Three-dimensional core-shell structure

Abstract

The present invention provides a novel three-dimensional structure containing epidermal keratinocytes, and a technique for producing the same.　The present invention provides a three-dimensional core-shell structure comprising a cell layer that has tight junctions present on the surface and that is configured at least from a plurality of epidermal keratinocytes, and a central part that is a microspherical object having an extracellular matrix component on the surface. The microspherical material is preferably a microcarrier or a cell mass.

Description

Three-dimensional core-shell structure

[0001] The present invention relates to a three-dimensional structure with a core-shell structure, a method for producing a three-dimensional structure with a core-shell structure, a method for evaluating skin homeostasis using a three-dimensional structure with a core-shell structure, and the like.

[0002] In recent years, attention has been focused on maintaining skin homeostasis, such as maintaining normal skin function and restoring weakened skin function. Skin tissue is composed of the epidermis, dermis, and subcutaneous tissue, each of which plays a different role in maintaining skin homeostasis. The epidermis, which is in contact with the outside world, is composed of epidermal keratinocytes, which have a multilayer structure consisting of layers at different stages of differentiation (specifically, the basal layer, spinous layer, granular layer, and stratum corneum). In the epidermis, epidermal keratinocytes differentiate from an undifferentiated state and ultimately peel off from the skin surface at the stratum corneum, a metabolic cycle that occurs at regular intervals. This metabolic cycle is called turnover. The epidermis, which is in contact with the outside world, also plays a role in barrier functions, preventing moisture evaporation from the body and the invasion of pathogens, etc. into the body. Meanwhile, the dermis supports the skin with collagen, elastin, etc., and plays a role in maintaining its shape and elasticity.

[0003] In recent years, as an alternative to model animal experiments, sheet-like three-dimensional cultured skin models with a three-dimensional structure including the dermis and epidermis similar to animal skin tissue have been used, and skin homeostasis functions such as skin barrier function and turnover have been evaluated in wells containing these sheet-like three-dimensional cultured skin models. In order to study such skin homeostasis, various markers have been used in addition to measurements using skin moisture measuring devices.

[0004] The following three-dimensional cultured skin models have been proposed. For example, Patent Document 1 proposes a "three-dimensional cultured skin model having an artificially created dermis (dermis equivalent) layer, comprising the following structures (1) to (3): (1) a dermis equivalent layer obtained by culturing a mixture of collagen and fibroblasts; (2) a layer of epidermal keratinocytes; and (3) melanocytes engrafted between the dermis equivalent layer (1) and the epidermal keratinocyte layer (2)." Patent Document 2 also discloses a three-dimensional cultured human skin model comprising, from bottom to top, a support layer A containing fibroblasts, a support layer B containing dendritic cells, and an epidermal keratinocyte layer C, in which the epidermal keratinocytes in C are in the form of a sheet seeded on the support layer B containing dendritic cells.

[0005] In addition to sheet-like structures, the following three-dimensional structures have also been proposed. For example, Non-Patent Document 1 proposes the production of three-dimensional dermal papilla (DP) cells by sequentially seeding dermal papilla (DP) cells, HaCaT keratinocytes, and human dermal fibroblast (HDF) cells into three-dimensional (3D) microwells made from polyethylene glycol diacrylate hydrogel. Non-Patent Document 2 also proposes three-dimensional cell culture using a clinostat (a condition similar to a microgravity environment) to reproduce changes in skin caused by microgravity, thereby enabling understanding of the effects of microgravity on human health before space exploration.

[0006] JP 2010-193822 A JP 2006-333763 A JP 2023-018632 A

[0007] Justin JY Tan et al., Cell Prolif. 2019 Sep ; 52(5):e12668.Dong Hyun Choi et al., npj Microgravity volume 7, Article number: 20 (2021).Sergio Cortez Ghio et al., Int. J. Mol. Sci. 2018, 19(8), 2174.Okita K. et.al., Nature Methods, 2011,8:409-12.Ido H, et al., J Biol Chem. 2007, 282, 11144-11154.

[0008] Thus, skin models are useful for evaluating skin homeostasis functions such as skin barrier function. The inventors considered that, compared with sheet-like skin models, approximately spherical three-dimensional structures in which cells aggregate to form masses, such as spheroids, can be obtained using fewer cells, fewer steps, and a shorter culture period, and that the obtained approximately spherical three-dimensional structures can be used to easily perform tests such as screening, evaluation, analysis, and measurement under a variety of conditions.

[0009] However, while Non-Patent Document 1 proposes three-dimensional dermal papilla cells, it does not disclose a three-dimensional structure with a core-shell structure in which epidermal keratinocytes cover the outside of a core of a fibroblast mass. Furthermore, Non-Patent Document 2 discloses a three-dimensional cell culture technique under microgravity using a special device and a special three-dimensional structure produced thereby, and aims to examine the effects of microgravity on human health before space exploration. Therefore, the technique disclosed in Non-Patent Document 2 is not a culture technique under Earth's gravity, as is commonly done in cell culture. Furthermore, there has been a demand for a roughly spherical three-dimensional structure that can also be used to effectively evaluate skin homeostasis functions, such as skin barrier function.

[0010] Therefore, a main object of the present invention is to provide a novel three-dimensional structure containing epidermal keratinocytes and a technique for producing the same.

[0011] The present inventors believed that the presence of tight junctions in a three-dimensional structure would enable more accurate testing of skin homeostasis functions (e.g., skin barrier function, etc.). However, there is insufficient knowledge about approximately spherical three-dimensional structures containing tight junctions and epidermal keratinocytes. Furthermore, there has been a demand for approximately spherical three-dimensional structures that form tight junctions, which are responsible for the barrier function, an important function of the epidermis, and that can evaluate the effects of drugs on these tight junctions. Therefore, the present inventors' second objective was to provide a three-dimensional structure containing tight junctions and a technique for producing the same.

[0012] As a result of extensive research, the present inventors have found that a novel three-dimensional structure with a core-shell structure comprising a cell layer containing a plurality of epidermal keratinocytes and a plurality of tight junctions can be provided by co-culturing microspheres and seeded epidermal keratinocytes.

[0013] The present invention can provide a three-dimensional structure with a core-shell structure comprising a cell layer having tight junctions present on the surface and composed of at least a plurality of epidermal keratinocytes, and a core portion that is a microsphere having extracellular matrix components on the surface.

[0014] The present invention provides a method for producing a three-dimensional structure with a core-shell structure by co-culturing microspheres and seeded epidermal keratinocytes, and a method for evaluating skin homeostasis using the three-dimensional structure.

[0015] The microspheres may be microcarriers or cell aggregates. The epidermal keratinocytes may be epidermal keratinocytes derived from pluripotent stem cells. The three-dimensional structure may be used to evaluate skin homeostasis. When culturing the microspheres and the seeded epidermal keratinocytes, a medium containing at least an epidermal keratinocyte medium, or a medium containing an epidermal keratinocyte medium and a fibroblast medium, may be used. The three-dimensional structure may be cultured so that the size of the three-dimensional structure is 1.05 to 1.5, where the size of the microspheres is taken as 1.

[0016] The present invention can provide a novel three-dimensional structure containing epidermal keratinocytes and a technique for producing the same.

[0017] 1 shows a schematic diagram of an example of a three-dimensional core-shell structure according to the present embodiment, which is composed at least of a cell layer in the shell portion containing multiple tight junctions and multiple epidermal keratinocytes, and a central portion in the core portion containing multiple cells (e.g., fibroblasts), but the present invention is not limited thereto. 1 shows a schematic diagram of an example of a three-dimensional core-shell structure according to the present embodiment, which is composed at least of a cell layer in the shell portion containing multiple tight junctions and multiple epidermal keratinocytes, and a central portion in the core portion containing microcarriers, but the present invention is not limited thereto. 1 shows an example of a manufacturing example of a three-dimensional core-shell structure according to the first embodiment, in which a cell cluster is present in the core, but the present invention is not limited thereto. 1 shows an example of a manufacturing example of a three-dimensional core-shell structure according to the second embodiment, in which a microcarrier is present in the core, but the present invention is not limited thereto. 1 shows an optical microscope photograph of a three-dimensional core-shell structure using cell clusters as the core, which was manufactured according to the first embodiment, but the present invention is not limited thereto. 1 shows a grayscale photograph of a fluorescent observation of a three-dimensional core-shell structure using cell clusters as the core, which was manufactured according to the first embodiment, but the present invention is not limited thereto. The grayscale photographs also show how to calculate the occupancy rate of Occludin-positive cells. Sample 1: Total (1): 3.907 / Occludin-positive cell region (2): 1.035 / Occludin-positive cell region: 1.035 / Occludin-positive cell region: 1.035 / 3.907 × 100 = 26.5%. Sample 2: Total (1): 6.384 / Occludin-positive cell region (1-2): 5.843 / Occludin-positive cell region: 5.843 / 6.384 × 100 = 91.5%. The grayscale photographs show fluorescence microscopy (Occludin-stained images) of cell aggregates obtained using the production method of the first embodiment, with the epidermal keratinocyte culture medium and fibroblast culture medium being mixed at ratios (for epidermal cells:for fibroblasts) of 100:0, 75:25, 50:50, 25:75, and 0:100. 1 is a gray photograph of a fluorescent microscope (Occludin stained image) showing cell masses obtained when the NHDF:iKC ratio was 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, or 1:8 in the production method of the first embodiment. FIG. 2 is a conceptual diagram showing the structure of the skin and the localization of epidermis-specific markers.

[0023] Figure 1 is a gray photograph showing the staining and localization during the culture period in the obtained three-dimensional structure with a core-shell structure of the first embodiment, when using Ki-67 antibody, Occludin antibody, and Collagen XVII antibody, as confirmed by a fluorescence microscope. Figure 2 is a gray photograph showing the time-dependent changes in the localization of the seeded epidermal keratinocyte types (iKC, HPEK) and epidermis-specific markers (Occludin, Ki-67) in the obtained three-dimensional structure with a core-shell structure of the first embodiment, as confirmed by a fluorescence microscope. Figure 3 is a gray photograph showing the time-dependent changes in the localization of the seeded epidermal keratinocyte types (HPEK) and epidermis-specific markers (Occludin, Ki-67) in the obtained three-dimensional structure with a core-shell structure of the second embodiment, as confirmed by a fluorescence microscope. In addition to Occludin staining and Ki-67 staining, Hoechst staining was also observed in the three-dimensional core-shell structure of the second embodiment. （Ｒ） Also shown are grayscale photographs of nucleic acid stained and Collagen XVII stained structures. Grayscale photographs showing that the core of the obtained core-shell three-dimensional structure of this first embodiment has a fibroblast cell cluster in the core portion, and the surface of the cell cluster has extracellular matrix (Col1 or Col4). The upper photograph shows the three-dimensional structure after three weeks of culture, stained with an antibody against Col IV (4), a type of extracellular matrix. The lower photograph shows the three-dimensional structure after three weeks of culture, stained with an antibody against Col I (1), a type of extracellular matrix.

[0018] Preferred embodiments for implementing the present technology will be described below. Note that the embodiments described below are examples of typical embodiments of the present technology, and the scope of the present technology will not be narrowly interpreted by them. Note that percentages in this specification are expressed by volume (volume / volume%) unless otherwise specified. In addition, the upper limit (or less) and the lower limit (or more) of each numerical range (to) can be arbitrarily combined as desired.

[0019] 1. Three-dimensional structure according to this embodiment This embodiment can provide a three-dimensional structure with a core-shell structure comprising epidermal keratinocytes and microspheres having extracellular matrix components on their surfaces. This embodiment can provide a three-dimensional structure with a core-shell structure comprising at least a cell layer composed of at least a plurality of epidermal keratinocytes and a central portion that is a microsphere, and it is preferable that the microspheres have extracellular matrix components on their surfaces. In this embodiment, it is more preferable that the central portion is surrounded by the cell layer as a surface layer of the three-dimensional structure. In this embodiment, it is preferable that the surface of the cell layer has tight junctions, and it is preferable that tight junctions are present between adjacent differentiated epidermal keratinocytes present on the surface of the cell layer that contacts the outside.

[0020] The "microsphere" in the microsphere having an extracellular matrix component on its surface is preferably a microcarrier (preferably acellular microcarrier) or a cell cluster (preferably a fibroblast cluster), and each of these three-dimensional structures with a core-shell structure is also referred to as the first embodiment (see FIG. 1A) and the second embodiment (see FIG. 1B), and the surface of the central portion preferably has an extracellular matrix component (e.g., collagen).

[0021] For example, in the case of the first embodiment, the first embodiment provides a three-dimensional structure with a core-shell structure containing a plurality of epidermal keratinocytes and a plurality of fibroblasts. The first embodiment can preferably provide a three-dimensional structure with a core-shell structure at least comprising a cell layer composed of at least a plurality of epidermal keratinocytes and a central portion composed of at least a plurality of fibroblasts, and the surface of the central portion comprises extracellular matrix components (e.g., collagen).

[0022] For example, the second embodiment provides a three-dimensional core-shell structure comprising a plurality of epidermal keratinocytes and one microcarrier. This second embodiment preferably provides a three-dimensional core-shell structure comprising at least a cell layer composed of at least a plurality of epidermal keratinocytes and a core composed of one microcarrier, with the surface of the core comprising extracellular matrix components (e.g., collagen). Advantages of the second embodiment include, for example, simplified operation because only one type of cell, epidermal keratinocytes, is required; evaluation does not require a special cell isolation process, allowing pure evaluation of only epidermal keratinocytes, which constitute the epidermis and are important for the skin barrier; and the use of iPS cell-derived epidermal keratinocytes provides a biomimetic model that purely reflects individual characteristics, allowing evaluation of each individual's skin characteristics and drug responsiveness. Furthermore, an advantage of the second embodiment is that, like the first embodiment, epidermal keratinocytes are present in the surface layer, and microcarriers are used in the core, which is believed to reduce the influence of other types of cells when evaluating the surface layer. Furthermore, compared to the first embodiment, the second embodiment is considered to have advantages over the first embodiment in terms of manufacturing method, such as reducing labor hours and costs, because it uses microcarriers, and is therefore considered to make it easier to improve production efficiency and mass production.

[0023] In this embodiment, the central portion is also referred to as the core portion, and the surface portion is also referred to as the shell portion. Furthermore, it is more preferable that the three-dimensional structure has a contact state between the inner layer of the shell portion (surface portion) toward the center and the outer layer of the core portion (central portion) toward the outer diameter, and it is more preferable that an extracellular matrix component (e.g., collagen) is present in this contact state. For example, in the first embodiment, the core portion is preferably a fibroblast mass, and in the second embodiment, the core portion is preferably a microcarrier.

[0024] In this embodiment, the cell layer is preferably a cell layer composed of at least a plurality of epidermal keratinocytes having tight junctions on their surface, and / or the central portion is preferably a microsphere, and more preferably the microsphere has an extracellular matrix component on the surface of the central portion. The central portion is preferably a cell cluster or a microcarrier, and in the case of a cell cluster, the first embodiment may be used, and in the case of a microcarrier, the second embodiment may be used. The central portion is preferably a single fibroblast cluster composed of at least a plurality of fibroblasts, or a single microcarrier.

[0025] In addition, a preferred aspect of this embodiment provides a three-dimensional structure with a core-shell structure comprising a cell layer composed at least of a plurality of tight junctions present between cells in the surface cell layer and a plurality of epidermal keratinocytes, and a central portion consisting of microspheres containing extracellular matrix components. Preferred aspects of the microspheres in this case can be appropriately adopted from the first or second embodiment described above. In addition, a preferred aspect of this embodiment is that the cell layer is composed of at least a differentiated layer containing differentiated epidermal keratinocytes and an undifferentiated layer toward the center containing undifferentiated epidermal keratinocytes, and / or that the tight junctions are present between adjacent differentiated epidermal keratinocytes present in the surface cell layer of the three-dimensional structure.

[0026] Another preferred aspect of this embodiment is a three-dimensional structure with a core-shell structure, in which the surface layer of the three-dimensional structure is composed at least of a plurality of epidermal keratinocytes and tight junctions between adjacent epidermal keratinocytes in the outermost layer, and the center of the three-dimensional structure is composed at least of microspheres (e.g., a plurality of fibroblasts, one microcarrier, etc.).

[0027] Another preferred aspect of this embodiment is a three-dimensional core-shell structure comprising: (a) a cell layer composed at least of tight junctions present between cells on the cell layer surface and a plurality of epidermal keratinocytes; and (b) a microsphere having an extracellular matrix component on its surface as a central portion. The microsphere is preferably a single cell cluster having an extracellular matrix component coating layer composed of at least a plurality of cells of a certain type, or a single microcarrier having an extracellular matrix component coating layer. These may be referred to as the first embodiment having a cell cluster and the second embodiment having a microcarrier, respectively. The cells of the certain type used in the first embodiment may be the same or a combination of different types, but are preferably the same type, and more preferably fibroblasts. The microcarriers used in the second embodiment are preferably non-cellular.

[0028] Another preferred aspect of this embodiment is a three-dimensional structure having a core-shell structure, comprising: (a) a cell layer in the surface portion, the cell layer including at least a plurality of epidermal keratinocytes and tight junctions present on the surface of the three-dimensional structure between adjacent epidermal keratinocytes; and (b) microspheres (e.g., non-cellular or cellular) in the center. It is more preferred that the microspheres (preferably, microcarriers or fibroblast cell clusters) serve as a core portion, and that the core portion is covered with a layer containing epidermal keratinocytes. The preferred (b) is preferably (b1) a fibroblast cell cluster in the center containing a plurality of fibroblasts, or (b2) acellular spheres and acellular microcarriers in the center. These may be referred to as the first embodiment having a cell cluster and the second embodiment having a microcarrier, respectively.

[0029] The three-dimensional structure according to this embodiment is suitable for use in evaluating skin homeostasis functions such as skin barrier function, etc. The three-dimensional structure according to this embodiment can be manufactured by the method described later in "2. Manufacturing method of the three-dimensional structure according to this embodiment."

[0030] In the description of this embodiment, explanations of the components and methods of epidermal keratinocytes, fibroblasts, microspheres, microcarriers, core-shell structures, pluripotent stem cells, cell culture equipment, etc. that overlap with the explanations of "2," "3," "4," etc. described below will be omitted as appropriate, but the explanations of "2," "3," "4," etc. described below also apply to this embodiment and can be adopted as appropriate.

[0031] <Core-shell structure> In the present specification, the core-shell structure preferably has a microsphere as the central part, and a cell layer formed by a plurality of cells surrounding the central part as the surface layer, more preferably a multi-layered cell layer, and more preferably, the microspheres contain extracellular matrix components. The microspheres may be cell masses formed from a plurality of cells or microcarriers.

[0032] In this first embodiment, it is more preferable that the cells constituting the cell mass and the cells constituting the cell layer are different cells. In this second embodiment, it is more preferable that the cells constituting the cell layer are cell-free microcarriers. The microspheres are also called nuclei or cores, and the surface layer or cell layer of the surface layer is also called a shell. Furthermore, the position of the microspheres in the three-dimensional structure is preferably such that a core-shell structure can be formed, and does not have to be at the center of the three-dimensional structure, but may be at the center (center or its periphery). Furthermore, it is preferable that the cell layer of the three-dimensional structure is located outward from the center. In this embodiment, the outward direction is preferably the outer diameter direction.

[0033] For example, the three-dimensional structure of this embodiment is not limited to Figures 1A and 1B, and a structure having a core portion and a shell portion thereof as shown in Figures 1A and 1B is also referred to as a core-shell structure. For example, a suitable example of the three-dimensional structure of this embodiment as shown in Figures 1A and 1B is a three-dimensional structure of a core-shell structure including (a) a core such as a fibroblast mass or a microcarrier, and (b) a shell such as an epidermal keratinocyte layer including a differentiated epidermal keratinocyte layer including an outermost layer and an innermost layer, and an undifferentiated epidermal keratinocyte layer. For example, the three-dimensional structure 1 with a core-shell structure according to the first embodiment is composed of at least a core portion of a fibroblast mass 10a containing fibroblasts 11 and a shell portion of a cell layer 20 of epidermal keratinocytes, and the cell layer 20 includes tight junctions 30, an undifferentiated epidermal keratinocyte layer 21, and a differentiated epidermal keratinocyte layer 22 and an outermost layer 23, the undifferentiated epidermal keratinocyte layer 21 including undifferentiated epidermal keratinocytes 201, and the differentiated epidermal keratinocyte layer 22 and outermost layer 23 including differentiated epidermal keratinocytes 202. For example, the three-dimensional structure 1 with a core-shell structure according to the second embodiment has a core portion of the three-dimensional structure 1 with a core-shell structure according to the first embodiment, which is a microcarrier 10b instead of a fibroblast mass 10a, and includes this microcarrier 10b and a shell portion of the cell layer 20 of epidermal keratinocytes that has the same configuration as the shell portion of the cell layer 20 of epidermal keratinocytes according to the first embodiment.

[0034] <Center of Three-Dimensional Structure> In this embodiment, the center of the three-dimensional structure is preferably, but not limited to, a microsphere such as a microcarrier or a cell cluster, and more preferably a microsphere having an extracellular matrix component on its surface. The microsphere is preferably, but not limited to, a cell cluster (preferably a fibroblast cluster) or a microcarrier (preferably a cell-free system). Of these, a microcarrier (preferably a cell-free system) is preferred, as this allows for the evaluation of skin homeostasis function in the cell layer to be performed without using cells other than epidermal keratinocytes, thereby reducing the influence of other cells.

[0035] <Extracellular Matrix Components> The extracellular matrix components are not particularly limited, and examples thereof include extracellular matrices such as collagen (e.g., types I to XIX), hyaluronic acid or a salt thereof (sodium, etc.), fibronectin, vitronectin, laminin (e.g., types 1 to 15 (e.g., laminin 511E8, etc.)), tenascin, thrombospondin, various elastins, and various proteoglycans; and extracellular matrix substitute materials such as poly-D,L-lactide-co-glycolide, n-isopropylacrylamide, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, alginate gel, and various hydrogels, and one or more types may be selected from these. As the extracellular matrix component, an extracellular matrix and / or an extracellular matrix substitute material can be used, with an extracellular matrix being preferred. The extracellular matrix component may be a non-cellular substance that fills the gap between cells, and is preferably a component that exerts or has a cell adhesive effect.

[0036] The extracellular matrix component present on the surface of the microspheres is preferably collagen, more preferably type 1 collagen and / or type 4 collagen, but is not limited thereto. Furthermore, when confirming the presence of an extracellular matrix component in the microspheres, collagen (preferably type 1 or type 4 collagen) can be confirmed. When confirming the exemplary extracellular matrix component, a corresponding marker capable of detection can be appropriately used, and commercially available products (e.g., reagent kits) can be used. Furthermore, in this embodiment, the surface of the microspheres is preferably a three-dimensional structure in which the presence of an extracellular matrix component can be confirmed using an extracellular matrix component detection marker. Among the extracellular matrix component detection markers, collagen is preferred, and more preferably, the three-dimensional structure is one in which the presence of one or both of these can be confirmed using at least Col 1 and / or Col IV. The confirmation method is not particularly limited as long as it can confirm the expression of the target gene or protein. Examples include staining and expression level, and methods used in the evaluation methods described below can be appropriately adopted.

[0037] In this first embodiment, the center of the three-dimensional structure contains a plurality of fibroblasts, and is preferably a fibroblast mass composed at least of these fibroblasts, and preferably has extracellular matrix components on its surface. The fibroblasts are preferably culturable fibroblasts, more preferably normal fibroblasts and / or fibroblasts derived from pluripotent stem cells, and even more preferably normal fibroblasts.

[0038] In this second embodiment, the center of the three-dimensional structure is preferably composed of a single spherical microcarrier, and the surface of the microcarrier preferably has extracellular matrix components. The microcarrier is preferably acellular. Noncellular cells include living or dead cells, cells that do not grow or divide, and preferably are not derived from animals.

[0039] <Microcarrier> The core used in this embodiment may be a single spherical microcarrier or a single mass composed of multiple microcarriers. However, it is preferable that a single spherical microcarrier forms the core of the microsphere, from the viewpoints of ease of workability, reduction in the number of steps, and cost. One mass microcarrier may be formed by appropriately combining multiple microcarriers of the same or different shapes, the same or different particle diameters, etc. The microcarrier used in this embodiment may be a commercially available product, and examples thereof include collagen-coated PVA microcarriers (Kuraray PVA microcarriers (collagen type 1) and Corning R Collagen-coated microcarriers, etc.), microcarriers (Corning R Examples of suitable microcarriers include untreated microcarriers, and also include microcarriers as described below, and one or more of these may be used. Microcarriers may also be produced by reference to known production methods.

[0040] The materials used for microcarriers may be organic, inorganic, or composite materials thereof, or may be hydrophobized materials or materials to which hydrophobic groups have been introduced. Microcarriers can be formed from these materials. From the viewpoint of cytocompatibility, it is preferable that the microcarrier contains an organic material. Cell culture may be performed using microcarriers formed from polymers having hydrophobic moieties as the organic material. Examples of hydrophobic groups include linear or cyclic hydrocarbon groups, and aromatic hydrocarbon groups.

[0041] Examples of organic substances include, but are not limited to, synthetic polymers (resins) such as polyvinyl alcohol, polylactic acid, polyglycolic acid, polytetrafluoroethylene, polystyrene, polyester, polyurethane, polyethylene, polypropylene, (meth)acrylic polymers, (meth)acrylamide polymers, silicone polymers, and epoxy resins; natural polymers (proteins or peptides) such as collagen and gelatin; polygalacturonic acids such as pectin and pectinate salts, alginic acid, cellulose, cross-linked agarose, dextran, chitosan, and polysaccharides such as salts thereof (e.g., alkali metals such as Na, K, and Li, and alkaline earth metals such as Ca and Mg). Examples of inorganic substances include glass, ceramic, metal, alloy, and metal oxide. Among resins, flexible resins, thermoplastic resins, and thermoplastic elastomers are preferred. Among organic substances, resins such as polyvinyl alcohol, polystyrene, and polyester are preferred, with polyvinyl alcohol being preferred. The term "(meth)acrylic acid polymer" refers to an acrylic acid polymer, a methacrylic acid polymer, or a mixture thereof, and may be a polymer or copolymer formed from a (meth)acrylic acid monomer or the like.

[0042] From the viewpoint of cytocompatibility, microcarriers formed from hydrogels may be used. Examples of such hydrogels include polyvinyl alcohol, sodium salts of (meth)acrylic polymers, copolymers with a large amount of hydrophilic groups introduced, and gelatin. Hydrogels in which hydrophobic groups are introduced into hydrophilic polymers may also be used.

[0043] To promote cell adhesion, an extracellular matrix component (preferably a cell adhesive polymer) may be disposed on the surface of the microcarrier. Examples of the extracellular matrix component (preferably a cell adhesive polymer) include collagen, gelatin, alginic acid, Matrigel™ (BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, elastin, heparan sulfate, dextran, dextran sulfate, chondroitin sulfate, and salts thereof. One or more selected from these may be used. Of these, collagen is preferred.

[0044] To promote cell adhesion, cationic functional groups may be introduced onto the surface of the microcarrier, such as dimethylamino, diethylamino, and groups containing substituted or unsubstituted cationic amino groups, such as amino groups.

[0045] Examples of the shape of the microcarrier include spherical, flat, cylindrical, plate-like, and prismatic shapes. The microcarrier preferably includes a spherical microcarrier. The microcarrier may be a porous microcarrier having internal pores, a microcarrier without internal pores, or a hollow microcarrier.

[0046] The microcarriers may be coated with a coating material to enhance cell adhesion, and the coating material is preferably one related to extracellular matrix components. The coating material is not particularly limited, but examples thereof include poly-D,L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, types I to XIX collagen, fibronectin, vitronectin, laminin-1 to -12, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, the immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Sepharose, alginate gel, and various hydrogels, and one or more selected from these may be used.

[0047] The average particle size (D50) of the microcarriers is not particularly limited from the viewpoint of promoting cell proliferation, but is, for example, 50 to 1,000 μm. A suitable lower limit is preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more. A suitable upper limit is preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 or 250 μm or less. A more preferred range is 100 to 500 μm, more preferably 120 to 250 μm, and even more preferably 150 to 250 μm. The average particle size (D50) of the microcarriers is the value measured as the median diameter (D50) in physiological saline or culture medium. The average particle size of the microcarriers can be measured using a laser diffraction / scattering particle size distribution analyzer. Microcarriers used for cell culture are preferably larger than the size of the cells used in cell culture.

[0048] <Cell Layer of Three-Dimensional Structure> In this embodiment, the epidermal keratinocyte layer in the cell layer (preferably the superficial layer) of the three-dimensional structure preferably contains a plurality of epidermal keratinocytes and is at least composed of these. The cell layer is preferably an epidermal keratinocyte layer. The epidermal keratinocytes are preferably culturable epidermal keratinocytes, more preferably normal epidermal keratinocytes and / or epidermal keratinocytes derived from pluripotent stem cells, and even more preferably epidermal keratinocytes derived from pluripotent stem cells. Using epidermal keratinocytes derived from pluripotent stem cells as the epidermal keratinocytes of the three-dimensional structure has the advantage of enabling evaluation or testing of skin homeostasis function over a longer period of time.

[0049] <Cell Layer: Tight Junctions> In this embodiment, tight junctions are preferably present in the gaps between cells present on the surface of the cell layer (preferably the epidermal keratinocyte layer) that contacts the outside. These tight junctions exist to seal the gaps between cells and can exert a barrier function that restricts the free passage of substances through the intercellular spaces. Furthermore, in this embodiment, tight junctions are preferably present between adjacent cells in multiple differentiated epidermal keratinocytes present in the epidermal keratinocyte layer on the surface of the three-dimensional structure, and more preferably between differentiated epidermal keratinocytes present in the outermost layer and in contact with the outside. As shown in the Examples below, in this embodiment, three-dimensional structures have been confirmed in which tight junctions are abundant in the outermost layer, and the amount and localization of tight junctions can be evaluated using TJ markers. In this embodiment, preferred tight junctions appear as dots when viewed in a cross-section of the approximate center of the three-dimensional structure, and may appear as a mesh when viewed on the surface of the three-dimensional structure (see Figure 1, etc.).

[0050] Generally, tight junctions refer to intercellular bonds that bond cells together, and are said to connect adjacent epithelial cells and function as a barrier to restrict the permeation of various substances (compounds, ions, water, etc.) through intercellular spaces. Skin tight junctions are formed by cells (SG2 cells) in the second layer of the granular layer inside the stratum corneum, forming a TJ barrier with a honeycomb-like mesh pattern on the surface, which is said to be responsible for the skin's barrier function.

[0051] <Cell Layer: Epidermal Keratinocyte Layer> In this embodiment, the epidermal keratinocyte layer is preferably a multilayer composed of, from the center outward, at least an undifferentiated cell layer containing undifferentiated epidermal keratinocytes and a differentiated cell layer containing differentiated epidermal keratinocytes. Furthermore, the epidermal keratinocyte layer is preferably a layer composed of at least a differentiated cell layer composed at least of differentiated epidermal keratinocytes located outward, and an undifferentiated cell layer composed at least of undifferentiated epidermal keratinocytes located in the central direction. Furthermore, it is more preferable that the epidermal keratinocyte layer comprises a differentiated cell layer including an outermost layer and an innermost layer, and an undifferentiated cell layer.

[0052] <Epidermal Keratinocyte Layer: Undifferentiated Cell Layer> In a preferred embodiment of the three-dimensional structure of this embodiment, epidermal keratinocytes in contact with microspheres (e.g., fibroblast clumps, microcarriers, etc.) remain undifferentiated, and the layer containing undifferentiated epidermal keratinocytes is also referred to as the basal cell layer of epidermal keratinocytes. It is preferred that the epidermal keratinocytes in contact with the microspheres form an undifferentiated epidermal keratinocyte layer covering the core microspheres. This undifferentiated epidermal keratinocyte layer is preferably a layer contained in the epidermal keratinocyte layer, more preferably a layer located toward the center of the epidermal keratinocyte layer and / or a layer located beyond the center of the three-dimensional structure toward the outside. It is preferred that these undifferentiated epidermal keratinocytes or their layer divide to form new cells, and the newly divided epidermal keratinocytes are sequentially pushed outward (preferably radially) to form differentiated epidermal keratinocytes. The presence of such a superficial epidermal keratinocyte layer, which sequentially pushes epidermal keratinocytes outward, allows for a test to be performed under conditions similar to normal skin turnover, thereby enabling more accurate evaluation of skin homeostasis functions such as skin turnover. The amount and localization of such undifferentiated cells can be evaluated using basal cell markers. Furthermore, the amount and localization of such undifferentiated and then differentiated cells can be evaluated using proliferation markers.

[0053] <Epidermal Keratinocyte Layer: Differentiated Cell Layer> In a preferred embodiment of the three-dimensional structure of this embodiment, a differentiated epidermal keratinocyte layer is preferably formed to cover an undifferentiated epidermal keratinocyte layer. The differentiated epidermal keratinocyte layer is preferably a layer contained in the epidermal keratinocyte layer, and more preferably a layer located outward from the undifferentiated cell layer. The differentiated epidermal keratinocytes preferably form multiple layers outward, and more preferably form an outermost layer of differentiated epidermal keratinocytes that contacts the exterior. Furthermore, it is even more preferred that the differentiated cell layer be two or more layers, including an outermost layer of differentiated epidermal keratinocytes and an innermost layer of differentiated epidermal keratinocytes that contacts the undifferentiated epidermal keratinocyte layer. It is more preferred that the outermost layer contacts the exterior, and the innermost layer contacts the undifferentiated epidermal keratinocyte layer.

[0054] This embodiment can provide a three-dimensional structure that resembles the state of actual skin, has a cell layer in which epidermal keratinocytes are in appropriate undifferentiated and differentiated states in appropriate layers, and has tight junctions at appropriate positions in the cell layer. Therefore, the three-dimensional structure of this embodiment is suitable for various tests, such as evaluation of skin homeostasis. The three-dimensional structure of this embodiment has the advantage that fewer cells are used in various tests than conventional sheet-like three-dimensional cultures, making it more compatible with screening experiments.

[0055] <Origin of Fibroblasts and Epidermal Keratinocytes Used in Three-Dimensional Structure> The origin of the cells used in preparing the three-dimensional structure of this embodiment is not particularly limited, and examples include cells derived from normal cells (e.g., normal epidermal keratinocytes, normal fibroblasts, etc.) and cells derived from differentiation-induced cells derived from pluripotent stem cells (preferably iPS cells), and the like, and one or more types selected from these may be used. In a preferred embodiment, the epidermal keratinocyte layer of the three-dimensional structure is preferably prepared using epidermal keratinocytes, more preferably formed at least from epidermal keratinocytes derived from pluripotent stem cells and / or normal cells, and even more preferably formed from epidermal keratinocytes derived from seeded pluripotent stem cells. In a preferred embodiment, when the center of the three-dimensional structure is a fibroblast mass, the mass is preferably formed at least from fibroblasts derived from normal cells. Furthermore, the origin of these cells is not particularly limited, but is preferably derived from mammals. Examples of mammals include, but are not limited to, humans, pigs, rodents (such as mice and rats), and pet animals (such as rabbits, dogs, and cats). One or more of these may be used, and of these, cells derived from humans are preferred.

[0056] <Shape, Size, etc. of Three-Dimensional Structure> The shape of the three-dimensional structure or microspheres (e.g., cell clusters, microcarriers, etc.) of this embodiment is not particularly limited, but is preferably a cluster or approximately spherical shape (e.g., an oval sphere or a regular sphere). The shape and size of this embodiment can be measured from a micrograph or the like. Specifically, the size (long axis or diameter) of the three-dimensional structure and spheroids (cell clusters) can be measured using images captured by optical microscopy in a measurement mode installed in image analysis software. The average long axis measured from the photograph is approximately 500 μm. In a more preferred embodiment, the size of the three-dimensional structure can be measured using ImageJ using images captured with a confocal laser microscope. In the case of approximately spherical three-dimensional structures, it is preferable to measure the size using a midline cross-sectional image. The thickness of the cell layer can be calculated from the formula: cell layer thickness = size of the three-dimensional structure - size of the microspheres (e.g., fibroblast clusters, cell clusters, microcarriers, etc.).

[0057] The size (major axis) of the three-dimensional structure of this embodiment is not particularly limited, but the preferred lower limit is preferably 300 μm or more, more preferably 350 μm or more, and even more preferably 400 μm or more, and the preferred upper limit is preferably 700 μm or less, more preferably 600 μm or less or 500 μm or less, and a more preferred numerical range is preferably 300 to 700 μm, more preferably 400 to 600 μm. The size (major axis) of the three-dimensional structure of this embodiment can be measured using the image analysis of the optical microscope observation described above. The size (major axis) of the three-dimensional structure in this second embodiment is not particularly limited, but the suitable upper and lower limits of the size (major axis) of the three-dimensional structure of this embodiment described above may be appropriately combined or adopted. The suitable lower limit is preferably 300 μm or more, more preferably 350 μm or more, and even more preferably 400 μm or more, and the suitable upper limit is preferably 700 μm or less, more preferably 600 μm or less or 500 μm or less. The size (major axis) of the three-dimensional structure in this second embodiment is approximately 300 to 500 μm. This size can be measured using the image analysis of optical microscope observation described above.

[0058] The size (major axis or diameter) of the microspheres is not particularly limited, but may be, for example, 50 to 1,000 μm, with the lower limit being preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more, and the upper limit being preferably 600 or 560 μm or less, more preferably 500 or 480 μm or less, even more preferably 400 or 300 μm or less, and even more preferably 250 μm or less. This size can be measured using the image analysis of optical microscope observation described above. The size (major axis or diameter) of the microcarrier is not particularly limited, but is, for example, 50 to 1,000 μm, with a suitable lower limit of preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more, and a suitable upper limit of preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 or 250 μm or less, with a more preferred numerical range being 100 to 500 μm, more preferably 120 to 250 μm, and even more preferably 150 to 250 μm. This size can be measured using image analysis of optical microscope observation as described above.

[0059] The size (long axis or diameter) of the cell clusters (preferably fibroblast clusters) in the three-dimensional structure in this first embodiment is not particularly limited, but a suitable lower limit is preferably 240 μm or more, more preferably 320 μm or more, and a suitable upper limit is preferably 560 μm or less, more preferably 480 μm or less, and a more suitable numerical range is preferably 240 to 560 μm, more preferably 320 to 480 μm. This size can be measured using image analysis of optical microscope observation as described above.

[0060] The size (major axis or diameter) of the microcarriers in the three-dimensional structure in this second embodiment is not particularly limited, but is, for example, 50 to 1,000 μm, with a suitable lower limit of preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more, and a suitable upper limit of preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 or 250 μm or less, with a more suitable numerical range of preferably 100 to 500 μm, preferably 120 to 250 μm. This size can be measured using the image analysis of optical microscope observation described above.

[0061] The size (thickness) of the cell layer containing epidermal keratinocytes in the three-dimensional structure in this embodiment is not particularly limited, but a suitable lower limit is preferably 60 μm or more, more preferably 80 μm or more, and a suitable upper limit is preferably 140 μm or less, more preferably 120 μm or less, and a more suitable numerical range is preferably 60 to 140 μm, more preferably 80 to 120 μm. The size (thickness) of the cell layer can be applied whether the central portion is a cell mass or a microcarrier, and can be appropriately adopted.

[0062] In this embodiment, when a cell cluster (preferably a fibroblast cluster) is used in the center, the number of cells (preferably the number of fibroblast cells) in the three-dimensional structure in this embodiment is not particularly limited, but is preferably 1 × 10 ２ ~1 x 10 ５ cell / three-dimensional structure, more preferably 1 x 10 ３ ~2 x 10 ４ The number of epidermal keratinocytes in the three-dimensional structure in this embodiment is not particularly limited, but is preferably 1 x 10 ２ ~1 x 10 ５ cell / three-dimensional structure, more preferably 1 x 10 ３ ~2 x 10 ４ cell / three-dimensional structure.

[0063] The size of the three-dimensional structure in this embodiment is not particularly limited, but when the size (major axis) of the central microsphere is taken as 1, the preferred lower limit is preferably 1.05 or more, more preferably 1.1 or more, and the preferred upper limit is preferably 3.0 or less, more preferably 2.0 or less, and even more preferably 1.5 or less, with the preferred numerical range being preferably 1.05 to 1.5. Furthermore, in the first or second embodiment, the size of the three-dimensional structure when the size (major axis) of the central microsphere is taken as 1 can be appropriately determined by the preferred upper and lower limits of the "size of the three-dimensional structure in this embodiment" described above; for example, in the first embodiment, it is preferably 1.05 to 1.5, and in the second embodiment, it is preferably 1.05 to 1.5.

[0064] The ratio of the number of fibroblasts to the number of epidermal keratinocytes in the three-dimensional structure in this first embodiment (number of fibroblasts / number of epidermal keratinocytes) is not particularly limited, but a suitable lower limit is, from the viewpoint of appropriate tight junction formation in the epidermal keratinocyte layer, preferably 1 / 4 or more, more preferably 1 / 2 or more, even more preferably 1 or more, more preferably 4 or more, more preferably 5 or more, more preferably 6 or more, and more preferably 7 or 8 or more; and a suitable upper limit is not particularly limited, but, from the viewpoint of coverage of the entire fibroblast mass with epidermal keratinocytes, examples include 20, 15, 13, or 11 or less, and is preferably 10 or less, more preferably 9 or less, and even more preferably 8 or less.

[0065] In this embodiment, the number of fibroblasts and epidermal keratinocytes in the three-dimensional structure, and their content ratios, are preferably expressed in terms of the number of cells used for seeding when producing a certain three-dimensional structure (converted to the number of seeded cells: cells / mL), as will be explained in the method for producing a three-dimensional structure described below.

[0066] The three-dimensional structure of this embodiment allows for a longer culturing period, and therefore the culturing period can be extended, allowing various tests to be performed during this culturing period. These tests include evaluation, measurement, analysis, detection, observation, etc., and one or more selected from these can be used. The three-dimensional structure of this embodiment allows for an appropriate lengthening of the culturing period, and therefore the period for various tests can also be appropriately extended, which has the advantage of allowing more accurate and reliable test results to be obtained and allowing evaluation results, determination results, etc. to be derived based on the test results.

[0067] The culture period or culturable period for the three-dimensional structure of this embodiment is not particularly limited, but the preferred lower and upper limits can be appropriately determined from the explanation in "2. Method for Producing a Three-Dimensional Structure According to This Embodiment" below. The preferred range of values ​​is more preferably 3 to 35 days, and even more preferably 6 to 22 days. It is more preferable that the start of the culture period or culturable period is "after seeding of epidermal keratinocytes." It is preferable to culture the three-dimensional structure by replacing the medium with a co-culture medium every day. The method for preserving the three-dimensional structure is not particularly limited, but methods for preserving animal cells can be used, such as preserving the three-dimensional structure in liquid nitrogen in the presence of a cryoprotective agent such as dimethyl sulfoxide (DMSO) in a synthetic medium.

[0068] <Uses of Three-Dimensional Structure (Various Tests, Such as Evaluation of Skin Homeostasis Function)> The three-dimensional structure of this embodiment can be used in various tests, but is more preferably used to evaluate skin homeostasis function. Therefore, it preferably has a state, properties, or function that can accommodate various evaluations of skin homeostasis function. A more preferred embodiment of the three-dimensional structure is one in which, like actual skin, epidermal keratinocytes present in the basal layer proliferate and differentiate, and tight junctions are appropriately localized in the granular layer. A more preferred example of a three-dimensional structure is one in which the surface layer has tight junctions, the group or layer of epidermal keratinocytes in contact with the core portion is in an undifferentiated state, and the shell portion and the core portion are in contact. By using this three-dimensional structure, skin homeostasis functions, such as barrier function, can be evaluated more accurately, as if actual skin were used. For example, the three-dimensional structure of this embodiment can be used to conduct tests (research, exploration, etc.) on maintaining skin homeostasis and the physical properties of skin, and the three-dimensional structure of this embodiment can be used to conduct various tests (evaluation, research, development, exploration, screening, etc.) on substances related to regulating the maintenance of skin homeostasis, such as skin barrier function (inhibition or promotion, decrease / deterioration / improvement of function, etc.).

[0069] The three-dimensional structure preferably has tight junctions between adjacent cells. To date, there have been no reports of a three-dimensional core-shell structure composed of epidermal keratinocytes with tight junctions in the surface layer. Having tight junctions in the surface layer facilitates more accurate evaluation of barrier function.

[0070] Furthermore, the three-dimensional structure preferably has cell proliferation capacity in the cell layer, and more preferably, the epidermal keratinocytes in contact with the core portion are in an undifferentiated state. The cell proliferation capacity of the cells present in the cell layer of the three-dimensional structure allows for a longer evaluable period, making it possible to obtain maintenance and change states through longer-term observation, etc. Furthermore, the presence of undifferentiated epidermal keratinocytes in contact with the core portion offers the advantage of facilitating evaluation of skin homeostasis functions (e.g., cell proliferation capacity, barrier function, etc.), since actual skin is similar to this state, as the basal layer proliferates and differentiates. Furthermore, the use of microcarriers in the core portion in this embodiment offers the advantage of reducing the influence of other types of cells when various evaluations are performed on the cell layer composed of epidermal keratinocytes.

[0071] Markers used to assess or determine the state of three-dimensional structures can include, for example, one or more selected from TJ markers, stratum corneum formation-related markers, proliferating cell markers, differentiated cell markers, and basal cell (undifferentiated cell) markers. For example, staining targets for TJ markers include Occludin, ZO-1, and Claudin-1, with Occludin being preferred. Examples of stratum corneum formation-related markers include Filaggrin and Caspase 14, with Filaggrin being preferred. Examples of proliferating cell markers include Ki-67 and PCNA, with Ki-67 being preferred. Examples of differentiated cell markers include Keratin 10, Involucrin, and Loricrin, with Keratin 10 (early differentiation) and Loricrin (terminal differentiation) being preferred. Examples of basal cell (undifferentiated cell) markers include Keratin 5, Keratin 14, and Collagen XVII, with Collagen XVII being preferred. Since it is possible to confirm the appropriate gene expression state, appropriate staining property, and appropriate localization for each marker, it is possible to confirm the expression state of the properties or functions of the three-dimensional structure of this embodiment (preferably, tight junctions, cell proliferation ability, undifferentiated / differentiated state, and extracellular matrix on the core surface).

[0072] The three-dimensional structure can be used to evaluate skin homeostasis. Homeostasis is generally defined as maintaining tissue function at a constant, ideal state regardless of changes in internal or external environmental factors. Functions related to skin (epidermal) homeostasis using the three-dimensional structure of this embodiment include, for example, epidermal cell proliferation (Ki-67 (cell proliferation)), differentiation (collagen XVII (undifferentiated), keratin 10 (early differentiation), loricrin (terminal differentiation), etc.), and barrier function (occludin (TJ), filaggrin (stratum corneum)), etc. Examples of evaluation methods include analyses using extracted messenger RNA, such as Northern blotting, real-time PCR, microarrays, and RNA sequencing; analyses using extracted proteins, such as Western blotting, ELISA, mass spectrometry-based proteomics, and multiplex assays; and histological analyses, such as in situ hybridization and fluorescent immunostaining. One or more of these may be used.

[0073] The evaluation of skin homeostasis function can be determined using various markers that can evaluate skin homeostasis function. The markers used are not particularly limited, and known techniques or commercially available kits, etc., can be used. It is preferable to use a measurement or detection method that can determine the expression level and localization of the marker used. It is preferable to appropriately adopt a measurement method or detection method appropriate for the marker used as the measurement or detection method. Examples include fluorescence microscopy (laser scanning, confocal laser (e.g., confocal laser scanning, etc.), multiphoton excitation (two-photon laser, etc.), light sheet fluorescence), and gene amplification methods (PCR, LAMP, real-time RT-PCR, etc.).

[0074] Taking this expression state into consideration, the three-dimensional structure of this embodiment can be used for more appropriate evaluation of skin homeostasis function, thereby enabling more appropriate evaluation.

[0075] The three-dimensional structure preferably has a skin homeostasis maintenance function, and the use of such a three-dimensional structure having a skin homeostasis maintenance mechanism can be used to explore the state of skin tissue (e.g., function, properties, etc.). Furthermore, the three-dimensional structure of this embodiment can be used to predict the behavior of a test substance when it is brought into contact with actual living skin, and can also be used to determine the suitability of the test substance, assess the skin condition, etc. This embodiment can also produce pluripotent stem cells based on individual or individual somatic cells, and produce a three-dimensional structure using epidermal keratinocytes derived from individual or individual pluripotent stem cells. Therefore, this embodiment can use differentiation-induced cells derived from pluripotent stem cells to explore the skin condition of an individual or individual, and can also explore test substances that are appropriate or inappropriate for an individual or individual.

[0076] 2. Method for manufacturing a three-dimensional structure according to this embodiment

[0077] This embodiment provides a method for producing a three-dimensional core-shell structure, which comprises co-culturing microspheres and seeded epidermal keratinocytes, preferably one microsphere, and the microspheres used for co-culturing may have extracellular matrix components on their surfaces when the co-culturing is initiated.

[0078] In the production method of this embodiment, it is preferable to use cell clusters or microcarriers as the microspheres. A suitable cell cluster is preferably a cell cluster prepared from seeded cells. A suitable microcarrier is preferably a microcarrier whose surface is coated with extracellular matrix components. When cell clusters are used as cores for co-culture, the surface of the cell clusters at this time may not be coated with extracellular matrix components, and it is preferable that the extracellular matrix components exist between the cell clusters (surface layer) and the cell layer (inner layer) in the prepared three-dimensional structure.

[0079] As a preferred aspect, the first embodiment can provide a method for producing a three-dimensional structure with a core-shell structure, which includes co-culturing a fibroblast mass prepared from seeded fibroblasts with seeded epidermal keratinocytes.As a preferred aspect, the second embodiment can provide a method for producing a three-dimensional structure with a core-shell structure, which includes co-culturing a microcarrier with seeded epidermal keratinocytes.

[0080] This embodiment can also provide a method for producing a three-dimensional structure with a core-shell structure, which includes seeding and co-culturing epidermal keratinocytes on a cell culture substrate containing microspheres. This embodiment can also provide a method for producing a three-dimensional structure with a core-shell structure, which includes seeding and co-culturing epidermal keratinocytes in a medium containing microspheres. As another aspect of this embodiment, there can be provided a method for producing a three-dimensional structure with a core-shell structure, which includes co-culturing microspheres and seeded epidermal keratinocytes.

[0081] Furthermore, by the manufacturing method of this embodiment, a three-dimensional structure having a core-shell structure as described above in "1. Three-dimensional structure according to this embodiment" can be obtained.

[0082] In the description of this embodiment, explanations of the components and methods of epidermal keratinocytes, microspheres, microcarriers, fibroblasts, core-shell structures, pluripotent stem cells, cell culture equipment, etc. that overlap with the explanations in "1." above and "3." and "4." below will be omitted as appropriate; however, the explanations in "1." above and "3." and "4." below also apply to this embodiment and can be adopted as appropriate.

[0083] As another aspect of this embodiment, there is provided a method for producing a three-dimensional structure with a core-shell structure, which includes further seeding epidermal keratinocytes onto microspheres to prepare a surface layer. The microspheres may be either cell clusters or spherical microcarriers. A suitable production method may also include seeding fibroblasts to prepare a fibroblast cluster that will become the center, as a pre-step of the co-culture step. A suitable production method may also include coating spherical microcarriers with extracellular matrix components to prepare extracellular matrix component-coated spherical microcarriers that will become the center, as a pre-step of the co-culture step. As another aspect of this first embodiment, there is provided a method for producing a three-dimensional structure with a core-shell structure, which includes co-culturing seeded fibroblasts and seeded epidermal keratinocytes.

[0084] Another aspect of this embodiment provides a method for producing a three-dimensional core-shell structure, comprising placing microspheres on the cell-adhesion-inhibiting treated surface of a mortar-shaped depression, seeding epidermal keratinocytes, and co-culturing them. A preferred production method involves seeding and culturing fibroblasts on the cell-adhesion-inhibiting treated surface of the mortar-shaped depression before co-culturing, or placing microcarriers on the cell-adhesion-inhibiting treated surface of the mortar-shaped depression before co-culturing. A preferred aspect of this embodiment provides a method for producing a three-dimensional core-shell structure, in which the co-culturing forms a core-shell structure such that the epidermal keratinocyte layer covers the microspheres (e.g., fibroblast clusters or spherical microcarriers). A more preferred aspect of this embodiment provides a method for producing a three-dimensional core-shell structure, in which the cell layer comprises an undifferentiated epidermal keratinocyte layer, a differentiated epidermal keratinocyte layer, and multiple tight junctions present between the differentiated epidermal keratinocytes in the outermost layer.

[0085] In another aspect of this embodiment, a method for producing a three-dimensional structure with a core-shell structure can be provided, which includes seeding a plurality of epidermal keratinocytes onto microspheres and co-culturing them to cover the fibroblast cell clusters with the epidermal keratinocytes, thereby preparing a surface layer of a three-dimensional structure in which a plurality of tight junctions are present. When preparing fibroblast cell clusters as the microspheres, the method may include seeding a plurality of fibroblasts and culturing them to prepare a fibroblast cell cluster that is the core of the three-dimensional structure. The microspheres may be spherical microcarriers coated with extracellular matrix components.

[0086] In a preferred aspect of the manufacturing method of this embodiment, when producing a three-dimensional structure, the fibroblasts and epidermal keratinocytes used for seeding are preferably cells derived from normal cells and / or differentiation-induced cells derived from pluripotent stem cells.

[0087] In a preferred aspect of the manufacturing method of this embodiment, when producing a three-dimensional structure, the cells to be seeded are cultured in a cone-shaped depression (e.g., a U-shaped, V-shaped depression, etc.), and it is more preferable that the surface of the depression is a surface treated to inhibit cell adhesion, and it is even more preferable that the treatment is a hydrophilization treatment.

[0088] In a preferred embodiment of the manufacturing method of this embodiment, when producing a three-dimensional structure, a medium containing at least an epidermal keratinocyte medium is used to culture the microspheres and seeded epidermal keratinocytes. Alternatively, a medium containing an epidermal keratinocyte medium and a fibroblast medium may be used. Furthermore, after the preparation of the three-dimensional structure, a medium containing at least an epidermal keratinocyte medium or a medium containing an epidermal keratinocyte medium and a fibroblast medium may be used for the medium used in evaluation, taking into consideration the microspheres and epidermal keratinocytes used. In a preferred embodiment of the manufacturing method of the first embodiment, when producing a three-dimensional structure, a medium containing an epidermal keratinocyte medium and a fibroblast medium is used to culture the fibroblast mass and seeded epidermal keratinocytes. In a preferred embodiment of the manufacturing method of the second embodiment, when producing a three-dimensional structure, a medium containing an epidermal keratinocyte medium is used to culture the spherical microcarriers and seeded epidermal keratinocytes.

[0089] In a preferred aspect of the production method of this embodiment, when producing a three-dimensional structure, the culture period after seeding the epidermal keratinocytes is 6 to 22 days. For this reason, the possible culture period for using a three-dimensional structure for evaluation is preferably 6 days or more after seeding the epidermal keratinocytes.

[0090] In addition, as a preferred aspect of the production method of this embodiment, when producing a three-dimensional structure, it is preferable to culture the three-dimensional structure so that the size of the microspheres is 1.05 to 1.5, where the size of the microspheres is 1, and the size can be confirmed by observation under an optical microscope, etc. In addition, as a preferred aspect of the production method of the first embodiment, when producing a three-dimensional structure, the seeding ratio of the fibroblasts to the epidermal keratinocytes is preferably 1:4 or more, more preferably 1:4 to 8:1.

[0091] As another preferred aspect of this embodiment, a test method or a method for evaluating skin homeostasis function can be provided, which uses the three-dimensional structure according to this embodiment or a three-dimensional structure obtained by the manufacturing method of this embodiment. As another preferred aspect of this embodiment, the method for evaluating skin homeostasis function may include the manufacturing method of this embodiment described above, and as a preferred example, a method for evaluating skin homeostasis function may be provided, which includes performing the manufacturing method of the three-dimensional structure according to this embodiment and performing a test (preferably evaluation of skin homeostasis function) using the manufactured three-dimensional structure.

[0092] The manufacturing method of this embodiment can provide a three-dimensional structure with a core-shell structure that includes microspheres (preferably fibroblast clusters or microcarriers) in the center and epidermal keratinocytes and tight junctions in the surface layer, and as a more preferred embodiment, the three-dimensional structure with a core-shell structure according to this embodiment described in "1." above can be obtained. The three-dimensional structure is preferably a three-dimensional structure with a core-shell structure that includes a cell layer composed at least of a plurality of tight junctions present between cells in the surface cell layer and a plurality of epidermal keratinocytes, and a central microsphere (preferably fibroblast clusters or microcarriers) composed at least of a plurality of fibroblasts, and more preferably a three-dimensional structure with a core-shell structure that includes an epidermal keratinocyte layer containing an undifferentiated cell layer and a differentiated cell layer as the shell portion.

[0093] <Preferable Manufacturing Example of the Method for Manufacturing a Three-Dimensional Structure According to the Present Embodiment> An example of a manufacturing example according to the present embodiment is shown below, but the method for manufacturing a three-dimensional structure according to the present embodiment is not limited to this. The method for manufacturing a three-dimensional structure having a core-shell structure according to the present embodiment preferably includes coating epidermal keratinocytes onto microspheres (preferably fibroblast clumps or microcarriers).

[0094] A preferred example of the production method of this embodiment includes the steps of (a) placing microspheres in a well having a cone-shaped depression and culturing them in a predetermined medium for a predetermined period of time as a pretreatment, and (b) seeding epidermal keratinocytes into the well having a cone-shaped depression containing the microspheres and culturing them in a co-culture medium or a medium for epidermal keratinocytes to prepare a three-dimensional structure with a core-shell structure. The microspheres are preferably fibroblast cell aggregates or microcarriers. Furthermore, if the microspheres are non-cellular, the predetermined period of culturing may not be necessary in the pretreatment step (a).

[0095] Furthermore, the method for producing a three-dimensional structure with a core-shell structure according to the first embodiment preferably includes preparing a fibroblast cluster and coating the prepared fibroblast cluster with epidermal keratinocytes. A preferred production example of the method of the first embodiment preferably includes coating a fibroblast cluster obtained from seeded fibroblasts with epidermal keratinocytes. A more preferred production example of the method of this embodiment preferably includes (a) seeding fibroblasts in a well having a mortar-shaped depression and culturing them in a fibroblast medium to prepare a fibroblast cluster, and (b) seeding epidermal keratinocytes in the well having a mortar-shaped depression containing the prepared fibroblast cluster and culturing them in a co-culture medium to prepare a three-dimensional structure with a core-shell structure.

[0096] A more preferred production example of the method of the first embodiment preferably further comprises adjusting the number of fibroblasts and / or epidermal keratinocytes to be seeded before carrying out the preparations of (a) and / or (b), and more preferably comprises growing the cells to be seeded as a pretreatment, thereby allowing the desired number of cells and seeded cells to be obtained as needed, so as to achieve the desired seeding number, the desired ratio of the number of fibroblasts to the number of epidermal keratinocytes to be seeded, etc.

[0097] <a. Adjustment of Cells and Cell Number for Seeding> The cells to be seeded in the method of this embodiment can be cultured using known cell culture methods to obtain or recover the desired number of cells and seeding number. For example, the seeded cells can be cultured under specified culture conditions, the proliferated cells recovered after culture, and the cell number adjusted to prepare a cell suspension with the desired number of cells to be seeded. As a more specific example, cells can be seeded onto one or more cell culture substrates, cultured under specified culture conditions, and after approximately 2 to 7 days of culture, washed with PBS(-), detached from the cell culture substrate with a detachment agent, centrifuged, and the supernatant removed. The detached cells can then be added to a culture medium or the like to obtain a cell suspension. The desired number of cells to be seeded can be obtained by adjusting the amount of solution added, such as culture medium, after centrifugation and supernatant removal.

[0098] When growing epidermal keratinocytes, a general method for culturing epidermal keratinocytes (e.g., 37°C, 5% CO ２ The culture can be carried out according to a general method for culturing fibroblasts (for example, a medium for epidermal keratinocytes, a medium for epidermal keratinocytes, etc.). Collagen, laminin, or fragments thereof is preferred as the coating agent, and trypsin or a trypsin-like enzyme is preferred as the cell detachment agent. When fibroblasts are to be grown, a general method for culturing fibroblasts (for example, a medium for epidermal keratinocytes, a medium for epidermal keratinocytes, etc.) can be used. ２ The culture medium can be prepared according to the following method (e.g., medium for fibroblasts, etc.).

[0099] This allows cells to be obtained or prepared for use in core production and / or shell production. The cell culture substrate used in the step of adjusting the number of cells to be seeded in this embodiment is preferably one that is generally used for expansion culture. For example, the wells into which seeding is performed in steps such as the step of adjusting the number of cells to be seeded are preferably wells with flat bottoms, such as petri dishes, and / or wells treated with a culture substrate (coating agent). The number of days for culture is not particularly limited, but a suitable lower limit is preferably 1 day or more, more preferably 2 days or more, and even more preferably 3 days or more. The suitable upper limit is preferably 7 days or less, more preferably 6 days or less, and even more preferably 5 days or less.

[0100] The cell number can be counted using methods commonly used in cell culture, such as automated cell counting (e.g., cell counters using image data, Coulter counters, flow cytometers, etc.) and manual cell counting, with Coulter counters being preferred. Manual cell counting can be calculated using an optical microscope and a hemocytometer as follows: cell concentration (cells / mL) = ([average cell number per compartment] × dilution factor] / volume of liquid per compartment (mL)).

[0101] <Culture Conditions> The "predetermined culture conditions" in the method of this embodiment are not particularly limited, and the culture temperature and atmospheric conditions are not particularly limited, but general culture conditions are preferred. The culture temperature is preferably a general culture temperature, preferably 36 to 38°C, more preferably 36.5 to 37.5°C. The culture atmosphere is preferably a general atmosphere, preferably 4 to 6% CO ２ or lower, more preferably 4.5 to 5.5% CO ２ More preferred culture conditions are preferably 36.5 to 37.5°C and 4.5 to 5.5% CO ２ and more preferably at 37°C and 5% CO ２ (an atmosphere of 5% carbon dioxide and 95% air). The "predetermined culture conditions" can be used as the "predetermined culture conditions" in the above-mentioned <a. Adjustment of the number of cells to be seeded>, the below-mentioned <b. Preparation of fibroblast clusters>, and <c. Coating of cell clusters with epidermal keratinocytes>.

[0102] <Culture medium> The medium used in this embodiment is not particularly limited, and a medium for the cells to be cultured can be used depending on the cells. For example, a medium for fibroblasts and a medium for epidermal keratinocytes can be used, and one or more selected from these can be used.

[0103] The fibroblast medium may be one or more selected from media obtained by known production methods or commercially available products (e.g., Fibroblast Medium (Takara Bio), HFDM-1 Medium (Cell Science Institute), Fibrolife S2 Medium Complete Kit (Lifeline Cell Technology), etc.). More specifically, DMEM (Dulbecco's Modified Eagle Medium) is preferred, and DMEM / F-12 medium, which is a 1:1 mixture of DMEM (Dulbecco's Modified Eagle Medium) and Ham's F-12 medium, is even more preferred.

[0104] The epidermal keratinocyte medium may be one or more selected from media obtained by known production methods or commercially available products (e.g., Keratinocyte Growth Medium 2 (Promocell), MCDB153 Medium (Cosmo Corporation), HuMedia-KG2 (Kurabo Industries, Ltd.), etc.). When creating a three-dimensional structure, it is preferable to use an epidermal keratinocyte 3D-differentiation medium (a medium for a three-dimensional epidermal keratinocyte model that does not contain animal- or human-derived components). Note that characteristic medium components of a medium for epidermal keratinocytes include, for example, ckDME-Ham medium, which contains, for example, 24.3 mg / L of adenine, 5 mg / L of insulin, and 0.01 mg / L of EGF (Non-Patent Document 3 (Reference): Sergio Cortez Ghio et al., Int. J. Mol. Sci. 2018, 19(8), 2174; https: / / doi.org / 10.3390 / ijms19082174).

[0105] When fibroblasts are grown for seeding, it is preferable to use a medium for fibroblasts, and when epidermal keratinocytes are grown for seeding, it is preferable to use a medium for epidermal keratinocytes.

[0106] Note that a medium may be selected for the same purpose in the following sections of <a. Adjustment of Cells and Cell Number for Seeding>. A fibroblast medium may also be used in <b. Preparation of Fibroblast Masses> described below. In <c. Coating of Microspheres with Epidermal Keratinocytes> and <d. Evaluation of Skin Homeostasis Function> described below, it is preferable to use a medium containing at least an epidermal keratinocyte medium (e.g., epidermal keratinocyte medium, co-culture medium) as described below. As the co-culture medium, for example, a mixed culture medium containing, for example, CnT-PR-3D for epidermal keratinocyte culture and, for example, DMEM / F12 (supplemented with 1 mM CaCl2 and 5% FBS) for fibroblast culture may be used.

[0107] <Cells Used for Seeding (Fibroblasts, Epidermal Keratinocytes)> The epidermal keratinocytes and fibroblasts used for seeding in the method of this embodiment are not particularly limited, and examples thereof include normal cells (e.g., normal epidermal keratinocytes, normal fibroblasts, etc.), differentiation-induced cells derived from pluripotent stem cells (e.g., epidermal keratinocytes, fibroblasts, etc.), and the like, and one or more types selected from these may be used. Using differentiation-induced cells derived from pluripotent stem cells to produce a three-dimensional structure has the advantage of enabling evaluation or testing of skin homeostasis function over a longer period than when normal cells are used. These cells are preferably derived from a mammal, and the mammal is not particularly limited, but is preferably derived from a human. Furthermore, prior to co-culture, pre-culture to increase the number of fibroblasts to be seeded is preferably performed using a general fibroblast proliferation method, and more preferably, culture is performed using a fibroblast medium. Furthermore, prior to co-culture, it is preferable to use a general method for growing epidermal keratinocytes as a pre-culture to increase the number of epidermal keratinocytes to be seeded, and more preferably, culture is performed using a medium for epidermal keratinocytes.

[0108] <Normal Cells Used for Seeding> Normal cells used for seeding in the method of this embodiment are preferably normal fibroblasts and / or normal epidermal keratinocytes. Normal dermal fibroblasts are not particularly limited and can be obtained by known production methods or commercially available products. Examples include neonatal foreskin dermal fibroblasts, adult dermal fibroblasts, and adult oral fibroblasts. One or more types selected from these can be used. Normal epidermal keratinocytes are also not particularly limited and can be obtained by known production methods or commercially available products. For example, in the case of human origin, examples include neonatal foreskin epidermal keratinocytes, normal human pediatric epidermal keratinocytes, and normal human adult epidermal keratinocytes. One or more types selected from these can be used.

[0109] <Pluripotent stem cell-derived differentiation-induced cells used for seeding> Furthermore, preferred pluripotent stem cell-derived cells are epidermal keratinocytes and fibroblasts. By using somatic cells of the target animal when producing pluripotent stem cells, it is possible to evaluate the maintenance of skin homeostasis and physical properties of the skin specific to the target animal, and to provide topical skin preparations and pharmaceuticals that are more suited to the target animal.

[0110] Pluripotent Stem Cells The pluripotent stem cells used in this embodiment are stem cells that have pluripotency, meaning they can differentiate into all cells present in a living organism, and also have the ability to proliferate. Examples of such stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos by nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and somatic stem cells derived from skin, bone marrow, fat, etc. (e.g., Muse cells and mesenchymal stem cells). One or more types selected from these may be used. These cells can be produced by known production methods or obtained commercially or from public institutions. Preferably, the cells are derived from mammalian cells, particularly human cells.

[0111] Embryonic stem cells (ES cells) are stem cells established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice. Spermatogonial stem cells are pluripotent stem cells derived from spermatozoa and are the cells that serve as the source of spermatogenesis. Embryonic germ cells are cells established from primordial germ cells during the fetal stage.

[0112] Induced pluripotent stem cells (iPS cells) can be produced by known production methods, and can also be purchased commercially or as custom-made products (see, for example, Patent Document 3 (Reference): JP 2023-018632 A, paragraphs

[0021] to

[0027] , etc.).

[0113] The pluripotent stem cells are preferably ES cells and / or iPS cells, more preferably iPS cells. The iPS cells are preferably derived from mammalian (preferably human) cells. Generally, the cells used for producing iPS cells are not particularly limited, and somatic cell-derived iPS cells are preferred.

[0114] In this embodiment, differentiated cells are preferred as somatic cells used to generate iPS cells. Among these differentiated cells, skin cells are preferred. Of these, epidermal keratinocytes (also referred to as "keratinocytes," "skin keratinocytes," or "keratinocytes") and dermal fibroblasts ("fibroblasts," "fibroblasts") are more preferred. Furthermore, in this embodiment, iPS cells generated using somatic epidermal keratinocytes or dermal fibroblasts are preferred from the perspective of evaluating skin homeostasis. Note that iPS cells derived from epidermal keratinocytes and dermal fibroblasts can be obtained using known methods for producing iPS cells derived from epidermal keratinocytes (e.g., Non-Patent Document 4 (Reference): Okita K. et al., Nature Methods, 2011, 8:409-12).

[0115] The somatic cells used in this embodiment refer to any animal cells (preferably mammalian cells including humans) excluding germline cells such as eggs, oocytes, sperm, etc., or totipotent cells such as ES cells, etc. Somatic cells include, but are not limited to, fetal (offspring) somatic cells, newborn (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, subculture cells, and established cell lines.

[0116] More specific examples of somatic cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; tissue progenitor cells; and differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts, epidermal keratinocytes, hair cells, stem cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells, brain cells, embryonic cells, kidney cells, and adipocytes; and one or more types selected from these can be used. Among these somatic cells, it is preferable to use fibroblasts and / or epidermal keratinocytes, which are the same as the cells obtained by inducing differentiation of pluripotent stem cell-derived cells.

[0117] <b. Preparation of Microspheres> The preparation step for microspheres in the method of this embodiment is not particularly limited, and they can be prepared with reference to known methods, or commercially available products can be obtained and used as appropriate. When the microspheres are cell clusters, the cell clusters may be prepared with reference to <b-1. Preparation of Fibroblast Clusters> below. Of the cell clusters, fibroblast clusters are preferred. When the microspheres are microcarriers (preferably spherical microcarriers), examples include the microcarriers described above in <Center Portion of Three-Dimensional Structure> and <Microcarriers>, and one or more of these can be used. Furthermore, known coating methods can be appropriately employed as a method for coating microcarriers with extracellular matrix components. As an example of a collagen coating method, the collagen coating method for resin culture dishes can be used. Microcarriers are coated with a predetermined collagen concentration (e.g., 0.01%, 0.05%, or 0.1%) at room temperature (approximately 20-37°C) for a coating time (removed immediately after addition, or approximately 0.25-1.5 hours, or 10-14 hours). If necessary, a dilution solvent such as dilute hydrochloric acid (0.01N) or acetic acid solution (5 mM) may be used. After coating, the cells may be washed one or more times (2-5 times) with sterile water or PBS. The type of collagen is not particularly limited; for example, type 1 or type 4 may be used.

[0118] <b-1. Preparation of fibroblast cell clusters> In the fibroblast cell cluster preparation step of the method of this embodiment, it is preferable to culture the seeded multiple fibroblasts in a cell culture vessel under predetermined culture conditions. To prepare fibroblast cell clusters (spheroids), it is preferable to use a cell culture vessel with one or more cone-shaped recessed bottoms, and / or to use a cell culture vessel with recesses that have been treated to inhibit cell adhesion. The number of cells to be seeded in the cell culture vessel is not particularly limited, but is preferably 1 x 10 ２ ~1 x 10 ５ Generally, a spheroid refers to a roughly spherical cell aggregate formed by the aggregation and aggregation of cells.

[0119] Furthermore, it is preferable to use a medium for fibroblasts as the medium used in the step of preparing the fibroblast mass. More preferably, the medium used in the step of preparing the fibroblast mass is prepared as a cell suspension containing fibroblasts and a culture medium, and then the cell suspension is seeded. It is preferable that the fibroblasts to be seeded are grown and cultured by a known culture method, recovered, and adjusted to the desired seeding number.

[0120] The culture period in the fibroblast cell mass preparation step is not particularly limited, but the preferred lower limit is preferably 0.5 days or more, more preferably 1 day or more, and the preferred upper limit is preferably 7 days or less, more preferably 4 days or less, and even more preferably 2 days or less. Furthermore, the number of days of culture from cell seeding in the fibroblast cell mass preparation step until cell masses are obtained is not particularly limited, but the preferred lower limit is preferably 0.25 days or more, more preferably 0.5 days or more, and the preferred upper limit is preferably 2 days or less, more preferably 1 day or less.

[0121] <c. Coating of Microspheres with Epidermal Keratinocytes> In the step of coating microspheres (preferably spherical microcarriers or fibroblast cell clusters) with epidermal keratinocytes in the method of this embodiment, one or more microspheres (preferably one) and a plurality of seeded epidermal keratinocytes are preferably cultured in a cell culture vessel under predetermined culture conditions. The cell culture vessel used in the coating step may be the same vessel used in and above, or a vessel equivalent to the vessel used in and above may be used. This allows for the production of three-dimensional structures or spheroids in which epidermal keratinocytes are coated on microspheres. A more preferred three-dimensional structure is a core-shell three-dimensional structure or spheroid, with microspheres (preferably spherical microcarriers or fibroblast cell clusters) as the core and epidermal keratinocytes as the shell. Generally, a spheroid refers to a spherical cell aggregate formed by the aggregation and aggregation of cells.

[0122] The size (major axis or diameter) of the microspheres (preferably spherical microcarriers or fibroblast aggregates) used for coating is not particularly limited, but the preferred lower limit is preferably 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 240 μm or more, or 320 μm or more, and the preferred upper limit is preferably 560 μm or less, more preferably 480 μm or less, with more preferred numerical ranges being 240 to 560 μm, and more preferably 320 to 480 μm. In the case of spherical microcarriers, for example, the size is 50 to 1,000 μm, and the preferred upper limit is preferably 300 μm or less or 250 μm or less, preferably 100 μm to 500 μm, more preferably 120 μm to 250 μm, and even more preferably 150 μm or 200 μm to 250 μm. The size of the resulting three-dimensional structure, cell layer, center, etc. can be adjusted to be the size described above in "2. Three-dimensional structure according to this embodiment" as appropriate.

[0123] To prepare a three-dimensional structure coated with epidermal keratinocytes, it is preferable to use a cell culture substrate having one or more recesses with a cone-shaped bottom, and / or a cell culture substrate having recesses that have been treated to inhibit cell adhesion. The number of epidermal keratinocytes to be seeded on the cell culture substrate is not particularly limited, but is preferably 5 x 10 ２ ~5 x 10 ４ is.

[0124] Furthermore, the medium used in the step of preparing a three-dimensional structure coated with epidermal keratinocytes is preferably a medium containing an epidermal keratinocyte medium, more preferably either an epidermal keratinocyte medium or a co-culture medium, and the co-culture medium is preferably a mixed medium of a fibroblast medium and an epidermal keratinocyte medium. When a non-cell-based microcarrier is used as the core, an epidermal keratinocyte medium may be used, and when a fibroblast cluster is used as the core, a co-culture medium is preferred. The epidermal keratinocytes used in this case are preferably epidermal keratinocytes derived from pluripotent stem cells induced to differentiate from pluripotent stem cells, more preferably iPS cell-derived epidermal keratinocytes induced to differentiate from iPS cells.

[0125] Furthermore, the culture period for obtaining the three-dimensional structure of this embodiment is preferably 2 or 3 days or more, more preferably 6 days or more, after seeding epidermal keratinocytes so that the obtained three-dimensional structure can be used for evaluation, etc. Furthermore, the preferred upper limit of this period is not particularly limited, but from the perspective of the culturing period, it is preferably 14 days or less, more preferably 10 days or less, and may be approximately 6 days. This allows for the production of a three-dimensional structure having a core-shell structure in which microspheres are coated with epidermal keratinocytes. The culture period for obtaining the three-dimensional structure may be the manufacturing period of the three-dimensional structure, or the description of the culturing period below may be appropriately adopted. During the culture period in the three-dimensional structure preparation step, the "3. Method for evaluating skin homeostasis function using the three-dimensional structure according to this embodiment" described below may be performed as "d. Step for evaluating skin homeostasis function."

[0126] The culture period or culturable period for the three-dimensional structure of this embodiment is not particularly limited. However, as the culture period after seeding of epidermal keratinocytes, a suitable lower limit is preferably 4 days or more, more preferably 5 days or more, and even more preferably 6 days or more. A suitable upper limit is not particularly limited, and the upper limit can be adjusted by adjusting the culture conditions. Examples of suitable upper limits include 40, 38, 35, 34, 33, 32, 31, 30, or 29 days, and are preferably 35 days or less, more preferably 28 days or less, and even more preferably 22 days or less. The suitable range of values ​​is preferably 4 to 35 days, more preferably 6 to 22 days. It is preferable to culture the three-dimensional structure by replacing the medium with a co-culture medium for epidermal keratinocytes every day.

[0127] In a more preferred embodiment, multiple epidermal keratinocytes are seeded and cultured on a cell culture vessel containing microspheres. For coating the microspheres with epidermal keratinocytes, it is preferable to use a cell culture vessel with one or more cone-shaped recessed bottoms, and / or a cell culture vessel with recesses that have been treated to inhibit cell adhesion. The cell culture vessel before seeding with epidermal keratinocytes may contain a medium containing an epidermal keratinocyte medium (e.g., an epidermal keratinocyte medium or co-culture medium). Alternatively, after preparing cell clusters, the medium may be removed from the cell culture vessel, and the cell suspension containing the epidermal keratinocytes to be seeded may be used as the medium. This liquid is preferably an epidermal keratinocyte medium or co-culture medium. When the microspheres are microcarriers, an epidermal keratinocyte medium or co-culture medium is preferred. When the microspheres are fibroblast clusters, a mixed medium containing a fibroblast medium and an epidermal keratinocyte medium (hereinafter also referred to as a "co-culture mixed medium") is preferred.

[0128] The fibroblast masses used to cover the epidermal keratinocytes are preferably the fibroblast masses obtained in the above-mentioned <b-1. Preparation of fibroblast masses>, and in a more preferred embodiment, are fibroblast masses prepared from seeded fibroblasts. More preferably, a cell suspension containing fibroblasts and a culture medium is prepared, and then the cell suspension is seeded. The fibroblasts to be seeded are preferably cultured and grown by a known culture method, recovered, and adjusted to the desired seeding number.

[0129] The microcarrier used to coat the epidermal keratinocytes is preferably one of the microcarriers described above in <Microcarriers>, more preferably a PVA microcarrier, and a more preferred embodiment is a coated microcarrier (preferably a PVA microcarrier) coated with an extracellular matrix component, the extracellular matrix component being preferably collagen, and examples of such collagen include type 1 and type 4.

[0130] <Co-culture medium> When preparing a three-dimensional structure comprising microspheres coated with epidermal keratinocytes, the medium is not particularly limited and may be, for example, a single medium or a mixed medium prepared by mixing single media. A co-culture medium is preferred, and this co-culture medium preferably contains various components so that fibroblasts and epidermal keratinocytes can be co-cultured. For example, the co-culture medium can be prepared by mixing a medium for fibroblasts and a medium for epidermal keratinocytes, and more preferably is a mixed medium containing a medium for fibroblasts and a medium for epidermal keratinocytes. Furthermore, the co-culture medium is preferably used when co-culturing fibroblasts and epidermal keratinocytes and / or when co-culturing fibroblast clumps and epidermal keratinocytes.

[0131] The co-culture medium used in this embodiment can use as its composition components medium components that allow fibroblasts and epidermal keratinocytes to grow, and the composition components of the co-culture medium may be prepared using these commercially available products or components contained therein.

[0132] More preferably, the co-culture medium is adjusted to contain animal- or human-derived components and / or calcium. Calcium chloride (preferably CaCl ) in the co-culture medium is preferably adjusted. ２ The concentration of ) is preferably 0.8 to 4 mM, more preferably 0.9 to 3.0 mM, and even more preferably 1.0 to 2.0 mM.

[0133] The mixing ratio of the epidermal keratinocyte medium and the fibroblast medium in the co-culture mixed medium used in this embodiment is not particularly limited, but is preferably 4:1 to 1:4, more preferably 3:1 to 1:3, even more preferably 2:1 to 1:2, and even more preferably 1:1. In the case of the first embodiment in which cell clusters are present in the core, the mixing ratio of the epidermal keratinocyte medium and the fibroblast medium in the co-culture mixed medium may be appropriately combined by adopting the upper and lower limits of the above-mentioned mixing ratios, but a preferred range is more preferably 3:1 to 1:3, even more preferably 2:1 to 1:2, and even more preferably 1:1. In the case of the second embodiment in which microcarriers are present in the core, the mixing ratio of the epidermal keratinocyte medium and the fibroblast medium in the co-culture mixed medium may be appropriately combined by adopting the upper and lower limits of the above-mentioned mixing ratios, but a preferred range is more preferably 3:1 to 1:3, even more preferably 2:1 to 1:2, and even more preferably 1:1. In the second embodiment, 100% epidermal keratinocyte medium may be used without using a fibroblast medium. The mixing ratio of the epidermal keratinocyte medium and the fibroblast medium may be 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more of the epidermal keratinocyte medium when the total amount of the epidermal keratinocyte medium and the fibroblast medium is 100%. By using a co-culture medium, or in some cases, an epidermal keratinocyte medium, epidermal keratinocytes can be formed around microspheres (preferably fibroblast clumps or microcarriers), resulting in a three-dimensional structure that has the proliferation and differentiation ability of epidermal keratinocytes and skin barrier function. Furthermore, the co-culture medium, or in some cases, an epidermal keratinocyte medium, can be used when evaluating skin homeostasis using a three-dimensional structure, and the culture period (evaluable period) of the three-dimensional structure can be extended.

[0134] <Number of fibroblasts and epidermal keratinocytes seeded> The number of fibroblasts and epidermal keratinocytes seeded in this embodiment is not particularly limited. The number of fibroblasts seeded in the cell culture vessel in <b. Preparation of fibroblast masses> is not particularly limited, but a preferable lower limit is 1 x 10 ２ or more, more preferably 5 × 10２ More preferably, 1×10 ３ The preferable upper limit is 1×10 ５ Less than or equal to 5 × 10, more preferably ４ More preferably, 2 × 10 ４ or less, and even more preferably 1×10 ４ The preferred range is preferably 1×10 ３ ~1 x 10 ４ is.

[0135] In the step of coating the fibroblast mass with epidermal keratinocytes, the number of epidermal keratinocytes to be seeded on the cell culture vessel is not particularly limited, but a preferable lower limit is 5 × 10 ２ or more, more preferably 1×10 ３ The upper limit is preferably 5×10 ４ Less than 2 × 10, more preferably ４ More preferably, 1×10 ４ The preferred range is preferably 1×10 ３ ~1 x 10 ４ is.

[0136] The ratio of the number of fibroblasts to the number of epidermal keratinocytes (number of fibroblasts / number of epidermal keratinocytes) used in this embodiment is not particularly limited, but a suitable lower limit is, from the viewpoint of appropriate tight junction formation in the epidermal keratinocyte layer, preferably ¼ or more, more preferably ½ or more, even more preferably 1 or more, more preferably 4 or more, and more preferably 8 or more. Also, a suitable upper limit is not particularly limited, but from the viewpoint of covering the entire area of ​​the fibroblast mass with epidermal keratinocytes, examples include 10 or less, preferably 9 or less, and more preferably 8 or less. Note that with regard to <Fibroblasts and epidermal keratinocytes and the number of each seeded>, the configuration of "the ratio of the number of fibroblasts to the number of epidermal keratinocytes (number of fibroblasts / number of epidermal keratinocytes)" described above in <Shape, size, etc. of three-dimensional structure> may be appropriately adopted.

[0137] The seeding number of fibroblasts is preferably the number of fibroblasts seeded in one well of the cell culture equipment when preparing a fibroblast mass, and the seeding number of epidermal keratinocytes is preferably the number of epidermal keratinocytes seeded in one well of the cell culture equipment when covering the prepared fibroblast mass with the epidermal keratinocytes.

[0138] <d. Evaluation of skin homeostasis function> After obtaining a three-dimensional structure with a core-shell structure according to this embodiment in the above step <c. Coating of epidermal keratinocytes on fibroblast clusters>, the below-described "3. Method for evaluating skin homeostasis function using a three-dimensional structure according to this embodiment" may be performed, including the step <d. Evaluation of skin homeostasis function>, or <d.> may be performed after the above step <c.>. This makes it possible to search for or select substances that have a skin homeostasis function, and more preferably, to search for or select substances related to skin homeostasis that are suitable for an individual. Note that <d. Evaluation of skin homeostasis function> will be described in detail in the below-described "3. Method for evaluating skin homeostasis function using a three-dimensional structure according to this embodiment," and the contents of this description may be adopted as appropriate.

[0139] <Others> <Cell Culture Equipment> The cell culture equipment used in the method of this embodiment is not particularly limited, but is preferably a cell culture equipment that can be typically used for cell culture. Examples include, but are not limited to, flasks, dishes, petri dishes, bottles, plates, tubes, and centrifuge tubes. Disposable products are also preferred. Examples of materials for the equipment include, but are not limited to, glass and plastic resins (preferably polystyrene resins). One or more materials selected from these examples can be used. The material (or material) of the cell culture equipment is not particularly limited, but is preferably one or more materials selected from synthetic resins (preferably plastics), such as styrene resins (polystyrene or styrene copolymers, etc.), polycarbonate, polyolefin resins (polyethylene, polypropylene, polyester, ethylene copolymers, etc.), (meth)acrylic resins, silicone resins, amino resins, fluororesins, and polyimide resins, and glass substrates. Of these, plastics (more preferably styrene-based resins, polyolefin-based resins (polyethylene, polypropylene), styrene-based resins, and polycarbonates) are preferred, and of these, styrene-based resins (more preferably polystyrene) are even more preferred.

[0140] As described above, in the method of this embodiment, in the step of producing approximately spherical objects such as three-dimensional structures, cell aggregates, or spheroids, it is preferable to culture the seeded cells on a cell adhesion-inhibiting surface (more preferably a hydrophilized surface) of a conical depression. In the proliferation step of increasing the number of cells, it is preferable to culture the cells on a culture substrate-treated surface of a flat depression such as a petri dish.

[0141] In producing a substantially spherical object, the bottom of the cone-shaped recess is preferably shaped so that multiple cells can gather in the central region of the bottom of the recess. It is more preferable that the bottom of the recess is V-shaped or U-shaped, which makes it easier for cells seeded in a V-shaped or U-shaped depression (such as a substantially conical or truncated conical shape) to gather, thereby enabling a better shape of substantially spherical cell clusters, which has the advantage of facilitating the formation of a fibroblast cell cluster that will serve as the nucleus of a three-dimensional structure, or facilitating the formation of a shell cell layer that covers the nucleus of a three-dimensional structure.

[0142] In producing the approximately spherical objects, the wells into which the cells are seeded are preferably subjected to a cell adhesion inhibitory treatment. This surface treatment can inhibit cell adhesion to the well surface, making it easier to detach the three-dimensional structure after culture. This surface treatment allows the seeded cells to gather in the central region of the well, facilitating cell adhesion, resulting in a better shape of the approximately spherical objects (e.g., fibroblast cell clusters, cell clusters with a core-shell structure). Examples of the cell adhesion inhibitory treatment include, but are not limited to, hydrophilic surface treatment. In producing the approximately spherical objects, it is more preferable to subject the wells to a hydrophilic surface treatment. Examples of hydrophilic treatment include, but are not limited to, plasma treatment, corona discharge treatment, oxidizing agent treatment, and coating with a hydrophilic substance (preferably, a cell adhesion inhibitor such as polyethylene glycol). Examples of hydrophilic substance coatings include, but are not limited to, high molecular weight polymer coatings. One or more of these can be used.

[0143] <Cell Release Agent> The method of this embodiment uses a cell release agent. The cell release agent is preferably a cell release component typically used to release cultured cells from cell cultureware during animal cell culture, or an agent containing such a component. Components used for cell release include serine proteases such as trypsin-based proteases (trypsin, trypsin-like proteases, etc.) and chymotrypsin; metalloproteases such as dispase; Ca-dependent proteases such as actinase; neutral proteases; proteases with protease and collagenase activity; enzyme-free release systems such as ethylenediaminetetraacetic acid (EDTA) and sodium citrate; TrypLE (trademark), etc. One or more of these may be used, but are not limited thereto. Commercially available cell release agents may also be used as the cell release agent. Furthermore, physical cell release means may also be used, such as a cell scraper (manufactured by Corning) or other cell culture release equipment.

[0144] Among the cell detachment agents, enzymes or those containing enzymes are preferred. Protease enzymes are particularly preferred, and trypsin-based enzymes are even more preferred. Trypsin-based proteases such as trypsin, trypsin-like proteases, and TrypLE™ Select enzymes are even more preferred. TrypLE™ Select enzymes are also preferred. Recombinant proteins with trypsin-like activity (more preferably recombinant microbial trypsin-like proteases) are also preferred. "Trypsin-based enzymes" encompass both trypsin and trypsin-like enzymes. "Trypsin-like" refers to enzymes that exhibit kinetics and cleavage properties similar to those of trypsin, and are more preferred for directly replacing trypsin in cell detachment without modifying the protocol. Trypsin's cleavage properties include its ability to hydrolyze peptide bonds at the carboxyl side of basic amino acids (lysine, arginine). The enzyme may be derived from either an animal or a microorganism, but is preferably derived from a microorganism, more preferably from a genetically modified microorganism that has been genetically modified to be able to produce protease or trypsin, and even more preferably from a genetically modified fungus.

[0145] The cell detachment agent preferably contains a protease and / or a TrypLE™ Select enzyme, and the protease is preferably one or more selected from trypsin, trypsin-like proteases, and TrypLE™ Select enzymes, and is even more preferably trypsin and / or TrypLE™ Select enzymes. Suitable cell detachment agents include, for example, trypsin, TrypLE™ Select enzymes, and cell detachment agents containing the TrypLE™ Select enzymes and 0.5 to 1.5 mM EDTA, and one or more selected from these may be used.

[0146] It is preferable to use a cell detachment agent in combination with a physical treatment to detach adherent cells from cell cultureware. Examples of physical treatment include water jet generation using pipetting (suction, spraying, etc.) and scraping methods such as a spatula-shaped scraper that can detach adherent cells. However, water jet generation (preferably, recovery of detached cells after detachment by suction and spraying) is preferred from the viewpoint of reducing cell damage. Furthermore, the recovered detached cells may be subjected to a separation method such as centrifugation, which allows further recovery of single cells for use in subsequent cell culture.

[0147] <Culture Substrate (Coating Agent)> In the method of this embodiment, a coating agent containing a culture substrate may be used, if necessary, to coat the portion of the cell culture substrate where the cells come into contact with the cell culture substrate. The culture substrate is preferably one that enhances or improves the adhesiveness of cells to the surface of the cell culture substrate, and coating the surface of the cell culture substrate with the culture substrate can enhance or improve the adhesiveness of cells to the surface of the cell culture substrate. Examples of coating treatments include pouring a solution containing the culture substrate into the cell culture substrate and then appropriately removing the solution, but the present invention is not limited to this, and any known coating treatment can be used as appropriate.

[0148] The culture substrate is not particularly limited, but includes extracellular matrix components such as laminin, collagen, fibronectin, vitronectin, Matrigel, fibrin, and thrombin; amino acid polymers such as poly-L-lysine and poly-D-lysine; and fragments thereof, and one or more selected from these can be used. Among the culture substrates, one or more selected from collagen and fragments thereof, and laminin and fragments thereof are preferred, with laminin and fragments thereof being more preferred. Laminin derived from humans is preferred. Laminin and fragments thereof are preferred, with a binding activity with integrin α6B1 exhibiting a dissociation constant of 10 nM or less. Commercially available collagen or collagen fragments, laminin or laminin fragments are preferred.

[0149] Examples of laminin fragments include the E8 fragment (also referred to as laminin 511E8 fragment or laminin 511E8), which is a fragment obtained by digesting laminin 511 with elastase (Non-Patent Document 5 (Reference): Ido H, et al., J. Biol. Chem. 2007, 282, 11144-11154), and recombinant human laminin 511E8 fragment expressed from genetically modified silkworm cocoons.

[0150] Among laminin and fragments thereof, laminin fragments are preferred, more preferably laminin 511 fragments, even more preferably laminin 511E8 fragments, and even more preferably those derived from humans.

[0151] <e. Three-dimensional structure with core-shell structure obtained by the manufacturing method of this embodiment> The manufacturing method of this embodiment can produce a three-dimensional structure with core-shell structure comprising a cell layer composed at least of a plurality of tight junctions present between cells in the surface cell layer and a plurality of epidermal keratinocytes, and microspheres having extracellular matrix components on their surface, which form the central part of the three-dimensional structure. More preferably, the three-dimensional structure with core-shell structure comprises an epidermal keratinocyte layer comprising an undifferentiated cell layer and a differentiated cell layer as the shell portion.

[0152] The resulting three-dimensional structure preferably has properties that allow for appropriate staining and localization of TJ markers, proliferation markers, extracellular matrix component markers, and basal cell markers. The size (long axis or diameter) of the resulting three-dimensional structure is preferably 300 to 700 μm, the size (long axis or diameter) of the fibroblast clusters in the three-dimensional structure is preferably 240 to 560 μm, and the size (thickness) of the cell layer containing epidermal keratinocytes in the three-dimensional structure is preferably 60 to 140 μm. These sizes may also be appropriately determined using the configurations described in <Shape, Size, etc. of the Three-Dimensional Structure>. Furthermore, the manufacturing method according to this embodiment can be used to obtain the above-mentioned "1. Three-dimensional Structure According to This Embodiment."

[0153] The preferable lower and upper limits of the culturable period of the obtained three-dimensional structure can be appropriately selected from the explanation in "2. Method for producing a three-dimensional structure according to this embodiment" above, and the preferable range of values ​​is more preferably 4 to 35 days, and even more preferably 6 to 22 days. The starting point of the culture period or culturable period is more preferably "after seeding of epidermal keratinocytes." Note that, when culturing the three-dimensional structure, it is preferable to replace the medium with a co-culture medium every day.

[0154] In this specification, "something" such as "culturing" may be expressed as a "process" or a "step," and a "process" may be expressed as "something" or a step, and a "step" may be expressed as "something" or a "process." In addition, in this embodiment, a "process" such as a "process" may be expressed as "an apparatus or part configured to perform the process," an "apparatus" may be expressed as "a mechanism or part," and a "part" may be expressed as "a part or apparatus to be provided in a mechanism, apparatus, system, or the like."

[0155] 3. Method for Evaluating Skin Homeostasis Function Using a Three-Dimensional Structure According to the Present Embodiment The method according to the present embodiment can provide a method for evaluating skin homeostasis function using a three-dimensional structure according to the present embodiment or a three-dimensional structure obtained by the method for producing a three-dimensional structure according to the present embodiment. The period during which the three-dimensional structure according to the present embodiment can be evaluated is not particularly limited, but is preferably 6 to 22 days after seeding epidermal keratinocytes on the fibroblast cluster. The three-dimensional structure can be formed approximately 3 days after seeding epidermal keratinocytes on the fibroblast cluster.

[0156] In the description of this embodiment, explanations of the components and processing methods of epidermal keratinocytes, microspheres, spherical microcarriers, fibroblasts, core-shell structures, pluripotent stem cells, cell culture equipment, etc. that overlap with the explanations in "2." and "3." above and "4." below will be omitted as appropriate, but the explanations in "2." and "3." above and "4." below also apply to this embodiment and can be adopted as appropriate.

[0157] By subjecting the three-dimensional structure to various physical or chemical treatments, it is possible to discover new physical properties (firmness, elasticity, blemishes, roughness, etc.) and chemical processing capabilities (test substance, acid or alkaline components, sebum, etc.) not found in conventional models (animal skin models or three-dimensional skin models). In this embodiment, cells derived from pluripotent stem cells can be used, and somatic cells of the target animal can be used to produce a custom-made three-dimensional structure and confirm its physical, chemical, or physiological / biochemical state. Tests on the three-dimensional structure can be performed using one or more of the following: physical tests (e.g., pressure / vacuum adjustment, vibration adjustment, pressure adjustment, light irradiation such as ultraviolet light or natural light), physiological / biochemical tests (markers, fluorescent staining, etc.), and chemical tests (searching for test substances, etc.).

[0158] By using or contacting a test substance with the three-dimensional structure according to this embodiment, it is possible to evaluate or select a test substance that has an effect on the skin. In a preferred embodiment, the three-dimensional structure is cultured in a culture medium with or without the test substance. In this case, when evaluating the test substance, the results of the test substance may be compared with the results of a control (e.g., a no-addition control, a positive control, or a negative control). The concentration of the test substance may be adjusted, and the skin homeostasis function may be evaluated based on the concentration change (increase / decrease, gradient).

[0159] In the method for evaluating skin homeostasis, a three-dimensional structure is used, and therefore the culture conditions used in <c. Coating of epidermal keratinocytes on microspheres> are preferably used. The medium used is preferably a co-culture medium or a medium for epidermal keratinocytes (preferably a co-culture medium, and in some cases a medium for epidermal keratinocytes), and it is preferable to change the co-culture medium every day. Alternatively, a co-culture medium or a medium for epidermal keratinocytes containing a test substance may be used. The evaluation period is preferably the culturable period of the three-dimensional structure, more preferably, for example, 6 to 22 days, and this culturable period may be used as the evaluable period or usable period of the three-dimensional structure.

[0160] Another aspect of the method of this embodiment can be to provide a substance that exhibits skin homeostasis maintenance function, or an active ingredient for skin homeostasis maintenance function, and / or a skin homeostasis maintenance functional agent. This can provide technology related to skin homeostasis maintenance function, and specifically, it can provide a substance that has a skin homeostasis maintenance effect, or an active ingredient for skin homeostasis maintenance function, and / or a skin homeostasis maintenance functional agent. This can provide a drug for maintaining skin homeostasis. The drug may be a composition.

[0161] The substance selected by the method for evaluating and / or selecting a skin homeostasis maintaining functional agent of this embodiment (hereinafter also referred to as "selected substance") can be provided as a substance having a skin homeostasis maintaining effect or as an active ingredient of a skin homeostasis maintaining functional agent. Furthermore, the method for evaluating and / or selecting a skin homeostasis maintaining functional agent of this embodiment may be a device or system for evaluating and / or selecting a skin homeostasis maintaining functional agent of this embodiment.

[0162] Examples of skin homeostasis maintenance effects include, but are not limited to, skin barrier function maintenance, skin turnover maintenance, anti-inflammatory effect, antioxidant effect, and skin color regulation effect. More specifically, examples include moisturizing effect due to skin barrier function maintenance effect, and rough skin prevention effect due to anti-inflammatory effect. One or more of these can be selected. Therefore, the selected substance obtained by the method for evaluating and / or selecting a skin homeostasis maintenance agent of this embodiment has a skin homeostasis maintenance function, and therefore, its effects or influences on skin barrier function, turnover, moisturizing, rough skin prevention, etc. can be confirmed.

[0163] The selected substance is not particularly limited and may be either naturally or artificially derived, and may be either a single substance or a mixture. The selected substance is preferably one or more substances selected from compounds, microorganisms or their cultures, extracts, mixtures thereof, and compositions. The compound may be either an inorganic compound or an organic compound.

[0164] More preferred specific selected substances include lipids, vitamins, carotenoids, etc., and further include, for example, phospholipids or derivatives thereof (such as LPA).

[0165] The selected substance can be contained as an active ingredient in a skin homeostasis maintenance agent (preferably a skin barrier function promoter, moisturizing or turnover promoter), an agent for preventing, improving or treating rough skin, etc. (hereinafter also referred to as "skin homeostasis maintenance agent, etc."), or can be used in the skin homeostasis maintenance agent, etc. The agent may be a composition. The selected substance can also be used to manufacture a skin homeostasis maintenance agent, etc. The present embodiment can also provide the selected substance or use thereof for maintaining skin homeostasis or for use in maintaining skin homeostasis. The present embodiment can also provide a method for maintaining skin homeostasis using the selected substance, or a method for maintaining skin homeostasis using an agent containing the selected substance.

[0166] <Diseases and Symptoms> Because the selected substance exerts its physiological activity, it can be used in methods for preventing, improving, or treating symptoms such as diseases or rough skin caused by a decrease in skin homeostasis function or sunburn, etc. Examples of symptoms of diseases or rough skin caused by skin roughness or sunburn, etc., include, but are not limited to, eczema, pigmentation, wrinkles, sagging, acne, pimples, dry skin, etc., and one or more of these can be selected from the group consisting of these. Furthermore, uses of this embodiment include, but are not limited to, the prevention, improvement, or treatment of skin barrier function, dry skin, wrinkles, acne, etc.

[0167] In this embodiment, "prevention" refers to preventing or delaying the onset of symptoms or diseases in a subject, or reducing the risk of developing symptoms or diseases in a subject. In this technology, "improvement" refers to improving or maintaining the disease, symptoms, or condition in a subject; preventing or delaying deterioration; or reversing, preventing, or delaying progression.

[0168] In this embodiment, the selected substance can be used in, for example, cosmetics, topical skin preparations, quasi-drugs, foods and beverages, feeds, etc., but is not particularly limited to these. Furthermore, the "skin homeostasis maintaining functional agent, etc." can be used in, for example, cosmetics, topical skin preparations, quasi-drugs, foods and beverages, feeds, etc., but is not particularly limited to these. Among these, cosmetics, topical skin preparations, pharmaceuticals, quasi-drugs, etc. are preferred, and applications for affecting the epidermal layer and / or dermal layer of the skin are preferred.

[0169] In the case where this embodiment is, for example, a cosmetic or topical skin preparation, the selected substance or skin homeostasis maintenance agent can be blended into various forms of cosmetic or topical skin preparation, such as, but not limited to, emulsion, cream, lotion, pack, cleanser, makeup cosmetic, dispersion, ointment, liquid, aerosol, patch, cataplasm, liniment, etc.

[0170] When the present embodiment is, for example, a pharmaceutical product or a quasi-drug, the selected substance or skin homeostasis maintenance agent can be incorporated into, for example, oral preparations such as tablets, capsules, granules, powders, liquids, and suspensions; external preparations such as dermatological preparations, patches, eye drops, nasal drops, oral preparations, and suppositories; and parenteral preparations such as infusions and injections, but are not limited to these.

[0171] The selected substance or the total secretion regulator can be produced by using a known production method. The selected substance may be a commercially available substance.

[0172] The content of the selected substance is not particularly limited, and the selected substance can be contained in an amount of preferably 0.001 to 99% by mass, more preferably 0.01 to 50% by mass, and even more preferably 0.1 to 10% by mass of the total amount of the agent or composition.

[0173] The selected substance or skin homeostasis maintenance agent can be applied to humans and non-human animals (e.g., pets, livestock, etc.), etc. Among these, humans and pets are preferred, and humans are more preferred.

[0174] Methods for using the selected substance or skin homeostasis maintaining agent, etc. include, but are not limited to, administration such as transdermal administration, oral administration, administration by injection, oral ingestion, application to the skin, etc. The amount of the selected substance to be used or administered is not particularly limited as long as it is an amount that can achieve the effects of the present invention, and may be appropriately adjusted depending on the dosage form of the preparation, the application site, age, sex, etc.

[0175] The skin homeostasis maintaining agent, etc. can be produced using a known production method. In addition to the selected substance, the skin homeostasis maintaining agent, etc. can also contain optional components such as various additives, if necessary.

[0176] As the optional ingredient, ingredients acceptable for cosmetics, topical skin preparations, pharmaceuticals, foods and beverages, feeds, etc. can be appropriately blended. For example, one or more selected from excipients, colorants, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, moisturizers, pH adjusters, etc. may be appropriately used, thereby allowing the desired dosage form to be obtained.

[0177] 4. The present technology may also adopt the following other aspects, configurations, or technical features as appropriate. [1] A three-dimensional structure with a core-shell structure comprising a cell layer having tight junctions present on its surface and composed at least of a plurality of epidermal keratinocytes, and a central portion that is a microsphere having an extracellular matrix component on its surface. It is preferable that the structure is composed of at least a plurality of tight junctions present between the cells of the cell layer on the surface of the cell layer and a plurality of epidermal keratinocytes. It is preferable that the cell layer is a layer composed at least of a differentiated layer containing differentiated epidermal keratinocytes and an undifferentiated layer toward the center containing undifferentiated epidermal keratinocytes. It is also preferable that the tight junctions are present between adjacent differentiated epidermal keratinocytes present in the cell layer on the surface of the three-dimensional structure. [2] The three-dimensional structure according to [1], wherein the microspheres are microcarriers or cell aggregates. [3] A three-dimensional structure with a core-shell structure comprising a cell layer having tight junctions present on the surface and composed of at least a plurality of epidermal keratinocytes, and a fibroblast mass in the center of the three-dimensional structure composed of at least a plurality of fibroblasts. [4] A three-dimensional structure with a core-shell structure comprising a cell layer having tight junctions present on the surface and composed of at least a plurality of epidermal keratinocytes, and a spherical microcarrier in the center of the three-dimensional structure having extracellular matrix components on its surface. [5] The three-dimensional structure according to any one of [1] to [4] above, wherein the epidermal keratinocytes or the fibroblasts are one or more types selected from normal cells (normal epidermal keratinocytes, normal fibroblasts), and epidermal keratinocytes and fibroblasts derived from pluripotent stem cells. [6] The three-dimensional structure according to any one of [1] to [5] above, used for evaluating skin homeostasis.

[0178] [7] A method for producing a three-dimensional structure with a core-shell structure by co-culturing microspheres and seeded epidermal keratinocytes. The microspheres are preferably microcarriers or cell clusters, and more preferably are approximately spherical. The microcarriers are preferably microcarriers coated with extracellular matrix components. The cell clusters are preferably fibroblast clusters prepared from a plurality of fibroblasts. [8] A method for producing a three-dimensional structure with a core-shell structure by co-culturing microspheres and seeded epidermal keratinocytes using a cell culture vessel. The microspheres are preferably fibroblast clusters prepared from seeded fibroblasts or microcarriers coated with extracellular matrix components. [9] A method for producing a three-dimensional structure with a core-shell structure by seeding and culturing epidermal keratinocytes in a cell culture vessel or medium containing microspheres. The cells used are preferably cultured on the cell adhesion-inhibiting treated surface of the cone-shaped depression, and the cells used are preferably fibroblast clusters and / or epidermal keratinocytes. It is also preferable to use a cell cultureware having one or more wells containing a co-culture medium.

[10] A method for producing a three-dimensional core-shell structure, comprising: placing microspheres on the cell adhesion-inhibiting treated surface of the cone-shaped depression; seeding epidermal keratinocytes on the surface; and co-culturing the microspheres; wherein an epidermal keratinocyte layer covering the microspheres forms a shell; the cell layer is formed to include an undifferentiated epidermal keratinocyte layer, a differentiated epidermal keratinocyte layer, and multiple tight junctions present between the differentiated epidermal keratinocytes in the outermost layer. The microspheres may be cell clusters containing extracellular matrix components obtained by seeding and culturing fibroblasts, or they may be microcarriers coated with extracellular matrix components.

[11] The method for producing a three-dimensional structure according to any one of [7] to

[10] above, wherein a medium containing at least a medium for epidermal keratinocytes, or a medium containing a medium for epidermal keratinocytes and a medium for fibroblasts, is used when culturing the microspheres and the seeded epidermal keratinocytes.

[12] The method for producing a three-dimensional structure according to any one of [7] to

[11] above, wherein the culture period after seeding of the epidermal keratinocytes is preferably 6 days or more, more preferably 6 to 22 days. The culture period may be a culturing period, a test period, or an evaluative period, and tests, evaluations, etc. may be performed using the three-dimensional structure according to any one of [7] to

[11] above during such a period.

[13] The method for producing a three-dimensional structure according to any one of [7] to

[12] above, wherein the three-dimensional structure is cultured so that the size of the three-dimensional structure becomes 1.05 to 1.5, where the size of the microspheres is taken as 1.

[14] The method for producing a three-dimensional structure according to any one of [7] to

[13] above, wherein the seeding ratio of the fibroblasts to the epidermal keratinocytes is preferably 1:4 or more, more preferably 1:4 to 8:1.

[15] The method for producing a three-dimensional structure according to any one of [7] to

[14] , wherein the epidermal keratinocytes or fibroblasts are one or more types selected from normal cells (normal epidermal keratinocytes, normal fibroblasts) and epidermal keratinocytes and fibroblasts derived from pluripotent stem cells.

[16] The method for producing a three-dimensional structure according to any one of [7] to

[15] , wherein the seeded epidermal keratinocytes or fibroblasts are one or more types selected from normal cells (normal epidermal keratinocytes, normal fibroblasts) and cells derived from pluripotent stem cells (epidermal keratinocytes, fibroblasts), preferably cells derived from pluripotent stem cells, more preferably epidermal keratinocytes derived from pluripotent stem cells (more preferably epidermal keratinocytes derived from iPS cells). It is more preferable that the iPS cells are iPS cells derived from somatic cells derived from the target animal.

[17] The method for producing a three-dimensional structure according to any one of [7] to

[16] above, wherein the microcarrier is derived from an organic material, an inorganic material, or a composite material thereof.The microcarrier is preferably a resin, and is preferably one or more selected from polyvinyl alcohol, polylactic acid, polyglycolic acid, polytetrafluoroethylene, polystyrene, polyester, polyurethane, polyethylene, polypropylene, (meth)acrylic polymers, (meth)acrylamide polymers, silicone polymers, and epoxy resins.

[18] The method for producing a three-dimensional structure according to any one of [7] to

[17] above, wherein the microcarrier is a microcarrier coated with an extracellular matrix component. The extracellular matrix component is preferably collagen (e.g., types 1 to 19), more preferably collagen types 1 and 4.

[0179]

[19] A method for producing a three-dimensional structure or a test method using a three-dimensional structure, comprising (1) preparing microspheres (preferably microcarriers or fibroblast clusters), and / or (2) obtaining a three-dimensional structure with a core-shell structure based on microspheres and epidermal keratinocytes.

[20] The method according to

[19] , comprising, prior to (1) and / or (2), growing cells to be seeded.

[21] The method according to any one of

[19] to

[20] , comprising, after (2), (3) conducting a test using the three-dimensional structure. Preferably, the test is a test for evaluating skin homeostasis.

[22] The method according to

[21] , wherein the test (3) is one or more types selected from a physical test, a physiological / biochemical test, and a chemical test on the three-dimensional structure.

[23] The method according to any one of

[19] to

[22] above, comprising contacting a test substance with the three-dimensional structure and culturing the structure, and determining the function of the test substance after the contact.

[0180]

[24] A method for evaluating skin homeostasis function, using the three-dimensional structure described in any one of [1] to [6] above, or a three-dimensional structure obtained by any one of the production methods described in [7] to

[18] above.

[25] A method for evaluating or selecting a substance having skin homeostasis function (positive or negative), using the three-dimensional structure described in

[24] above.

[26] The method described in

[24] or

[25] above, using a medium (epidermal keratinocyte medium or co-culture medium) or co-culture medium containing at least an epidermal keratinocyte medium. When the microspheres are microcarriers, a medium containing at least an epidermal keratinocyte medium is preferred, and when the microspheres are fibroblast clumps, a co-culture medium is preferred.

[27] A mixture containing a medium for fibroblasts and a medium for epidermal keratinocytes for use in the method according to

[24] or

[25] above, or use of a medium for fibroblasts and a medium for epidermal keratinocytes for producing a medium for use in the method according to

[24] or

[25] above.

[0181] Hereinafter, the present embodiment will be described in more detail based on examples, etc. Note that the examples, etc. described below are examples of typical examples, etc. of the present technology, and are not to be construed as narrowing the scope of the present technology.

[0182] <<Production Example 1 [Fibroblast Clusters]: Three-dimensional structure with core-shell structure and method for producing same>> A three-dimensional structure with a core-shell structure, comprising a fibroblast cluster as the core and epidermal keratinocytes as the shell, was produced as follows (see Figure 3), to provide the three-dimensional structure with a core-shell structure of Production Example 1. Specifically, fibroblasts were seeded into a well and cultured for a certain period of time to produce the core of the fibroblast cluster, and then epidermal keratinocytes to form the shell were seeded into the same well, followed by static culture.

[0183] <Preparation of the Core of the Three-Dimensional Structure> Fibroblast cell clusters to serve as the core of the three-dimensional structure were produced as follows. [Adjustment of the Seeding Number of Fibroblasts] Normal human fibroblasts (NHDF) (LIFELINE CELL TECHNOLOGY, model number FC-0001 (neonatal foreskin)) were cultured for 3 days at 37°C and 5% CO2 in FibroLife S2 medium (low-serum liquid medium for the growth of normal human dermal fibroblasts (phenol red-free), FibroLife S2 Comp kit LIFELINE CELL TECHNOLOGY, model number LFC-LL0011). Polystyrene tissue culture plates (cylindrical wells, coating agent: laminin 511E8 fragment) were used for culture. On the second day of culture, the medium was replaced with FibroLife S2 medium.

[0184] [FibroLife S2 Comp kit (LFB-LM0001 and LFK-LS1038)] LFB-LM0001: FibroLife BM: Basal medium for skin fibroblasts* (phenol red, antibacterial free) 500mL LFK-LS1038: FibroLife S2 LifeFactors: Additive set for skin fibroblast proliferation, low serum type (L-glutamine (7.5 mM), hFGF-B (5 ng / mL), insulin (5 μg / mL), ascorbic acid (50 μg / mL), hydrocortisone (1 μg / mL), FBS (2% V / V), gentamicin (30 μg / mL), amphotericin B (15 ng / mL)) (final concentrations in kit medium)

[0185] The fibroblasts were cultured for 3 days to prepare a cell suspension, and in the preparation of fibroblast cell clusters, fibroblasts were seeded using this cell suspension to prepare the cores that would become the nuclei of the three-dimensional structure. The fibroblasts were washed with PBS(-) and then incubated with TrypLE. (TM) The cells were detached using Select Enzyme (Gibco, product number 12563011). The detached cells were separated by centrifugation and diluted to 1 × 10 in DMEM / F12 medium containing 5% FBS (Sigma-Aldrich) and 1% CaCl. 5The cells were suspended at a concentration of 1000 cells / mL. The cells were counted using a Coulter counter (CDA-1000, Sysmex Corporation).

[0186] [Preparation of fibroblast cell aggregates] Fibroblast cell suspension (1 × 10 5 100 μL / well of normal fibroblasts (1 × 10 cells / mL) were seeded into a V-bottom 96-well multi-well plate and cultured at 37°C and 5% CO2 for one day. After culturing for one day after seeding, the cells gathered at the bottom of the V-bottom well to form a mass. In this way, a fibroblast mass was prepared, which was used as the core of the three-dimensional structure. The number of normal fibroblasts seeded per well was 1 × 10 4 The V-bottom 96-well multi-well plate is made of PS (polystyrene), has a cell culture surface treatment (hydrophilic surface treatment of high molecular weight polymer), is nonpyrogenic, and is sterilized. The culture area per well (cm 2 ): 0.38, Maximum volume per well (mL): 0.32, Dimensions (mm): 127.8 x 85.5 x 16.5.

[0187] <Preparation of the shell of a three-dimensional structure> Epidermal keratinocytes induced from human iPS cells were seeded on the core of a fibroblast mass present in each well of a V-bottom 96-well multiwell plate and co-cultured to produce the three-dimensional structure of Production Example 1. Note that a three-dimensional structure can also be produced by co-culture using normal human epidermal keratinocytes instead of human iPS cells.

[0188] [Adjusting the seeding number of human iPS cell-derived epidermal keratinocytes] Human iPS cell-derived epidermal keratinocytes were cultured in CnT-07 (CELLnTEC) medium supplemented with 20 ng / mL EGF (MACS) and 10 μM Y-27632 at 37°C and 5% CO2. Polystyrene tissue culture plates (cylindrical wells, coating agent: laminin 511E8 fragment) were used for culture. On day 2 of culture, the medium was replaced with CnT-07 (CELLnTEC) medium supplemented with 20 ng / mL EGF (MACS) and 10 μM Y-27632.

[0189] For the co-culture of epidermal keratinocytes and fibroblasts, a co-culture medium (hereinafter also referred to as "Half medium") was prepared by mixing DMEM / F12 medium (medium for fibroblasts) supplemented with 5% FBS (Sigma-Aldrich) and 1 mM CaCl2 with CnT-PR-3D medium (CELLnTEC) (medium for epidermal keratinocytes) in a 1:1 (volume:volume) mixture.

[0190] For seeding of epidermal keratinocytes, cells were washed with PBS(-) and then added to TrypLE. (TM) The epidermal keratinocytes were detached using Select Enzyme. The detached epidermal keratinocytes were separated by centrifugation and then cultured in the above-mentioned co-culture medium until the epidermal keratinocytes reached 5 × 10 4 The cells were suspended at a concentration of 100 μL / well. The fibroblast medium was removed by suction from each well of the 96-well multiwell plate, leaving the fibroblast clumps behind. After removing the medium by suction from each well, a cell suspension of epidermal keratinocytes was seeded at 100 μL / well into each well where the fibroblast clumps remained. Each well contained 1 fibroblast clump and 5×10 epidermal keratinocytes. 3 There was co-culture medium containing cells / mL.

[0191] The core-shell 3D constructs containing fibroblasts and epidermal keratinocytes (Production Example 1) prepared as described above were cultured at 37°C under 5% CO2. The medium was replaced daily with 100 μL / well of co-culture medium. The 3D constructs, consisting of at least a core of normal fibroblasts and a shell of iPS cell-derived, differentiation-induced epidermal keratinocytes, could be cultured for 22 days in the co-culture medium.

[0192] [Various markers] Occludin was selected as a tight junction (TJ) marker. Ki67 was selected as a proliferation marker. Collagen XVII was selected as a basal cell marker. These were detected by known methods or by commercially available antibodies. Commercially available antibodies include: Occludin antibody: anti-occludin antibody clone 1C17 ZooMAb （R）Rabbit monoclonal (Sigma-Aldrich), Ki67 antibody: anti-Ki67 antibody clone 1O15 ZooMAb （R） Examples include rabbit monoclonal antibody (Sigma-Aldrich), collagen XVII antibody: anti-COL17A1 [NC16a-3] antibody (GeneTex).

[0193] [Sampling (RNA)] 1. Collect spheroids under the same conditions together with the medium into a sterilized 1.5 mL tube. 2. Remove as much medium as possible. 3. Add 400 μL of TRIzol. 4. Vortex until no spheroids are visible. 5. Store at -80°C.

[0194] [Sampling (staining)] 1. Transfer the spheroids under the same conditions onto a slide glass; 2. Fix the spheroids with ethanol; 3. After blocking, perform immunostaining using the desired primary and secondary antibodies.

[0195] Experimental Example 1: Study of Medium Mixing Ratios. The medium used was a mixture of DMEM / F12 medium supplemented with 5% FBS and 1 mM CaCl2 and CnT-PR-3D medium at the ratios listed in Table 1. Epidermal keratinocytes (iKCs) differentiated from iPS cells and NHDF fibroblasts were used as epidermal keratinocytes. The core-shell three-dimensional structures of Example 1 were obtained according to the procedure described above in "Core-shell Three-dimensional Structures and Their Production Methods." Three weeks after seeding with iKCs, the three-dimensional structures were immunostained with anti-Occludin antibodies and photographed using a confocal laser scanning microscope (LSM900, ZEISS). The total area of ​​the three-dimensional structures and the manually designated area of ​​Occludin-positive cells were measured using ImageJ. The ratio of the area of ​​the Occludin-positive cell area to the total area of ​​the three-dimensional structure was calculated as the occupancy rate of the entire three-dimensional structure (Figure 4).

[0196] - Tight junctions: Stained using an antibody against Occludin, a component of the junctions. They exist in a mesh-like structure, covering the outermost layer of the three-dimensional structure. - Fibroblast clusters are spherical and less transparent than the epidermal keratinocyte layer. - Epidermal keratinocyte layer: A spherical structure made up of fibroblast clusters uniformly wrapped around it.

[0197] <Size of the obtained three-dimensional core-shell structure> The size of the obtained three-dimensional core-shell structure (spheroid) of Production Example 1 varied depending on the culture conditions (number of cells seeded, number of days of culture, type of medium, etc.), but was generally within the range of 300 to 700 μm, and the major axis of the three-dimensional core-shell structure of Production Example 1 was, for example, 442, 460, or 470 μm. The major axis of the three-dimensional structure was measured using ImageJ using a midline cross-section image taken with a confocal laser scanning microscope (LSM900, ZEISS).

[0198] <Results of Test Example 1>

[0199]

[0200] <Evaluation criteria for Occludin staining> Based on the occupancy rate of Occludin-positive cells in the entire three-dimensional structure (see Figure 4), the evaluation was made according to the following criteria: ○ (Excellent): 80% or more △ (Pass): 50% or more, less than 80% × (Fail): Less than 50%

[0201] <Mixed Culture Medium Ratio and Occludin Stainability> The 50:50 mixture of keratinocyte culture medium and fibroblast culture medium had the highest occupancy rate of Occludin-positive cells relative to the entire three-dimensional structure (91.5%) and was rated as excellent. The next highest occupancies were 25:75 (73.9%) and 75:25 (53.8%), and were rated as pass (see Figure 5).

[0202] Test Example 2: Study of the seeding ratio of fibroblasts and epidermal keratinocytes NHDF and iKC were seeded at the cell numbers listed in Table 2, and culture was performed using Half medium. The core-shell three-dimensional structure of Production Example 1 was obtained according to the procedure described above in <Three-dimensional structure with core-shell structure and its production method>. Three weeks after seeding with iKC, the three-dimensional structure was immunostained with an anti-occludin antibody and photographed using a confocal laser microscope (LSM900, ZEISS). Calculation of the occupancy rate of occludin and evaluation of pass / fail were performed using the same methods and criteria as in Example 1.

[0203]

[0204]

[0205] <Results of Test Example 2> The occupancy rate of Occludin-positive cells in the entire three-dimensional structure of this Production Example 1 was highest (94.2%) when the seeding ratio of fibroblasts to iKCs was 8:1, followed by 4:1 (81.3%), and was rated as excellent. The ratios of 2:1 (76.7%), 1:1 (75.2%), 1:2 (76.9%), and 1:4 (54.2%) were rated as pass (see Figure 6).

[0206] <Test Example 3: Examination of culturing period and confirmation of epidermal keratinocyte-specific markers> 1.0 x 10 4 NHDF, 5.0 x 10 ３ Using iKCs and half medium, a three-dimensional core-shell structure of Production Example 1 was obtained according to the above-mentioned <Three-dimensional structure with core-shell structure and its production method>. 6, 14, and 22 days after seeding the iKCs, the three-dimensional structures were collected, immunostained with various antibodies, and then photographed using a confocal laser microscope (LSM900, ZEISS). The pass / fail evaluation criteria used in <Test Example 1> were also used, along with evaluation criteria using epidermal keratinocyte proliferation markers and basal cell markers.

[0207]

[0208]

[0209] <Evaluation criteria when using proliferation markers> <Stainability> ○ (Pass) Proliferation of epidermal keratinocytes is confirmed in patches × (Fail) Staining of proliferation markers is not confirmed <Localization> ○ (Pass) Proliferation markers are confirmed in epidermal keratinocytes that are in direct contact with the fibroblast cluster × (Fail) Proliferation markers are confirmed in patches throughout the epidermal keratinocyte layer

[0210] <Evaluation criteria when using basal cell markers> <・Staining> ○ (Pass) Staining of basal cell markers is observed × (Fail) Staining of basal cell markers is not observed <・Localization> ○ (Pass) Basal cell markers are observed only in epidermal keratinocytes that are in direct contact with the fibroblast cluster × (Fail) Basal cell markers are observed throughout the entire epidermal keratinocyte layer

[0211] <Evaluation criteria for overall evaluation> ○ (Excellent): Staining of each marker is confirmed in the appropriate localization. △ (Pass): Staining of each marker is confirmed, but the localization is inappropriate in some areas. × (Fail): Staining of each marker is not confirmed.

[0212] <Results of Test Example 3> As described above, the undifferentiated / differentiated state of the epidermal keratinocyte layer could be reliably distinguished. The undifferentiated layer was a collagen XVII-positive cell layer, and the differentiated layer was a collagen XVII-negative cell layer. Furthermore, occludin, which is expressed in the granular layer (Figure 7), which represents the late differentiation stage of epidermal keratinocytes, was confirmed in the outermost (most) surface layer of the three-dimensional structure, suggesting that the epidermal keratinocyte layer undergoes a stepwise differentiation process from the inside to the outside. Furthermore, by culturing spheroids with a core-shell structure in which the core of the fibroblast cluster was covered with iKC in co-culture medium, the expression of tight junctions and the differentiation marker occludin was observed throughout the outermost layer of the spheroids. Furthermore, in the spheroids cultured for 14 days and 22 days, the localization of epidermal keratinocyte proliferation markers and undifferentiation markers (Ki67, collagen XVII) was confirmed in the single epidermal keratinocyte layer directly in contact with the fibroblast mass (dermis layer), just as in the living body (see Figure 8).

[0213] Test Example 4: Investigation of epidermal keratinocyte type The three-dimensional core-shell structure of Production Example 1 was obtained in the same manner as Test Example 3 above, except that the type of epidermal keratinocytes seeded on the fibroblast mass was changed. The epidermal keratinocyte types used were normal human epidermal keratinocytes (HPEK) and iKC. The pass / fail evaluation criteria of Test Example 3 were used.

[0214]

[0215] <Results of Test Example 4> The results are shown in Table 5 and Figure 9. In spheroids cultured for 8 days, expression of the tight junction marker Occludin and the proliferation marker Ki67 was observed regardless of the type of epidermal keratinocytes seeded in the core of the fibroblast cluster. On the other hand, in spheroids cultured for 22 days, only spheroids using iKCs were positive for the tight junction marker and proliferation marker at the appropriate positions (see Figure 9).

[0216] Test Example 5: Evaluation of Selected Substances The three-dimensional core-shell structure of Production Example 1 was obtained in the same manner as in Test Example 3 above. Five and six days after seeding epidermal keratinocytes, lysophosphatidic acid (LPA), known as a tight junction-enhancing component, was added at concentrations of 0, 1, 10, and 100 μg / mL. Seven days later, samples were collected, RNA was extracted, cDNA was synthesized, and qPCR was performed using each primer.

[0217]

[0218]

[0219]

[0220]

[0221] <Results of Test Example 5> An LPA concentration-dependent increase in expression was observed for the TJ marker genes claudin 1 (CLDN1) and occludin (OCLN). An increase in expression was observed for loricrin (LOR), a terminal differentiation marker, and filaggrin (FLG), a keratinization-related marker, at 100 μg / mL of LPA. These findings suggest that LPA has the effects of strengthening TJ barrier function, promoting terminal differentiation of epidermal keratinocytes, and promoting keratinization.

[0222] <<Production Example 2 [Microcarrier]: Three-dimensional structure with core-shell structure and its production method>> A three-dimensional structure with a core-shell structure, comprising PVA microspheres as the core and epidermal keratinocytes as the shell, was produced as follows (see Figure 1B), to provide the three-dimensional structure with a core-shell structure of Production Example 2. Specifically, one PVA microsphere was placed in a well, and epidermal keratinocytes to form the shell were seeded in the same well, followed by static culture. The structure could be produced in the same manner as Production Example 1, except that the fibroblast cell mass was replaced with PVA microspheres.

[0223] <PVA Microspheres> Particle size (D50): 200-250 μm Surface treatment: Collagen Type I Surface area per 1 g dry: 2600 cm 2 Swelling degree (when soaked in PBS): 10 Recommended amount for 1 liter of MSC culture: 1.54 g Sterilization: gamma irradiation

[0224] 1. Limit dilution of KURARAY collagen-coated PVA microspheres (1) Take a small amount of powdered PVA microspheres and dilute them with an appropriate amount of half medium (DMEM / F12 supplemented with 5% FBS and 1 mM CaCl2: CnT-PR-3D = 1:1)* (2) Seed the diluted solution from (1) above at 50 μL / well into a V-bottom 96-well plate. (3) Find a well containing only one PVA microsphere under a microscope, and use it to obtain the three-dimensional core-shell structure of Production Example 2.

[0225] <2. Seeding HPEK on PVA microspheres seeded by limiting dilution> (1) Remove the medium from the HPEK cultured in a T75 flask and wash twice with PBS(-). (2) Add 1 mL of the detachment agent TrypLE Select to the HPEK. (3) Incubate at 37°C for 10 min. (4) During incubation, place 3 mL of half medium in a 15 mL centrifuge tube. (5) Add 2 mL of PBS(-) to the incubated cells, detach the cells by pipetting, and collect them in (4) above. (6) Add 2 mL of PBS(-) to the T75 flask again, pipette several times, and then collect them in (4) above. (7) Centrifuge the cells (190 x g, 5 min.). (8) Remove the supernatant and resuspend the precipitated cells in 2 mL of half medium. (9) Measure the cell concentration of the cell suspension. (10) Dilute with half medium to 5 x 10^4 cells / mL. (11) Seed the above (10) at 100 μL / well into a V-bottom 96-well plate containing PVA microspheres. (12) Incubate at 37°C and 5% CO2. Change the medium every day with 100 μL / well half of the medium and culture for 2 weeks.

[0226] <3. Immunostaining of HPEK-PVA microsphere spheroids> (1) Place several HPEK-PVA microsphere spheroids in a 1.5 mL tube and remove as much medium as possible. (2) Fix using cold ethanol. (3) Remove ethanol and wash with PBS(-). (4) Transfer spheroids to glass slides and perform blocking. (5) Immunostain using a diluted solution of primary antibodies. Dilutions of antibodies specific to COL17, Ki67, and Occludin. (6) Wash with PBS(-). (7) Immunostain using a diluted solution of secondary antibodies. A reaction solution containing antibodies that recognize each primary antibody and a nuclear staining reagent. (8) Wash with PBS(-). (9) Mount the spheroids on the glass slide using a mounting medium.

[0227] Test Example 6 The time-dependent changes in the localization of the three-dimensional core-shell structure obtained in Production Example 2 were observed under a fluorescence microscope using antibodies to Hoechest, Collagen XVII (a basal cell marker), Ki67, and Occludin (a tight junction (TJ) marker). These markers were used in accordance with the [Various Markers] and the evaluation criteria for Occludin stainability, evaluation criteria when using proliferation markers, and evaluation criteria when using basal cell markers described in Production Example 1 above. Grayscale photographs are shown in Figure 10, showing the time-dependent changes in the localization of the seeded epidermal keratinocyte types (iKC, HPEK) and the epidermis-specific marker (OccludinKi-67) in the three-dimensional core-shell structure obtained in Production Example 2, observed under a fluorescence microscope.

[0228] The size of the obtained three-dimensional core-shell structures (spheroids) of Production Example 2 varied depending on the culture conditions (cell seeding number, culture days, type of medium, etc.), but was generally within the range of 300 to 500 μm, and the major axis of the three-dimensional core-shell structures of Production Example 2 was, for example, 312, 370, or 400 μm. The major axis of the three-dimensional structures was measured using ImageJ using midline cross-sectional images taken with a confocal laser scanning microscope (LSM900, ZEISS).

[0229] These results demonstrate that Production Example 2, like Production Example 1, can stably distinguish between undifferentiated and differentiated epidermal keratinocyte layers. The undifferentiated layer is a collagen XVII-positive cell layer, and the differentiated layer is a collagen XVII-negative cell layer. Furthermore, because occludin, which is expressed in the granular layer, which represents the later differentiation stage of epidermal keratinocytes, was confirmed in the outermost layer (outermost layer) of the three-dimensional structure, it can be inferred that the epidermal keratinocyte layer undergoes a stepwise differentiation process from the inside to the outside.

[0230] <Test Example 7> We examined whether factors constituting extracellular matrix components (Col 1 or Col 4) were present on the surface of the fibroblast cell clusters in the core portions of the three-dimensional structures obtained in Production Example 1. As a result, it was verified that Col 1 and Col 4 were present around the fibroblast cell clusters (see Figure 11). This confirmed that at least collagen and extracellular matrix components were present on the surface of the core portions of Production Examples 1 and 2.

[0231] <Comprehensive Study> As a result, a three-dimensional structure with a core-shell structure was obtained in one well, with a fibroblast cluster as the core and epidermal keratinocytes as the shell of that core. The three-dimensional structure obtained in Production Example 1 had a fibroblast cluster composed of multiple fibroblasts in the center, and multiple epidermal keratinocytes covering the fibroblast cluster in a layer-like manner, with multiple tight junctions between the epidermal keratinocytes present on the surface in contact with the outside. The epidermal keratinocytes in contact with the fibroblast cluster were undifferentiated epidermal keratinocytes and formed a first layer, which corresponds to the basal cell layer. In the outer diameter direction of this first layer, a second layer, a third layer, and so on, multiple differentiated epidermal keratinocytes were present in the second layer and beyond, forming a differentiated epidermal keratinocyte layer (single layer or multilayer).

[0232] As described above, it was confirmed that spheroids (masses) could be produced by seeding and culturing epidermal keratinocytes on cell cultureware containing fibroblasts prepared from seeded fibroblasts. These spheroids (masses) had a three-dimensional core-shell structure, with (a) a fibroblast mass in the center composed of at least a plurality of fibroblasts, and (b) a shell composed of at least a plurality of tight junctions present between cells in the surface cell layer and a plurality of epidermal keratinocytes, including an undifferentiated layer and a differentiated layer.

[0233] Furthermore, a core-shell three-dimensional structure was obtained in one well, with a microcarrier as the core and epidermal keratinocytes as the shell. The three-dimensional structure obtained in Production Example 2 has a single PVA microsphere at the center, covered with multiple layers of epidermal keratinocytes, and has multiple tight junctions between the epidermal keratinocytes present on the surface in contact with the outside. The epidermal keratinocytes in contact with the fibroblast mass are undifferentiated epidermal keratinocytes and form a first layer, which corresponds to the basal cell layer. From the outer diameter of this first layer, there are second, third, and so on layers, and from the second layer onwards, multiple differentiated epidermal keratinocytes are present, forming a differentiated epidermal keratinocyte layer (single layer or multilayer).

[0234] As described above, it was confirmed that spheroids (masses) could be produced by seeding and culturing epidermal keratinocytes on a cell cultureware containing a single PVA microcarrier. The spheroids (masses) had a three-dimensional core-shell structure, with (a) a single microcarrier at the center and (b) a shell composed of at least multiple tight junctions between cells in the surface cell layer and multiple epidermal keratinocytes, and including an undifferentiated layer and a differentiated layer.

[0235] The present inventors have considered that the core portion of a three-dimensional core-shell structure may be either a cell mass or a microcarrier, and that it is preferable that these have extracellular matrix components on their surface and a cell layer of epidermal keratinocytes surrounding the cell mass. Thus, they have been able to provide a three-dimensional core-shell structure comprising a cell layer having tight junctions present on the surface and at least composed of a plurality of epidermal keratinocytes, and a central portion that is a microsphere having extracellular matrix components on its surface.

[0236] 1 Three-dimensional structure with a core-shell structure; 1a Three-dimensional structure of the first embodiment, 1b Three-dimensional structure of the second embodiment; 10a Fibroblast mass, 10b Microcarrier; 11 Fibroblast; 20 Cell layer of epidermal keratinocyte; 21 Undifferentiated epidermal keratinocyte layer; 22 Differentiated epidermal keratinocyte layer; 23 Outermost layer; 201 Undifferentiated epidermal keratinocyte; 202 Differentiated epidermal keratinocyte; 30 Tight junction

Claims

1. A three-dimensional core-shell structure comprising a cell layer having tight junctions on its surface and composed at least of a plurality of epidermal keratinocytes, and a central portion which is a microsphere having extracellular matrix components on its surface.

2. The three-dimensional structure of claim 1, wherein the microspheres are microcarriers or cell clusters.

3. The three-dimensional structure described in claim 1 or 2, wherein the epidermal keratinocytes are epidermal keratinocytes derived from pluripotent stem cells.

4. A three-dimensional structure according to claim 1 or 2, used for evaluating skin homeostasis function.

5. A method for producing a three-dimensional core-shell structure by co-culturing microspheres with seeded epidermal keratinocytes.

6. The method for producing a three-dimensional structure according to claim 5, wherein a medium containing at least a medium for epidermal keratinocytes or a medium containing a medium for epidermal keratinocytes and a medium for fibroblasts is used when culturing the microspheres and the seeded epidermal keratinocytes.

7. A method for producing a three-dimensional structure according to claim 5 or 6, wherein the three-dimensional structure is cultured so that the size of the three-dimensional structure is 1.05 to 1.5, assuming that the size of the microspheres is 1.

8. A method for evaluating skin homeostasis maintenance function using the three-dimensional structure described in claim 1.

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