fig. 4(b), filaggrin (not shown), a component of keratohyalin granules, was confirmed to be expressed in the granular layer 16 (granules within granular cells), and claudin 1 (not shown), a tight junction-forming adhesion molecule, was confirmed to be expressed in the spinous layer 16 (around the cells of spinous cells). From Fig. 4(a) and (b), the morphological characteristics of the three-dimensional skin model could be confirmed from the localization of differentiation marker molecules.

[0081] [Analysis of the barrier function of the three-dimensional cultured skin model 1] (Analysis method) Using the above method, a sodium dodecyl sulfate (SDS) solution, a surfactant prepared at a concentration of 0 - 0.5% (in 0.1% increments), was applied to the epidermal surface of the three-dimensional skin model cultured in the gas phase for 12 days, and cultured in a CO2 incubator at 37°C for 1 hour. After culturing, the SDS solution remaining on the epidermal surface was removed, the remaining SDS solution was washed away with phosphate-buffered saline (PBS)(-), and further recovery culture was performed in a CO2 incubator at 37°C for 24 hours. The cytotoxicity after culturing was measured using a Cytotoxicity LDH Assay Kit-WST (manufactured by Dojindo Laboratories) with the activity of cytoplasmic redox enzyme (LDH) leaked into the medium as an index.

[0082] (Results) As shown in Fig. 5, the cytotoxicity of the three-dimensional skin model showed an increasing trend as the concentration of the SDS solution increased. Therefore, it was confirmed that the three-dimensional skin model has a barrier function in the epidermis.

[0083] [Analysis of the barrier function of the three-dimensional cultured skin model 2] (Analysis method) Using the above method, normal human skin fibroblasts and normal human epidermal keratinocytes were seeded in the insert, and the transepithelial electrical resistance (TEER) of the three-dimensional skin model was measured over time with the day when the gas-phase culture was started set as day 0.

[0084] (Results) As shown in Fig. 6, it was confirmed that the barrier function was enhanced with the growth of the three-dimensional skin model and was maintained even after the maturation of the three-dimensional skin model.

[0085] [Analysis of the responsiveness of the three-dimensional skin model to physiological stimuli 1] (Analysis method) Using the above method, a 10 μM solution of retinoic acid (ATRA) was applied to the epidermis (surface) of the three-dimensional skin model that had been cultured in the gas phase for 12 days, and the cells were cultured at 37°C for 1 hour. After the culture, the retinoic acid solution was removed, and after further culturing for 5 hours, RNA was extracted from the skin tissue and gene expression analysis was performed using the real-time PCR method.

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

[0087] [Analysis of the responsiveness of the three-dimensional skin model to physiological stimuli 2] (Analysis method) Using the above method, transforming growth factor β (TGFβ) prepared to a final concentration of 10 nM was added to the medium of the three-dimensional skin model cultured in the gas phase for 12 days, followed by culturing for 48 hours, and gene expression analysis of the reconstructed three-dimensional skin model was performed. In addition, immunohistochemical staining was performed using an antibody against a protein whose localization in the dermis was clarified.

[0088] (Results) As shown in FIGS. 8(a) and 8(b), induction of gene expression of dermal collagen fibers and elastic fibers such as COL1A1 (type I collagen gene) and FBN1 (fibrillin-1 gene) could be confirmed.

[0089] As shown in FIGS. 9(a) and 9(b), when the expression of type I collagen (41) and fibrillin-1 (42) in the dermal fibroblast layer 14 (dermis) of immunohistochemical staining was confirmed, in the three-dimensional skin model (40) cultured with the addition of TGFβ, an increase in the expression and a change in the localization of the fibrous protein could be confirmed. Thereby, it was also confirmed that the reconstructed skin model has responsiveness to physiological stimuli.

[0090] [Analysis of Responsiveness of Three-Dimensional Skin Model to Cosmetics] (Analysis Method) Using the above method, commercially available cosmetics (skin care products) 1 to 5 were applied to the epidermal surface of the three-dimensional skin model cultured in the gas phase for 12 days, and cultured in a CO2 incubator at 37°C for 1 hour. After culturing, the cosmetics were removed, and after further culturing for 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 gene (hyaluronic acid synthase 2 (HAS2)) was analyzed using real-time PCR.

[0091] (Results) As shown in FIGS. 10(a) to 10(d) and FIGS. 11(a) to 11(d), it was confirmed that different responsiveness to Cosmetics 1 to 5 was exhibited depending on the type of epidermal gene. Thus, it was confirmed that the functional evaluation of a product (such as cosmetics) is possible by analyzing a three-dimensional skin model to which the product (such as cosmetics) has been applied.

[0092] [Protein Expression Analysis of Three-Dimensional Skin Model] (Analysis Method) Using the above method, only the epidermal layer of the three-dimensional skin model cultured in the gas phase for 14 days was excised, and an extracted protein solution was prepared using a general cell lysate. Using the prepared extracted protein solution, protein expression analysis of the skin tissue was performed by Western Blotting method.

[0093] (Results) As shown in FIGS. 12(a) and 12(b), synthesis and metabolism of filaggrin and caspase 14 were confirmed from the granular layer by Western Blotting method.

[0094] [Responsiveness Analysis of Three-Dimensional Cultured Skin Model to Cosmetics of the Same Dosage Form] (Analysis Method) To perform functional evaluation within the same category (dosage form) of skin care products, commercially available cosmetics (skin care products) such as essence (S001 - S016), lotion (L001 - L012), emulsion (E001 - E011), or cream (C001 - C012) were applied to the epidermal surface of a three - dimensional skin model cultured for 12 days in a gas - phase culture. After culturing at 37°C for 1 hour in a CO2 incubator, the cosmetics on the epidermal surface were removed, and after further culturing for 23 hours or 47 hours, RNA was extracted from the skin tissue and the expression of various epidermal genes (glucosylceramidase (GBA) / serine palmitoyltransferase long - chain base subunit 1 (SPTLC1) / elongation of very long - chain fatty acids protein 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 alpha chain 1 (COL1A1) / fibrillin (FBN) / fibulin 5 (FBLN5) / latent TGF - beta binding protein 4 (LTBP4) / hyaluronan synthase 2 (HAS2), etc.) was analyzed using real - time PCR method.

[0095] (Results) As shown in (a) - (f) of Fig. 13 to (a) - (f) of Fig. 16, it was confirmed that different responsiveness was shown for each product of essence (S001 - S016), lotion (L001 - L012), emulsion (E001 - E011), or cream (C001 - C012) depending on the types of epidermal genes and dermal genes. Thus, it was confirmed that the functional evaluation of skin care products is possible by analyzing a three - dimensional skin model to which commercially available cosmetics (skin care products) of the same category (dosage form) are applied.

Industrial Applicability

[0096] According to the present invention, since it can suppress the contraction of the dermis layer and maintain the structure of the epidermis and dermis over a long period, it is effective for efficient sample evaluation of cosmetics, etc., which is carried out using a culture container having a large number of wells.

Explanation of Symbols

[0097] 10, 30, 40 three-dimensional skin models 11 The first layer, the epidermis 12 The second layer, the dermis 13 Intermediate membrane 14 Dermal fibroblast layer 15 Stratum corneum 16 Granular layer 17 Spinous layer 18 Basal layer 20 Culture vessel 21 Well 22 Support part 23 Culture solution 41 Type I collagen 42 Fibrin-1 43 Nucleus

Claims

1. Using a culture vessel comprising a plurality of wells for containing a culture solution, an epidermis layer, a dermis layer, an intervening membrane intervening between the epidermis layer and the dermis layer, having, the epidermis layer has a stratum corneum, a granular layer, a spinous layer, and a basal layer, The dermis has a multi-layer structure and has a cell density of 2000 cells / cm ２ or more and 10000 cells / cm ２ or less, and culturing a three-dimensional skin model; a step of administering a sample to the three-dimensional skin model during 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 the sample administration, including, the culture vessel has 12, 24, 48, or 96 of the wells, the culturing step is performed within a range of 6 days or more and 30 days or less, A method for evaluating a sample.

2. The method for evaluating a sample according to claim 1, wherein the three-dimensional skin model is formulated with a collagen hydrogel in an amount of 0 w / v% or more and less than 0.6 w / v%, and the v means the volume of the dermis layer.

3. The method for evaluating a sample according to claim 1 or 2, wherein the evaluating step includes a step of processing the three-dimensional skin model, a step of administering the same or different samples two or more times, or a step of removing the administered sample during the culture period.

4. The method for evaluating a sample according to claim 1 or 2, wherein at least the three-dimensional skin model to which no sample is administered has an epidermis layer having a stratum corneum, a granular layer, a spinous layer, and a basal layer, and a dermis layer having a multilayer structure after the culture period has elapsed.

5. The method for evaluating a sample according to claim 1 or 2, wherein the relationship between the planar area of the epidermis layer and the planar area of the dermis layer of the three-dimensional skin model after the culture period has elapsed satisfies the planar area of the epidermis layer ≦ the planar area of the dermis layer, or the planar area of the dermis layer after the culture period has elapsed is 0.7 times or more the planar area of the dermis layer before the culture period has elapsed.

6. The method for evaluating a sample according to claim 1 or 2, wherein the analysis includes gene expression, protein expression, histochemical analysis, or skin barrier function analysis.

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