Method for producing three-dimensional skin tissue, and three-dimensional skin tissue

Abstract

To provide a novel technique for producing layered three-dimensional skin tissue in which epidermal and dermal layers are laminated, from stem cells.SOLUTION: The present invention, which solves the above problems, is a method for producing three-dimensional skin tissue, including a skin tissue formation step of producing three-dimensional skin tissue in which a dermal layer and an epidermal layer are laminated. The skin tissue formation step includes a three-dimensional culture step of placing a three-dimensional culture obtained by suspension culture of stem cells on a membrane, which is permeable to a culture liquid, within a culture vessel, without incision, and culturing it to obtain a layered three-dimensional skin tissue in which at least the dermal layer and the epidermal layer are laminated.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a technique for producing three-dimensional skin tissue from stem cells. [Background technology]

[0002] Technologies for producing skin organoids are being developed for use as compositions for skin transplantation and as screening tools for active ingredients in pharmaceuticals or cosmetics. To date, techniques have been proposed for producing organoids similar to the epithelial structure in the body using undifferentiated cells or cells with introduced mutations (Non-Patent Document 1), for producing organoids that have both epithelium and stroma (Non-Patent Document 2), and for producing organoids that have an internal vascular network (Non-Patent Document 3, Non-Patent Document 4, Patent Document 1). In particular, Non-Patent Document 5 and Patent Document 2 propose techniques for producing skin organoids having a multi-layered structure of an epithelial layer and a dermal layer.

[0003] In addition, Non-Patent Document 6 discloses a technique in which spherical skin organoids with an epidermal layer formed on the inside, obtained by the method of Non-Patent Document 5, are dissected and cultured on a cell culture insert. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2013 / 047639 [Patent Document 2] International Publication No. 2017 / 070506 [Non-patent literature]

[0005] [Non-Patent Document 1] Sato T et al.,Nature 459,pp262-265,2009 [Non-Patent Document 2] Spence JR et al.,Nature 470,pp105-109,2011 [Non-Patent Document 3] Takebe T et al.,Nature 499,pp481-484,2013 [Non-Patent Document 4] Takebe T et al.,Cell Stem Cell 16,pp556-565,2015 [Non-Patent Document 5] J Lee et al.,Nature 582,pp399-404,2020 [Non-Patent Document 6] Song-yi J et la.,iScience.25(10),105150,2022 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above-mentioned prior art, an objective of the present invention is to provide a novel technique for producing layered three-dimensional skin tissue having a laminated epidermal layer and a laminated dermal layer from stem cells. [Means for solving the problem]

[0007] The present invention for solving the above problems and its preferred embodiments are as follows. [1] A method for producing three-dimensional skin tissue, comprising a skin tissue formation step of producing three-dimensional skin tissue having a laminated dermis layer and an epidermis layer, The skin tissue formation step is a method for producing three-dimensional skin tissue, which includes a three-dimensional culture step in which a three-dimensional culture obtained by culturing stem cells in suspension is placed, without incision, on a membrane of a culture vessel equipped with a membrane permeable to a culture medium, and cultured to obtain a layered three-dimensional skin tissue having at least a dermis layer and an epidermis layer stacked thereon.

[0008] By culturing three-dimensional cultures obtained by culturing stem cells on a membrane without incision, three-dimensional skin tissue with laminated dermis and epidermis layers can be easily obtained.

[0009] [2] The method for producing a three-dimensional skin tissue described in [1], wherein the three-dimensional culture step is carried out 1 to 20 days after the start of differentiation induction of the stem cells. The above-mentioned form can promote the formation of a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are stacked.

[0010] [3] The method for producing a three-dimensional skin tissue described in [1] or [2], wherein the three-dimensional culture step is performed by air-liquid interface culture and / or two-layer liquid interfacial culture in which different culture media are filled above and below the membrane. By adopting the above-mentioned embodiment, three-dimensional skin tissue having a layered structure of a dermis layer and an epidermis layer can be efficiently obtained.

[0011] [4] The method for producing a three-dimensional skin tissue described in [3], wherein in the two-layer liquid culture, an epithelial cell medium is filled above the membrane, and the upper part of the three-dimensional culture is brought into contact with the epithelial cell medium for culture. By adopting the above-mentioned form, differentiation of cells that constitute the epidermis layer can be promoted in the upper part of the three-dimensional culture, thereby efficiently obtaining three-dimensional skin tissue having a layered structure of the dermis layer and the epidermis layer.

[0012] [5] The method for producing a three-dimensional skin tissue described in any one of [1] to [4], wherein the three-dimensional skin tissue is obtained in the three-dimensional culture step in which an epidermis layer is layered above a dermis layer. In the three-dimensional skin tissue of this type, the epidermis layer is formed above the dermis layer, and the epidermis layer is in contact with the outside world. Therefore, the three-dimensional skin tissue of the above type can be suitably used as a clinical or test tool.

[0013] [6] The skin tissue forming step includes: A first induction step of inducing the formation of non-neural ectodermal epithelium by culturing a spheroid containing stem cells in a first differentiation medium containing one or more factors selected from an antagonist of transforming growth factor β (TGFβ) signaling, an agonist of fibroblast growth factor (FGF) signaling, and an agonist of bone morphogenetic protein (BMP) signaling; A method for producing a three-dimensional skin tissue described in any one of [1] to [5], comprising a step of culturing the three-dimensional culture that has undergone the first induction step in a second differentiation medium containing an agonist of fibroblast growth factor (FGF) signaling and / or an antagonist of bone morphogenetic protein (BMP) signaling, thereby inducing the three-dimensional culture into a higher-order structure. By adopting the above-mentioned form, differentiation of the culture into skin tissue can be induced in the skin tissue formation step.

[0014] [7] The method for producing a three-dimensional skin tissue described in any one of [1] to [6], wherein the membrane of the culture vessel is coated with an extracellular matrix component. The above-mentioned form can promote the culture to form a layer structure of a dermis layer and an epidermis layer.

[0015] [8] The method for producing a three-dimensional skin tissue described in any one of [1] to [7], wherein the three-dimensional skin tissue is derived from a human.

[0016] [9] A three-dimensional skin tissue produced by the method for producing three-dimensional skin tissue described in any one of [1] to [8].

[0017]

[10] A method for producing three-dimensional skin tissue described in any one of [1] to [8], wherein in the three-dimensional culture process, the three-dimensional culture placed on the membrane has a maximum diameter of 2 mm or less.

[0018]

[11] The method for producing a three-dimensional skin tissue described in any one of [1] to [8] and

[10] , wherein in the three-dimensional culture step, the three-dimensional culture placed on the membrane does not have hair follicles.

[0019]

[12] The method for producing a three-dimensional skin tissue described in any one of [1] to [8] and

[10] to

[11] , wherein in the three-dimensional culture step, the three-dimensional culture placed on the membrane contains undifferentiated mesenchymal cells or the layer containing the mesenchymal cells has a thickness of 200 μm or less.

[0020]

[13] The method for producing a three-dimensional skin tissue described in any one of [1] to [8] and

[10] to

[12] , wherein the three-dimensional skin tissue obtained in the three-dimensional culture step has hair follicles.

[0021]

[14] A three-dimensional skin tissue prepared by the method for preparing three-dimensional skin tissue described in any one of [1] to [8] and

[10] to

[13] .

[15] The three-dimensional skin tissue described in

[14] having hair follicles. Effect of the Invention

[0022] According to the present invention, a layered three-dimensional skin tissue in which an epidermis layer and a dermis layer are stacked can be produced. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a diagram showing a preferred embodiment of the process flow of the method for producing three-dimensional skin tissue of the present invention. [Diagram 2] FIG. 13 is a diagram showing a schematic diagram of the air-liquid interface culture in the three-dimensional culture step S24. [Diagram 3] FIG. 13 is a diagram showing a schematic diagram of two-layer liquid interphase culture in three-dimensional culture step S24. [Figure 4] 1 is a fluorescent photograph showing the results of immunohistochemical staining of three-dimensional skin tissue on day 27 from the start of induction in Example 1 of Test Example 1. The scale bar is 200 μm. [Diagram 5] 5 is a fluorescent photograph showing the results of immunohistochemical staining of three-dimensional skin tissue having hair follicles in Test Example 2. Figure 5 (a) is Example 10 on the 116th day from the start of induction, (b) is Example 11 on the 116th day from the start of induction, and (c) is Example 12 on the 117th day from the start of induction. The scale bar is 100 μm. The dashed line indicates the position of the membrane of the culture insert, and * indicates the position of the hair follicle. [Figure 6] Fluorescence photographs after immunohistochemical staining, showing the process of inducing differentiation of spheroids into skin tissue in Test Example 3. The scale bar is 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to the creation of three-dimensional skin tissue from stem cells. Hereinafter, one embodiment of the present invention will be described. Note that the present invention is not limited to the embodiment described below, and can be appropriately modified within the scope of the invention.

[0025] In this embodiment, the method for producing three-dimensional skin tissue of the present invention is roughly divided into a spheroid formation step S1 for obtaining stem cell spheroids, and a skin tissue formation step S2 for culturing the spheroids to form three-dimensional skin tissue. The skin tissue formation step S2 includes a step of culturing the spheroids using an induction medium (a first induction step S21 for culturing in a first differentiation medium, optionally a second induction step S22 for culturing in a second differentiation medium, and optionally a third induction step S23 for inducing into a more advanced structure), and a three-dimensional culture step S24 for forming a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated.

[0026] FIG. 1 shows four preferred embodiments of the method for culturing three-dimensional skin tissue of the present invention.

[0027] In the first embodiment, a spheroid formation step S1, a first induction step S21, a second induction step S22, and a three-dimensional culture step S24 are carried out in this order (FIG. 1(a)). The three-dimensional culture step S24 in the first embodiment includes an induction step S241 of culturing in a second differentiation medium, and a maturation step S242 of inducing a higher-order structure in which the dermis layer and the epidermis layer are laminated.

[0028] In the second embodiment, the spheroid formation step S1, the first induction step S21, the second induction step S22, and the three-dimensional culture step S24 are performed in this order (FIG. 1(b)). The three-dimensional culture step S24 in the second embodiment includes the maturation step S242.

[0029] In the third embodiment, the spheroid formation step S1, the first induction step S21, the second induction step S22, the third induction step S23, and the three-dimensional culture step S24 are carried out in this order (FIG. 1(c)). The three-dimensional culture step S24 in the third embodiment includes the maturation step S242.

[0030] In the fourth embodiment, the spheroid formation step S1, the first induction step S21, and the three-dimensional culture step S24 are performed in this order (FIG. 1(d)). The three-dimensional culture step S24 in the fourth embodiment includes the induction step S241 and the maturation step S242.

[0031] In this embodiment, an embodiment including the spheroid formation step S1 is exemplified, but the present invention may be an embodiment in which the process starts from the skin tissue formation step S2 using stem cell spheroids prepared in advance.

[0032] The stem cells used in the present invention and the method for culturing them, as well as preferred embodiments of each step, will be described below.

[0033] <1> Stem cells and their culture method Stem cells used in the present invention include pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells), and somatic stem cells.

[0034] The origin of the stem cells is not particularly limited, and for example, cells from mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats, and pigs can be used.

[0035] The three-dimensional skin tissue produced by the method of the present invention is effective for clinical application to humans and as an evaluation tool for human pharmaceuticals, cosmetics, etc. Therefore, it is preferable to use human stem cells in the present invention. When the three-dimensional skin tissue is used clinically, the stem cells may be autologous or allogeneic, but autologous cells are preferably used.

[0036] iPS cells are artificial stem cells derived from somatic cells that have almost the same characteristics as ES cells, such as pluripotency and the ability to proliferate through self-renewal, and can be produced by introducing a certain nuclear reprogramming factor into a somatic cell in the form of a nucleic acid or protein encoding the factor, or by increasing the expression of the endogenous mRNA and / or protein of the factor using a drug (K. Takahashi and S. Yamanaka (2006), Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; M. Nakagawa et al. (2008), Nat. Biotechnol., 26:101-106).

[0037] The nuclear reprogramming factor may be a gene specifically expressed in ES cells, or a gene or its gene product that plays an important role in maintaining the undifferentiated state of ES cells, and is not particularly limited, but examples thereof include Oct3 / 4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, and Esrrg. These nuclear reprogramming factors may be used in combination when establishing iPS cells. For example, the combination includes at least one of the above nuclear reprogramming factors, preferably two or more, and more preferably three or more.

[0038] In carrying out the present invention, an established human iPS cell line may be used as a cell line. For example, a human iPS cell line selected from ChiPSC7, ChiPSC11, ChiPSC12, ChiPSC19, ChiPSC20, ChiPSC21, ChiPSC22, ChiPSC23, 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, and 836B3 may be cultured by the method of the present invention. The above-mentioned human iPS cell lines are available from Cellartis, iPS Academia Japan, Inc., or the Kyoto University iPS Research Institute.

[0039] ES cells are stem cells with pluripotency and the ability to proliferate through self-renewal, established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently ES cell lines were established in humans, monkeys, and other primates.

[0040] ES cells can be established by extracting the inner cell mass from the blastocyst of the fertilized egg of the target animal and culturing the inner cell mass on a fibroblast feeder. In addition, the maintenance of the cells by subculture can be performed using a medium supplemented with substances such as LIF and bFGF. Methods for establishing and maintaining human and monkey ES cells are described, for example, in H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932 and H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585. In addition, some research institutes distribute ES cells. For example, human ES cell lines KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0041] Examples of somatic stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells. Examples of somatic stem cells include mesenchymal stem cells. In a broad sense, mesenchymal stem cells refer to a group of stem cells or precursor cells that can differentiate into all or some of the mesenchymal cells, such as osteoblasts, chondroblasts, and adipblasts. More specific examples of mesenchymal stem cells include bone marrow-derived mesenchymal stem cells, umbilical cord matrix-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and hair follicle mesenchymal stem cells.

[0042] These stem cells may be primarily obtained or established, or may be commercially available.

[0043] The method of culturing the stem cells before subjecting them to the spheroid formation step S1 is not particularly limited as long as they can be cultured while maintaining their differentiation potential. The stem cells are preferably cultured by an adherent culture method. Although the stem cells may be cultured by adherent culture on a culture surface on which feeder cells such as MEF cells are laid, it is preferable to perform the adherent culture under feeder-free conditions.

[0044] When stem cells are cultured in an adherent manner under feeder-free conditions, it is preferable to coat the culture surface with various culture substrates. Examples of the culture substrates include extracellular matrices such as laminin, collagen, fibronectin, vitronectin, matrigel, fibrin, and thrombin; amino acid polymers such as poly-L-lysine and poly-D-lysine, and fragments thereof, and one or more selected from these may be used. Among the culture substrates, laminin and its fragments are preferred.

[0045] As laminin and its fragments, it is preferable to use laminin 511 (laminin consisting of α5 chain, β1 chain, and γ1 chain) and its fragments. Laminin is a major cell adhesion molecule in the basement membrane, and is a heterotrimer consisting of three subunit chains, α chain, β chain, and γ chain, and is a huge glycoprotein with a molecular weight of about 800,000 Da. It is a heterotrimeric molecule in which the three subunit chains associate at the C-terminus side to form a coiled-coil structure and are stabilized by disulfide bonds. Thus, laminin 511 means laminin whose α chain is α5, whose β chain is β1, and whose γ chain is γ1.

[0046] The laminin may be a mutant, and is not particularly limited as long as it has integrin binding activity. Laminin derived from human is preferable. Laminin and its fragments are preferable to have a binding activity with integrin α6B1 with a dissociation constant of 10 nM or less. It is preferable to use a commercially available product as laminin or laminin fragment.

[0047] Examples of laminin fragments include the E8 fragment (also called laminin 511E8 fragment or laminin 511E8), which is a fragment obtained by digesting laminin 511 with elastase (Reference 2: Ido H, et al., J. Biol. Chem. 2007, 282, 11144-11154), and recombinant human laminin 511E8 fragment expressed from genetically modified silkworm cocoons. Among laminin and fragments thereof, laminin fragments are preferred, more preferably laminin 511 fragments, even more preferably laminin 511E8 fragments, and even more preferably those derived from humans.

[0048] Examples of cell culture equipment to be coated with the culture substrate include, but are not limited to, flasks, dishes, petri dishes, bottles, plates, etc. The material of the cell culture equipment is not particularly limited, but is preferably one or more selected from synthetic resins (preferably plastics) such as styrene resins (polystyrene or styrene copolymers, etc.), polycarbonate, polyolefin resins (polyethylene, polypropylene, polyester, ethylene copolymers, etc.), (meth)acrylic resins, silicone resins, amino resins, fluororesins, and polyimide resins, and glass substrates.

[0049] The medium used for culturing stem cells is not limited. For example, StemFit TM AK02N (Ajinomoto), StemSure TM hPSC (Fujifilm Wako Pure Chemical), mTeSR TM 1 (Stemcell Technologies), TeSR TM -E6 (Stemcell Technologies), TeSR TM -E8 (Stemcell Technologies), StemFlex TM (Thermo Fisher Scientific), Essential6 TM Medium (Thermo Fisher Scientific), Essential8 TM Medium (Thermo Fisher Scientific), Essential8 TM It is preferable to use Flex Medium (Thermo Fisher Scientific). In addition, these media may contain Penicillin-Streptmycin (Thermo Fisher Scientific) or normocin (Normo Fisher Scientific) as necessary. TM Antibiotics such as (Invivogen) can be added. In addition, homemade media suitable for pluripotent stem cell culture can be used by adding necessary growth factors such as FGF, the antibiotics mentioned above, and various proteins such as HSA and BSA to common cell culture basal media such as αMEM and DMEM.

[0050] iMatrix coated StemFit TM AK03N (Ajinomoto) is preferred, and Essential 8 is preferred when coated with vitronectin. TM Flex Medium (Thermo Fisher Scientific) is preferred, and mTeSR when coated with Matrigel. TM (Stemcell Technologies) is preferred.

[0051] Furthermore, on feeder cells such as MEF cells, an example of the medium is Dulbecco's modified Eagle's medium / F12 medium containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, and the like.

[0052] <2> Spheroid formation process S1 The specific embodiment of the spheroid-forming step S1 is not limited as long as it can form stem cell spheroids. The spheroid-forming step S1 is preferably performed by suspension culture of stem cells.

[0053] Suspension culture refers to growing cells in a suspended state in a medium. The means of suspension culture are not particularly limited, but examples include agitation culture, static culture, or a combination of agitation culture and static culture. Furthermore, in static culture, microcarriers can be used as necessary.

[0054] Agitated culture is a culture in which cells are suspended in the medium rather than attached to the surface of the culture substrate (container). There are two methods of agitated culture: one in which the culture solution is stirred with a stirrer or impeller, and another in which the culture container itself is driven to indirectly move the culture solution inside. An example of the former is the culture method using a spinner flask.

[0055] Culturing using microcarriers refers to culturing in a state in which cells are attached to the surface of the microcarriers. As the microcarrier, one made of a synthetic polymer or a natural polymer can be used. The microcarrier may or may not be coated with an adhesive substrate such as collagen or laminin, but is preferably coated. When forming spheroids using microcarriers, it is preferable to perform static culture as described below.

[0056] An example of a method for static culture is a method in which stem cells are seeded in a U-bottom 96-well plate for spheroid formation (e.g., #174925, Nunclon Sphera: Thermo Scientific). The culture substrate for spheroid formation used in this static culture is sufficient as long as cells do not adhere to it, the bottom shape is U-shaped, etc., and the seeded cells naturally gather at the bottom. The shape of the bottom is not limited to U-shape, and any shape such as V-shape, M-shape, flat surface, etc. can be used, and further, it is not limited to 96 wells.

[0057] When using U-bottom plates, centrifugation may be performed after seeding the stem cells, as the centrifugal force will cause the stem cells to collect at the bottom of the plate and promote aggregation.

[0058] When the culture for maintaining stem cells performed before the spheroid formation step S1 is performed by adherent culture, the stem cells are detached from the culture surface using any detachment agent such as trypsin or accutase, and then subjected to the spheroid formation step S1.

[0059] The amount of medium and the number of stem cells used in the spheroid formation step S1 are not limited and can be appropriately designed. For example, when using a U-bottom 96-well plate for spheroid formation, the amount of medium is preferably 50 to 200 μl / well, more preferably 80 to 120 μl / well, and the stem cells are seeded at preferably 1000 to 6000 cells / well, more preferably 2000 to 5000 cells / well.

[0060] The number of live cells may be adjusted to a number suitable for forming the above-mentioned spheroids by distinguishing between live and dead cells and counting them. The distinction between live and dead cells can be easily carried out by using a staining reagent specific to live or dead cells. Trypan blue can be used as a nuclear staining reagent specific to dead cells.

[0061] The culture period in the spheroid formation step S1 is not limited as long as it is sufficient for the formation of spheroids. The culture period can be, for example, 1 to 10 days, preferably 1 to 4 days.

[0062] The medium used in the spheroid formation step S1 can be the medium for stem cell culture described above. More specifically, Essential 8 TM It is preferable to use Flex Medium (Thermo Fisher Scientific).

[0063] In the spheroid formation step S1, a component for promoting the formation of spheroids may be added to the medium. An example of the component for promoting the formation of spheroids is a Rho-kinase (ROCK) inhibitor. ROCK inhibitors are known to have the effect of protecting single cells and small aggregates of cells (see, for example, Watanabe K, et al., "A ROCK inhibitor permits survival of dissociated human embryonic stem cells," Nat. Biotechnol. 25: 681-686 (2007)).

[0064] Examples of ROCK inhibitors that can be suitably used in the spheroid formation step S1 include (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine dihydrochloride (informal name: H-1152), 1-(5-isoquinolinesulfonyl)piperazine hydrochloride (informal name: HA-100), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (informal name: H-7), 1-(5-isoquinolinesulfonyl)-3-methylpiperazine (informal name: IsoH-7), N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide dihydrochloride (informal name: H-8), N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (informal name: H-9), N-[2 Examples include, but are not limited to, N-(p-bromo-cinnamylamino)ethyl)-5-isoquinolinesulfonamide dihydrochloride (informal name: H-89), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (informal name: H-1004), 1-(5-isoquinolinesulfonyl)homopiperazine dihydrochloride (informal name: H-1077), (S)-(+)-2-methyl-4-glycyl-1-(4-methylisoquinolinyl-5-sulfonyl)homopiperazine dihydrochloride (informal name: Glycyl H-1152), and (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride (informal name: Y-27632).

[0065] The concentration of the ROCK inhibitor in the medium is preferably 1 to 20 μM, more preferably 3 to 15 μM, and further preferably 5 to 12 μM.

[0066] <3> Skin tissue formation process S2 The skin tissue formation step S2 is a step of inducing differentiation of stem cells and forming three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated from stem cell spheroids. The skin tissue formation step S2 will be explained below. Note that the first day of the skin tissue formation step S2, that is, the day when differentiation induction is started, is set as "day 0" and the culture period and the like will be explained.

[0067] In this embodiment, the skin tissue formation step S2 includes steps of inducing differentiation of stem cell spheroids (first induction step S21, second induction step S22, and third induction step S23), and a three-dimensional culture step S24 of obtaining layered three-dimensional skin tissue having at least a dermis layer and an epidermis layer stacked thereon from the three-dimensional culture that has undergone these induction steps. In this embodiment, the start date of the first inducing step S21 in the skin tissue formation step S2 corresponds to "day 0".

[0068] In the differentiation induction process, the first induction step S21, the second induction step S22, and the third induction step S23 have different differentiation induction conditions. Each step will be described below.

[0069] <3-1>First induction step S21 The first induction step S21 is a step of inducing the formation of non-neural ectodermal epithelium. More specifically, the cells are cultured in a first differentiation medium containing one or more factors selected from an antagonist of transforming growth factor β (TGFβ) signaling, an agonist of fibroblast growth factor (FGF) signaling, and an agonist of bone morphogenetic protein (BMP) signaling.

[0070] More specifically, the culture is carried out in a first differentiation medium containing an antagonist of TGFβ signaling and an agonist of FGF signaling, and optionally containing an agonist of bone morphogenetic protein (BMP) signaling.

[0071] In one embodiment, from day 0, stem cell spheroids are cultured in a first differentiation medium containing an antagonist of TGFβ signaling, an agonist of BMP signaling, and an agonist of FGF signaling.

[0072] In another embodiment, a 1-1 differentiation medium and a 1-2 differentiation medium are used as the 1st differentiation medium. Specifically, in this embodiment, on day 0, stem cell spheroids are cultured in a 1-1 differentiation medium containing an antagonist of TGFβ signaling and an agonist of FGF signaling, and at any timing between day 0 and day 1, they are further cultured in a 1-2 differentiation medium containing an agonist of BMP signaling.

[0073] The first differentiation medium can be prepared by adding the above-mentioned factors to a medium suitable for culturing stem cells as described above. The base of the first differentiation medium is not particularly limited as long as it is a medium suitable for culturing stem cells, but Essential 6 TM Medium (Thermo Fisher Scientific) is a particularly suitable example.

[0074] Antagonists of TGFβ signaling include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), SB525334 (6-[2-tert-butyl-5-(6-methylpyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline), SD-208 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), and LDN-193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl)-pyrazole-1-carbothioamide). ]-quinoline hydrochloride), E-616452 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), LY2157299 (2-(6-methyl-pyridin-2-yl)-3-[6-amido-quinolin-4-yl]-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole), LY2109761 (4-[5,6-dihydro-2-(2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-7-[2-(4-morpholinyl)ethoxy]-quinoline), and other low molecular weight compounds, RNAi nucleic acids specific to TGFβ receptors, and anti-TGFβ antibodies. In the present invention, SB431542 is particularly suitable.

[0075] When the above-mentioned low molecular weight compound is used as an antagonist of TGFβ signaling, the concentration of the low molecular weight compound in the medium in the first induction step S21 is preferably 1 to 50 μM, more preferably 3 to 30 μM, and even more preferably 5 to 20 μM.

[0076] Examples of the agonist of BMP signaling include one or more selected from the group consisting of BMP4, BMP2, and BMP7, and conjugates or fragments thereof. In particular, BMP4 or a conjugate or fragment thereof is a suitable example.

[0077] In the first induction step S21, the concentration of the above-mentioned BMP signaling agonist in the first differentiation medium is preferably 0 to 30 ng / ml, more preferably 0.1 to 20 ng / ml, more preferably 0.5 to 10 ng / ml, more preferably 1 to 7 ng / ml, and more preferably 2 to 6 ng / ml.

[0078] Examples of agonists of FGF signaling include proteins belonging to the FGF family or conjugates or fragments thereof. In the present invention, preferred examples of proteins belonging to the FGF family include bFGF, FGF4, FGF9, etc., and among these, bFGF is particularly preferred.

[0079] In the first induction step S21, the concentration of the above-mentioned agonist of FGF signaling in the first differentiation medium is preferably 0.1 to 20 ng / ml, more preferably 1 to 10 ng / ml, and even more preferably 2 to 6 ng / ml.

[0080] The first differentiation medium may further contain an extracellular matrix component. The extracellular matrix component may be a basement membrane extract (BME). Specific examples of the extracellular matrix component include laminin, collagen IV, entactin (Nightgen), and heparan sulfate proteoglycan. Matrigel (BD Biosciences, Corning, etc.) can be used as a reagent containing these extracellular matrix components.

[0081] In the present invention, Matrigel refers to a soluble preparation obtained from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. For the preparation of Matrigel, DMEM is preferably used, for example, DMEM (containing 1 g / L glucose) can be suitably used.

[0082] The concentration of the extracellular matrix component in the first differentiation medium is preferably 0.1 to 5 mass %, more preferably 0.3 to 3 mass %, and even more preferably 0.5 to 2 mass %.

[0083] In the first induction step S21, the spheroids formed in the spheroid formation step S1 are subjected to suspension culture in a first differentiation medium. Although the specific form of suspension culture is not particularly limited, it is preferable to carry out the first induction step S21 by the above-mentioned static culture.

[0084] In a preferred embodiment, the first differentiation medium is dispensed into a 96-well U-bottom plate, preferably at 50 to 200 μl / well, more preferably at 80 to 150 μl / well, and one spheroid is individually cultured in each well.

[0085] The period during which the first induction step S21 is performed is preferably 1 day or more, more preferably 2 days or more, and even more preferably 3 days or more. By performing the first induction step S21 for the above period, non-neural ectodermal epithelium can be formed more satisfactorily. Moreover, the period for which the first induction step S21 is performed is preferably 7 days or less, more preferably 5 days or less, and even more preferably 4 days or less. The above-mentioned period is sufficient for the formation of non-neural ectodermal epithelium. In a particularly preferred embodiment, the duration of the first induction step S21 is three days.

[0086] During the first induction step S21, replacement of the first differentiation medium is optional. When the first induction step S21 is performed during the above period, replacement of the first differentiation medium is not necessary.

[0087] <3-2>Second induction step S22 The second induction step S22 is a step of inducing the three-dimensional culture that has undergone the first induction step S21 to a higher-order structure. In the second induction step S22, it is preferable to perform suspension culture of the three-dimensional culture. In addition, in the second induction step S22, it is preferable to perform culture using a second differentiation medium containing an agonist of fibroblast growth factor (FGF) signaling and / or an antagonist of bone morphogenetic protein (BMP) signaling.

[0088] In one embodiment, the second differentiation medium comprises an antagonist of BMP signaling and optionally an agonist of FGF signaling.

[0089] In another embodiment, the second differentiation medium comprises an agonist of FGF signaling and an antagonist of BMP signaling, which can improve the efficiency of forming the three-dimensional skin tissue.

[0090] The agonist of FGF signaling used in the second induction step S22 may be the above-mentioned protein belonging to the FGF family or a conjugate or fragment thereof. In the present invention, suitable examples of the protein belonging to the FGF family include bFGF, FGF4, FGF9, etc., among which bFGF is particularly suitable. It is preferable that the FGF signaling agonist used in the first induction step S21 and the second induction step S22 is the same, and more preferably bFGF is used in both steps.

[0091] The concentration of the FGF signaling agonist in the second differentiation medium is preferably 1 to 1000 ng / ml, more preferably 10 to 800 ng / ml, even more preferably 50 to 500 ng / ml, and still more preferably 100 to 300 ng / ml.

[0092] Examples of antagonists of BMP signal transduction include proteinaceous inhibitors such as Chordin, Noggin, and Follistatin, Dorsomorphin (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), its derivatives (PBYu et al. (2007), Circulation, 116:II_60; PBYu et al. (2008), Nat.Chem.Biol., 4:33-41; J.Hao et al. (2008), PLoS ONE, 3(8):e2904) and LDN-193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). Preferably, LDN-193189.

[0093] The concentration of the antagonist of BMP signaling in the second differentiation medium is preferably 0 to 4000 nM, more preferably 50 to 3000 nM, even more preferably 100 to 2000 nM, and still more preferably 500 to 1500 nM.

[0094] The second differentiation medium can be prepared by adding the above-mentioned factors to a medium suitable for culturing stem cells as a base. The base of the second differentiation medium is not particularly limited, but may be selected from Essential 6 TM Medium (Thermo Fisher Scientific) is a particularly suitable example.

[0095] The second induction step S22 may be performed by removing the entire first differentiation medium used in the first induction step S21 and replacing it with the second differentiation medium.

[0096] In one embodiment, the second induction step S22 is started by further adding a second differentiation medium to the first differentiation medium in which the suspension culture is carried out in the first induction step S21.

[0097] In this case, on the day the first induction step S21 ends (the day the second induction step S22 starts), the second differentiation medium is added in an amount of preferably 0.01 to 1 times, more preferably 0.05 to 0.5 times, even more preferably 0.1 to 0.3 times, and even more preferably 0.15 to 0.25 times, the first differentiation medium.

[0098] In a more preferred embodiment, the second induction step S22 is started by further adding a second differentiation medium to the first differentiation medium in which the suspension culture is carried out in the first induction step S21. During the second induction step S22, the three-dimensional culture is cultured while adding a fresh medium that does not contain an agonist of FGF signaling and an antagonist of BMP signaling to the above-mentioned mixed medium, or while replacing a part or all of the mixed medium with the fresh medium. In this embodiment, the concentration of the components of the second differentiation medium is changed stepwise during the second induction step S22. By performing the second induction step S22 so that the medium composition is changed stepwise in this manner, the efficiency of forming three-dimensional skin tissue can be improved.

[0099] The period during which the second induction step S22 is performed is preferably 1 day or more, and can be 3 days or more, 4 days or more, or 5 days or more. In another embodiment, the period during which the second induction step S22 is performed can be preferably 7 days or more. By carrying out the second induction step S22 during this period, it is possible to promote the formation of layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated in the subsequent three-dimensional culture step S24.

[0100] The upper limit of the implementation period of the second induction step S22 is not limited, and can be, for example, 14 days or less, more preferably 10 days or less, and even more preferably 9 days or less. The upper limit of the implementation period of the second induction step S22 can be 5 days or less, 4 days or less, or 3 days or less. By carrying out the second induction step S22 during the above period, it is possible to promote the formation of layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated in the subsequent three-dimensional culture step S24.

[0101] Here, in the first embodiment of the present invention (FIG. 1(a)), the period during which the second induction step S22 is carried out is preferably 1 to 14 days, more preferably 1 to 10 days, more preferably 1 to 8 days, more preferably 1 to 5 days, more preferably 1 to 4 days, and more preferably 1 to 3 days. In the second and third embodiments (FIGS. 1(b) to (c)), the period during which the second induction step S22 is carried out is preferably 3 to 14 days, more preferably 4 to 10 days, more preferably 5 to 10 days, and more preferably 7 to 9 days. By carrying out the second induction step S22 during the above period, it is possible to promote the formation of layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated in the subsequent three-dimensional culture step S24.

[0102] Furthermore, in one embodiment (for example, the fourth embodiment), the second induction step S22 may not be performed, and the three-dimensional culture step S24 may be performed after the first induction step S21.

[0103] <3-3>Third induction step S23 The skin tissue formation step S2 may optionally include a third induction step S23 for further maturing the three-dimensional culture that has undergone the second induction step S22. Specifically, this corresponds to the third embodiment of the present invention (FIG. 1(c)).

[0104] In the third induction step S23, suspension culture is preferably performed. The specific form of such suspension culture is not limited, and preferred examples include a form in which organoids are suspension cultured in a culture vessel such as a flask, dish, petri dish, bottle, plate, etc. filled with a maturation medium. The culture vessel preferably has a low-adhesion surface.

[0105] The third induction step S23 may be performed by leaving the culture vessel stationary, or by placing the culture vessel on a shaker and shaking the culture medium. An example of the shaker is an orbital shaker that shakes in a two-dimensional orbit. The shaking speed of the orbital shaker is, for example, 50 to 80 rpm.

[0106] The maturation medium used in the third induction step S23 can be any cell culture medium without limitation. For example, Advanced DMEM / F-12 (Thermo Fisher Scientific), Neurobalsal TM MEDIUM (Thermo Fisher Scientific), or a mixed medium thereof is a suitable example.

[0107] The period during which the third induction step S23 is carried out is preferably 3 days or more, more preferably 4 days or more, even more preferably 5 days or more, and even more preferably 6 days or more.

[0108] The upper limit of the period during which the third induction step S23 is carried out is preferably 8 days or less, and more preferably 7 days or less.

[0109] The period during which the third induction step S23 is carried out is preferably 3 to 8 days, more preferably 4 to 8 days, even more preferably 5 to 8 days, and even more preferably 6 to 7 days.

[0110] The medium used in the third induction step S23 may further contain an extracellular matrix component. The extracellular matrix component may be a basement membrane extract (BME). Specific examples of the extracellular matrix component include laminin, collagen IV, entactin (Nightgen), and heparan sulfate proteoglycan. Matrigel (BD Biosciences, Corning, etc.) can be used as a reagent containing these extracellular matrix components.

[0111] The concentration of the extracellular matrix components in the medium used in the third induction step S23 is preferably 0.05 to 5 mass %, more preferably 0.1 to 2 mass %, and even more preferably 0.3 to 1 mass %.

[0112] <3-4> Three-dimensional culture process S24 The three-dimensional culture step S24 is a step of culturing the three-dimensional culture obtained by suspension culture to obtain a layered three-dimensional skin tissue in which at least a dermis layer and an epidermis layer are laminated.

[0113] The three-dimensional culture step S24 is preferably started after differentiation induction in the first differentiation medium in the first induction step S21.

[0114] The three-dimensional culture step S24 is preferably started 1 to 20 days after the start of differentiation induction, more preferably 1 to 18 days, even more preferably 3 to 18 days, even more preferably 3 to 15 days, and particularly preferably 4 to 12 days after the start of differentiation induction. Also preferred is an embodiment in which the three-dimensional culture step S24 is started 5 to 20 days, more preferably 5 to 18 days, and even more preferably 6 to 18 days after the start of differentiation induction. In one embodiment, the three-dimensional culture step S24 is started preferably 1 day or more, more preferably 2 days or more, and even more preferably 3 days or more after the start of differentiation induction. In one embodiment, the three-dimensional culture step S24 is started preferably 20 days or less, more preferably 18 days or less, even more preferably 15 days or less, and even more preferably 12 days or less after the start of differentiation induction.

[0115] That is, in a preferred embodiment, the three-dimensional culture step S24 is initiated preferably on the 1st to 20th day, more preferably on the 3rd to 18th day, even more preferably on the 3rd to 15th day, and particularly preferably on the 4th to 12th day from the start of differentiation induction.

[0116] By setting the start time of differentiation induction as described above, it is possible to promote the formation of a three-dimensional skin tissue having a structure in which the epidermis layer is laminated above the dermis layer and at least the epidermis layer is open to the outside world. Furthermore, it is possible to promote the formation of hair follicles in the three-dimensional skin tissue. In the present invention, the term "a structure in which at least the epidermis layer is open to the outside world" does not refer to a "structure in which the dermis layer surrounds the epidermis layer and thereby contains the epidermis layer", but rather to a structure in which the epidermis layer is exposed to the outside world.

[0117] For example, in the first embodiment (FIG. 1(a)), the three-dimensional culture step S24 is initiated on the 5th to 9th day, more preferably the 5th to 8th day, and even more preferably the 6th to 7th day from the start of induction. In a preferred embodiment, the three-dimensional culture step S24 may be initiated on the 3rd to 12th day, more preferably the 4th to 11th day from the start of induction. This configuration can promote the formation of a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated, and can also promote the formation of a three-dimensional skin tissue in which the epidermis layer is laminated above the dermis layer and at least the epidermis layer is open to the outside world.

[0118] In the second embodiment (FIG. 1(b)), the three-dimensional culture step S24 may be initiated on the 10th to 13th day, more preferably the 12th to 13th day, from the start of induction. In the third embodiment (FIG. 1(c)), the three-dimensional culture step S24 may be initiated on the 15th to 19th day, more preferably the 17th to 18th day, from the start of induction. In the fourth embodiment (FIG. 1(d)), the three-dimensional culture step S24 may be initiated on the 1st to 3rd day, more preferably the 2nd to 3rd day, from the start of induction.

[0119] In the three-dimensional culture step S24, preferably, the three-dimensional culture after the first induction step S21, the second induction step S22 or the third induction step S23 is placed, without being cut, on a membrane of a culture vessel equipped with a membrane that is permeable to the culture medium.

[0120] In the present invention, a three-dimensional culture having a three-dimensional shape is placed on the membrane of the culture vessel. That is, the present invention does not place single isolated cells on the membrane. In a preferred embodiment, the three-dimensional culture is placed on the membrane while maintaining the three-dimensional shape without being cut or subjected to a process for isolating cells before being placed on the membrane. A treatment for separating cells in a three-dimensional culture is, for example, a treatment for degrading components of the extracellular matrix of the three-dimensional culture, and specifically includes treating the three-dimensional culture with a proteolytic enzyme and / or a collagenolytic enzyme (collagenase), or further with a proteolytic enzyme and a collagenolytic enzyme. The present invention, in which the three-dimensional culture is arranged on a membrane, can eliminate the need for enzyme treatment for separating cells, and can more easily obtain a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated. In addition, by adopting such a form, the epidermis layer is laminated above the dermis layer, and at least the epidermis layer has a structure open to the outside world, and it is possible to promote the formation of a three-dimensional skin tissue having hair follicles.

[0121] The three-dimensional culture placed on the membrane is not particularly limited in the differentiation state of cells or tissues, as long as it is a culture obtained through the suspension culture of stem cells and has a three-dimensional shape.For example, the three-dimensional culture may be in a state in which the cells forming the dermis layer and the cells forming the epidermis layer are undifferentiated, or the dermis layer and the epidermis layer are already formed.When the dermis layer and the epidermis layer are already formed, the three-dimensional culture may be in a form in which the epidermis layer is on the inside and the dermis layer is on the outside. In one embodiment, the three-dimensional culture placed on the membrane may be in the form of a sphere in which only epithelial cells are differentiated, or in the form of a sphere in which a layer containing mesenchymal cells is formed so as to surround a layer containing epithelial cells, and the layer containing epithelial cells is encapsulated in the layer containing mesenchymal cells.

[0122] The three-dimensional culture obtained through the first induction step S21 has non-neurectodermal epithelium formed on the surface layer of the culture. That is, in one embodiment, in the three-dimensional culture step S24, preferably the three-dimensional culture having non-neurectodermal epithelium formed is placed on a membrane. In addition, the three-dimensional culture to be placed on the membrane may contain one or more, preferably two or more, more preferably three or more, more preferably four or more, and even more preferably all five types selected from non-neurectodermal epithelium, epidermal progenitor cells, cranial placode, neuroectoderm, and mesenchymal cells.

[0123] In another embodiment, in the three-dimensional culture step S24, a three-dimensional culture containing epidermal keratinocytes and / or fibroblasts can be placed on a membrane.

[0124] In one embodiment, the three-dimensional culture placed on the membrane is preferably one that does not have hair follicles, i.e., the three-dimensional culture placed on the membrane is preferably one in which hair follicles are not differentiated. By using an immature three-dimensional culture of undifferentiated hair follicles, it is possible to easily obtain three-dimensional skin tissue having a structure in which the epidermis layer is layered above the dermis layer and at least the epidermis layer is open to the outside world, simply by culturing the three-dimensional culture on a membrane.

[0125] In one embodiment, the three-dimensional culture placed on the membrane is preferably spherical in shape. The spherical three-dimensional culture comprises an approximately spherical shape formed by the aggregation of cells. When a spherical three-dimensional culture is used, the maximum diameter of the three-dimensional culture is preferably 2 mm or less, more preferably 1.5 mm or less, more preferably 1.3 mm or less, and even more preferably 1 mm or less. By adopting such a form, it is possible to promote the formation of a three-dimensional skin tissue having a structure in which the epidermis layer is laminated above the dermis layer and at least the epidermis layer is open to the outside world, simply by culturing the three-dimensional culture on a membrane.Furthermore, it is possible to promote the formation of hair follicles in the three-dimensional skin tissue. The maximum diameter of the three-dimensional culture can be measured by image analysis.

[0126] When a spherical three-dimensional culture is used, the lower limit of the maximum diameter of the three-dimensional culture is not particularly limited, but may be 100 μm or more, or 300 μm or more.

[0127] In one embodiment, the three-dimensional culture placed on the membrane is preferably such that the mesenchymal cells are undifferentiated or the layer having the mesenchymal cells is less than a certain thickness. The thickness of the layer having the mesenchymal cells is preferably 200 μm or less, more preferably 180 μm or less, more preferably 150 μm or less, more preferably 130 μm or less, more preferably 100 μm or less, more preferably 90 μm or less, more preferably 80 μm or less, more preferably 70 μm or less, more preferably 60 μm or less, even more preferably 50 μm or less, and particularly preferably 40 μm or less. By adopting such a form, it is possible to promote the formation of a three-dimensional skin tissue having a structure in which the epidermis layer is laminated above the dermis layer and at least the epidermis layer is open to the outside world, simply by culturing the three-dimensional culture on a membrane.Furthermore, it is possible to promote the formation of hair follicles in the three-dimensional skin tissue. In one embodiment, the mesenchymal cells may be cranial neural crest cells (CNCCs).

[0128] The number of three-dimensional cultures to be placed on the membrane is not particularly limited, and for example, one or more three-dimensional cultures obtained through the first induction step S21 may be placed per space (for example, per well in the case of a multi-well plate) separated by a culture vessel. When multiple three-dimensional cultures are placed, two or more, five or more, or ten or more three-dimensional cultures may be placed per space (per well).

[0129] The membrane provided in the culture vessel used in the three-dimensional culture step S24 is a membrane that allows the culture solution to pass through, and a porous membrane can be preferably used. A preferred example of a culture vessel provided with such a membrane is a cell culture plate (e.g., a 24-well plate) in which a transwell insert or a cell culture insert provided with a porous membrane is placed. Alternatively, the culture vessel itself may be provided with a porous membrane.

[0130] The porous membrane provided in the culture vessel has a pore size of preferably 0.1 to 8 μm, more preferably 0.4 to 5 μm, and even more preferably 0.4 to 3 μm.

[0131] The thickness of the membrane used in the present invention is not particularly limited and can be, for example, 10 to 1000 μm. The thickness of the membrane can also be adjusted by the thickness of the coating with an extracellular matrix component or the like, which will be described later.

[0132] The material of the membrane provided in the culture vessel is not particularly limited, but hydrophilic polytetrafluoroethylene (PTFE), mixed cellulose ester, polycarbonate, polyethylene terephthalate, etc. can be appropriately used.

[0133] The membrane provided in the culture vessel may be a membrane having micropores and made of an extracellular matrix component. In the membrane made of the extracellular matrix component, the extracellular matrix component forms a mesh structure. The extracellular matrix component constituting the membrane is preferably collagen, more preferably collagen I, and even more preferably atelocollagen obtained from collagen I. A preferred example of such a culture vessel is one in which FibColl (registered trademark) highly permeable atelocollagen insert for 24 wells (Koken Co., Ltd.) is placed on a cell culture plate.

[0134] The membrane provided on the culture vessel is preferably coated with an extracellular matrix component. The extracellular matrix component used is preferably collagen, more preferably collagen I. Culturing on a membrane coated with an extracellular matrix component can promote the formation of a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are stacked, and can also promote the formation of a three-dimensional skin tissue in which the epidermis layer is stacked above the dermis layer and at least the epidermis layer is open to the outside world.

[0135] When collagen is used as an extracellular matrix component, it is preferable that the collagen is in a gel state.

[0136] The membrane provided in the culture vessel may have feeder cells laid on it. As the feeder cells, adipose-derived mesenchymal stem cells are preferably used.

[0137] In addition, in the three-dimensional culture step S24, it is preferable to fill the medium under the membrane and culture the three-dimensional culture on the membrane. That is, it is preferable to contact the medium filled under the membrane with the three-dimensional culture through the membrane, and to supply the medium to the three-dimensional culture through the membrane. By adopting such a form, it is possible to promote the formation of a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated.

[0138] The three-dimensional culture may be maintained in a state of being placed on the membrane, i.e., the three-dimensional culture may be attached to the membrane, or may not be completely attached to the membrane and may be suspended from the membrane by pipetting or shaking. When feeder cells are laid on the membrane, the three-dimensional culture may be maintained in a state in which it is placed on the feeder cells on the membrane.

[0139] In the three-dimensional culture step S24, the upper part of the membrane is preferably a culture environment that induces differentiation of epithelial cells. In a preferred embodiment, in the three-dimensional culture step S24, air-liquid interface culture and / or two-layer liquid interphase culture is performed on the three-dimensional culture. Preferably, in the three-dimensional culture step S24, air-liquid interface culture is performed. By adopting such a form, three-dimensional skin tissue having a layered structure of a dermis layer and an epidermis layer can be efficiently obtained.

[0140] Fig. 2 is a schematic diagram showing the state of air-liquid interface culture, and Fig. 3 is a schematic diagram showing two-layer liquid interfacial culture. Figs. 2 and 3 show a state in which a three-dimensional culture C obtained by the suspension culture process is placed on a cell culture insert 11 installed in a cell of a cell culture plate 10 (a), and a state in which a dermis layer L1 and an epidermis layer L2 are formed by continuing the culture (b).

[0141] In air-liquid interface culture, culture is performed by adding culture medium to the lower part of a membrane M and not adding medium to the upper part (Figure 2). Specifically, after placing a three-dimensional culture C obtained by suspension culture on membrane M, the medium on membrane M is removed, and the three-dimensional culture is cultured with the upper part exposed to the gas phase and the lower part in contact with the culture medium. This promotes differentiation of cells that constitute the epidermal layer L2 in the upper part of the three-dimensional culture C, making it possible to efficiently obtain three-dimensional skin tissue having a layered structure of the dermis layer L1 and the epidermal layer L2.

[0142] The culture medium used is not particularly limited as long as it is a medium suitable for maturation of the three-dimensional culture C, but a preferred example is the maturation medium used in the third induction step S23. An extracellular matrix component may be added to the maturation medium. The preferred form of the extracellular matrix component to be added is the same as that of the medium used in the third induction step S23, and specifically, Matrigel can be used.

[0143] Two-layer intercellular liquid culture is a method in which different culture media are filled above and below the membrane M (Figure 3). In two-layer intercellular liquid culture, the upper part of the membrane M is filled with epithelial cell medium, and the upper part of the three-dimensional culture C is brought into contact with the epithelial cell medium for culture. This promotes differentiation of cells that constitute the epidermal layer L2 in the upper part of the three-dimensional culture C, making it possible to efficiently obtain three-dimensional skin tissue having a layered structure of the dermis layer L1 and the epidermal layer L2.

[0144] The epithelial cell medium to be used is not particularly limited, and examples thereof include Keratinocyte serum free medium (KSFM, Thermo Fisher Scientific), EpiLife TM Examples of suitable medium include, but are not limited to, Minimum Essential Medium (MEM) containing about 5-20% fetal bovine serum, Dulbecco's Modified Eagle Medium (DMEM), RPMI 1640 medium, 199 medium, F12 medium, etc. In addition, suitable components (calcium, growth factors, etc.) for culturing epithelial cells may be added as appropriate.

[0145] A maturation medium is preferably filled below the membrane M. A preferred embodiment of the maturation medium is the same as that of the air-liquid interface culture described above.

[0146] In the present invention, the step of culturing using a maturation medium in the three-dimensional culture step S24 is also referred to as a maturation step S242. In the maturation step S242, the culture may be started in a medium containing an extracellular matrix component, and then cultured in a medium not containing an extracellular matrix component.

[0147] Here, in a preferred embodiment, the maturation step S242 includes both a step of culturing in a maturation medium to which extracellular matrix components have been added, and a step of culturing in a maturation medium to which no extracellular matrix components have been added. In such an embodiment, the maturation step S242 is initiated with a medium containing extracellular matrix components, and one to four days later, all or part of the existing medium is replaced with a medium not containing extracellular matrix components, and then the culture is continued while replacing all or part of the existing medium with a medium not containing extracellular matrix components every one to four days.

[0148] In addition, the maturation step S242 in the third embodiment (Figure 1(c)) can be configured to include a step of culturing in a maturation medium to which no extracellular matrix components have been added, but not to include a step of culturing in a maturation medium to which extracellular matrix components have been added.

[0149] Moreover, the three-dimensional culture step S24 is preferably started after differentiation induction in the first differentiation medium and during differentiation induction in the second differentiation medium. Moreover, the three-dimensional culture step S24 may be started after differentiation induction in the first differentiation medium and may include a differentiation induction step in the second differentiation medium. In such a form, for example, in the first embodiment (Figure 1(a)), if the period during which the second induction step S22 is performed is less than 6 to 10 days and the three-dimensional culture used at the start of the three-dimensional culture step S24 is immature, or in the fourth embodiment (Figure 1(d)), if the second induction step S22 is not performed, it is preferable to perform a step (induction step S241) of culturing using the second differentiation medium used in the second induction step S22 before culturing in the above-mentioned maturation medium when culturing at the start of the three-dimensional culture step S24. In such a case, it is preferable that the medium filled below the membrane in the air-liquid interface culture or two-layer liquid interphase culture in the induction step S241 is a second differentiation medium.

[0150] When the inducing step S241 is performed in the three-dimensional culture process S24, the period during which the inducing step S241 is performed can be appropriately changed depending on the differentiation state of the culture. In the present invention, the total culture period of the culture using the second differentiation medium in the second induction step S22 and the induction step S241 is preferably 2 days or more, more preferably 5 days or more, and even more preferably 7 days or more. In addition, the total culture period of the culture using the second differentiation medium in the second induction step S22 and the induction step S241 can be, for example, 14 days or less, preferably 13 days or less, more preferably 10 days or less, and even more preferably 9 days or less. Specifically, the total culture period of the culture using the second differentiation medium in the second induction step S22 and the induction step S241 can be, for example, 2 to 14 days, preferably 5 to 13 days, more preferably 5 to 10 days, even more preferably 7 to 10 days, and even more preferably 7 to 9 days.

[0151] In another embodiment, the three-dimensional culture step S24 may be started after differentiation induction in the second differentiation medium. In this embodiment, for example, in the second embodiment (FIG. 1(b)), the three-dimensional culture step S24 may be started after the second induction step S22 and may not include a culture step in the second differentiation medium.

[0152] Furthermore, the three-dimensional culture step S24 may be started during the culture in the maturation medium after differentiation induction in the second differentiation medium. In such a case, the three-dimensional culture step S24 is preferably started after the culture in the maturation medium containing the extracellular matrix components is completed.

[0153] In the three-dimensional culture step S24, the three-dimensional culture may be cultured by only one of the culture methods of air-liquid interface culture and two-layer liquid interfacial culture, or the two culture methods may be performed in any order.

[0154] The period during which the three-dimensional culture step S24 is carried out is preferably 8 days or more, more preferably 10 days or more, even more preferably 14 days or more, and even more preferably 20 days or more. Furthermore, when obtaining three-dimensional skin tissue having hair follicles, the period for which the three-dimensional culture step S24 is carried out is preferably 90 days or more, more preferably 100 days or more, and even more preferably 105 days or more.

[0155] The upper limit of the period for carrying out the three-dimensional culture step S24 is not particularly limited, but can be 200 days or less, 150 days or less, 100 days or less, or 80 days or less. The period during which the three-dimensional culture step S24 is carried out can be, for example, 8 to 200 days, 10 to 150 days, 14 to 100 days, or 20 to 80 days.

[0156] By setting the period of the three-dimensional culture step S24 within the above range, it is possible to promote the formation of a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are stacked.

[0157] In the three-dimensional culture step S24, the three-dimensional culture is cultured on a membrane without incision before the three-dimensional culture has a highly differentiated skin tissue structure after the start of induction, unlike Non-Patent Document 6. Therefore, the procedure for producing the three-dimensional skin tissue can be simplified, and the culture period required to obtain the desired three-dimensional skin tissue can be shortened. Furthermore, the present invention having such a three-dimensional culture step S24 can be obtained by a simple method without cutting the culture, and therefore can be suitably applied to industrial production such as mass culture.

[0158] In the skin tissue formation step S2, as described above, culture is performed using the second differentiation medium in the second induction step S22 and / or induction step S241. In one embodiment, the culture in the second differentiation medium in the second induction step S22 and the induction step S241 can be in the following form.

[0159] For example, in an embodiment in which the culture in the second differentiation medium is carried out for two days or more, fresh medium not containing an agonist of FGF signaling or an antagonist of BMP signaling may be added 2 to 3 days after the start of the culture in the second differentiation medium, preferably in an amount of 0.3 to 1 times, more preferably 0.4 to 0.8 times, and even more preferably 0.5 to 0.7 times the amount of the existing differentiation medium. Thereafter, after an interval of 1 to 3 days, preferably 30 to 70%, more preferably 40 to 60%, of the existing differentiation medium is replaced with the fresh medium, and the culture in the second differentiation medium is continued.

[0160] In addition, in the culture using the second differentiation medium, the three-dimensional culture is cultured in a medium containing an antagonist of BMP signaling at a concentration of preferably 1 to 1000 nM, more preferably 5 to 500 nM, and even more preferably 10 to 250 nM. When culturing in the second differentiation medium while changing the concentration stepwise, the concentration can be changed within the above concentration range.

[0161] In addition, in the culture using the second differentiation medium, the three-dimensional culture is cultured in a medium containing an FGF signaling agonist at a concentration of preferably 0.5 to 250 ng / ml, more preferably 2 to 150 ng / ml, and even more preferably 5 to 75 ng / ml. When culturing in the second differentiation medium while changing the concentration stepwise, the concentration can be changed within the above concentration range.

[0162] The concentration of the agonist of FGF signaling in the medium during culture in the second differentiation medium in the second induction step S22 and / or induction step S241 is preferably adjusted to be higher than the concentration in the medium in the first induction step S21. Specifically, the concentration of the agonist of FGF signaling in the medium during culture in the second differentiation medium in the second induction step S22 and / or induction step S241 is preferably 1.1 to 20 times, more preferably 2 to 15 times, the concentration in the medium in the first induction step S21. When the second induction step S22 and / or induction step S241 are performed while gradually changing the concentration of the agonist for FGF signaling, the concentration may be adjusted so as to satisfy the above condition at least at the start of the second induction step S22.

[0163] In this way, by gradually adjusting the concentration of the second differentiation medium in the second induction step S22 and / or induction step S241, it is possible to promote the formation of layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are stacked.

[0164] Furthermore, in the induction step S241 in the three-dimensional culture process S24, after culturing in the first differentiation medium, the second differentiation medium may be added to the first differentiation medium to start culturing in the second differentiation medium. That is, the medium used in the induction step S241 may contain both the factors contained in the first differentiation medium and the factors contained in the second differentiation medium. A preferred embodiment of this form is the same as the "form in which the second induction step S22 is started by further adding the second differentiation medium to the first differentiation medium in which the suspension culture is carried out in the first induction step S21" in the above-mentioned second induction step S22.

[0165] Furthermore, in the present invention, in the three-dimensional culture step S24, the three-dimensional culture obtained by culturing stem cell spheroids may be cultured in a medium containing an agonist of Wnt signaling, but it can also be cultured in a medium not containing an agonist of Wnt signaling. In the method described in Non-Patent Document 6, the size of the obtained spherical skin organoids is increased by culturing in a medium containing an agonist of Wnt signaling, making the skin organoids easier to dissect. On the other hand, in the present invention, a three-dimensional culture obtained by culturing a spheroid of stem cells is placed on a membrane without dissection. Therefore, in the present invention, as shown in Non-Patent Document 6, a desired layered three-dimensional skin tissue can be formed on a membrane without including a step of culturing in a medium containing an agonist of Wnt signaling. Examples of the agonist of Wnt signaling include GSK3β inhibitors, specifically CHIR9902.

[0166] <Three-dimensional skin tissue> According to the present invention, a layered three-dimensional skin tissue in which an epidermis layer and a dermis layer are laminated can be produced. The present invention also relates to the three-dimensional skin tissue itself produced by the above-mentioned production method. A preferred embodiment of the three-dimensional skin tissue of the present invention that can be produced by the above-mentioned production method will be described below.

[0167] In a preferred embodiment, the three-dimensional skin tissue of the present invention has an epidermal layer in direct contact with the dermal layer, which is more suitable for clinical or testing applications since it has a structure similar to that of a living skin.

[0168] In a preferred embodiment, the three-dimensional skin tissue of the present invention has a structure in which an epidermis layer is laminated above a dermis layer. Such a three-dimensional skin tissue having a normal multi-layer structure is suitable for clinical applications and as a testing tool.

[0169] Furthermore, the three-dimensional skin tissue of the present invention preferably has an epidermis layer laminated above the dermis layer, with at least the epidermis layer being open to the outside world. In such a three-dimensional skin tissue, the epidermis layer is formed on the top of the membrane, and the epidermis layer is exposed to the outside world. Therefore, the obtained three-dimensional skin tissue can be used as a biomaterial or a test tool as it is. Such a form of three-dimensional skin tissue is distinguished from a spherical form in which the dermis layer surrounds the epidermis layer, thereby encapsulating the epidermis layer within the dermis layer.

[0170] The epidermis layer of the three-dimensional skin tissue of the present invention may also include epidermal keratinocytes, and the dermis layer of the three-dimensional skin tissue of the present invention may also include dermal fibroblasts.

[0171] In a preferred embodiment, the three-dimensional skin tissue of the present invention may comprise one or more cells selected from the group consisting of mesenchymal stem cells, dermal papilla cells, dermal sheath cells and follicular epidermal stem cells.

[0172] In a preferred embodiment, the three-dimensional skin tissue of the present invention has a hair follicle. That is, in a preferred embodiment, the three-dimensional skin tissue is a hair follicle organoid. The hair follicle preferably forms a hair bulb having hair papilla cells in the dermis layer.

[0173] <Application> The three-dimensional skin tissue of the present invention can be used in clinical applications as a skin composition for transplantation. That is, the present invention also relates to a skin composition for transplantation, which comprises the above-mentioned three-dimensional skin tissue.

[0174] The three-dimensional skin tissue of the present invention can also be used as a testing tool. Specifically, by applying a test substance to the three-dimensional skin tissue of the present invention and observing the anatomical and molecular biological reactions, it is possible to screen active ingredients of medicines and cosmetics. Methods for applying the test substance to the three-dimensional skin tissue include coating and injection. That is, the present invention also resides in a screening method for active ingredients of pharmaceuticals or cosmetics, which comprises applying a test substance to the above-mentioned three-dimensional skin tissue. EXAMPLES

[0175] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0176] 1. Test Example 1 <Cell line culture> The stem cells used in the test were Cellartis human iPS cell line (Passage 21-47) purchased from Takara Bio Inc.

[0177] The cell lines were tested for mycoplasma prior to the experiment and found to be negative. The cells were incubated with vitronectin recombinant human protein (Invitrogen) at 0.5 μg / cm 2 The cells were cultured on 6-well plates coated with 100% β-lactam IgG at a concentration of 1:1.

[0178] Stem cells were cultured in Essential 8 supplemented with 100 μg / ml Normocin. TM The cells were maintained in Flex (Thermo Fisher Scientific) medium (hereafter referred to as E8 medium). The medium was replenished every other day or every day depending on the degree of cell confluency. The cells were passaged when approximately 80% confluent (usually every 4 to 5 days).

[0179] CTS is used for cell detachment during cell passaging. TM TrypLE TM Select Enzyme (hereinafter, TrypLE Select; Thermo Fisher Scientific) was used.

[0180] Furthermore, during passaging, E8 medium containing 10 μM Y27632 (ROCK inhibitor, apoptosis inhibitor; Semgent) was used, and 24 hours after passaging, the medium was replenished with fresh E8 medium not containing Y27632.

[0181] <Spheroid formation process> Stem cells were detached from the culture dish using TrypLE Select. After detachment, they were collected as a single cell suspension in E8 medium containing 10 μM Y27632 (hereafter referred to as E8-10Y). To distinguish between dead and live cells, Trypan blue (Thermo Fisher Scientific) was used at a ratio of 1:1 (Trypan blue:cell suspension) to count the number of live cells.

[0182] An appropriate number of live cells required for the experiment was transferred to E8 medium containing 20 μM Y27632 (hereinafter referred to as E8-20Y). The E8-20Y cell suspension was dispensed into a 96-well U-bottom plate (ThermoFisher Scientific). At this time, the cells were distributed so that 100 μl / well and 3500 cells / well were obtained.

[0183] The plate containing the cells was centrifuged at 110 g for 6 minutes to promote cell aggregation. After 24 hours of culture, 100 μl of fresh E8 medium was added to each well to dilute the Y27632 and promote cell proliferation and growth of aggregates.

[0184] <Skin tissue formation process> In the skin tissue formation process, conditions such as the start time of the three-dimensional culture process and the culture form were examined (Examples 1 to 9).

[0185] [Example 1] (1st induction step) Differentiation induction of the stem cell spheroids obtained in the spheroid formation step was started. In the following description, the day when differentiation induction started (the day when the skin tissue formation step (first induction step) started) is referred to as "day 0".

[0186] On day 0, to initiate differentiation, all spheroids were individually harvested and transferred to a new 96-well U-bottom plate filled with 100 μl of differentiation medium per well, and the spheroids were cultured in suspension to initiate non-neural ectoderm formation. Differentiation medium is Essential6 TM (hereinafter referred to as "E6 medium") and contains 2% Matrigel (Corning), 10 μM SB431542 (Stemgent), 4 ng / ml bFGF (hereinafter referred to as FGF; PeproTech), and 5.0 ng / ml BMP4 (PeproTech).

[0187] (Second induction process) On day 3, to induce the formation of cranial neural crest-like cells (CNC cells), E6 medium containing 1 μM LDN-193189 (BMP inhibitor, Stemgent) and 250 ng / ml FGF was added in a volume of 25 μl per well, resulting in a final volume of 125 μl per well.

[0188] (Three-dimensional culture process) On the sixth day, 75 μl of fresh E6 medium was added per well to the 96-well U-bottom plate, and 200 μl of the resulting medium was transferred to a 24-well plate. In addition, 200 μl of new E6 medium was prepared with Matrigel, SB431542, FGF, BMP4, and LDN-193189 added to the 24-well plate to obtain the same composition, and the medium volume per well was 400 μl (400 μl of medium contains 1% Matrigel, 5 μM SB431542, 2.5 ng / ml BMP4, 125 nM LDN-193189, and 33.25 ng / ml FGF). Furthermore, a culture insert (millicell, diameter 12 mm; membrane pore size 3 μm, made of polycarbonate, hereinafter referred to as "CI") was placed. The CI used had a membrane coated with collagen.

[0189] Collagen-coated CIs were prepared by gelling collagen solution by adding 1N NaOH (0.023 times the volume of collagen) to Collagen I rat tail (Corning) diluted with PBS to 2 mg / mL, and placing 50 μL of the gel on the CI and leaving it at 37°C and 5% CO2 for 30 minutes.

[0190] Next, the cultures obtained in the second induction step were placed one by one on the CI, and the medium on the CI was removed. After that, no medium was added above the CI, and air-liquid interface culture was performed with the bottom of the culture in contact with the medium through the porous membrane of the CI.

[0191] Half of the medium was changed on days 8 and 10 (200 μl of spent medium was removed and 200 μl of fresh E6 medium was added).

[0192] On day 12, the entire amount of spent medium was removed, and 400 μl of maturation medium containing 1% Matrigel (Corning) was added to each well, and air-liquid interface culture was continued.

[0193] The maturation medium was Advanced DMEM / F-12 (Thermo Fisher Scientific) and Neurobasal TM The medium is a 1:1 mixture of Thermo Fisher Scientific GlutaMax 1× and GlutaMax 2×. TM (Thermo Fisher Scientific), 0.5x B-27 minus vitamin A (Thermo Fisher Scientific) and 0.5x N-2 (Thermo Fisher Scientific) as supplements, and contains 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific) and 100 μg / ml normocin.

[0194] On day 15, half of the spent medium was replaced with fresh maturation medium containing 1% Matrigel (200 μl spent medium was removed and 200 μl fresh medium was added). From day 18 onwards, half of the medium was replaced with fresh maturation medium without Matrigel every 3 days to continue the air-liquid interface culture.

[0195] [Example 2] The three-dimensional culture step was carried out in the same manner as in Example 1 above, except that two-layer liquid interfacial culture was carried out instead of air-liquid interface culture.

[0196] In the two-layer liquid interfacial culture, the medium on the CI was removed, and then keratinocyte serum free medium (KSFM, Thermo Fisher Scientific) was added to the CI. That is, the culture was performed in a state where the CI was filled with KSFM on the upper side and E6 medium (having the same composition as that used in the air-liquid interface culture in Example 1) on the lower side through the CI membrane.

[0197] Medium changes were performed on days 8 and 10. For the lower CI, half of the spent medium (200 μl) was removed and 200 μl of fresh E6 medium was added. For the upper CI, the entire spent medium was removed and 200 μl of KSFM was added to the top of each CI.

[0198] On the 12th day, the entire amount of spent medium was removed, and 400 μl of maturation medium containing 1% Matrigel was added to the lower CI portion and 200 μl of KSFM was added to the upper CI portion for each well, and bilayer interphase culture was continued.

[0199] On day 15, spent medium was replaced: for the lower CI, half the spent medium (200 μl) was removed and 200 μl of fresh maturation medium containing 1% Matrigel was added. For the upper CI, the entire spent medium was removed and 200 μl of KSFM was added to the top of each CI.

[0200] From day 18 onwards, the medium was changed every 3 days. Half of the medium in the lower part of the CI was replaced with fresh maturation medium without Matrigel, and the entire amount of the used medium in the upper part of the CI was replaced, and the bilayer intercellular culture was continued.

[0201] [Example 3] In the three-dimensional culture step, the procedure was the same as in Example 2, except that the start time of the three-dimensional culture step on the CI was changed from day 6 to day 12 after the start of induction. The changes are described below.

[0202] (Second induction process) The steps up to the sixth day were the same as those in Examples 1 and 2. On day 6, 75 μl of fresh E6 medium was added to bring the final volume to 200 μl. On days 8 and 10, half of the medium was changed (100 μl of spent medium was removed and 100 μl of fresh E6 medium was added).

[0203] (Three-dimensional culture process) On the 12th day, the entire amount of spent medium was removed, and 400 μl of maturation medium containing 1% Matrigel was added to the lower CI and 200 μl of KSFM was added to the upper CI for each well to perform bilayer interphase culture. The procedure for the two-layer liquid interphase culture was the same as that in Example 2 from day 12 onwards.

[0204] [Example 4] In the three-dimensional culture process, the start time of the three-dimensional culture process on the CI was changed from the 12th day to the 18th day after the start of induction. The procedure up to the start of the 12th day was the same as in Example 3. The changes will be described below.

[0205] (Third induction process) On day 12, to induce self-organization, all cultures were transferred to maturation medium containing 1% Matrigel filled in individual wells of a 24-well low-adhesion plate (ThermoFisher Scientific) at 500 μl.

[0206] To maintain constant medium circulation while culturing the organoids in suspension in maturation medium, the 24-well plates were placed on an orbital shaker shaking at 65 rpm in a 37°C incubator with 5% CO .

[0207] On day 15, half of the spent medium was replaced with fresh maturation medium containing 1% Matrigel (250 μl spent medium was removed and 250 μl fresh medium was added).

[0208] (Three-dimensional culture process) On the 18th day, half of the medium was replaced with fresh maturation medium without Matrigel, and the replaced medium was added to a 24-well plate at 400 μl per well, and collagen-coated CIs were placed on the plate. The culture obtained in the third induction step was then placed on the CI, one per well, and cultured at the air-liquid interface in the same manner as in Example 1.

[0209] [Example 5] The three-dimensional culture step was carried out in the same manner as in Example 4 above, except that two-layer liquid interfacial culture was carried out instead of air-liquid interface culture.

[0210] Specifically, on the 18th day, half of the medium was replaced with fresh maturation medium without Matrigel, and 400 μl of the replaced medium was added to the lower part of the CI for each well. After placing one culture on the CI per well, 200 μl of KSFM was added to the upper part of the CI to perform two-layer liquid interstitial culture. Thereafter, the medium was replaced every 3 days. The method of medium replacement was the same as that of Example 2.

[0211] [Examples 6 to 9] Examples 6 to 8 were the same as Examples 1 to 3 except that CIs without collagen coating were used. Example 9 was also carried out in which air-liquid interface culture was carried out using CIs without collagen coating in the three-dimensional culture process in Example 3. Table 1 lists the culture conditions for Examples 1 to 9. The "timing of starting the three-dimensional culture process" is stated as the number of days from the start date of differentiation induction (the start date of differentiation induction is considered to be day 0).

[0212] [Table 1]

[0213] <Immunostaining> For the skin tissues of Examples 1 to 9, immunohistochemical staining was performed for the following markers (primary antibodies) on the 27th day after the start of induction. E-cadherin (an intercellular adhesion molecule marker for epithelial cells) ·PDGFRα (mesenchymal cell marker)

[0214] Immunostaining was carried out according to the following procedure. Using the obtained skin tissue, frozen sections were cut parallel to the top and bottom direction of the CI so as to include the upper and lower regions of the skin tissue by the usual method, and the sections were washed with PBS-T. Then, 10% Gоat Serum was added for blocking, and the primary antibody (see above) was reacted at 4℃. After the reaction, the sections were washed with PBS-T, and the secondary antibodies (Alexa488 Antimouse and Alexa647 Antirabbit) were reacted at room temperature. After the reaction, the skin tissue was washed with PBS and mounted in a slide with ProLong Gold Antifade Mountant with DAPI (Invitrogen). The obtained skin tissue was subjected to a fluorescence microscope (Keyence Corporation) to capture stained images of the skin tissue.

[0215] <Result> The three-dimensional cultures of Examples 1 to 9 were observed to have a laminated dermis layer and epidermis layer. That is, by culturing an undeveloped three-dimensional culture on a porous membrane such as CI without incision, a layered three-dimensional skin tissue in which the dermis layer and the epidermis layer are laminated could be obtained.

[0216] 4, an epidermal layer expressing E-cadherin was observed to be laminated on a dermal layer expressing PDGFRα in Example 1. Moreover, the skin tissue obtained in Example 1 did not have the epidermal layer incorporated inside the tissue, but had the epidermal layer laminated flatly above the dermal layer.

[0217] 2. Test Example 2 After carrying out the <cell line culture> and <spheroid formation step> in the same manner as in Test Example 1, Examples 10 to 12 were carried out according to the following procedures.

[0218] [Example 10] As in Example 1, the first induction step was carried out on the day induction started, and the second induction step was carried out on the third day after induction started. Next, on the fourth day after the start of induction, the culture obtained in the second induction step was placed on the CI at 10 cells per well. In Example 10, FibColl (registered trademark) highly permeable atelocollagen insert for 24 wells (Koken Co., Ltd.) was used as the CI, and collagen coating of the CI was not performed. Subsequent culture was carried out in the same manner as in Example 1, and the three-dimensional skin tissue of Example 10 was obtained.

[0219] [Example 11] In Example 11, the same atelocollagen insert as in Example 10 was used as the CI, and 10 cultures obtained in the second induction step were placed per well. The three-dimensional skin tissue of Example 11 was obtained in the same manner as in Example 1, except for the type of CI and the number of cultures placed.

[0220] [Example 12] Cultivation was carried out in the same manner as in Example 9 to obtain the three-dimensional skin tissue of Example 12.

[0221] <Immunostaining> Immunohistochemical staining was performed using the following primary and secondary antibodies on the skin tissues of Examples 10 to 12. Immunohistochemical staining was performed on the 116th day after the start of induction for Examples 10 and 11, and on the 117th day after the start of induction for Example 12. Immunohistochemical staining was performed in the same manner as in Test Example 1 above.

[0222] Epithelial cell adhesion factor marker (Examples 10-11) Primary antibody: E-cadherin, Secondary antibody: Alexa488 Antimouse Mesenchymal cell markers (Examples 10-12) Primary antibody: PDGFRα, secondary antibody: Alexa647 Antirabbit -Dermal papilla cell marker Primary antibody: SOX2 (Examples 10 to 12), secondary antibody: Alexa555 Antirat (Examples 10 to 11), Alexa488 Antimouse (Example 12)

[0223] 5, in the three-dimensional skin tissues of Examples 10 to 12, a dermis layer expressing PDGFRα and an epidermis layer expressing E-cadherin were laminated in this order above the membrane indicated by the dashed line. Furthermore, it was confirmed that hair follicles were formed at the positions marked with * in the three-dimensional skin tissues. These results demonstrated that by initiating culture on CI when the cells are immature, approximately 12 days after the start of induction, the epidermal layer is layered above the dermal layer so that it is open to the outside world, and layered three-dimensional skin tissue containing hair follicles is formed.

[0224] 3. Test Example 3 The progress of differentiation induction of the spheroids obtained in the above <spheroid formation step> was confirmed by immunohistochemical staining. Specifically, the cultures on the 3rd, 5th, 6th, 8th, 10th, 12th, 15th, and 18th days from the start of induction were subjected to immunohistochemical staining using the following antibodies. The cultures were cultured in the same manner as in Examples 4 and 5 above, and on the 18th day from the start of induction, staining was performed without transition to CI. The procedure of immunohistochemical staining was the same as in Test Example 1.

[0225] -Epithelial cell adhesion molecule marker Primary antibody: E-cadherin, Secondary antibody: Alexa488 Antimouse Mesenchymal cell markers Primary antibody: PDGFRα, secondary antibody: Alexa647 Antirabbit

[0226] Figure 6 shows the results of immunohistochemical staining for each marker after each day of induction. E-cadherin fluorescence was confirmed on day 3 after the start of induction. Then, over the course of 10 days after the start of induction, the outer shell of the aggregates became clearer as the culture grew. On day 12 after the start of induction, PDGFRα fluorescence was confirmed outside the layer containing epithelial cells, and a layer containing mesenchymal cells had been formed surrounding the layer containing epithelial cells. On the 15th day after the start of induction, the layer containing mesenchymal cells had increased in thickness. On the 18th day after the start of induction, the layer containing mesenchymal cells had become even thicker than on the 15th day after the start of induction. However, no hair follicle formation was confirmed on the 18th day after the start of induction.

[0227] Considering the results of Test Examples 1 to 3, in order to obtain three-dimensional skin tissue in which the epidermal layer is layered above the dermal layer and the epidermal layer is open to the outside world, particularly skin tissue having hair follicles, it can be said that it is important to transfer and culture the three-dimensional culture onto a CI at an immature stage when few days have passed since the start of induction and differentiation has not progressed. [Industrial Applicability]

[0228] The present invention can be applied to techniques for producing skin tissue useful for clinical use or as a screening tool. [Explanation of symbols]

[0229] 10 Cell Culture Plates 11 Cell Culture Inserts M membrane C Three-dimensional culture L1 dermal layer L2 epidermal layer

Claims

1. A method for producing three-dimensional skin tissue, comprising a skin tissue formation step of producing three-dimensional skin tissue in which a dermis layer and an epidermis layer are laminated, The skin tissue formation step is a method for producing three-dimensional skin tissue, which includes a three-dimensional culture step in which a three-dimensional culture obtained by culturing stem cells in suspension is placed, without incision, on a membrane of a culture vessel equipped with a membrane permeable to a culture medium, and cultured to obtain a layered three-dimensional skin tissue having at least a dermis layer and an epidermis layer stacked thereon.

2. The method for producing a three-dimensional skin tissue according to claim 1 , wherein the three-dimensional culture step is carried out 1 to 20 days after the start of differentiation induction of the stem cells.

3. The method for producing a three-dimensional skin tissue according to claim 1 or 2, wherein the three-dimensional culture process comprises air-liquid interface culture and / or two-layer liquid interfacial culture in which different culture media are filled above and below the membrane.

4. The method for producing a three-dimensional skin tissue according to claim 3, wherein in the two-layer liquid culture, an epithelial cell medium is filled above the membrane, and the upper part of the three-dimensional culture is cultured in contact with the epithelial cell medium.

5. The method for producing a three-dimensional skin tissue according to any one of claims 1 to 4, wherein in the three-dimensional culture step, the three-dimensional skin tissue is obtained in which an epidermis layer is layered above a dermis layer.

6. The skin tissue forming step includes: A first induction step of inducing the formation of a non-neural ectodermal epithelium by culturing a spheroid containing stem cells in a first differentiation medium containing one or more factors selected from an antagonist of transforming growth factor β (TGFβ) signaling, an agonist of fibroblast growth factor (FGF) signaling, and an agonist of bone morphogenetic protein (BMP) signaling; The method for producing a three-dimensional skin tissue according to any one of claims 1 to 5, further comprising a step of culturing the three-dimensional culture that has undergone the first induction step in a second differentiation medium containing an agonist of fibroblast growth factor (FGF) signaling and / or an antagonist of bone morphogenetic protein (BMP) signaling, thereby inducing the three-dimensional culture into a higher-order structure.

7. The method for producing three-dimensional skin tissue according to any one of claims 1 to 6, wherein in the three-dimensional culture process, the three-dimensional culture placed on the membrane has a maximum diameter of 2 mm or less.

8. The method for producing three-dimensional skin tissue according to any one of claims 1 to 7, wherein in the three-dimensional culture step, the three-dimensional culture placed on the membrane does not have hair follicles.

9. The method for producing a three-dimensional skin tissue according to any one of claims 1 to 8, wherein in the three-dimensional culture step, the three-dimensional culture placed on the membrane contains undifferentiated mesenchymal cells or the thickness of a layer containing the mesenchymal cells is 200 µm or less.

10. The method for producing a three-dimensional skin tissue according to any one of claims 1 to 9, wherein the three-dimensional skin tissue is derived from a human.

11. The method for producing a three-dimensional skin tissue according to any one of claims 1 to 10, wherein the three-dimensional skin tissue obtained in the three-dimensional culture process has hair follicles.

12. A three-dimensional skin tissue produced by the method for producing three-dimensional skin tissue according to any one of claims 1 to 11.

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