Three-dimensional structure of core-shell structure

Abstract

Provided are a novel three-dimensional structure comprising epidermal keratinocytes and a technique for producing the same. Provided is a three-dimensional structure having a core-shell structure, comprising: a cell layer comprising at least a plurality of epidermal keratinocytes and having a tight bond present on the surface; and a central part which is a microsphere having an extracellular matrix component on the surface thereof. The microspheres are preferably microcarriers or cell masses.

Description

Technical Field

[0001] This invention relates to core-shell three-dimensional structures, methods for manufacturing core-shell three-dimensional structures, and methods for evaluating the skin homeostasis maintenance function of core-shell three-dimensional structures. Background Technology

[0002] In recent years, maintaining skin homeostasis, including preserving normal skin function and restoring the function of deteriorated skin, has received increasing attention. Skin tissue consists of the epidermis, dermis, and subcutaneous tissue, each contributing to skin homeostasis through its distinct functions. The epidermis, in contact with the external environment, is composed of epidermal keratinocytes, forming a stratified structure of different differentiation stages (specifically, the basal layer, spinous layer, granular layer, and stratum corneum). Furthermore, the differentiation of undifferentiated epidermal keratinocytes into the stratum corneum and their eventual shedding from the skin surface as part of a metabolic cycle occurs periodically; this metabolic cycle is called cell turnover. Additionally, the epidermis, in contact with the external environment, also serves as a barrier, preventing water evaporation and the invasion of pathogens. On the other hand, the dermis, through collagen and elastin, supports the skin, maintaining its shape and elasticity.

[0003] In recent years, sheet-like three-dimensional cultured skin models have begun to be used as an alternative to animal model experiments. These models employ a three-dimensional structure similar to animal skin tissue, including the dermis and epidermis. By inserting these sheet-like three-dimensional cultured skin models into wells, the skin's barrier function, turnover rate, and other homeostatic maintenance functions are evaluated. To study this skin homeostasis, in addition to using skin moisture measuring devices, various biomarkers are also being used.

[0004] The following three-dimensional cultured skin model was proposed.

[0005] For example, Patent Document 1 proposes "a three-dimensional cultured skin model comprising the structures described in (1) to (3) below, having an artificially produced dermal (dermal equivalent) layer. (1) A dermal equivalent layer obtained by culturing a mixture of collagen and fibroblasts. (2) A layer of epidermal keratinocytes. (3) Melanocytes, which are colonized between (1) the dermal equivalent layer and (2) the epidermal keratinocyte layer."

[0006] In addition, Patent Document 2 discloses a sheet-like three-dimensional cultured human skin model, characterized in that it comprises, from bottom to top: A, a support layer containing fibroblasts; B, a support layer containing dendritic cells; and C, an epidermal keratinocyte layer, which is a sheet-like material obtained by seeding the epidermal keratinocytes in C onto the support layer containing dendritic cells in B.

[0007] In addition to sheet-like structures, the following three-dimensional structures have also been proposed.

[0008] For example, Non-Patent Literature 1 proposes to prepare three-dimensional dermal papilla (DP) cells by sequentially seeding dermal papilla (DP) cells, HaCaT keratinocytes, and human dermal fibroblasts (HDF) cells into three-dimensional (3D) micropores made of polyethylene glycol diacrylate hydrogel.

[0009] In addition, Non-Patent Document 2 proposes a three-dimensional cell culture for the following purpose: to reproduce skin changes caused by microgravity by using a three-dimensional cell culture with a gyroscope (a state close to a microgravity environment), thereby enabling the understanding of the effects of microgravity on human health before space exploration.

[0010] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2010-193822 Patent Document 2: Japanese Patent Application Publication No. 2006-333763 Patent Document 3: Japanese Patent Application Publication No. 2023-018632 Non-patent literature Non-patent literature 1: Justin JY Tan et al., Cell Prolif. 2019 Sep; 52(5):e12668. Non-patent literature 2: Dong Hyun Choi et al., npj Microgravity volume 7, Article number: 20 (2021). Non-patent literature 3: Sergio Cortez Ghio et al., Int. J. Mol. Sci. 2018, 19(8), 2174. Non-patent literature 4: Okita K. et al., Nature Methods, 2011, 8:409-12. Non-patent literature 5: Ido H, et al., J Biol Chem. 2007, 282, 11144-11154. Summary of the Invention

[0011] The problem that the invention aims to solve Therefore, skin models are beneficial in evaluating skin homeostasis maintenance functions such as skin barrier function. Furthermore, the inventors of this application believe that, compared to sheet-like skin models, spheroids, which are roughly spherical three-dimensional structures formed by cells agglomerating into blocks, can be obtained with fewer cells, fewer procedures, and a shorter culture period. Using the obtained roughly spherical three-dimensional structures, it is easy to conduct screening, evaluation, analysis, and measurement experiments under various conditions.

[0012] However, while Non-Patent Document 1 proposes three-dimensional dermal papillary cells, it does not disclose a three-dimensional structure with a core-shell structure, where epidermal keratinocytes cover the outer side of the nucleus of a fibroblast mass. Furthermore, Non-Patent Document 2 discloses a technique for three-dimensional cell culture under microgravity using a special device and a special three-dimensional structure based thereon, with the aim of studying the effects of microgravity on human health before space exploration. Therefore, the technique disclosed in Non-Patent Document 2 is not a conventional cell culture technique performed under Earth's gravity. Additionally, a generally spherical three-dimensional structure capable of effectively evaluating skin barrier function and other skin homeostasis maintenance functions is desired.

[0013] Therefore, the main objective of this invention is to provide a novel three-dimensional structure comprising epidermal keratinocytes and the technology for manufacturing it.

[0014] Methods for solving problems The inventors of this application believe that the presence of tight connections within a three-dimensional structure allows for more precise testing of skin homeostasis maintenance functions (e.g., skin barrier function). However, there is a lack of sufficient understanding regarding generally spherical three-dimensional structures with tight connections and containing epidermal keratinocytes. Furthermore, a generally spherical three-dimensional structure is desired that forms tight connections that perform the barrier function, a crucial function of the epidermis, and allows for the evaluation of the effects of pharmaceuticals. Therefore, the inventors of this application aim to provide a three-dimensional structure with tight connections and a technique for manufacturing it as a secondary objective.

[0015] Furthermore, the inventors of this application conducted in-depth research and discovered that by co-culturing microspheres and inoculated epidermal keratinocytes, it is possible to provide a novel three-dimensional structure comprising a core-shell structure with a cell layer containing multiple epidermal keratinocytes and multiple tight junctions. That is, the present invention is as follows.

[0016] This 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 multiple epidermal keratinocytes; and The central part consists of microspheres with extracellular matrix components on their surface.

[0017] The present invention provides a method for manufacturing a core-shell three-dimensional structure by co-culturing microspheres with inoculated epidermal keratinocytes.

[0018] The present invention can provide an evaluation method for the skin homeostasis maintenance function using the aforementioned three-dimensional structure.

[0019] The aforementioned microspheres can be microcarriers or cell blocks.

[0020] The aforementioned epidermal keratinocytes can be epidermal keratinocytes derived from pluripotent stem cells.

[0021] The aforementioned three-dimensional structure can be used to evaluate the skin's homeostasis maintenance function.

[0022] When culturing the aforementioned microspheres with inoculated epidermal keratinocytes, a culture medium containing at least a culture medium for epidermal keratinocytes, or a culture medium containing both a culture medium for epidermal keratinocytes and a culture medium for fibroblasts, can be used.

[0023] The microspheres can be cultured such that the size of the three-dimensional structure is between 1.05 and 1.5 when the size of the microspheres is set to 1.

[0024] The effects of the invention This invention provides novel three-dimensional structures containing epidermal keratinocytes and the technology for manufacturing them. Attached Figure Description

[0025] [ Figure 1A The diagram shows a simplified example of a three-dimensional structure with a core-shell structure in this embodiment. The three-dimensional structure is composed of at least a cell layer as a shell portion containing a plurality of tightly connected epidermal keratinized cells and a central portion as a core portion containing a plurality of cells (e.g., fibroblasts), but the invention is not limited thereto.

[0026] [ Figure 1B The diagram shows a simplified example of a three-dimensional structure of a core-shell structure in this embodiment, which is composed of at least a cell layer as a shell portion containing a plurality of tightly connected and a plurality of epidermal keratinized cells, and a central portion as a core portion containing microcarriers, but the invention is not limited thereto.

[0027] [ Figure 2A This invention illustrates an example of manufacturing a three-dimensional structure with a core-shell structure containing cell blocks in the nucleus, according to the first embodiment of this application, but the invention is not limited thereto.

[0028] [ Figure 2B This invention illustrates an example of manufacturing a three-dimensional structure with a core-shell structure containing microcarriers in the core, according to the second embodiment of this application, but the invention is not limited thereto.

[0029] [ Figure 3 The image shows an optical microscope photograph of a three-dimensional structure of a core-shell structure manufactured in the first embodiment of this application, which uses cell blocks as the core, but the invention is not limited thereto.

[0030] [ Figure 4 This image shows a grayscale photograph of fluorescence observation of a three-dimensional structure with a core-shell structure using cell blocks as the core, manufactured according to the first embodiment of this application, but the invention is not limited thereto. Additionally, the grayscale photograph also shows the method for determining the occupancy rate of occupant-positive cells. Sample 1: Overall (1): 3.907 / Occludin-positive cell region (2): 1.035 / Occupancy rate: 1.035 / 3.907 × 100 = 26.5%. Sample 2: Overall (1): 6.384 / Occludin-positive cell region (1-2): 5.843 / Occludin occupancy rate: 5.843 / 6.384 × 100 = 91.5%.

[0031] [ Figure 5 [Image] is a grayscale photograph of a cell block obtained by fluorescence microscopy (closed protein staining image) when the mixing ratio (for epidermal keratinocyte culture medium and for fibroblast culture medium, i.e., 100:0, 75:25, 50:50, 25:75, 0:100) of the manufacturing method of the first embodiment of this application is 100:0, 75:25, 50:50, 25:75, 0:100.

[0032] [ Figure 6 [Image] is a grayscale photograph of a cell block obtained by fluorescence microscopy (closed protein staining image) in the manufacturing method of the first embodiment of this application when the NHDF:iKC ratio is 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8.

[0033] [ Figure 7 [A conceptual diagram illustrating the structure of the skin and the local presence of epidermal-specific markers.]

[0034] [ Figure 8 [Image] is a grayscale photograph taken during the culture period of the three-dimensional structure of the core-shell structure of the first embodiment of this application obtained by using a fluorescence microscope to confirm the staining and local presence of the structure using Ki-67 antibody, closure protein antibody, and type XVII collagen antibody.

[0035] [ Figure 9 [Image] is a grayscale photograph taken using a fluorescence microscope to confirm the changes over time in the local presence of the three-dimensional structure of the core-shell structure of the first embodiment of this application, the types of inoculated epidermal keratinocytes (iKC, HPEK), and the local presence of epidermal-specific markers (closure protein, Ki-67).

[0036] [ Figure 10 [Image 1] is a grayscale photograph taken using a fluorescence microscope to confirm the changes over time in the local presence of seeded epidermal keratinocytes (HPEK) and epidermal-specific markers (closure protein, Ki-67) in the three-dimensional structure of the core-shell structure of the second embodiment of this application. It also shows Hoechst staining of the three-dimensional structure of the core-shell structure of the second embodiment of this application, excluding closure protein staining and Ki-67 staining. （R） Grayscale images of nucleic acid staining and type XVII collagen staining, respectively.

[0037] [ Figure 11 [This is a grayscale photograph showing a fibroblast block in the core portion of the three-dimensional structure of the core-shell structure obtained according to the first embodiment of this application, and the surface of the cell block having an extracellular matrix (Col1 or Col4). The upper image shows the three-dimensional structure after 3 weeks of culture, stained with an antibody of one of the extracellular matrix, namely Col IV (4). The lower image shows the three-dimensional structure after 3 weeks of culture, stained with an antibody of one of the extracellular matrix, namely Col I (1).] Detailed Implementation

[0038] The following describes suitable implementation methods for carrying out this technology. It should be noted that the embodiments described below illustrate one example of a representative implementation of this technology and are not intended to narrowly interpret the scope of this technology. It should also be noted that, unless otherwise specified, percentages in this specification are based on capacity (capacity / capacity%). Furthermore, the upper (below) and lower (above) values ​​of each numerical range (~) can be arbitrarily combined as desired.

[0039] 1. The three-dimensional structure involved in this embodiment This embodiment provides a core-shell three-dimensional structure comprising epidermal keratinocytes and microspheres having extracellular matrix components on their surface. Preferably, this embodiment provides a core-shell three-dimensional structure consisting of at least a cell layer and a central portion serving as microspheres. The cell layer is composed of at least a plurality of epidermal keratinocytes, and the microspheres preferably have extracellular matrix components on their surface. More preferably, in this embodiment, the surface portion of the aforementioned cell layer as the three-dimensional structure covers the area surrounding the aforementioned central portion. Preferably, this embodiment has tight connections on the surface of the cell layer, and preferably tight connections between adjacent differentiated epidermal keratinocytes present on the surface of the cell layer in contact with the outside.

[0040] The aforementioned "microspheres" having extracellular matrix components on their surface are preferably microcarriers (preferably non-cellular microcarriers) or cell blocks (preferably fibroblast cell blocks). The three-dimensional structures of each nucleoshell structure are also referred to as the first embodiment of this application (see [link to previous document]). Figure 1A ) and the second embodiment of this application (see Figure 1B The surface of the central portion preferably has extracellular matrix components (e.g., collagen).

[0041] For example, in the case of the first embodiment of this application, the first embodiment of this application provides a three-dimensional structure with a core-shell structure comprising a plurality of epidermal keratinocytes and a plurality of fibroblasts. The first embodiment of this application preferably provides a three-dimensional structure with a core-shell structure comprising at least a cell layer and a central portion, wherein the cell layer comprises at least a plurality of epidermal keratinocytes, the central portion comprises at least a plurality of fibroblasts, and the surface of the central portion has extracellular matrix components (e.g., collagen).

[0042] For example, in the case of the second embodiment of this application, the second embodiment of this application provides a three-dimensional structure comprising a core-shell structure containing a plurality of epidermal keratinocytes and a microcarrier. The second embodiment of this application preferably provides a three-dimensional structure consisting of a core-shell structure at least composed of a cell layer and a central portion, wherein the cell layer is composed of at least a plurality of epidermal keratinocytes, and the central portion is composed of a microcarrier, the surface of which has extracellular matrix components (e.g., collagen). Advantages of the second embodiment of this application include, for example: only epidermal keratinocytes are needed, thus simplifying the operation; no special cell separation process is required for evaluation, allowing evaluation solely of the epidermal keratinocytes that constitute the epidermis, which is important for the skin barrier; and when using epidermal keratinocytes derived from iPS cells, it becomes a biomimetic model that simply reflects individual characteristics, enabling evaluation of individual skin characteristics, responsiveness to drugs, etc. Furthermore, as an advantage of the second embodiment of this application, since it has epidermal keratinized cells in the surface layer, similar to the first embodiment, and uses microcarriers in the nucleus portion, it is believed that the influence of other cell types can be reduced when evaluating the surface layer. Moreover, compared to the first embodiment, the second embodiment, due to the use of microcarriers, is considered advantageous in terms of manufacturing methods such as reduced operating time and costs compared to cell block production, and is believed to be easier to achieve in terms of production efficiency and large-scale production.

[0043] In this embodiment, the central portion is also referred to as the core portion, and the surface portion as the shell portion. Furthermore, for a three-dimensional structure, a state in which the inner layer in the central direction of the shell portion (surface portion) contacts the outer layer in the outer diameter direction of the core portion (central portion) is more preferably present, and the presence of extracellular matrix components (e.g., collagen) in this contact state is even more preferable. For example, in the case of the first embodiment of this application, the core portion is preferably a fibroblast cell mass, and in the case of the second embodiment of this application, the core portion is preferably a microcarrier.

[0044] In addition, in this embodiment, it is preferable that the aforementioned cell layer is a cell layer composed of at least a plurality of epidermal keratinocytes with tightly connected surfaces, and / or that the aforementioned central portion is a microsphere. More preferably, the surface of the aforementioned central portion has an extracellular matrix component. The aforementioned central portion is preferably a cell block or a microcarrier; in the case of a cell block, it can be the first embodiment, and in the case of a microcarrier, it can be the second embodiment. The aforementioned central portion is preferably a fibroblast cell block composed of at least a plurality of fibroblasts, or a microcarrier.

[0045] Furthermore, as a suitable embodiment, a three-dimensional structure with a core-shell structure can be provided, comprising: a cell layer consisting of at least a plurality of tight junctions between cells present in the surface cell layer and a plurality of epidermal keratinocytes; and a central portion consisting of microspheres having extracellular matrix components. Regarding the preferred embodiment of the microspheres, the first or second embodiment described above may be appropriately employed.

[0046] In addition, as a suitable embodiment, the aforementioned cell layer is at least composed of a differentiated layer containing differentiated epidermal keratinocytes and an undifferentiated layer containing undifferentiated epidermal keratinocytes located in the central direction, and / or, the aforementioned differentiated epidermal keratinocytes are present between adjacent cells of the cell layer that are closely connected to the surface of the three-dimensional structure.

[0047] As another suitable embodiment of this invention, a core-shell structure can also be provided, wherein the surface portion of the three-dimensional structure is composed of at least a plurality of epidermal keratinocytes and tight connections between adjacent epidermal keratinocytes located in its outermost layer, and the central portion of the three-dimensional structure is composed of at least microspheres (e.g., a plurality of fibroblasts, a microcarrier, etc.).

[0048] In addition, as another suitable embodiment, a three-dimensional structure with a core-shell structure can be provided, comprising: (a) a cell layer consisting of at least tight junctions between cells present on the surface of the cell layer and a plurality of epidermal keratinocytes; and (b) a microsphere having extracellular matrix components on its surface as the central part.

[0049] The aforementioned microspheres are preferably: a cell block having an extracellular matrix coating layer composed of at least one type of cell and multiple cells, or a microcarrier having an extracellular matrix coating layer. Each of these can be configured as a first embodiment having a cell block and a second embodiment having a microcarrier.

[0050] The type of cells used in the first embodiment can be the same type or a combination of different types, preferably the same type, and more preferably fibroblasts.

[0051] In the second embodiment, the microcarrier used is preferably a non-cell line.

[0052] In addition, as another suitable embodiment, a three-dimensional structure with a core-shell structure can be provided, comprising: (a) a cell layer located in the surface portion, which includes at least a plurality of epidermal keratinocytes and tight connections between adjacent epidermal keratinocytes present on the surface of the three-dimensional structure; and (b) microspheres (e.g., non-cellular or cellular) located in the center portion.

[0053] Furthermore, it is more preferable to use microspheres (preferably microcarriers or fibroblast blocks) as the core portion, and the core portion is covered by a layer containing epidermal keratinocytes. Suitable of the above (b) is preferably (b1) a fibroblast block located in the center containing a plurality of fibroblasts; or (b2) a non-cellular microcarrier located in the center, which is a non-cellular sphere, and they can each be configured as a first embodiment having cell blocks and a second embodiment having microcarriers.

[0054] The three-dimensional structure involved in this embodiment is preferably used for evaluating skin homeostasis maintenance functions such as skin barrier function.

[0055] Furthermore, the three-dimensional structure involved in this embodiment can be manufactured using the method described later in "2. Manufacturing method of the three-dimensional structure involved in this embodiment".

[0056] It should be noted that in the description of this embodiment, descriptions of components and methods of epidermal keratinocytes, fibroblasts, microspheres, microcarriers, nucleoshell structures, pluripotent stem cells, cell culture equipment, etc., that are repeated with the descriptions in "2.", "3.", "4.", etc., described later, are appropriately omitted. The descriptions in "2.", "3.", "4.", etc., described later also apply to this embodiment and can be used appropriately.

[0057] <Core-shell structure> As the core-shell structure in this specification, a microsphere is preferably the central portion, and a cell layer formed by multiple cells further covering the periphery of the central portion is preferably the surface portion; more preferably, the cell layer is a multilayer structure; and even more preferably, the microsphere has an extracellular matrix component. The microsphere can be a cell block formed by multiple cells or a microcarrier.

[0058] In the first embodiment of this application, it is more preferable that the cells constituting the cell block and the cells constituting the cell layer are different cells. In the second embodiment of this application, it is more preferable that the cells are non-cellular microcarriers and the cells constituting the cell layer.

[0059] It should be noted that the microspheres are also referred to as the core, and the surface portion or the cell layer of the surface portion is also referred to as the shell. Furthermore, the location of the microspheres in the three-dimensional structure is preferably such that a core-shell structure can be formed; it does not have to be the center of the three-dimensional structure, but can be the central portion (center or its periphery). Additionally, the cell layer of the three-dimensional structure preferably exists from the center outwards. In this embodiment, the outward direction is preferably the outer diameter direction.

[0060] For example, the three-dimensional structure in this embodiment is not limited to Figure 1A and Figure 1B ,Will Figure 1A and Figure 1B Such a structure, having a core and a shell, is also called a core-shell structure. For example, as... Figure 1A and Figure 1BSuitable examples of the three-dimensional structures of this embodiment shown can be exemplified by the following core-shell structure comprising: (a) a core such as a fibroblast block or microcarrier; and (b) a shell such as an epidermal keratinocyte layer comprising differentiated epidermal keratinocyte layers (including an outermost and an innermost layer) and undifferentiated epidermal keratinocyte layers. For example, regarding the core-shell structure 1 of the first embodiment of this application, an example can be exemplified by: a core portion comprising at least a fibroblast block 10a containing fibroblasts 11, and a shell portion comprising a cell layer 20 of epidermal keratinocytes, the cell layer 20 comprising tight junctions 30, an undifferentiated epidermal keratinocyte layer 21, and differentiated epidermal keratinocyte layers 22 and an outermost layer 23, the undifferentiated epidermal keratinocyte layer 21 comprising undifferentiated epidermal keratinocytes 201, and the differentiated epidermal keratinocyte layers 22 and the outermost layer 23 comprising differentiated epidermal keratinocytes 202. For example, regarding the three-dimensional structure 1 with a core-shell structure according to the second embodiment of this application, the core portion of the three-dimensional structure 1 with a core-shell structure according to the first embodiment of this application is a microcarrier 10b instead of a fibroblast block 10a, and includes the microcarrier 10b and the shell portion of the cell layer 20 of epidermal keratinocytes which is the same as the shell portion of the cell layer 20 of epidermal keratinocytes according to the first embodiment of this application.

[0061] <Central part of a three-dimensional structure> In this embodiment, the central portion of the three-dimensional structure is not particularly limited, and is preferably a microsphere such as a microcarrier or cell block, more preferably a microsphere having extracellular matrix components on its surface. As for this microsphere, there is no particular limitation, but cell blocks (preferably fibroblast blocks) or microcarriers (preferably non-cellular systems) are preferred. Among these, microcarriers (preferably non-cellular systems) are preferred, thereby avoiding the use of cells other than epidermal keratinocytes when evaluating skin homeostasis maintenance functions related to the cell layer, thus reducing the influence of other cells.

[0062] <Extracellular matrix components> As components of the extracellular matrix, there are no particular limitations. Examples include collagen (e.g., types I to XIX), hyaluronic acid or its salts (sodium, etc.), fibronectin, hyalin, laminin (e.g., types 1 to 15, such as laminin 511E8, etc.), tendin, platelet-reactive proteins, various elastins, various proteoglycans, etc.; poly(D,L-lactide-co-glycolide), N-isopropylacrylamide, matrix gum, poly(D-lysine), poly(L-lysine), etc. - Lysine, chitin, deacetylated chitosan, agarose gel, alginate gel, various hydrogels, and other extracellular matrix substitutes, selected from one or more of these. As components of the extracellular matrix, extracellular matrix and / or extracellular matrix substitutes can be used, with extracellular matrix being preferred. It should be noted that extracellular matrix components can be non-cellular substances that fill the spaces between cells, preferably components that enable cell adhesion or have cell adhesion properties.

[0063] As an extracellular matrix component located on the surface of the microspheres, collagen is preferred, more preferably type I collagen and / or type IV collagen, but not limited thereto. Furthermore, if the presence of the extracellular matrix component of the microspheres is confirmed, collagen (preferably type I or type IV collagen) can be identified. When identifying an example of the extracellular matrix component, appropriate and verifiable markers can be used, such as commercially available products (kits, etc.).

[0064] Furthermore, in this embodiment, the microspheres are preferably three-dimensional structures whose surface can be confirmed by extracellular matrix component confirmation markers to indicate the presence of extracellular matrix components. Among these extracellular matrix component confirmation markers, collagen is preferred, and more preferably, a three-dimensional structure whose presence can be confirmed by at least Col 1 and / or Col IV, or both. It should be noted that this confirmation is not particularly limited to any method capable of confirming the expression of the target gene or protein; examples include staining and expression level measurements. The methods used in the evaluation methods described later can be appropriately employed.

[0065] In the first embodiment of this application, the central portion of the three-dimensional structure preferably comprises a plurality of fibroblasts, or at least a fibroblast mass composed of these fibroblasts, and preferably has an extracellular matrix component 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.

[0066] In the second embodiment of this application, the central portion of the three-dimensional structure is preferably composed of a spherical microcarrier, which preferably has an extracellular matrix component on its surface. This microcarrier is preferably a non-cellular lineage. Examples of non-cellular lines include living or dead cells, non-proliferating or non-dividing cells, etc., and are preferably not derived from animal cells.

[0067] <Microcarrier> In this embodiment, the central part can be a single spherical microcarrier or a block composed of multiple microcarriers. From the viewpoint of ease of operation, reduction of process steps or cost, it is preferable to form the core of the microsphere with a single spherical microcarrier.

[0068] A block-shaped microcarrier can be formed by appropriately combining multiple microcarriers of the same or different shapes, the same or different particle sizes.

[0069] The microcarriers used in this embodiment can be commercially available products, such as collagen-coated PVA microcarriers (Kuraray PVA microcarriers (type 1 collagen), Corning microcarriers, etc.). R Collagen-coated microcarriers, etc.), microcarriers (Corning R (Untreated microcarriers, etc.), and other microcarriers such as those described later can be used, and one or more of them can be used. In addition, microcarriers can be manufactured with reference to known manufacturing methods.

[0070] The materials used in microcarriers can be organic, inorganic, or composites thereof, or they can be materials obtained by hydrophobizing them or by incorporating hydrophobic groups. Microcarriers can be formed from these materials. From a cell compatibility point of view, microcarriers preferably contain organic materials. Cell culture can also be performed using microcarriers formed from polymers having hydrophobic portions as organic materials. Examples of hydrophobic groups include, for example, chain or cyclic hydrocarbon groups, aromatic hydrocarbon groups, etc.

[0071] As organic materials, there are no particular limitations. Examples include synthetic polymers (resins) such as polyvinyl alcohol, polylactic acid, polyglycolic acid, polytetrafluoroethylene, polystyrene, polyester, polyurethane, polyethylene, polypropylene, (meth)acrylic acid polymers, (meth)acrylamide polymers, organosilicon polymers, and epoxy resins; natural polymers (proteins or peptides) such as collagen and gelatin; and polysaccharides such as pectin, pectinates, polygalacturonic acid, alginic acid, cellulose, cross-linked agarose, dextran, deacetylated chitosan, and their salts (e.g., alkali metals such as Na, K, and Li, and alkaline earth metals such as Ca and Mg). As inorganic materials, examples include glass, ceramics, metals, alloys, and metal oxides. Among resins, flexible resins, thermoplastic resins, and thermoplastic elastomers are preferred. Among organic materials, resins such as polyvinyl alcohol, polystyrene, and polyesters are preferred, with polyvinyl alcohol being the most preferred. "(Meth)acrylic polymers" refers to acrylic polymers or methacrylic polymers or mixtures thereof, which may be polymers or copolymers formed from (meth)acrylic monomers, etc.

[0072] From a cell compatibility perspective, microcarriers can be made from hydrogels. Examples of such hydrogels include polyvinyl alcohol, sodium salts of (meth)acrylic acid polymers, copolymers with a large number of hydrophilic groups, and gelatin. Hydrogels formed by introducing hydrophobic groups into hydrophilic polymers can also be used.

[0073] From the viewpoint of promoting cell adhesion, an extracellular matrix component (preferably a cell adhesion polymer) can be disposed on the surface of the microcarrier. Examples of such extracellular matrix components (preferably cell adhesion polymers) include collagen, gelatin, alginate, Matrigel (trademark) (BD Biosciences), hyaluronic acid, laminin, fibronectin, hyaluronic acid, elastin, heparan sulfate, dextran, dextran sulfate, chondroitin sulfate, and their salts; one or more selected from these may be used. Collagen is preferred.

[0074] From the perspective of promoting cell attachment, cationic functional groups can be introduced onto the surface of microcarriers. Examples of cationic functional groups include groups containing substituted or unsubstituted cationic amino groups such as dimethylamino, diethylamino, and amino.

[0075] Examples of microcarrier shapes include spherical, flat, cylindrical, plate-like, and prismatic shapes. Spherical microcarriers are preferred. Microcarriers can be porous microcarriers with internal pores, microcarriers without internal pores, or hollow microcarriers.

[0076] For reasons such as improving cell adhesion, microcarriers can be coated with a coating material, preferably one related to the extracellular matrix components. There are no particular limitations on the coating material; examples include poly-D,L-lactic-co-glycolic acid, sodium hyaluronate, N-isopropylacrylamide, type I to XIX collagen, fibronectin, hyalin, laminin-1 to XIX, tendinin, platelet-reactive protein, von Willebrand factor, osteopontin, fibrinogen, various elastins, various proteoglycans, various cadherins, desmocollin, desmoglein, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrix gel, poly-D-lysine, poly-L-lysine, chitosan, deacetylated chitosan, agarose gel, alginate gel, and various hydrogels. One or more of these can be used.

[0077] From the viewpoint of promoting cell proliferation, the average particle size (D50) of the microcarriers is not particularly limited, for example, it is 50 to 1,000 μm. As a suitable lower limit, it is preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more. As a suitable upper limit, it is preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 or 250 μm or less. As a more suitable numerical range, it is preferably 100 to 500 μm, more preferably 120 to 250 μm, and even more preferably 150 to 250 μm. In the case of the average particle size (D50) of the microcarriers, it is set as the value measured as the median particle size (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 measuring device.

[0078] The microcarriers used in cell culture are preferably larger than the cells supplied for cell culture.

[0079] <Cellular layer of a three-dimensional structure> In this embodiment, the epidermal keratinocyte layer located in the cell layer (preferably the surface layer) of the three-dimensional structure preferably contains a plurality of epidermal keratinocytes, and is at least composed of them. This 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. By using epidermal keratinocytes derived from pluripotent stem cells in this three-dimensional structure, it has the advantage of enabling evaluation or testing of skin homeostasis maintenance function over a longer period.

[0080] <Cellular layer: tight junctions> In this embodiment, it is more preferable that tight junctions exist in the intercellular spaces of the cell layer (preferably the epidermal keratinized cell layer) on the surface in contact with the outside. These tight junctions exist in a way that fills the spaces between cells, thus acting as a barrier to restrict 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 keratinized cells on the surface of the three-dimensional structure, and more preferably between differentiated epidermal keratinized cells in the outermost layer that are in contact with the outside. As shown in the [Examples] described later, in this embodiment, a large number of tight junctions were found in the outermost three-dimensional structure, and the amount and local presence of these tight junctions can be evaluated using TJ markers. Regarding suitable tight junctions in this embodiment, there are cases where they appear as dots when observing the approximate central cross-section of the three-dimensional structure, and as a mesh-like structure when observing the surface of the three-dimensional structure (see Figure 1, etc.).

[0081] It should be noted that tight junctions are generally considered to be intercellular bonds that allow cells to adhere to each other. They connect adjacent epithelial cells and function as a barrier to prevent various substances (compounds, ions, water, etc.) from passing through the intercellular spaces. In the context of skin tight junctions, the cells of the second granular layer (SG2 cells) located inside the stratum corneum are considered to form the TJ barrier, which presents a honeycomb-like mesh pattern on the surface and is responsible for the skin's barrier function.

[0082] <Cellular layer: Epidermal keratinocyte layer> In this embodiment, the epidermal keratinocyte layer is preferably a multilayer consisting of at least an undifferentiated cell layer containing undifferentiated epidermal keratinocytes and a differentiated cell layer containing differentiated epidermal keratinocytes, extending outward from the center. Furthermore, this epidermal keratinocyte layer is preferably a layer consisting of at least a differentiated cell layer in the outward direction consisting of at least differentiated epidermal keratinocytes and an undifferentiated cell layer in the central direction consisting of at least undifferentiated epidermal keratinocytes. More preferably, the epidermal keratinocyte layer comprises: a differentiated cell layer including an outermost and an innermost layer; and an undifferentiated cell layer.

[0083] <Epidermal keratinocyte layer: undifferentiated cell layer> As a suitable way for the three-dimensional structure of this embodiment, the epidermal keratinocytes in contact with the microspheres (e.g., fibroblast blocks, microcarriers, etc.) remain in an undifferentiated state, and the layer containing the undifferentiated epidermal keratinocytes is also referred to as the basal cell layer of epidermal keratinocytes. The epidermal keratinocytes in contact with the microspheres preferably form an undifferentiated epidermal keratinocyte layer by covering the microspheres that form the nuclei. This undifferentiated epidermal keratinocyte layer is preferably a layer contained within the epidermal keratinocyte layer, more preferably a layer existing in the central direction within the epidermal keratinocyte layer, and / or a layer existing in the outward direction beyond the center of the three-dimensional structure. Preferably, new cells are formed by the division of the undifferentiated epidermal keratinocytes or their layers, and the newly divided epidermal keratinocytes are successively pushed in the outward direction (preferably the outer diameter direction) to form differentiated epidermal keratinocytes. By establishing a layer of epidermal keratinocytes that sequentially pushes epidermal keratinocytes outward from the surface, an experiment can be designed to closely approximate the normal state of skin turnover. This allows for more precise evaluation of skin homeostasis maintenance functions, such as skin turnover. The quantity and local presence of these undifferentiated cells can be evaluated using basal cell markers. Furthermore, the quantity and local presence of these undifferentiated cells that subsequently differentiate can be evaluated using proliferation markers.

[0084] <Epidermal keratinocyte layer: differentiated cell layer> As a suitable form of the three-dimensional structure in this embodiment, it is preferable to form a differentiated epidermal keratinocyte layer that covers an undifferentiated epidermal keratinocyte layer. This differentiated epidermal keratinocyte layer is preferably a layer contained within the epidermal keratinocyte layer, more preferably a layer extending outward from the undifferentiated cell layer. This differentiated epidermal keratinocyte layer preferably forms multiple layers in the outward direction, more preferably an outermost layer of differentiated epidermal keratinocytes that contacts the outside. Furthermore, the differentiated cell layer is more preferably two or more layers including the outermost layer of differentiated epidermal keratinocytes and the innermost layer of differentiated epidermal keratinocytes that contacts the undifferentiated epidermal keratinocyte layer. More preferably, the outermost layer contacts the outside, and the innermost layer contacts the undifferentiated epidermal keratinocyte layer.

[0085] This embodiment provides a three-dimensional structure that resembles actual skin, comprising a cell layer in which epidermal keratinocytes are in appropriate undifferentiated and differentiated states, and with tight junctions located in appropriate positions within this cell layer. Therefore, the three-dimensional structure of this embodiment is suitable for various experiments, such as evaluating skin homeostasis maintenance function. It should be noted that, compared to conventional sheet-like or other three-dimensional cultures, the three-dimensional structure of this embodiment has the advantages of requiring fewer cells for various experiments and having better compatibility with screening line experiments.

[0086] <Source of fibroblasts and epidermal keratinocytes used in three-dimensional structures> The source of cells used in preparing the three-dimensional structure of this embodiment is not particularly limited. For example, one or more sources selected from normal cells (e.g., normal epidermal keratinocytes, normal fibroblasts, etc.) and differentiation-induced cells derived from pluripotent stem cells (preferably iPS cells) can be used. As a suitable method, the epidermal keratinocyte layer of the three-dimensional structure is preferably prepared using epidermal keratinocytes, more preferably formed from at least epidermal keratinocytes derived from pluripotent stem cells and / or normal cells, and even more preferably formed from epidermal keratinocytes seeded from pluripotent stem cells. Furthermore, as a suitable method, if the central portion of the three-dimensional structure is a fibroblast block, this block is preferably formed from at least fibroblasts derived from normal cells.

[0087] Furthermore, the source of these cells is not particularly limited, but they are preferably derived from mammals. The mammals are not particularly limited, for example, humans, pigs, rodents (mice, rats, etc.), pets (e.g., rabbits, dogs, cats, etc.), etc. One or more of these can be used, with humans being the preferred source.

[0088] <Shape, size, etc. of three-dimensional structures> The shape of the three-dimensional structure or the shape of the microsphere (e.g., cell block, microcarrier, etc.) in this embodiment is not particularly limited, but it is preferred to be blocky or approximately spherical (e.g., ellipsoidal, perfect sphere, etc.).

[0089] It should be noted that the shape, size, etc., of this embodiment can be determined based on microscopic photographs, etc. Specifically, the size (major axis or diameter) of the three-dimensional structure and the spherical body (cell block) can be determined using images taken by observation through an optical microscope, in a measurement mode loaded into image analysis software. The average major axis measured from the photograph is approximately 500 μm. As a more suitable method, for the three-dimensional structure, etc., the size can be determined using images taken by a confocal laser microscope, and in the case of a roughly spherical three-dimensional structure, a mid-section image is preferably used to determine the size. It should be noted that the thickness of the cell layer can be calculated as: cell layer thickness = size of the three-dimensional structure - size of the microsphere (e.g., fibroblast blocks, cell blocks, microcarriers, etc.).

[0090] The size (major axis) of the three-dimensional structure in this embodiment is not particularly limited. As a suitable lower limit, it is preferably 300 μm or more, more preferably 350 μm or more, and even more preferably 400 μm or more. As a suitable upper limit, it is preferably 700 μm or less, more preferably 600 μm or less or 500 μm or less. As a more suitable numerical range, it is preferably 300 to 700 μm, more preferably 400 to 600 μm. The size (major axis) of the three-dimensional structure in this embodiment can be determined using image analysis observed under an optical microscope as described above.

[0091] It should be noted that the size (major axis) of the three-dimensional structure in the second embodiment of this application is not particularly limited. Appropriate upper and lower limits of the size (major axis) of the three-dimensional structure in this embodiment can be appropriately combined or adopted. As the appropriate lower limit, it is preferably 300 μm or more, more preferably 350 μm or more, and even more preferably 400 μm or more. As the appropriate upper limit, it 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 the second embodiment of this application is approximately 300 to 500 μm. This size can be determined using image analysis observed using an optical microscope as described above.

[0092] The size (major axis or diameter) of the microspheres is not particularly limited, but can be 50 to 1,000 μm, for example. 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 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 determined using image analysis observed under an optical microscope as described above.

[0093] The size (major axis or diameter) of the microcarrier is not particularly limited, for example, 50 to 1,000 μm. As a suitable lower limit, it is preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more. As a suitable upper limit, it is preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 or 250 μm or less. As a more suitable numerical range, it is preferably 100 to 500 μm, more preferably 120 to 250 μm, and even more preferably 150 to 250 μm. This size can be determined using image analysis observed by optical microscopy as described above.

[0094] The size (long axis or diameter) of the cell blocks (preferably fibroblast blocks) in the three-dimensional structure of the first embodiment of this application is not particularly limited. As a suitable lower limit, it is preferably 240 μm or more, more preferably 320 μm or more. As a suitable upper limit, it is preferably 560 μm or less, more preferably 480 μm or less. As a more suitable numerical range, it is preferably 240 to 560 μm, more preferably 320 to 480 μm. This size can be determined using image analysis observed by the optical microscope described above.

[0095] The size (major axis or diameter) of the microcarriers in the three-dimensional structure in the second embodiment of this application is not particularly limited, but is, for example, 50 to 1,000 μm. As a suitable lower limit, it is preferably 100 μm or more, more preferably 120 μm or more, and even more preferably 150 μm or more. As a suitable upper limit, it is preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 or 250 μm or less. As a more suitable numerical range, it is preferably 100 to 500 μm, and more preferably 120 to 250 μm. This size can be determined using image analysis observed by the optical microscope described above.

[0096] The size (thickness) of the cell layer containing epidermal keratinocytes in the three-dimensional structure of this embodiment is not particularly limited. As a suitable lower limit, it is preferably 60 μm or more, more preferably 80 μm or more. As a suitable upper limit, it is preferably 140 μm or less, more preferably 120 μm or less. As a more suitable numerical range, it is preferably 60 to 140 μm, more preferably 80 to 120 μm. Regarding the size (thickness) of the cell layer, either a cell block or a microcarrier in the center is applicable and can be appropriately adopted.

[0097] In this embodiment, when a cell block (preferably a fibroblast cell block) is used in the central part, the number of cells (preferably fibroblasts) in the three-dimensional structure is not particularly limited, but is preferably 1×102 2 ~1×10 5 One cell / three-dimensional structure, more preferably 1×10 3 ~2×10 4 In this embodiment, the number of epidermal keratinocytes in the three-dimensional structure is not particularly limited, but is preferably 1×103. 2 ~1×10 5 One cell / three-dimensional structure, more preferably 1×10 3 ~2×10 4 Individual cells / three-dimensional structures.

[0098] The size of the three-dimensional structure in this embodiment is not particularly limited. When the size (major diameter) of the microsphere at the center is set to 1, a suitable lower limit value is preferably 1.05 or more, more preferably 1.1 or more. In addition, a suitable upper limit value is preferably 3.0 or less, more preferably 2.0 or less, and even more preferably 1.5 or less. As a suitable numerical range, it is preferably 1.05 or more and 1.5 or less.

[0099] Furthermore, in the first embodiment or the second embodiment of this application, the size of the three-dimensional structure when the size (major diameter) of the microspheres at the center is set to 1 can be appropriately adopted from the above-mentioned "size of the three-dimensional structure in this embodiment". For example, in the case of the first embodiment of this application, it is preferably 1.05 or more and 1.5 or less, and in the case of the second embodiment of this application, it is preferably 1.05 or more and 1.5 or less.

[0100] The ratio of fibroblasts to epidermal keratinocytes in the three-dimensional structure of the first embodiment of this application (number of fibroblasts / number of epidermal keratinocytes) is not particularly limited. As a suitable lower limit value, from the viewpoint of forming appropriate tight connections in the epidermal keratinocyte layer, it is preferably 1 / 4 or more, more preferably 1 / 2 or more, further preferably 1 or more, more preferably 4 or more, more preferably 5 or more, more preferably 6 or more, more preferably 7 or 8 or more. As for the suitable upper limit value, it is not particularly limited. From the viewpoint of the entire range of epidermal keratinocytes covering the fibroblast block, for example, 20, 15, 13, or 11 or less can be cited, preferably 10 or less, more preferably 9 or less, and further preferably 8 or less.

[0101] The number of fibroblasts and epidermal keratinocytes in the three-dimensional structure in this embodiment, and their proportions, are preferably as described in the manufacturing method of the three-dimensional structure described later, and are expressed as the conversion of the number of cells used for each inoculation when manufacturing a certain three-dimensional structure (conversion of inoculated cell number: cells / mL).

[0102] The three-dimensional structure of this embodiment has a longer culture period, thus extending the culture period and allowing for various experiments to be conducted during this period. Examples of these experiments include evaluation, measurement, analysis, detection, and observation; one or more of these methods can be used. Because the three-dimensional structure of this embodiment has a sufficiently extended culture period, the time for various experiments can also be appropriately extended, resulting in advantages such as obtaining more accurate and reliable experimental results, and being able to derive evaluation and judgment results based on these results.

[0103] The culture period or culturable period of the three-dimensional structure in this embodiment is not particularly limited. The appropriate lower and upper limits can be appropriately adopted from the description in "2. Method for manufacturing three-dimensional structures according to this embodiment" described later, and the appropriate numerical range is more preferably 3 to 35 days, and even more preferably 6 to 22 days. The starting point of this culture period or culturable period is more preferably "after inoculation of epidermal keratinocytes".

[0104] It should be noted that the culture medium for culturing three-dimensional structures is preferably changed daily using a co-culture medium. Furthermore, there are no particular limitations on the method for preserving three-dimensional structures; methods for preserving animal cells can be used, such as preservation in liquid nitrogen in a fully synthetic culture medium in the presence of cryoprotectants like dimethyl sulfoxide (DMSO).

[0105] <Applications of three-dimensional structures (e.g., various tests to evaluate skin homeostasis maintenance function)> The three-dimensional structure in this embodiment can be used for various tests, but is more preferably used for evaluating skin homeostasis maintenance function. Therefore, it is preferable to have states, characteristics, or functions that can cope with various evaluations related to skin homeostasis maintenance function. A more suitable configuration for the three-dimensional structure is that, like actual skin, epidermal keratinocytes proliferate and differentiate in the basal layer, and are appropriately locally present in the granular layer with tight junctions. A more suitable specific example of the three-dimensional structure has the following three characteristics: tight junctions in the superficial portion; the epidermal keratinocyte population or epidermal keratinocyte layer in contact with the nucleus portion is in an undifferentiated state; and the shell portion is in contact with the nucleus portion. By using this three-dimensional structure, skin homeostasis maintenance functions such as barrier function can be evaluated with greater precision, just as with actual skin.

[0106] For example, the three-dimensional structure of this embodiment can be used to conduct experiments (research, exploration, etc.) on skin homeostasis maintenance, skin physical properties, etc., or to conduct various experiments (evaluation, research, development, exploration, screening, etc.) on substances related to the regulation (inhibition, promotion, functional reduction, deterioration, improvement, etc.) of skin homeostasis maintenance such as skin barrier function.

[0107] Three-dimensional structures preferably have tight junctions between adjacent cells. Previously, no three-dimensional structures with tightly connected nucleoshell structures in the superficial layer composed of epidermal keratinocytes have been reported. Tight junctions in the superficial layer facilitate the evaluation of barrier function with greater precision.

[0108] Furthermore, the three-dimensional structure preferably has cell proliferation capacity in its cell layer, and more preferably, the epidermal keratinocytes in contact with the nucleus are in an undifferentiated state. By enabling cell proliferation in the cell layer of the three-dimensional structure, a longer evaluation period can be set, and maintenance and alteration states can be obtained based on longer-term observations. Additionally, the presence of undifferentiated epidermal keratinocytes in contact with the nucleus closely approximates the proliferation and differentiation state of the actual basal layer of skin, thus this embodiment has the advantage of easily evaluating skin homeostasis maintenance functions (e.g., cell proliferation capacity, barrier function, etc.). Furthermore, in this embodiment, by using microcarriers in the nucleus, the influence of other cell types can be reduced when performing various evaluations related to the cell layer composed of epidermal keratinocytes.

[0109] As markers used to determine or classify the state of three-dimensional structures, one or more can be selected from TJ markers, keratinization-related markers, proliferative cell markers, differentiated cell markers, and basal cell (undifferentiated cell) markers. For example, staining targets for TJ markers include closure protein, ZO-1, and claudin-1, with closure protein being preferred. As keratinization-related markers, filaggrin and caspase-14 are examples, with filaggrin being preferred. As proliferative cell markers, Ki-67 and PCNA are examples, with Ki-67 being preferred. As differentiated cell markers, keratin 10, involucrin, and naegrin are examples, with keratin 10 (initial differentiation) and naegrin (terminal differentiation) being preferred. As basal cell (undifferentiated cell) markers, keratin 5, keratin 14, and type XVII collagen are examples, with type XVII collagen being preferred. Since it is possible to identify the appropriate gene expression state, appropriate staining properties, and appropriate local presence corresponding to each marker, it is possible to identify the phenotypic or functional expression state of the three-dimensional structure of this embodiment (preferably tight junctions, cell proliferation capacity, undifferentiated / differentiated state, and extracellular matrix on the nuclear surface).

[0110] Three-dimensional structures can be used to evaluate the skin's homeostasis maintenance function.

[0111] Generally, steady-state maintenance is considered to mean that an organization's functions remain constant and maintain their proper state regardless of changes in internal and external environmental factors.

[0112] Functions related to the maintenance of skin (epidermal) homeostasis using the three-dimensional structure of this embodiment include, for example, the proliferative capacity of epidermal cells (Ki-67 (cell proliferation)), differentiation capacity (type XVII collagen (undifferentiated), keratin 10 (initial differentiation), naegrin (terminal differentiation), etc.), and barrier function (closing protein (TJ), filaggrin (keratin layer), etc.). Evaluation methods may include, for example, analysis using extracted messenger RNA such as Northern blotting, real-time PCR, microarray, and RNA sequencing; or analysis using extracted proteins such as Western blotting, ELISA, mass spectrometry-based proteomics, and multiplex detection; and histological analysis such as in situ hybridization and fluorescence immunostaining. One or more of these methods may be used.

[0113] The evaluation of skin homeostasis maintenance function can be performed using various biomarkers capable of assessing this function. There are no particular limitations on the biomarkers used; known methods or commercially available kits can be used. Preferably, a measurement or detection method that can determine the expression level and local presence of the biomarker is employed. The measurement or detection method used for the biomarker should preferably be an appropriate method corresponding to that biomarker. Examples include fluorescence microscopy (laser scanning, confocal laser (e.g., confocal laser scanning), multiphoton excitation (two-photon laser, light-sheet fluorescence), and gene amplification methods (PCR, LAMP, real-time RT-PCR, etc.).

[0114] This expression state can be considered, and the three-dimensional structure of this embodiment can be used for a more appropriate evaluation of skin homeostasis maintenance function, thereby obtaining a more appropriate evaluation.

[0115] The three-dimensional structure preferably has the function of maintaining skin homeostasis. By using the three-dimensional structure with this skin homeostasis maintenance mechanism, it is possible to explore the state of skin tissue (e.g., function, properties, etc.).

[0116] In addition, the three-dimensional structure of this embodiment can be used to predict the movement of the test substance when it comes into contact with the skin of an actual organism, or it can be used to determine whether the test substance is suitable or not, or to determine the condition of the skin.

[0117] Furthermore, this embodiment can also generate pluripotent stem cells based on an individual's somatic cells and use epidermal keratinocytes derived from pluripotent stem cells to create three-dimensional structures. Therefore, by using differentiation-inducing cells derived from pluripotent stem cells, this embodiment can explore the skin condition of an individual, and also explore test substances that are suitable or unsuitable for that individual.

[0118] 2. The manufacturing method of the three-dimensional structure involved in this embodiment This embodiment provides a method for manufacturing a core-shell three-dimensional structure, which includes co-culturing microspheres with seeded epidermal keratinocytes, preferably one microsphere. Furthermore, at the start of the co-culturing, the microspheres used in the co-culturing may have extracellular matrix components on their surface.

[0119] The manufacturing method in this embodiment preferably uses cell blocks or microcarriers as microspheres. Suitable cell blocks are preferably cell blocks prepared from inoculated cells. Suitable microcarriers are preferably microcarriers whose surfaces are coated with extracellular matrix components. It should be noted that when cell blocks are used as the core portion for co-culture, the surface of the cell blocks may not be coated with extracellular matrix components; preferably, extracellular matrix components exist between the cell blocks (surface layer) and the cell layer (inner layer) in the prepared three-dimensional structure.

[0120] As a suitable approach, the first embodiment of this application can provide a method for manufacturing a three-dimensional structure with a core-shell structure, which includes co-culturing a fibroblast block prepared from inoculated fibroblasts with inoculated epidermal keratinocytes.

[0121] As a suitable approach, the second embodiment of this application can provide a method for manufacturing a core-shell structured three-dimensional structure, which includes co-culturing microcarriers with inoculated epidermal keratinocytes.

[0122] Furthermore, this embodiment provides a method for manufacturing a three-dimensional structure with a core-shell structure, which includes seeding epidermal keratinocytes into a cell culture apparatus containing microspheres and co-culturing them. Additionally, this embodiment provides a method for manufacturing a three-dimensional structure with a core-shell structure, which includes seeding epidermal keratinocytes into a culture medium containing microspheres and culturing them. Furthermore, as another aspect of this embodiment, a method for manufacturing a three-dimensional structure with a core-shell structure can be provided, which includes co-culturing microspheres with seeded epidermal keratinocytes.

[0123] Furthermore, the manufacturing method of this embodiment can produce a three-dimensional structure with a core-shell structure as described in "1. Three-dimensional structure according to this embodiment".

[0124] In the description of this embodiment, descriptions of the components and methods of epidermal keratinocytes, microspheres, microcarriers, fibroblasts, nucleoshell structures, pluripotent stem cells, cell culture equipment, etc., that are repeated with the descriptions of “1.” above, “3.” and “4.” below, are appropriately omitted. The descriptions of “1.” above, “3.” and “4.” below, are also applicable to this embodiment and can be used appropriately.

[0125] Alternatively, as another embodiment of this invention, a method for manufacturing a core-shell three-dimensional structure is provided, which includes further seeding epidermal keratinocytes onto microspheres to prepare a surface layer. The microspheres can be any of cell blocks or spherical microcarriers.

[0126] Additionally, as a suitable manufacturing method, the following steps can be included as a pre-process of the co-culture process: inoculating fibroblasts to prepare a fibroblast block forming the center. As a suitable manufacturing method, the following steps can be included as a pre-process of the co-culture process: coating spherical microcarriers with extracellular matrix components to prepare spherical microcarriers with completed extracellular matrix component coating treatment forming the center. Furthermore, as another embodiment of the first application, a method for manufacturing a core-shell structured three-dimensional structure includes co-culturing inoculated fibroblasts with inoculated epidermal keratinocytes.

[0127] In addition, as another embodiment of this invention, a method for manufacturing a three-dimensional structure with a core-shell structure can also be provided, which includes: disposing microspheres on a cell adhesion inhibition treatment surface of a mortar-shaped recess, then inoculating epidermal keratinocytes and co-culturing them.

[0128] As a suitable manufacturing method, fibroblasts can be seeded and cultured on the cell adhesion inhibition treatment surface of the mortar-shaped recess before co-culturing. Alternatively, microcarriers can be disposed on the cell adhesion inhibition treatment surface of the mortar-shaped recess before co-culturing.

[0129] As a suitable approach, a method for manufacturing a three-dimensional structure with a core-shell structure can be provided, wherein the core-shell structure is formed by covering a layer of epidermal keratinocytes with microspheres (e.g., fibroblast blocks or spherical microcarriers) through the aforementioned co-culture. Alternatively, as a more suitable approach, a method for manufacturing a three-dimensional structure with a core-shell structure can be provided, wherein the aforementioned cell layer comprises: an undifferentiated layer of epidermal keratinocytes; a differentiated layer of epidermal keratinocytes; and a plurality of tight junctions existing between the outermost differentiated epidermal keratinocytes.

[0130] In addition, as another embodiment of this invention, a method for manufacturing a core-shell three-dimensional structure can also be provided, which includes: seeding a plurality of epidermal keratinocytes onto a microsphere and co-culturing them, thereby covering the fibroblast cell block with the epidermal keratinocytes to prepare a surface portion of a plurality of tightly connected three-dimensional structures. When preparing the fibroblast cell block as the aforementioned microsphere, the following steps may be included: seeding a plurality of fibroblasts and culturing them to prepare a fibroblast cell block as the core portion of the three-dimensional structure. As the aforementioned microsphere, a spherical microcarrier coated with extracellular matrix components can be used.

[0131] As a suitable method in the manufacturing method of this embodiment, when manufacturing a three-dimensional structure, the fibroblasts and epidermal keratinocytes used for inoculation are preferably cells derived from normal cells and / or differentiation-induced cells derived from pluripotent stem cells.

[0132] As a suitable method in the manufacturing method of this embodiment, when manufacturing a three-dimensional structure, the inoculated cells are cultured using a mortar-shaped recess (e.g., a U-shaped, V-shaped, or other recess), and the surface of the recess is more preferably a cell adhesion inhibition treatment surface. Furthermore, this treatment is more preferably a hydrophilic treatment.

[0133] As a suitable method in the manufacturing method of this embodiment, when culturing the microspheres and seeded epidermal keratinocytes during the manufacturing of the three-dimensional structure, a culture medium containing at least a culture medium for epidermal keratinocytes can be used, or a culture medium containing both a culture medium for epidermal keratinocytes and a culture medium for fibroblasts can be used. Furthermore, when evaluating the three-dimensional structure after its preparation, the culture medium used can also take into account the microspheres and epidermal keratinocytes used, and a culture medium containing at least a culture medium for epidermal keratinocytes, or a culture medium containing both a culture medium for epidermal keratinocytes and a culture medium for fibroblasts can be used.

[0134] As a suitable method in the manufacturing method of the first embodiment of this application, when manufacturing a three-dimensional structure, a culture medium containing a culture medium for epidermal keratinocytes and a culture medium for fibroblasts is used when culturing the aforementioned fibroblast block and the inoculated epidermal keratinocytes.

[0135] As a suitable method in the manufacturing method of the second embodiment of this application, when manufacturing a three-dimensional structure, a culture medium containing a culture medium for epidermal keratinocytes is used when culturing the aforementioned spherical microcarriers and the inoculated epidermal keratinocytes.

[0136] As a suitable method in the manufacturing method of this embodiment, when manufacturing a three-dimensional structure, the culture period after inoculation of the aforementioned epidermal keratinocytes is set to 6 to 22 days. Therefore, for the culturing period of the three-dimensional structure used in evaluation, it is preferable to set the culture period after inoculation of the aforementioned epidermal keratinocytes to 6 days or more.

[0137] In addition, as a suitable method in the manufacturing of this embodiment, when manufacturing a three-dimensional structure, it is preferable to cultivate it in a way that when the size of the microspheres is set to 1, the size of the three-dimensional structure is 1.05 or more and 1.5 or less, so that the size can be confirmed by observation using an optical microscope or the like while cultivating.

[0138] Furthermore, as a suitable method in the manufacturing method of the first embodiment of this application, when manufacturing a three-dimensional structure, the seeding ratio of the aforementioned fibroblasts to the aforementioned epidermal keratinocytes is preferably 1:4 or more, and more preferably 1:4 to 8:1.

[0139] Furthermore, as another suitable embodiment, a testing method or a method for evaluating skin homeostasis maintenance function can be provided, which uses the three-dimensional structure involved in this embodiment or a three-dimensional structure obtained by the manufacturing method of this embodiment. Additionally, as another suitable embodiment, the manufacturing method of this embodiment described above can be included in the method for evaluating skin homeostasis maintenance function. As a suitable example, a method for evaluating skin homeostasis maintenance function can be provided, which includes: performing a manufacturing method for the three-dimensional structure involved in this embodiment, and conducting a test (preferably an evaluation of skin homeostasis maintenance function) using the manufactured three-dimensional structure.

[0140] Using the manufacturing method of this embodiment, a three-dimensional structure with a core-shell structure, comprising microspheres (preferably fibroblast blocks or microcarriers) in the central portion and epidermal keratinocytes and tightly connected cells in the surface portion, can be obtained. More preferably, a three-dimensional structure with a core-shell structure as described in "1." of this embodiment can be obtained. This three-dimensional structure is preferably a core-shell structure comprising a cell layer and microspheres (preferably fibroblast blocks or microcarriers) in the central portion. The cell layer is composed of at least a plurality of tightly connected cells and a plurality of epidermal keratinocytes present on the surface, and the microspheres in the central portion are composed of at least a plurality of fibroblasts. More preferably, it is a core-shell structure comprising an epidermal keratinocyte layer as the shell portion, the epidermal keratinocyte layer comprising undifferentiated cell layers and differentiated cell layers.

[0141] <Suitable manufacturing examples of the manufacturing method for the three-dimensional structure involved in this embodiment> The following is an example of a manufacturing method according to this embodiment, but the manufacturing method of the three-dimensional structure involved in this embodiment is not limited to this.

[0142] The method for manufacturing a three-dimensional structure with a core-shell structure according to this embodiment preferably includes coating epidermal keratinocytes with microspheres (preferably fibroblast blocks or microcarriers).

[0143] As a suitable manufacturing example in the method of this embodiment, it preferably includes: (a) pretreatment by placing microspheres in wells with mortar-shaped recesses and culturing them for a predetermined time using a predetermined culture medium; and (b) inoculating epidermal keratinocytes in the wells with mortar-shaped recesses containing the microspheres and culturing them using a co-culture medium or an epidermal keratinocyte culture medium to prepare a three-dimensional structure with a core-shell structure. The microspheres are preferably fibroblast blocks or microcarriers. Alternatively, if the microspheres are not a cell line, the predetermined time of culturing in the aforementioned pretreatment (a) may be omitted.

[0144] Furthermore, the method for manufacturing a three-dimensional structure with a core-shell structure according to the first embodiment of this application preferably includes: preparing a fibroblast cell block; and covering the prepared fibroblast cell block with epidermal keratinocytes.

[0145] As a suitable manufacturing example in the method of the first embodiment of this application, it is preferable to include coating epidermal keratinocytes with fibroblast blocks obtained from inoculated fibroblasts.

[0146] As a more suitable manufacturing example in the method of this embodiment, it is preferable to include: (a) inoculating fibroblasts into a pore having a mortar-shaped recess and culturing them using a fibroblast culture medium to prepare a fibroblast block; and (b) inoculating epidermal keratinocytes into a pore having a mortar-shaped recess in which the prepared fibroblast block is present and culturing them using a co-culture medium to prepare a three-dimensional structure with a core-shell structure.

[0147] As a more suitable manufacturing example in the method of the first embodiment of this application, it is preferable to further adjust the number of fibroblasts and / or the number of epidermal keratinocytes used for inoculation before performing the preparation described in (a) and / or (b) above, and more preferably to include cell proliferation for inoculation as a pretreatment. Thus, the desired number of cells and the number of inoculated cells can be obtained as needed, in a manner that achieves the desired inoculation number, the ratio of the number of fibroblasts to the number of epidermal keratinocytes used, etc.

[0148] <a. Cells used for inoculation and regulation of cell number> Regarding the cells seeded in the method of this embodiment, known cell culture methods can be used to culture them to obtain or recover the desired number of cells and seeding density. For example, this can be achieved by culturing the seeded cells under specified culture conditions, recovering the proliferating cells after culture, adjusting the cell number, and preparing a cell suspension with the desired seeding density. More specifically, cells can be seeded into one or more cell culture apparatuses, cultured under specified culture conditions for approximately 2-7 days, washed appropriately with PBS (-), then detached from the cell culture apparatus using a stripping agent, centrifuged, and the supernatant removed. The detached cells are then added to culture medium to obtain a cell suspension. The desired cell suspension with the desired seeding density can be obtained by adjusting the amount of culture medium or other solution added after centrifugation and supernatant removal.

[0149] To induce the proliferation of epidermal keratinocytes, conventional epidermal keratinocyte culture methods can be followed (e.g., 37°C, 5% CO2, epidermal keratinocyte culture medium, etc.). Collagen or laminin, or fragments thereof, are preferred as the coating agent, and trypsin or trypsin-like enzymes are preferred as the cell stripping agent.

[0150] To promote fibroblast proliferation, the usual fibroblast culture methods can be followed (e.g., 37°C, 5% CO2, fibroblast culture medium, etc.).

[0151] Therefore, it is possible to obtain or prepare cells used in the production of the nucleus and / or cells used in the production of the shell. Regarding the cell culture equipment used in the cell number adjustment step of this embodiment, equipment typically used for proliferation culture is preferred. For example, in steps such as the cell number adjustment step, the recess for inoculation is preferably a flat-bottomed recess such as a culture dish, and / or preferably treated with a culture medium (coating agent).

[0152] There is no particular limitation on the number of cultivation days. As a suitable lower limit, it is preferred to be more than 1 day, more preferably more than 2 days, and even more preferably more than 3 days. As a suitable upper limit, it is preferred to be less than 7 days, more preferably less than 6 days, and even more preferably less than 5 days.

[0153] Cell counts can be performed using methods commonly used in cell culture, such as automated cell counting (e.g., image-based cell counters, Coulter counters, flow cytometers, etc.) and manual cell counting, with Coulter counters being preferred. Manual cell counting can be performed using an optical microscope and a hemocytometer, with the cell concentration (cells / mL) calculated as ([average number of cells per compartment] × dilution factor] / volume of liquid per compartment (mL)).

[0154] <Cultivation Conditions> The term "prescribed culture conditions" in this embodiment is not particularly limited, and the culture temperature and atmosphere are not particularly limited, but are preferably ordinary culture conditions. The culture temperature is preferably an ordinary culture temperature, preferably 36-38°C, more preferably 36.5-37.5°C. The culture atmosphere is preferably an ordinary atmosphere, preferably at 4-6% CO2, more preferably at 4.5-5.5% CO2. As more suitable culture conditions, 36.5-37.5°C and 4.5-5.5% CO2 are preferred, more preferably 37°C and 5% CO2 (an atmosphere of 5% carbon dioxide and 95% air). These "prescribed culture conditions" can be used as the "prescribed culture conditions" in <a. Adjustment of the number of cells for inoculation>, <b. Preparation of fibroblast cell blocks>, and <c. Covering of cell blocks with epidermal keratinocytes> described below.

[0155] <Cultivation medium> The culture medium used in this embodiment is not particularly limited. The culture medium used for the cells being cultured can be used according to the cells being cultured. For example, culture medium for fibroblasts and culture medium for epidermal keratinocytes can be used. One or more of these can be selected.

[0156] The culture medium for fibroblasts can be one or more selected from culture media obtained by known manufacturing methods or commercially available products (e.g., fibroblast culture medium (Takara Bio Inc.), HFDM-1 medium (Cell Science Research Institute Co., Ltd.), Fibrolife S2 Medium Complete Kit (Lifeline Cell Technology Co., Ltd.), etc.). More specifically, DMEM (Dupua New Guinea modified Eagle medium) is preferred, and DMEM / F-12 medium prepared by mixing DMEM (Dupua New Guinea modified Eagle medium) and Ham's F-12 medium in a 1:1 ratio is even more preferred.

[0157] The culture medium for epidermal keratinocytes can be one or more selected from culture media obtained by known manufacturing methods or commercially available products (e.g., Keratinocyte Growth Medium 2 (Promocell), MCDB153 medium (COSMO CO.,LTD.), HuMedia-KG2 (KURABO), etc.). Furthermore, when constructing three-dimensional structures, it is preferable to use a culture medium for 3D differentiation of epidermal keratinocytes (a culture medium for three-dimensional epidermal keratinocyte models that does not contain components derived from animals or humans). It should be noted that, in terms of the characteristic culture medium components of the culture medium for epidermal keratinocytes, for example, ckDME-Ham culture medium, for example, a culture medium containing 24.3 mg / L adenine, 5 mg / L insulin, 0.01 mg / L EGF, etc. (Non-Patent Literature 3 (Reference): Sergio Cortez Ghio et al., Int. J. Mol. Sci. 2018, 19(8), 2174; https: / / doi.org / 10.3390 / ijms19082174).

[0158] When fibroblasts are proliferated for inoculation, a fibroblast culture medium is preferred; when epidermal keratinocytes are proliferated for inoculation, an epidermal keratinocyte culture medium is preferred.

[0159] It should be noted that in the subsequent sections <a. Cells used for inoculation and adjustment of cell number>, the culture medium can be selected for the same purpose. Additionally, in <b. Preparation of fibroblast blocks> described later, a fibroblast culture medium can also be used. In <c. Coverage of epidermal keratinocytes with microspheres> and <d. Evaluation of skin homeostasis maintenance function> described later, it is preferable to use a culture medium containing at least an epidermal keratinocyte culture medium (e.g., epidermal keratinocyte culture medium, co-culture medium), as described later. As a co-culture medium, for example, a mixed culture medium obtained by combining, for example, CnT-PR-3D for epidermal keratinocyte culture with, for example, DMEM / F12 (supplemented with 1 mM CaCl2 and 5% FBS) for fibroblast culture can be used.

[0160] <Cells used in inoculation (fibroblasts, epidermal keratinocytes)> The epidermal keratinocytes and fibroblasts used for seeding in the method of this embodiment are not particularly limited. For example, normal cells (e.g., normal epidermal keratinocytes, normal fibroblasts, etc.) and differentiation-inducing cells derived from pluripotent stem cells (e.g., epidermal keratinocytes, fibroblasts, etc.) can be selected, and one or more of these can be chosen. By using differentiation-inducing cells derived from pluripotent stem cells in the manufacture of three-dimensional structures, it is advantageous to perform evaluations or experiments on skin homeostasis maintenance functions for a longer period of time compared to using normal cells. These cells are preferably derived from mammals, and while there is no particular limitation on the mammal, human sources are preferred.

[0161] Furthermore, prior to co-culture, as a pre-culture to increase the number of inoculated fibroblasts, it is preferable to use a conventional fibroblast proliferation method, and more preferably to use a fibroblast culture medium. Similarly, prior to co-culture, as a pre-culture to increase the number of inoculated epidermal keratinocytes, it is preferable to use a conventional epidermal keratinocyte proliferation method, and more preferably to use an epidermal keratinocyte culture medium.

[0162] <Normal cells used in inoculation> Normal fibroblasts and / or normal epidermal keratinocytes are preferred as the normal cells used for inoculation in the method of this embodiment.

[0163] There are no particular limitations on normal skin fibroblasts. They can be obtained by known manufacturing methods or commercially available products. Examples include neonatal foreskin skin fibroblasts, adult skin fibroblasts, and adult oral fibroblasts. One or more of these can be used.

[0164] In addition, as normal epidermal keratinocytes, there are no particular limitations, and they can be obtained by known manufacturing 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 of these can be used.

[0165] <Differentiation-inducing cells derived from pluripotent stem cells used in inoculation> Furthermore, the preferred cells derived from pluripotent stem cells are epidermal keratinocytes and fibroblasts derived from pluripotent stem cells. When manufacturing pluripotent stem cells, the use of somatic cells from the target animal allows for the evaluation of skin homeostasis maintenance and skin properties specific to that animal, and the provision of more suitable topical skin agents, pharmaceuticals, etc., for that animal.

[0166] Pluripotent stem cells The pluripotent stem cells used in this embodiment are stem cells that possess the ability to differentiate into all types of cells present in an organism and also have proliferative capacity. There are no particular limitations; examples include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos using nuclear transfer (ntES cells), sperm stem cells (GS cells), embryonic germ cells (EG cells), artificially pluripotent stem cells (iPS cells), and adult stem cells derived from skin, bone marrow, adipose tissue, etc. (Muse cells, mesenchymal stem cells, etc.). One or more types selected from these can be used. These cells can be manufactured using known methods or obtained from the market or public institutions. The cells are preferably mammalian cells, and particularly preferably human cells.

[0167] Embryonic stem cells (ES cells) are stem cells that develop from the internal cell blocks of early embryos (such as blastocysts) in mammals like humans and mice. Sperm stem cells are pluripotent stem cells derived from sperm and are the cells that originate to form sperm. Embryonic germ cells are cells that develop from primordial germ cells during the embryonic period.

[0168] Artificial pluripotent stem cells (iPS cells) can be manufactured using known manufacturing methods. Alternatively, commercially available or custom-made products can be purchased (for example, see Patent Document 3 (Reference): Japanese Patent Application Publication No. 2023-018632, paragraphs

[0021] to

[0027] , etc.).

[0169] The aforementioned 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 in the production of iPS cells are not particularly limited, but iPS cells derived from somatic cells are preferred.

[0170] In this embodiment, differentiated cells are preferred as the somatic cells used in the production of iPS cells. Among these differentiated cells, cells of the skin lineage are preferred, and epidermal keratinocytes (also known as "keratinocytes," "skin keratinocytes," or "keratinization cells") and skin fibroblasts ("fibroblasts") are more preferred. Furthermore, from the viewpoint of evaluating the maintenance of skin homeostasis, iPS cells produced using epidermal keratinocytes and skin fibroblasts as somatic cells are preferred in this embodiment. It should be noted that iPS cells derived from epidermal keratinocytes and skin fibroblasts can be obtained using known methods for producing iPS cells derived from epidermal keratinocytes (e.g., Non-Patent Literature 4 (Reference): Okita K. et al., Nature Methods, 2011, 8:409-12).

[0171] It should be noted that the somatic cells used in this embodiment refer to all animal cells (preferably animal cells of mammals, including humans) other than germline cells such as eggs, oocytes, and sperm, or differentiated totipotent cells such as ES cells. Somatic cells are not limited to any of the following: fetal (larval) somatic cells, neonatal (larval) somatic cells, and mature healthy or diseased somatic cells. Additionally, they include any of the following: primary cultured cells, passaged cultured cells, and lineaged cells.

[0172] Regarding somatic cells, more specific examples include tissue stem cells (adult stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; tissue progenitor cells; 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. One or more types selected from these can be used. Preferably, the somatic cells used are fibroblasts and / or epidermal keratinocytes that are identical to the cells obtained by inducing differentiation from pluripotent stem cells.

[0173] <b. Preparation of microspheres> The preparation step of the microspheres in the method of this embodiment is not particularly limited. It can be prepared by referring to known methods, or commercially available products can be used.

[0174] When the microspheres are cell blocks, the cell blocks can be prepared by referring to <b-1. Preparation of fibroblast cell blocks> below. Among the cell blocks, fibroblast cell blocks are preferred.

[0175] In addition, when the microsphere is a microcarrier (preferably a spherical microcarrier), the microcarriers described in the above-mentioned <Central part of three-dimensional structure> <Microcarrier> can be used, and one or more of these can be used.

[0176] Furthermore, known coating methods can be appropriately used for preparing microcarriers by coating them with extracellular matrix components. For example, a method for coating collagen in resin culture dishes can be referenced, where microcarriers are coated with a specified collagen concentration (e.g., 0.01%, 0.05%, 0.1%) at room temperature (approximately 20–37°C) for a specified coating time (removed immediately after addition, or approximately 0.25–1.5 h, or 10–14 h). Depending on the needs, dilute solvents such as 0.01 N hydrochloric acid or 5 mM acetic acid solution can be used, and sterile water or PBS can be used as washing buffers for single or multiple washings (2–5 times). Additionally, the type of collagen is not particularly limited; for example, type I or type IV collagen can be used.

[0177] <b-1. Preparation of fibroblast blocks> As a step in preparing fibroblast blocks in the method of this embodiment, it is preferable to have multiple seeded fibroblasts in a cell culture apparatus and culture them under specified culture conditions. In preparing the fibroblast blocks (spheroids), it is preferable to use a cell culture apparatus with one or more mortar-shaped recesses at the bottom, and / or preferably to use a cell culture apparatus with recesses treated to inhibit cell adhesion. The number of cells seeded in the cell culture apparatus is not particularly limited, but is preferably 1 × 10⁻⁶. 2 ~1×10 5 Generally, a spherical body refers to a roughly spherical aggregate of cells formed by the aggregation and condensation of cells together.

[0178] In addition, the culture medium used in the preparation process of fibroblast blocks is preferably a fibroblast culture medium.

[0179] As a culture medium used in the preparation of fibroblast blocks, it is more preferable to inoculate the cell suspension after preparing a cell suspension containing fibroblasts and culture medium. Regarding the inoculated fibroblasts, it is preferable to proliferate and culture them using known culture methods and then recover them, adjusting the inoculation number to the desired level.

[0180] The culture period for the preparation of fibroblast blocks is not particularly limited. As a suitable lower limit, it is preferably 0.5 days or more, more preferably 1 day or more. As a suitable upper limit, it is preferably 7 days or less, more preferably 4 days or less, and even more preferably 2 days or less.

[0181] Furthermore, in the process of preparing fibroblast cell blocks, there is no particular limitation on the number of days of culture from inoculation to obtaining cell blocks. As a suitable lower limit, it is preferably 0.25 days or more, more preferably 0.5 days or more. As a suitable upper limit, it is preferably 2 days or less, more preferably 1 day or less.

[0182] <c. Coverage of microspheres by epidermal keratinocytes> In the coating step of epidermal keratinocytes onto microspheres (preferably spherical microcarriers or fibroblast blocks) in the method of this embodiment, it is preferable to have one or more microspheres (preferably one) and multiple seeded epidermal keratinocytes in a cell culture apparatus and cultured under specified culture conditions. The cell culture apparatus used in this coating step can be the apparatus used in <a.>< / b.> described above, or an apparatus equivalent to the apparatus used in <a.>< / b.> described above. This results in a three-dimensional structure or sphere containing epidermal keratinocytes coated with microspheres. A more suitable three-dimensional structure is a core-shell structure or sphere with a microsphere (preferably a spherical microcarrier or fibroblast block) as the core and epidermal keratinocytes as the shell. Generally, a sphere refers to a spherical cell aggregate formed by the aggregation and condensation of cells.

[0183] The size (long axis or diameter) of the microspheres (preferably spherical microcarriers or fibroblast blocks) used for coating is not particularly limited. As a suitable lower limit, it 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. As a suitable upper limit, it is preferably 560 μm or less, more preferably 480 μm or less. As a more suitable numerical range, it is preferably 240 to 560 μm, more preferably 320 to 480 μm. In the case of spherical microcarriers, for example, it is 50 to 1,000 μm. As a suitable upper limit, it is preferably 300 μm or less or 250 μm or less, preferably 100 μm or more or 500 μm or less, more preferably 120 μm or more or 250 μm or less, and even more preferably 150 μm or 200 μm or more or 250 μm or less. It should be noted that the size of the obtained three-dimensional structure, cell layer, central part, etc., can be appropriately adopted from the size described in "2. Three-dimensional structure involved in this embodiment" above, and can be prepared in a way that achieves that size.

[0184] In the preparation of three-dimensional structures coated with epidermal keratinocytes, cell culture apparatus with a mortar-shaped base containing one or more recesses is preferred, and / or cell culture apparatus with recesses treated to inhibit cell adhesion is also preferred. The number of epidermal keratinocytes seeded into the cell culture apparatus is not particularly limited, but is preferably 5 × 10⁻⁶. 2 ~5×10 4 .

[0185] Furthermore, the culture medium used in the preparation process of the three-dimensional structure coated with epidermal keratinocytes is preferably a culture medium containing an epidermal keratinocyte culture medium, more preferably either an epidermal keratinocyte culture medium or a co-culture medium. As the co-culture medium, a mixed culture medium containing fibroblast culture medium and epidermal keratinocyte culture medium is preferred. When a non-cell-based microcarrier is used as the nucleus, an epidermal keratinocyte culture medium can be used; when a fibroblast block is used as the nucleus, a co-culture medium is preferred. The epidermal keratinocytes used in this case are preferably epidermal keratinocytes derived from pluripotent stem cells induced from differentiation of pluripotent stem cells, more preferably epidermal keratinocytes derived from iPS cells induced from differentiation of iPS cells.

[0186] Furthermore, regarding the culture period for obtaining the three-dimensional structure of this embodiment, in order to enable the obtained three-dimensional structure to be used for evaluation, it is preferable to co-culture for 2 or 3 days or more after inoculating epidermal keratinocytes, more preferably 6 days or more. This suitable upper limit is not particularly limited; from the viewpoint of culturable period, it is preferably 14 days or less, more preferably 10 days or less, and can be approximately 6 days. Thus, a core-shell structure of a three-dimensional structure formed by epidermal keratinocytes coated with microspheres can be obtained. The culture period for obtaining this three-dimensional structure can be the manufacturing period of the three-dimensional structure, or the culturable period described below can be appropriately adopted. The culture period in the preparation process of the three-dimensional structure can be implemented as "d. Skin homeostasis maintenance function evaluation process" and then described later as "3. Skin homeostasis maintenance function evaluation method using the three-dimensional structure according to this embodiment".

[0187] The culture period or culturable period of the three-dimensional structure in this embodiment is not particularly limited. Regarding the culture period after inoculation with 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. Furthermore, there is no particular limitation on the suitable upper limit; the upper limit can be adjusted by adjusting the culture conditions. For example, 40, 38, 35, 34, 33, 32, 31, 30, or 29 days are possible, preferably 35 days or less, more preferably 28 days or less, and even more preferably 22 days or less. As a suitable numerical range, 4 to 35 days is preferred, and 6 to 22 days is more preferred. It should be noted that the culture medium for the three-dimensional structure is preferably changed every day using a co-culture medium for epidermal keratinocytes.

[0188] Furthermore, as a suitable method, multiple epidermal keratinocytes are seeded into a cell culture apparatus containing microspheres for culture. Moreover, in the coating of epidermal keratinocytes with microspheres, it is preferable to use a cell culture apparatus with one or more mortar-shaped recesses at the bottom, and / or preferably to use a cell culture apparatus with recesses treated to inhibit cell adhesion.

[0189] The cell culture apparatus before inoculating epidermal keratinocytes may contain a culture medium for epidermal keratinocytes (e.g., epidermal keratinocyte culture medium, co-culture medium). Alternatively, the culture medium may be removed from the cell culture apparatus after preparing cell blocks, and a liquid containing a cell suspension of the inoculated epidermal keratinocytes may be used as the culture medium. This liquid is preferably epidermal keratinocyte culture medium or co-culture medium. When the microspheres are microcarriers, epidermal keratinocyte culture medium or co-culture medium is preferred. When the cell blocks are fibroblasts, a mixed culture medium containing fibroblast culture medium and epidermal keratinocyte culture medium (hereinafter also referred to as "co-culture mixed medium") is preferred.

[0190] The fibroblast cell block covered with epidermal keratinocytes is more preferably the fibroblast cell block obtained in <b-1. Preparation of fibroblast cell block> above, and more preferably, it is a fibroblast cell block prepared from seeded fibroblasts. More preferably, the cell suspension is inoculated after preparation of a cell suspension containing fibroblasts and culture medium. Regarding the seeded fibroblasts, it is preferable to proliferate and culture them using known culture methods and then recover them, adjusting the inoculation number to the desired level.

[0191] The microcarrier used to coat epidermal keratinocytes is preferably the microcarrier described in the above-mentioned <microcarrier>, more preferably a PVA microcarrier. As a more suitable method, it is preferred to use a microcarrier coated with extracellular matrix components (preferably a PVA microcarrier), and the extracellular matrix component is preferably collagen, such as type 1 or type 4 collagen.

[0192] <Co-culture medium> When preparing three-dimensional structures composed of microspheres coated with epidermal keratinocytes, the culture medium is not particularly limited. For example, it can be a single culture medium or a mixed culture medium composed of two single culture media. A co-culture medium is preferred, preferably one that contains various components in a manner capable of co-culturing fibroblasts and epidermal keratinocytes. For example, the co-culture medium can be prepared by mixing a fibroblast culture medium and an epidermal keratinocyte culture medium, and more preferably a mixed culture medium containing both fibroblast and epidermal keratinocyte culture media. Furthermore, it is preferable to use the co-culture medium for co-culturing fibroblasts and epidermal keratinocytes, and / or for co-culturing fibroblast blocks and epidermal keratinocytes.

[0193] The co-culture medium used in this embodiment can be composed of components of a culture medium that can proliferate fibroblasts and epidermal keratinocytes, or it can be prepared using commercially available products or components containing these components.

[0194] A more suitable co-culture medium preferably has an adjusted concentration of components derived from animals or humans and / or calcium concentration. The concentration of calcium chloride (preferably CaCl2) in the co-culture medium is preferably 0.8-4 mM, more preferably 0.9-3.0 mM, and even more preferably 1.0-2.0 mM.

[0195] In this embodiment, the mixing ratio of the epidermal keratinocyte culture medium and the fibroblast culture medium in the co-culture mixed culture medium 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 more preferably 1:1.

[0196] In the case of the first embodiment of this application where cell blocks are present in the nucleus, the mixing ratio of the culture medium for epidermal keratinocytes and the culture medium for fibroblasts in the co-culture mixed culture medium can be appropriately combined using the upper and lower limits of the above-mentioned mixing ratio. As a suitable range, it is more preferably 3:1 to 1:3, further preferably 2:1 to 1:2, and even more preferably 1:1.

[0197] In the case of the second embodiment of this application where microcarriers are present in the core, the mixing ratio of the culture medium for epidermal keratinocytes and the culture medium for fibroblasts in the co-culture mixed culture medium can be appropriately combined using the upper and lower limits of the above-mentioned mixing ratio. As a suitable range, it is more preferably 3:1 to 1:3, further preferably 2:1 to 1:2, and even more preferably 1:1.

[0198] It should be noted that, in the case of the second embodiment of this application, 100% epidermal keratinocyte culture medium may be used instead of fibroblast culture medium. Regarding the mixing ratio of epidermal keratinocyte culture medium and fibroblast culture medium, when the total amount of epidermal keratinocyte culture medium and fibroblast culture medium is set to 100%, the epidermal keratinocyte culture medium may be 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more.

[0199] By using a co-culture medium, or, depending on the situation, a keratinocyte culture medium, a three-dimensional structure can be obtained that surrounds microspheres (preferably fibroblast blocks or microcarriers) with epidermal keratinocytes, possesses the proliferative and differentiation capabilities of epidermal keratinocytes, and exhibits skin barrier function. Furthermore, the co-culture medium (depending on the situation, an epidermal keratinocyte culture medium) can be used for evaluating the skin homeostasis maintenance function of the three-dimensional structure, and the culture period (evaluable period) of the three-dimensional structure can be extended.

[0200] <Fibroblasts and epidermal keratinocytes and their seeding numbers> In this embodiment, there is no particular limitation on the number of fibroblasts and epidermal keratinocytes seeded.

[0201] In the aforementioned <b. Preparation of fibroblast blocks>, the number of fibroblasts seeded into the cell culture apparatus is not particularly limited; however, a suitable lower limit is preferably 1 × 10⁻⁶. 2 The above is preferred, with 5×10 being more ideal. 2 The above is further preferred to be 1×10 3 Furthermore, as a suitable upper limit value, 1×10 is preferred. 5 The following is more preferably 5×10 4 The following is a further preferred option: 2×10 4 The following is a further preferred option: 1×10 4 The preferred numerical range is 1×10⁻⁶. 3 ~1×10 4 .

[0202] In the aforementioned process of coating fibroblast blocks with epidermal keratinocytes, the number of epidermal keratinocytes seeded into the cell culture apparatus is not particularly limited; however, a suitable lower limit is preferably 5 × 10⁻⁶. 2 The above is preferred to be 1×10 3 Furthermore, as a suitable upper limit value, 5 × 10 is preferred. 4 The following is more preferably 2×10 4 Hereinafter, 1×10 is further preferred. 4 Hereinafter, a suitable numerical range is preferably 1×10⁻⁶.3 ~1×10 4 .

[0203] The ratio of fibroblasts to epidermal keratinocytes used in this embodiment (number of fibroblasts / number of epidermal keratinocytes) is not particularly limited. As a suitable lower limit, from the viewpoint of forming appropriate tight junctions in the epidermal keratinocyte layer, it is preferably 1 / 4 or more, more preferably 1 / 2 or more, further preferably 1 or more, more preferably 4 or more, and more preferably 8 or more. As for the suitable upper limit, it is not particularly limited. From the viewpoint of the epidermal keratinocytes covering the entire range of the fibroblast block, for example, 10 or less can be cited, preferably 9 or less, and more preferably 8 or less. It should be noted that, regarding the <number of fibroblasts and epidermal keratinocytes and the number of each>, the composition of "the ratio of fibroblasts to epidermal keratinocytes (number of fibroblasts / number of epidermal keratinocytes)" described in the <shape, size, etc. of the three-dimensional structure> can be appropriately adopted.

[0204] It should be noted that the preferred number of fibroblasts to be inoculated is the number of fibroblasts inoculated into one well of the cell culture apparatus when preparing fibroblast blocks, and the preferred number of epidermal keratinocytes to be inoculated is the number of epidermal keratinocytes inoculated into one well of the cell culture apparatus when covering the prepared fibroblast blocks with epidermal keratinocytes.

[0205] <d. Evaluation of skin homeostasis maintenance function> After obtaining the three-dimensional structure with the core-shell structure according to this embodiment in the above-described step <c. Covering fibroblast masses with epidermal keratinocytes>, the following "3. Method for evaluating skin homeostasis maintenance function using the three-dimensional structure according to this embodiment" can be implemented by including the step <d. Evaluation of skin homeostasis maintenance function>, or <d.> can be performed immediately after <c.>. Therefore, it is possible to explore or screen substances that have skin homeostasis function, and more preferably, to explore or screen substances related to skin homeostasis suitable for an individual. It should be noted that <d. Evaluation of skin homeostasis maintenance function> is described in detail in the following "3. Method for evaluating skin homeostasis maintenance function using the three-dimensional structure according to this embodiment," and its description can be appropriately adopted.

[0206] <Other> <Cell Culture Equipment> The cell culture equipment used in the method of this embodiment is not particularly limited. Generally, cell culture equipment suitable for cell culture is preferred, such as flasks, dishes, culture dishes, bottles, plates, tubes, centrifuge tubes, etc., but is not limited to these. In addition, disposable products are preferred. As for the material of the equipment, for example, glass, plastic resin (preferably polystyrene resin), etc., are preferred, but are not limited to these. One or more of these examples can be used.

[0207] The material (manufacture) of the cell culture apparatus is not particularly limited, but preferably one or more synthetic resins selected from styrene-based 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 (preferably plastics), and glass substrates. Among these, plastics are preferred (more preferably styrene-based resins, polyolefin resins (polyethylene, polypropylene), styrene-based resins, and polycarbonate), and styrene-based resins (more preferably polystyrene) are even more preferred.

[0208] Regarding the method of this embodiment, as described above, in the process of manufacturing generally spherical objects such as three-dimensional structures, cell blocks, or spheres, it is preferable to culture multiple inoculated cells on a cell adhesion inhibition-treated surface (more preferably a hydrophilic surface) of a mortar-shaped recess. Furthermore, in the proliferation process to increase the number of cells, it is preferable to culture on a planar recessed culture medium treated surface such as a culture dish.

[0209] In the manufacture of the generally spherical object, the bottom of the aforementioned mortar-shaped recess is preferably shaped such that multiple cells can aggregate in the central region of the bottom of the recess. The bottom of the recess is more preferably V-shaped or U-shaped, because cells inoculated into the V-shaped or U-shaped recess (generally conical, generally frustum-shaped, etc.) tend to aggregate more easily, thus having the following advantages: it is possible to form a better shape of generally spherical cell blocks, it is easy to form fibroblast cell blocks that become the nucleus of a three-dimensional structure, or it is easy to form a cell layer that covers the shell portion of a three-dimensional structure.

[0210] In the manufacture of the aforementioned generally spherical structures, it is preferable to perform a cell adhesion inhibition treatment on the recesses where cells are inoculated. This surface treatment inhibits cell adhesion to the surface of the recesses and also facilitates the removal of the three-dimensional structure after culture. Through this surface treatment, the inoculated cells aggregate in the central region of the recesses, and the cells adhere to each other more easily, thereby forming a better shape of the generally spherical structure (e.g., fibroblast blocks, nucleoshell cell blocks).

[0211] There are no particular limitations on the aforementioned cell adhesion inhibition treatments; for example, hydrophilic surface treatments can be cited.

[0212] In the manufacture of the aforementioned generally spherical object, a surface treatment that hydrophilizes the recess is more preferable. Examples of such hydrophilization include plasma treatment, corona discharge treatment, oxidant treatment, and coating treatment with a hydrophilic substance (preferably a cell adhesion inhibitor such as polyethylene glycol), but are not limited to these. Examples of hydrophilic substance coating include polymer coating. One or more of these can be used.

[0213] <Cell stripping agent> In the method of this embodiment, a cell stripping agent is used. Preferably, the cell stripping agent is a cell stripping component or a reagent containing the cell stripping component that is typically used to strip cell culture equipment and cultured cells when culturing animal cells.

[0214] Examples of components used in cell stripping include trypsin-based proteases (trypsin, trypsin-like proteases, etc.) and serine proteases such as chymotrypsin; metalloproteinases such as dispersants; Ca-dependent proteases such as streptomycin; neutral proteases; enzyme stripping systems such as proteases and proteases with collagenase activity; and enzyme-free stripping systems such as ethylenediaminetetraacetic acid (EDTA) and sodium citrate; TrypLE (trademark), etc. One or more of these can be used, but are not specifically limited to these. Commercially available cell stripping agents can be used as cell stripping agents. Additionally, physical cell stripping methods can be used as cell stripping techniques, such as cell scrapers (manufactured by Corning).

[0215] Among the aforementioned cell stripping agents, an enzyme or a cell stripping agent containing an enzyme is preferred. As the enzyme, protease-based enzymes are further preferred, and trypsin-based enzymes are even more preferred. Trypsin-based proteases such as trypsin, trypsin-like proteases, and TrypLE (trademark) Select enzyme are more preferred; TrypLE (trademark) Select enzyme; and recombinant proteins with trypsin-like activity (more preferably trypsin-like proteases derived from recombinant microorganisms). "Trypsin-based enzyme" refers to an enzyme containing both trypsin and trypsin-like enzymes.

[0216] "Trypsin-like" substances preferably exhibit the same or similar kinetics and cleavage properties as trypsin. More preferably, they can directly replace trypsin in cell dissection without changing the protocol. A key cleavage property of trypsin is its ability to hydrolyze the peptide bonds on the carboxyl side of basic amino acids (lysine, arginine).

[0217] In addition, the enzyme can be from any of animal or microbial sources, preferably from microbial sources, more preferably from recombinant microbial sources that have undergone genetic recombination in a manner capable of producing proteases or trypsin, and even more preferably from recombinant fungal sources.

[0218] The aforementioned cell stripping agent preferably comprises a protease and / or TrypLE (trademark) Select enzyme. More preferably, the protease is one or more selected from trypsin, trypsin-like protease, and TrypLE (trademark) Select enzyme, and even more preferably, trypsin and / or TrypLE (trademark) Select enzyme. Other preferred cell stripping agents include, for example, trypsin, TrypLE (trademark) Select enzyme, and cell stripping agents comprising the TrypLE (trademark) Select enzyme and 0.5-1.5 mM EDTA; one or more selected from these may be used.

[0219] It should be noted that the process of using a cell stripping agent to detach adherent cells from cell culture equipment is preferably combined with physical treatment. Examples of physical treatments include: methods that generate water flow using blowing and suction operations (suction and jetting, etc.), and scraping methods such as shovel-shaped scrapers that can detach adherent cells. From the viewpoint of reducing cell damage, methods that generate water flow are preferred (preferably the recovery of detached cells after detachment using suction and jetting).

[0220] In addition, the recovered and detached cells can be separated by centrifugation and other methods, thereby allowing for the further recovery of individual cells for use in the next cell culture.

[0221] <Culture substrate (coating agent)> In this embodiment, a coating agent containing a culture medium can be used as needed. Using this coating agent, the portion of the cell culture apparatus that comes into contact with the cells is coated with the culture medium. The culture medium is preferably a matrix that enhances or improves the adhesion of cells to the surface of the cell culture apparatus. By coating the surface of the cell culture apparatus with this culture medium, the adhesion of cells to the surface of the cell culture apparatus can be enhanced or improved. Examples of coating treatment include placing a solution containing a culture medium into the cell culture apparatus and then appropriately removing the solution, but this is not particularly limited to these methods; known coating treatments can be appropriately employed.

[0222] There are no particular limitations on the culture medium. Examples of extracellular matrix components include laminin, collagen, fibronectin, hydrin, matrix gel, fibrin, and thrombin; amino acid polymers such as poly-L-lysine and poly-D-lysine, and their fragments. One or more of these can be used.

[0223] The culture medium preferably contains one or more of collagen and its fragments, laminin and its fragments, more preferably laminin and its fragments. Laminin is preferably derived from human sources. Laminin and its fragments preferably exhibit a dissociation constant below 10 nM when binding to integrin α6B1. Commercially available collagen or collagen fragments, and laminin or laminin fragments are preferably used.

[0224] Examples of laminin fragments include the E8 fragment (also known as the laminin 511E8 fragment or laminin 511E8) obtained by digesting laminin 511 with elastase (Non-Patent Literature 5 (Reference): Ido H, et al., J Biol Chem. 2007, 282, 11144-11154), and the recombinant human laminin 511E8 fragment expressed using recombinant silkworm cocoons.

[0225] Among laminin and its fragments, laminin fragments are preferred, laminin 511 fragments are more preferred, laminin 511E8 fragments are even more preferred, and those derived from humans are even more preferred.

[0226] <e. Three-dimensional structures with core-shell structures obtained by the manufacturing method of this embodiment> Using the manufacturing method of this embodiment, a core-shell structured three-dimensional structure can be manufactured, comprising: a cell layer, the cell layer being composed of at least a plurality of tight junctions between cells present in the surface cell layer and a plurality of epidermal keratinocytes; and microspheres, the microspheres being the central part of the three-dimensional structure and having extracellular matrix components on their surface; more preferably, a core-shell structured three-dimensional structure containing an epidermal keratinocyte layer comprising an undifferentiated cell layer and a differentiated cell layer as the shell portion.

[0227] Furthermore, the obtained three-dimensional structure preferably possesses the following properties: appropriate staining characteristics and appropriate local presence can be confirmed using TJ markers, proliferation markers, extracellular matrix component markers, and basal cell markers. The size (major axis or diameter) of the obtained three-dimensional structure is preferably 300–700 μm, the size (major axis or diameter) of the fibroblast mass within the three-dimensional structure is preferably 240–560 μm, and the size (thickness) of the cell layer containing epidermal keratinocytes within the three-dimensional structure is preferably 60–140 μm. Additionally, regarding their size, the configuration described in <Shape, Size, etc. of Three-Dimensional Structures> can be appropriately adopted.

[0228] Furthermore, by utilizing the manufacturing method described in this embodiment, the aforementioned "1. Three-dimensional structure described in this embodiment" can be obtained.

[0229] Regarding the culturable period of the obtained three-dimensional structure, the appropriate lower and upper limits can be appropriately adopted from the description in "2. Method for Manufacturing Three-Dimensional Structures According to This Embodiment" above, and are preferably 4 to 35 days, and even more preferably 6 to 22 days. The starting point of this culturable period is more preferably "after inoculation of epidermal keratinocytes". It should be noted that the culture medium of the three-dimensional structure is preferably changed every day using co-culture medium.

[0230] In this specification, "events" such as "performing cultivation" can be designated as "processes" or "steps," "processes" can be designated as "events" or "steps," and "steps" can be designated as "events" or "processes." Furthermore, in this embodiment, "process" can refer to "an apparatus or part configured to perform a process," "apparatus" can refer to "a mechanism or part," and "part" can refer to "a mechanism, apparatus, or part or apparatus for use in a system, etc."

[0231] 3. The skin homeostasis maintenance function evaluation method using the three-dimensional structure involved in this embodiment. The method of this embodiment can provide a way to evaluate the skin homeostasis maintenance function using the three-dimensional structure involved in this embodiment, or a three-dimensional structure obtained by the manufacturing method of the three-dimensional structure involved in this embodiment.

[0232] The evaluation period for the three-dimensional structure involved in this embodiment is not particularly limited, but is preferably 6 to 22 days after inoculating the fibroblast block with epidermal keratinocytes. It should be noted that the three-dimensional structure can be formed approximately 3 days after inoculating the fibroblast block with epidermal keratinocytes.

[0233] In the description of this embodiment, descriptions of the components and processing methods of epidermal keratinocytes, microspheres, spherical microcarriers, fibroblasts, nucleoshell structures, pluripotent stem cells, cell culture equipment, etc., that are repeated with the descriptions of "2.", "3.", "4.", etc., described above, are also applicable to this embodiment and may be used as appropriate.

[0234] By applying various physical or chemical treatments to the three-dimensional structure, new physical properties (tension, elasticity, pigmentation, cracking, etc.) and chemical treatment capabilities (test substances, acid or alkali components, sebum, etc.) not present in previous models (animal skin models or three-dimensional skin models) can be exhibited. In this embodiment, cells derived from pluripotent stem cells can be used, thus enabling the creation of customized three-dimensional structures by using somatic cells from the target animal, and confirming their physical, chemical, or physiological / biochemical states. As tests for the three-dimensional structure, one or more can be selected from physical tests (e.g., pressure regulation, vibration regulation, pressure regulation, irradiation with ultraviolet light or natural light, etc.), physiological / biochemical tests (markers, fluorescent staining, etc.), and chemical tests (exploration of test substances, etc.).

[0235] By using or exposing the three-dimensional structure according to this embodiment to the test substance, it is possible to evaluate or select test substances that affect the skin. Preferably, the three-dimensional structure is cultured in a culture medium containing or without the test substance. In this case, when evaluating the test substance, the results can be compared with those of a control (e.g., a control without additive, a positive control, or a negative control). The concentration of the test substance can be adjusted to evaluate the skin homeostasis maintenance function based on concentration changes (increase, decrease, gradient).

[0236] In the method for evaluating skin homeostasis maintenance function, since a three-dimensional structure is used, the culture conditions under which <c. epidermal keratinocytes are coated with microspheres> are preferred. As the culture medium used, a co-culture medium or an epidermal keratinocyte culture medium (preferably a co-culture medium, but depending on the situation, an epidermal keratinocyte culture medium) is preferred, and the co-culture medium is preferably changed every day. Alternatively, a co-culture medium or an epidermal keratinocyte culture medium containing the test substance may also be used. The evaluation period is preferably the culturing period of the three-dimensional structure, for example, more preferably 6 to 22 days, or this culturing period may be set as the evaluation period or the usable period of the three-dimensional structure.

[0237] As another aspect of the method in this embodiment, it is also possible to provide substances that exert skin homeostasis maintenance functions, or effective ingredients for skin homeostasis maintenance functions, and / or skin homeostasis maintenance agents. Therefore, it is possible to provide technology related to skin homeostasis maintenance functions; specifically, it is possible to provide substances that have skin homeostasis maintenance effects, or effective ingredients for skin homeostasis maintenance functions, and / or skin homeostasis maintenance agents. Thus, it is possible to provide agents for maintaining skin homeostasis. It should be noted that the reagent can be a composition.

[0238] The substance selected by the evaluation and / or selection method of the skin homeostasis maintenance agent in this embodiment (hereinafter also referred to as "selected substance") can be provided as a substance with skin homeostasis maintenance function or as an effective component of the skin homeostasis maintenance agent.

[0239] In addition, the evaluation and / or selection method of the skin homeostasis maintenance agent can be the device or system for evaluating and / or selecting the skin homeostasis maintenance agent in this embodiment.

[0240] Examples of skin homeostasis maintenance functions include, but are not limited to, skin barrier function maintenance, skin turnover maintenance, anti-inflammatory effects, antioxidant effects, and skin tone adjustment. More specifically, examples include moisturizing effects based on skin barrier function maintenance and skin lesion prevention effects based on anti-inflammatory effects. One or more of these can be selected. Therefore, the selected substance obtained by the evaluation and / or selection method of the skin homeostasis maintenance agent of this embodiment has skin homeostasis maintenance function, and thus its effects or influences on skin barrier function, turnover, moisturizing, and skin lesion prevention can be confirmed.

[0241] The aforementioned selected substances are not particularly limited and can be any from natural or artificial sources, and can be any pure substance or mixture. Furthermore, the selected substances are preferably one or more selected from compounds, microorganisms or their cultures, extracts, mixtures thereof, and compositions thereof. Compounds can be any inorganic or organic compounds.

[0242] More suitable specific selections could include lipids, vitamins, carotenoids, etc., and phospholipids or their derivatives (e.g., LPA).

[0243] The aforementioned selected substances can be contained in or used as active ingredients in skin homeostasis maintenance agents (preferably skin barrier function promoters, moisturizing or replacement promoters), skin lesion prevention, improvement, and treatment agents (hereinafter also referred to as "skin homeostasis maintenance agents, etc."). It should be noted that the agent can be a composition.

[0244] Furthermore, the aforementioned selected substances can be used to manufacture skin homeostasis maintainers, etc. Additionally, this embodiment can also provide the aforementioned selected substances, or their applications, for maintaining skin homeostasis or for use in maintaining skin homeostasis.

[0245] This embodiment can also provide a method for maintaining skin homeostasis using the aforementioned selected substance, or a method for maintaining skin homeostasis using a reagent containing the aforementioned selected substance.

[0246] <Disease, Symptoms> The aforementioned selected substances, due to their physiological activity, can be used in methods for preventing, improving, or treating symptoms of diseases or skin lesions caused by decreased skin homeostasis, sun exposure, etc.

[0247] As a disease or symptom of skin lesions caused by skin lesions, sun exposure, etc., there are no particular limitations. For example, eczema, pigmentation, wrinkles, sagging, acne, pustules, dry skin, etc. can be mentioned. One or more of them can be selected from the group consisting of them.

[0248] In addition, as for the uses of this embodiment, for example, it can be used for the prevention, improvement or treatment of skin barrier function, dry skin, wrinkles, acne, etc., but is not limited to these.

[0249] In this embodiment, "prevention" refers to preventing or delaying the onset of symptoms or disease in the target population, or reducing the risk of symptom or disease onset in the target population. In this technology, "improvement" refers to: improvement or maintenance of the target population's disease, symptoms, or condition; prevention or delay of deterioration; reversal, prevention, or delay of progression.

[0250] This embodiment enables the aforementioned selected substances to be used in, for example, cosmetics, topical skin agents, quasi-pharmaceuticals, food and beverages, animal feed, etc., but is not particularly limited to these.

[0251] In addition, the aforementioned "skin homeostasis maintenance agents, etc." can also be used as cosmetics, topical skin agents, quasi-medicines, food and beverages, feed, etc., but are not specifically limited to these.

[0252] Among them, cosmetics, topical skin agents, pharmaceuticals, and quasi-pharmaceuticals are preferred for use in affecting the epidermis and / or dermis of the skin.

[0253] When this embodiment is used in cosmetics and topical skin agents, the aforementioned selected substances or skin homeostasis maintaining agents can be incorporated into various forms of cosmetics or topical skin agents, such as lotions, creams, lotions, masks, facial cleansers, makeup products, dispersions, ointments, liquids, aerosols, patches, poultices, and liniments, but are not limited to these.

[0254] In the case of pharmaceuticals and quasi-pharmaceuticals, the aforementioned selected substances or skin homeostasis maintenance agents may be incorporated into oral preparations such as tablets, capsules, granules, powders, liquids, and suspensions; topical preparations such as transdermal preparations, patches, eye drops, nasal drops, oral preparations, and suppositories; and non-oral preparations such as drops and injections, but are not limited to these.

[0255] The aforementioned selected substances or whole plasma secretion regulators can be manufactured using known manufacturing methods. The aforementioned selected substances can be commercially available products.

[0256] There is no particular limitation on the content of the aforementioned selected substances. The total amount of the reagent or composition may contain approximately 0.001 to 99% by mass, more preferably 0.01 to 50% by mass, and even more preferably about 0.1 to 10% by mass of the aforementioned selected substances.

[0257] Regarding the applicable objects of the aforementioned selected substances or skin homeostasis maintenance agents, they can be applied to humans and non-human animals (e.g., pets, livestock, etc.). Among them, humans and pets are preferred, and humans are more preferred.

[0258] Methods of using the aforementioned selected substances or skin homeostasis maintenance agents may include transdermal administration, oral administration, injection-based administration, oral ingestion, and application to the skin, but are not limited to these.

[0259] The amount of the selected substance used or applied can be any amount that achieves the effect of the present invention, and there are no particular limitations. It can be appropriately adjusted according to the dosage form of the preparation, the applicable site, age, gender, etc.

[0260] Skin homeostasis maintenance functions, etc., can be manufactured using known manufacturing methods. Furthermore, in addition to the aforementioned selected substances, various optional ingredients such as additives can be used as needed to maintain these skin homeostasis functions.

[0261] As the aforementioned optional ingredients, they can be appropriately combined with permissible ingredients found in cosmetics, topical skin agents, pharmaceuticals, food and beverages, or animal feed. For example, one or more of the following can be appropriately used: excipients, colorants, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, preservatives, humectants, pH adjusters, etc., thereby obtaining the desired dosage form.

[0262] 4. In addition, the present technology may also adopt other aspects, or the following structures or technical features as appropriate.

[0263] ·〔1〕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 multiple epidermal keratinocytes; and The central part consists of microspheres with extracellular matrix components on their surface.

[0264] Preferably, it consists of at least a plurality of tight junctions present between cells in the cell layer on the surface of the aforementioned cell layer, and a plurality of epidermal keratinocytes. The aforementioned cell layer is preferably a layer consisting of at least a differentiated layer containing differentiated epidermal keratinocytes and an undifferentiated layer containing undifferentiated epidermal keratinocytes located in the central direction. Furthermore, the aforementioned tight junctions are preferably present between adjacent cells of the differentiated epidermal keratinocytes in the cell layer present on the surface of the three-dimensional structure.

[0265] ·〔2〕The three-dimensional structure as described in 〔1〕 above, wherein the aforementioned microspheres are microcarriers or cell blocks.

[0266] ·〔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 multiple epidermal keratinocytes; and A fibroblast mass consisting of at least multiple fibroblasts, which is the central part of a three-dimensional structure.

[0267] ·〔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 multiple epidermal keratinocytes; and The spherical microcarriers with extracellular matrix components on their surface are the central part of the three-dimensional structure.

[0268] ·〔5〕A three-dimensional structure as described in any one of〔1〕 to 〔4〕 above, wherein the aforementioned epidermal keratinocytes or the aforementioned 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.

[0269] ·〔6〕A three-dimensional structure as described in any one of 〔1〕 to 〔5〕 above, used for evaluating the skin homeostasis maintenance function.

[0270] ·〔7〕A method for manufacturing a three-dimensional structure with a core-shell structure, wherein microspheres are co-cultured with inoculated epidermal keratinocytes.

[0271] The aforementioned microspheres are preferably microcarriers or cell blocks, more preferably generally spherical. The microcarriers are preferably coated with extracellular matrix components. The cell blocks are preferably fibroblast cell blocks prepared from multiple fibroblasts.

[0272] [8] A method for manufacturing a three-dimensional structure with a core-shell structure, wherein microspheres are co-cultured with inoculated epidermal keratinocytes using cell culture equipment. The microspheres are preferably fibroblast blocks prepared from inoculated fibroblasts or microcarriers coated with extracellular matrix components.

[0273] [9] A method for manufacturing a three-dimensional structure with a core-shell structure, wherein epidermal keratinocytes are seeded onto a cell culture apparatus or culture medium containing microspheres and cultured. Preferably, the cells used are cultured on a cell adhesion inhibition-treated surface with a mortar-shaped recess, and the cells used are preferably fibroblast blocks and / or epidermal keratinocytes. In addition, it is preferable to use a cell culture apparatus having one or more wells containing co-culture medium.

[0274] ·

[10] A method for manufacturing a three-dimensional structure with a core-shell structure, comprising placing microspheres on a cell adhesion inhibition treatment surface in a mortar-shaped recess, then inoculating epidermal keratinocytes and co-culturing them to form an epidermal keratinocyte layer covering the microspheres as a shell, the cell layer being formed to include: an undifferentiated epidermal keratinocyte layer; a differentiated epidermal keratinocyte layer; and a plurality of tight junctions between the differentiated epidermal keratinocytes present in the outermost layer.

[0275] The aforementioned microspheres can be cell blocks containing extracellular matrix components obtained by inoculating and culturing fibroblasts, or microcarriers coated with extracellular matrix components.

[0276] ·〔11〕A method for manufacturing a three-dimensional structure as described in any one of 〔7〕 to 〔10〕, wherein when culturing the aforementioned microspheres with inoculated epidermal keratinocytes, a culture medium containing at least a culture medium for epidermal keratinocytes, or a culture medium containing a culture medium for epidermal keratinocytes and a culture medium for fibroblasts, is used.

[0277] ·〔12〕The method for manufacturing a three-dimensional structure as described in any one of 〔7〕 to 〔11〕 above, wherein the culture period after inoculating the aforementioned epidermal keratinocytes is preferably 6 days or more, more preferably 6 to 22 days. This culture period can be a cultureable period, a testable period, or an evaluable period, during which tests and evaluations of the three-dimensional structure described in any one of 〔7〕 to 〔11〕 above can be conducted.

[0278] ·〔13〕The method for manufacturing a three-dimensional structure as described in any one of 〔7〕 to 〔12〕 above, wherein the structure is cultured such that the size of the three-dimensional structure is 1.05 or more and 1.5 or less when the size of the microsphere is set to 1.

[0279] ·〔14〕The method for manufacturing a three-dimensional structure as described in any one of 〔7〕 to 〔13〕, wherein the seeding ratio of the aforementioned fibroblasts to the aforementioned epidermal keratinocytes is preferably 1:4 or more, more preferably 1:4 to 8:1.

[0280] ·〔15〕The method for manufacturing a three-dimensional structure as described in any one of 〔7〕 to 〔14〕, wherein the aforementioned epidermal keratinocytes or the aforementioned 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.

[0281]

[16] The method for manufacturing a three-dimensional structure as described in any one of [7] to

[15] above, wherein the aforementioned seeded epidermal keratinocytes or the aforementioned seeded fibroblasts are one or more 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). The iPS cells are more preferably iPS cells based on somatic cells derived from the target animal.

[0282] ·〔17〕A method for manufacturing a three-dimensional structure as described in any one of〔7〕 to 〔16〕, wherein the aforementioned microcarrier is derived from organic matter, inorganic matter, or composite materials thereof.

[0283] The aforementioned microcarrier is preferably a resin, and more preferably one or more selected from polyvinyl alcohol, polylactic acid, polyglycolic acid, polytetrafluoroethylene, polystyrene, polyester, polyurethane, polyethylene, polypropylene, (meth)acrylic polymers, (meth)acrylamide polymers, organosilicon polymers, and epoxy resins.

[0284] ·〔18〕The method for manufacturing a three-dimensional structure as described in any one of 〔7〕 to 〔17〕 above, wherein the aforementioned microcarrier is a microcarrier coated with an extracellular matrix component. The aforementioned extracellular matrix component is preferably collagen (e.g., type 1 to type 19), more preferably type 1 or type 4 collagen.

[0285] ·〔19〕A method for manufacturing a three-dimensional structure or a method for testing a three-dimensional structure, comprising: (1) preparing microspheres (preferably microcarriers or fibroblast blocks); and / or (2) obtaining a core-shell structure based on the microspheres and epidermal keratinocytes.

[0286] ·

[20] The method described in

[19] above includes proliferating the cells to be inoculated prior to (1) and / or (2) above.

[0287] •

[21] The method as described in any one of

[19] to

[20] above, comprising (3) performing a test using the aforementioned three-dimensional structure after (2) above. The aforementioned test is preferably a test for evaluating the skin homeostasis maintenance function.

[0288] ·〔22〕The method described in 〔21〕as described above, wherein the aforementioned (3) test is one or more selected from physical tests, physiological and biochemical tests and chemical tests for the aforementioned three-dimensional structure.

[0289] ·

[23] The method as described in any one of

[19] to

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

[0290] ·

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

[18] above.

[0291]

[25] A method that uses the three-dimensional structure described above

[24] to evaluate or select substances that have a skin homeostasis maintenance function (positive or negative).

[0292] ·

[26] The method described in

[24] or

[25] above uses a culture medium (epidermal keratinocyte culture medium or co-culture medium) or a co-culture medium that contains at least a culture medium for epidermal keratinocytes. When the aforementioned microspheres are microcarriers, a culture medium containing at least a culture medium for epidermal keratinocytes is preferred; when fibroblast blocks are used, a co-culture medium is preferred.

[0293] ·

[27] Used in the method described in

[24] or

[25] above, for the preparation of a mixture comprising a culture medium for fibroblasts and a culture medium for epidermal keratinocytes; or, for the preparation of a culture medium for fibroblasts and a culture medium for epidermal keratinocytes in the manufacture of a culture medium used in the method described in

[24] or

[25] above.

[0294] Example The present embodiments will be described in further detail below, based on examples and the like. It should be noted that the examples described below represent only one example of a representative embodiment of the present technology and are not intended to narrowly interpret the scope of the present technology.

[0295] <<Example 1: Manufacturing of a Fibroblast Block: A Three-Dimensional Structure with a Core-Shell Structure and Its Manufacturing Method>> A three-dimensional structure with a core-shell structure, consisting of fibroblast masses as the core and epidermal keratinocytes as the shell, is manufactured in the following manner (see [reference]). Figure 3 This serves as a three-dimensional structure for the core-shell structure used in Example 1. Specifically, fibroblasts are seeded into wells and cultured for a certain period of time to create the nucleus of the fibroblast block. Then, epidermal keratinized cells used to form the shell are seeded into the same wells and then statically cultured.

[0296] <Nuclear Preparation of Three-Dimensional Structures> The fibroblast block of the nucleus, which is a three-dimensional structure, is manufactured in the following manner.

[0297] [Regulation of fibroblast seeding number] Normal human fibroblasts (NHDF, LIFELINE CELL TECHNOLOGY, model 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 proliferation of normal human skin fibroblasts (phenol red-free), FibroLife S2 Comp kit LIFELINE CELL TECHNOLOGY, model LFC-LL0011). Culture was performed using polystyrene tissue culture plates (cylindrical wells, coating agent: laminin 511E8 fragment). The medium was replaced with FibroLife S2 medium on day 2.

[0298] [FibroLife S2 Comp kit (LFB-LM0001 and LFK-LS1038)] LFB-LM0001: FibroLife BM: Basal Culture Medium for Skin Fibroblasts※ (Phenol Red and Antibacterial Agent Free) 500mL LFK-LS1038: FibroLife S2 LifeFactors: Skin Fibroblast Proliferation Additive Kit 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 concentration in kit medium) After culturing the fibroblasts for 3 days, a cell suspension was prepared. In the preparation of fibroblast cell blocks, the cell suspension was used to inoculate fibroblasts and create a nucleus that forms the core of a three-dimensional structure.

[0299] Fibroblasts were washed with PBS (-) and then subjected to TrypLE. （TM） The stripping process was performed using Select enzyme (Gibco, model 12563011).

[0300] The dissected cells were separated using a centrifuge and cultured in DMEM / F12 medium supplemented with 5% FBS (Sigma-Aldrich) and 1% CaCl2 to form 1×10⁻⁶ cells. 5Cells are suspended in a manner that uses cells per mL.

[0301] It should be noted that the cell counting was performed using a Coulter counter (CDA-1000, Sysmex Corporation).

[0302] [Preparation of fibroblast cell blocks] Fibroblast cell suspension (1×10) 5 Cells (100 μL / well) were seeded at 100 μL / well in a 96-well V-shaped plate and cultured for 1 day at 37°C and 5% CO2. After one day of post-seeding culture, the cells aggregated on the bottom surface of the V-shaped base of the wells, forming clumps. In this way, fibroblastic cell clumps can be prepared and used as the core of three-dimensional structures.

[0303] It should be noted that the normal seeding number of fibroblasts is 1 × 10⁶ per well. 4 Cells. The 96-well multi-well plate with a V-shaped bottom is as follows: Material: PS (polystyrene), completed cell culture surface treatment (hydrophilization surface treatment of polymer), pyrogen-free, sterilized, culture area per well (cm²). 2 ): 0.38, Maximum capacity per well (mL): 0.32, Dimensions (mm): 127.8×85.5×16.5.

[0304] <Shell Preparation of Three-Dimensional Structures> Epidermal keratinocytes induced by human iPS cells were seeded into the nuclei of fibroblast blocks present in each well of a 96-well V-bottom multi-well plate and co-cultured to fabricate the three-dimensional structure of Example 1. It should be noted that normal human epidermal keratinocytes can also be used instead of human iPS cells to fabricate three-dimensional structures through co-culture.

[0305] [Regulation of the number of epidermal keratinocytes derived from human iPS cells] Epidermal keratinocytes derived from human iPS cells were cultured at 37°C and 5% CO2 in CnT-07 (CELLnTEC) medium supplemented with 20 ng / mL EGF (MACS) and 10 μM Y-27632. Culture was performed using polystyrene tissue culture plates (cylindrical wells, coated with laminin 511E8 fragment). 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.

[0306] The co-culture of epidermal keratinocytes and fibroblasts used a co-culture medium prepared by mixing DMEM / F12 medium (fibroblast medium) supplemented with 5% FBS (Sigma-Aldrich) and 1mM CaCl2 with CnT-PR-3D medium (CELLnTEC) (epidermal keratinocyte medium) at a 1:1 (volume:volume) ratio (hereinafter also referred to as "half medium").

[0307] Regarding the seeding of epidermal keratinocytes, after washing the cells with PBS (-), TrypLE was used. （TM） Select enzyme was used for peeling. The peeled epidermal keratinocytes were separated using a centrifuge and cultured in the aforementioned co-culture medium until the epidermal keratinocytes reached a density of 5 × 10⁻⁶ cells / cm². 4 Epidermal keratinocytes were suspended at a rate of 1 cell / mL. While retaining fibroblast blocks, fibroblasts were removed from each well of the 96-well multi-well plate using culture medium. After removing the culture medium from each well, the epidermal keratinocyte suspension was seeded at 100 μL / well into each well containing the fibroblast blocks. Each well contained one fibroblast block and 5 × 10⁶ cells / mL. 3 One cell / mL of co-culture medium containing epidermal keratinocytes.

[0308] The three-dimensional structure (Example 1) containing the nucleus-shell structure of fibroblasts and epidermal keratinocytes, manufactured as described above, was cultured and maintained at 37°C and 5% CO2. The co-culture medium was replaced daily at a rate of 100 μL / well. The three-dimensional structure, consisting of at least the nucleus of a normal fibroblast and the shell of an epidermal keratinocyte derived from iPS cells after differentiation induction, was successfully cultured continuously in the co-culture medium for 22 days.

[0309] [Various signs and markers] As a marker of tight junctions (TJ), occludin was chosen. As a proliferation marker, Ki67 was chosen. As a basal cell marker, type XVII collagen was chosen. These were performed using known methods or commercially available antibodies. Commercially available examples include occludin antibodies: anti-occludin (Occludin) antibody clone 1C17 ZooMAb. （R） Rabbit monoclonal antibody (Sigma-Aldrich), Ki67 antibody: anti-Ki67 antibody clone 1O15 ZooMAb （R） Rabbit monoclonal antibody (Sigma-Aldrich), type XVII collagen antibody: anti-COL17A1 [NC16a-3] antibody (GeneTex), etc.

[0310] [Regarding RNA sampling] 1. Collect the spheroids along with the culture medium from the same conditions into a sterilized 1.5 mL tube; 2. Remove as much culture medium as possible; 3. Add 400 μL of TRIzol; 4. Vortex until the spheroids disappear; 5. Store at -80°C.

[0311] [Regarding sampling (staining)] 1. Transfer the spherical bodies under the same conditions onto a glass slide; 2. Fix the spherical bodies with ethanol; 3. After blocking, perform immunostaining using the primary and secondary antibodies of the target.

[0312] <Experimental Example 1: Study on the Mixing Ratio of Culture Media> Regarding the culture medium, a medium prepared by mixing DMEM / F12 medium supplemented with 5% FBS and 1mM CaCl2 with CnT-PR-3D medium at the ratios listed in Table 1 was used. Regarding the cells, epidermal keratinocytes (iKCs) differentiated from iPS cells were used as epidermal keratinocytes, and NHDF was used as fibroblasts. Following the above-described <Three-Dimensional Structures of Core-Shell Structures and Their Manufacturing Methods>, the three-dimensional structure of this manufacturing example 1 was obtained. Three weeks after iKC inoculation, the three-dimensional structure was immunostained with anti-closure protein antibody and photographed using a confocal laser microscope (LSM900, ZEISS). Regarding the obtained images, ImageJ was used to measure the overall area of ​​the three-dimensional structure and the area of ​​manually specified closure protein-positive cell regions. The area of ​​the closure protein-positive cell regions / the overall area of ​​the three-dimensional structure was taken as the occupancy rate of closure protein-positive cells relative to the overall three-dimensional structure. Figure 4 ).

[0313] • Tight junctions: Staining was performed using antibodies targeting the closing proteins that are building blocks. They exist in a mesh-like manner, covering the outermost layer of the three-dimensional structure.

[0314] • State of fibroblast masses: fibroblast masses are spherical and have low transparency compared to the epidermal keratinocyte layer.

[0315] • The state of the epidermal keratinocyte layer: a spherical structure formed by uniformly wrapped fibrous cell blocks.

[0316] <Size of the obtained three-dimensional core-shell structure> The size of the three-dimensional core-shell structure (sphere) obtained in this Manufacturing Example 1 varies depending on the culture conditions (cell seeding number, culture days, culture medium type, etc.), ranging from approximately 300 to 700 μm. The major axis of the three-dimensional core-shell structure in this Manufacturing Example 1 is, for example, 442, 460, and 470 μm. Regarding the major axis of the three-dimensional structure, the length was measured using ImageJ based on the mid-section image obtained from a confocal laser microscope (LSM900, ZEISS).

[0317] <Results of Experiment 1> [Table 1] <Evaluation Criteria for the Staining Properties of Closure Proteins> Based on the occupancy of closure protein-positive cells relative to the overall three-dimensional structure (see...) Figure 4 The following criteria shall be used for judgment.

[0318] ○ (Excellent): 80% or higher △ (Qualified): 50% or more but less than 80% × (Unacceptable): Less than 50% <Ratio of mixed culture medium and staining properties of closure proteins> When the mixing ratio of epidermal keratinocyte culture medium to fibroblast culture medium is 50:50, the percentage of cells positive for closure protein relative to the overall three-dimensional structure is the highest (91.5%), which is considered excellent. The percentages of 25:75 (73.9%) and 75:25 (53.8%) are the second highest, and are considered acceptable (see [link to relevant documentation]). Figure 5 ).

[0319] <Experimental Example 2: Study on the ratio of seeding numbers of fibroblasts and epidermal keratinocytes> The cells were inoculated with NHDF and iKC at the numbers listed in Table 2, and cultured using half-volume culture medium. Otherwise, the three-dimensional core-shell structure of this manufacturing example 1 was obtained according to the above-described <Three-Dimensional Structures of Core-Shell Structures and Their Manufacturing Methods>. Three weeks after inoculation with iKC, the three-dimensional structure was immunostained with an anti-closure protein antibody and photographed using a confocal laser microscope (LSM900, ZEISS). The calculation of the closure protein occupancy and the evaluation of its suitability were performed using the same methods and criteria as in Example 1.

[0320] [Table 2] [Table 3] <Results of Experiment 2> With a fibroblast to iKC seeding ratio of 8:1, the percentage of closure protein-positive cells relative to the overall three-dimensional structure of Example 1 was the highest (94.2%), followed by 4:1 (81.3%), which was deemed excellent. For 2:1 (76.7%), 1:1 (75.2%), 1:2 (76.9%), and 1:4 (54.2%), the results were deemed acceptable (see [link to documentation]). Figure 6 ).

[0321] <Experimental Example 3: Study on the culture period and confirmation of epidermal keratinocyte-specific markers> For 1.0 x 10 4 NHDF, using 5.0 x 10 3 Using iKC and half a volume of culture medium, and following the above-described method for manufacturing three-dimensional structures of core-shell structures, the three-dimensional structure of this manufacturing example 1 was obtained. Six, 14, and 22 days after iKC inoculation, the three-dimensional structure was recovered, immunostained with various antibodies, and then photographed using a confocal laser microscope (LSM900, ZEISS). Regarding the evaluation criteria for pass / fail, in addition to the criteria for pass / fail in <Experiment 1>, evaluation criteria using epidermal keratinocyte proliferation markers and basal cell markers were also flexibly utilized.

[0322] [Table 4-1] [Table 4-2] <Evaluation Criteria for Using Proliferation Markers> <Staining properties> ○ (Pass) Mottled confirmation of epidermal keratinocyte proliferation. × (Unqualified) No staining of proliferation markers detected. <Local Existence> ○ (Pass) Proliferation markers were identified in epidermal keratinocytes in direct contact with fibroblast masses. × (Unacceptable) Proliferation markers were mottledly identified throughout the epidermal keratinocyte layer. <Evaluation Criteria for Using Basal Cell Markers> <Staining properties> ○ (Pass) Staining of basal cell markers was observed. × (Unacceptable) No staining of basal cell markers was observed. <Local Existence> ○ (Pass) Basal cell markers were only detected in epidermal keratinocytes in direct contact with fibroblast masses. × (Unacceptable) Basal cell markers were detected throughout the epidermal keratinocyte layer. <Comprehensive Evaluation Criteria> ○ (Excellent) Staining of each marker was confirmed by appropriate local presence. △ (Pass) Although staining of various markers was observed, some local areas showed inappropriate staining. × (Unacceptable) No staining of any markers was observed. <Results of Experiment 3> As described above, it is possible to reliably distinguish between undifferentiated and differentiated cells within the epidermal keratinocyte layer. The undifferentiated layer is a type XVII collagen-positive cell layer, while the differentiated layer is a type XVII collagen-negative cell layer. Furthermore, in the outermost layer of the three-dimensional structure, cells expressed in the granular layer, which represents the later differentiation stage of epidermal keratinocytes, were successfully identified. Figure 7 The presence of closure proteins suggests that the epidermal keratinocyte layer undergoes a phased differentiation process from the inside to the outside.

[0323] Furthermore, by culturing spheroids with nuclei covered by iKC from fibroblast blocks in a co-culture medium, the expression of tight junctions and differentiation markers, namely closure proteins, was observed comprehensively in the outermost layer of the spheroids. Additionally, for spheroids cultured for 14 days and 22 days, similar to the organism, the local presence of epidermal keratinocyte proliferation markers and undifferentiated markers (Ki67, type XVII collagen) was confirmed in the epidermal keratinocyte layer in direct contact with the fibroblast block (dermis) (see [link to original text]). Figure 8 ).

[0324] <Experimental Example 4: Study on the types of epidermal keratinocytes> Except for changing the type of epidermal keratinocytes seeded into the fibroblast block, the same procedure as in <Experimental Example 3> was followed to obtain the three-dimensional structure with a core-shell structure of this Manufacturing Example 1. The types of epidermal keratinocytes used in this case were normal human epidermal keratinocytes (HPEK) and iKC. The evaluation criteria for pass / fail were the same as those in <Experimental Example 3>.

[0325] [Table 5] <Results of Experiment 4> The results are shown in Table 5 and Figure 9Regarding 8-day-old globules, expression of closure protein (a marker of tight junctions) and Ki67 (a marker of proliferation) was observed regardless of the type of epidermal keratinocytes seeded onto the nucleus of the fibroblast block. On the other hand, regarding 22-day-old globules, only globules treated with iKC showed positive results for tight junction markers and proliferation markers at appropriate locations (see [link to relevant documentation]). Figure 9 ).

[0326] <Experimental Example 5: Evaluation of Selected Substances> The same procedure as in <Experimental Example 3> was followed to obtain the three-dimensional core-shell structure of this Manufacturing Example 1. After inoculation with epidermal keratinocytes, lysophosphatidic acid (LPA), a known tight junction enhancer, was added at concentrations of 0, 1, 10, and 100 μg / mL on days 5 and 6. The sample was then recovered on day 7, RNA was extracted, cDNA was synthesized, and qPCR was performed using the appropriate primers.

[0327] [Table 6] [Table 7] [Table 8] [Table 9] <Results of Experiment 5> Increased LPA expression in a concentration-dependent manner was observed in sealing protein 1 (CLDN1) and closure protein (OCLN), genes that serve as TJ markers. Increased LPA expression was also observed at a concentration of 100 μg / mL in lobe-rhizoid protein (LOR), a marker of terminal differentiation, and filaggrin (FLG), a marker of keratinization. Based on these findings, it can be concluded that LPA enhances TJ barrier function, promotes terminal differentiation of epidermal keratinocytes, and promotes keratinization.

[0328] <<Example 2 [Microcarrier]: Three-dimensional Core-Shell Structures and Their Manufacturing Methods>> A three-dimensional structure with a core-shell structure, consisting of PVA microspheres as the core and epidermal keratinocytes as the shell, is manufactured in the following manner (see [link to documentation]). Figure 1B This serves as a three-dimensional structure for the core-shell structure in Manufacturing Example 2. Specifically, one PVA microsphere is placed in a well, and epidermal keratinized cells for shell formation are seeded into the same well, followed by static culture. The same method as in Manufacturing Example 1 can be used, except that the cell blocks of fibroblasts are replaced with PVA microspheres.

[0329] <PVA microspheres> Particle size (D50): 200-250μm Surface treatment: Type I collagen Surface area per 1g of dried mass: 2600 cm² 2 Swelling degree (when soaked in PBS): 10 Recommended feed intake for 1 liter of MSC culture: 1.54g Sterilization: Gamma ray irradiation <1. Limited dilution of KURARAY collagen-coated PVA microspheres> (1) Weigh a small amount of powdered PVA microspheres and dilute them with an appropriate amount of half-volume culture medium (DMEM / F12 : CnT-PR-3D = 1:1 supplemented with 5% FBS and 1mM CaCl2). (2) The diluted solution from (1) was seeded at 50 μL / well into a V-bottom 96-well plate. (3) Find a hole under a microscope that contains only one PVA microsphere and use it to obtain the three-dimensional structure of the core-shell structure of Example 2.

[0330] <2. Inoculate HPEK onto PVA microspheres that have been inoculated with limiting dilution> (1) Remove the culture medium from the HPEK cultured on the T75 flask and wash twice with PBS (-). (2) Add 1 mL of the stripping agent TrypLE Select to HPEK. (3) Incubate at 37℃ for 10 min. (4) During incubation, transfer 3 mL of half the culture medium into a 15 mL centrifuge tube. (5) Add 2 mL of PBS (-) to the incubated cells, peel the cells off by blowing and aspiration, and return them to the above (4). (6) Add 2 mL of PBS (-) to the T75 flask again, and after several blow-aspiration cycles, return it to the solution in (4) above. (7) Centrifuge the cells (190×g, 5 minutes) (8) Remove the supernatant and resuspend the precipitated cells in 2 mL of half-volume culture medium. (9) Measure the cell concentration of the cell suspension. (10) to become 5×10 4 Dilute with half the volume of culture medium at a rate of 1 cell / mL. (11) The above (10) was seeded at 100 μL / well into a V-bottom 96-well plate containing PVA microspheres. (12) Incubate at 37℃ and 5% CO2. Replace the medium daily with half the amount of medium at 100 μL / well for 2 weeks.

[0331] <3. Immunostaining of HPEK-PVA microspheres> (1) Place several HPEK-PVA microspheres in a 1.5 mL test tube and remove as much culture medium as possible. (2) Fixation using cold ethanol (3) Remove ethanol and wash with PBS (-). (4) Transfer the spherical body onto a glass slide and seal it. (5) Immunostaining was performed using a dilution of the primary antibody. Diluent for antibodies specific to COL17, Ki67, and the closing protein. (6) Wash with PBS (-) (7) Perform immunostaining using a dilution of the secondary antibody. A reaction solution containing antibodies that recognize each primary antibody and nuclear staining reagent. (8) Wash with PBS (-) (9) Seal the spherical body into the glass slide with a sealing agent. <Experimental Example 6> For the three-dimensional core-shell structure obtained in Manufacturing Example 2, the localized changes over time in the three-dimensional structure were confirmed using fluorescence microscopy using antibodies against Hoechest, type XVII collagen (basal cell marker), Ki67, and closure protein (tight junction (TJ) marker). These markers were used according to the various markers described in Manufacturing Example 1 above, as well as the evaluation criteria for closure protein staining, the evaluation criteria for using proliferation markers, and the evaluation criteria for using basal cell markers.

[0332] This is a grayscale photograph taken using a fluorescence microscope to confirm the changes over time in the localized presence of the types of inoculated epidermal keratinocytes (iKC, HPEK) and epidermal-specific markers (closure protein Ki-67) in the three-dimensional structure of the core-shell structure obtained in Example 2 of this manufacture (see [link]). Figure 10 ).

[0333] The size of the three-dimensional core-shell structure (sphere) obtained in this Manufacturing Example 2 varies depending on the culture conditions (cell seeding number, culture days, culture medium type, etc.), and is approximately in the range of 300-500 μm. The major axis of the three-dimensional core-shell structure in this Manufacturing Example 2 is, for example, 312, 370, and 400 μm. Regarding the major axis of the three-dimensional structure, the length was measured using ImageJ based on the mid-section image obtained from a confocal laser microscope (LSM900, ZEISS).

[0334] Based on these results, it can be seen that, like in Manufacturing Example 1, Example 2 can stably distinguish between undifferentiated and differentiated cells in the epidermal keratinocyte layer. The undifferentiated layer is a type XVII collagen-positive cell layer, and the differentiated layer is a type XVII collagen-negative cell layer. Furthermore, a closing protein expressed in the granular layer, which represents the later differentiation stage of epidermal keratinocytes, was successfully identified in the outermost layer of the three-dimensional structure. Therefore, it can be inferred that the epidermal keratinocyte layer undergoes a phased differentiation process from the inside to the outside.

[0335] <Experimental Example 7> The presence of factors (Col 1 or Col 4) constituting extracellular matrix components on the surface of the fibroblast block of the nuclear portion of the three-dimensional structure obtained in this manufacturing example 1 was confirmed.

[0336] The results confirmed the presence of Col 1 and Col 4 around the fibroblast mass (see [link to article]). Figure 11 ).

[0337] Therefore, it can be seen that at least collagen and extracellular matrix components are present on the surface of the core portion in Manufacturing Example 1 and Manufacturing Example 2.

[0338] <Comprehensive Study> Thus, a three-dimensional structure with a core-shell structure, consisting of a fibroblast mass as the core and epidermal keratinocytes as the shell, was successfully obtained in a single well. The central part of the three-dimensional structure obtained in Example 1 is a fibroblast mass composed of multiple fibroblasts, with multiple epidermal keratinocytes covering this fibroblast mass in layers. The epidermal keratinocytes present on the surface in contact with the outside are tightly connected to each other. The epidermal keratinocytes in contact with the fibroblast mass are undifferentiated, forming a first layer, which corresponds to the basal cell layer. A second layer, a third layer, and so on exist in the outer diameter direction of this first layer, with multiple differentiated epidermal keratinocytes forming differentiated epidermal keratinocyte layers (single or multiple layers).

[0339] As shown above, it has been successfully confirmed that spherical bodies (blocks) can be manufactured by inoculating epidermal keratinocytes into a cell culture apparatus containing fibroblasts prepared from the inoculated fibroblasts and culturing them.

[0340] Furthermore, the spherical body (block) is a three-dimensional block with a core-shell structure, wherein (a) the central part is a block of fibroblasts composed of at least a plurality of fibroblasts, and (b) the shell part is a cell layer consisting of at least a plurality of tight junctions between cells in the cell layer present on the surface and a plurality of epidermal keratinocytes, including an undifferentiated layer and a differentiated layer.

[0341] Furthermore, a three-dimensional structure with a core-shell structure, using a microcarrier as the core and epidermal keratinocytes as the shell, was successfully obtained in a single well. Regarding the three-dimensional structure obtained in Example 2, the central part is a PVA microsphere covered by multiple layers of epidermal keratinocytes, with multiple tightly connected epidermal keratinocytes on the surface in contact with the outside. The epidermal keratinocytes in contact with the fibroblast mass are undifferentiated, forming a first layer, which corresponds to the basal cell layer. Along the outer diameter of this first layer, there are second, third, and so on layers, with multiple differentiated epidermal keratinocytes forming differentiated epidermal keratinocyte layers (single or multiple layers).

[0342] As shown above, it has been successfully confirmed that spheroids (blocks) can be produced by seeding epidermal keratinocytes into a cell culture apparatus containing a PVA microcarrier and culturing them.

[0343] Furthermore, the spherical body (block) is a three-dimensional block with a core-shell structure, wherein (a) the central part is a microcarrier, and (b) the shell part is a cell layer consisting of at least a number of tight connections between cells in the cell layer present on the surface and a number of epidermal keratinocytes, including an undifferentiated layer and a differentiated layer.

[0344] The inventors of this application believe that, for a three-dimensional structure with a core-shell structure, it is preferred that the core portion can be any of cell blocks and microcarriers, which have extracellular matrix components on their surface and a cell layer of epidermal keratinocytes around them.

[0345] Thus, a three-dimensional structure with a core-shell structure was successfully provided, comprising: a cell layer having tight junctions present on the surface and consisting of at least a plurality of epidermal keratinocytes; and a central portion consisting of microspheres having extracellular matrix components on the surface.

[0346] Explanation of reference numerals in the attached figures 1. A three-dimensional structure with a core-shell structure; 1a. A three-dimensional structure according to the first embodiment; 1b. A three-dimensional structure according to the second embodiment; 10a. A fibroblast block; 10b. A microcarrier; 11. Fibroblasts; 20. A cell layer of epidermal keratinocytes; 21. An undifferentiated layer of epidermal keratinocytes; 22. A differentiated layer of epidermal keratinocytes; 23. The outermost layer; 201. Undifferentiated epidermal keratinocytes; 202. Differentiated epidermal keratinocytes; 30. Tight junctions

Claims

1. 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 multiple epidermal keratinocytes; and The central part consists of microspheres with extracellular matrix components on their surface.

2. The three-dimensional structure as described in claim 1, wherein, The microspheres are microcarriers or cell blocks.

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

4. The three-dimensional structure as described in claim 1 or 2, used for evaluating skin homeostasis maintenance function.

5. A method for manufacturing a three-dimensional structure with a core-shell structure, wherein microspheres are co-cultured with inoculated epidermal keratinocytes.

6. The method for manufacturing a three-dimensional structure as described in claim 5, wherein, When culturing the microspheres with inoculated epidermal keratinocytes, a culture medium containing at least a culture medium for epidermal keratinocytes, or a culture medium containing both a culture medium for epidermal keratinocytes and a culture medium for fibroblasts, is used.

7. The method for manufacturing a three-dimensional structure as described in claim 5 or 6, wherein, The microspheres are cultured such that the size of the three-dimensional structure is between 1.05 and 1.5 when the size of the microspheres is set to 1.

8. A method for evaluating the skin's homeostasis maintenance function, which uses the three-dimensional structure described in claim 1.

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