JP2017201979A - Method for producing hair follicle tissue in vitro - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for easily producing hair follicle tissues or peripheral tissues thereof (hair-follicle tissues and the like) in vitro, or kits for use in such methods; to provide methods for easily evaluate whether formation of hair follicle tissues and the like is promoted or inhibited, or kits for use in such methods.SOLUTION: By preparing and culturing three layers of gel comprising approximately spherical gel (epidermal cell gel) embedding epidermal cells derived from a mammal, approximately spherical gel (pluripotent stem cell gel) coated with such epidermal cell gel and embedding pluripotent stem cells derived from a mammal, and approximately spherical gel (cell growth factor gel) coated with such pluripotent stem cell gel and containing cell growth factor, hair-follicle tissues and the like can be produced and whether formation of the hair follicle tissues and the like is promoted or inhibited can be evaluated.SELECTED DRAWING: None

Description

  The present invention evaluates the formation of a hair follicle tissue or a surrounding tissue (hereinafter sometimes referred to as “hair follicle tissue or the like”), a kit used for such a method, or a hair follicle tissue or the like in vitro. The present invention relates to a method or a kit used in such a method.

  Hair follicle tissue is a mammalian skin appendage that produces hair and repeats self-renewal for almost a lifetime in a mature organism. Although the hair follicle tissue itself is not directly necessary for maintaining life activity, there are not a few people who suffer from thinning hair, hair loss, or unwanted hair. From the viewpoint of QOL (Quality Of Life), these hair follicle tissue-related diseases Prevention and treatment of alopecia and hirsutism are needed.

  The development of a hair restorer or a hair suppressant requires an assay system that can accurately evaluate the hair restorer or the effect of hair growth. For example, a screening method is known in which a target substance having the proliferation activity of human hair follicle epithelial cultured cells is further evaluated for hair-growth effect by human head hair follicle organ culture method (Patent Document 1). However, since it is difficult to obtain human hair follicle tissue itself, there is a problem in terms of convenience, and it is not suitable for comprehensive screening. In addition, screening methods for organ-culturing hair follicles derived from mice or pigs instead of humans are also known (Patent Documents 2 and 3). However, it cannot be said that the reliability of screening results is high in that model animals other than humans are used.

  On the other hand, with recent advances in research related to cell engineering and stem cells, hair regeneration in vivo by cell transplantation has been actively promoted. For example, when a cell cluster is prepared and transplanted from a dental pulp-derived mesenchymal stem cell (Non-patent Document 1) or an induced pluripotent stem cell (iPS cell) (Non-patent Document 2) in vitro (in vitro), It has been reported that hair regenerates in the body (in vivo). In addition, it has been reported that hair follicle tissue is regenerated in vivo when dermal papilla cells are cultured and transplanted in vitro (Patent Document 4). However, all of these methods regenerate hair follicle tissue in vivo, and no method for producing hair follicle tissue in vitro has been reported so far.

JP 11-49647 A JP 2002-62289 A JP 2009-139353 A JP 2007-274949 A

Toyoshima, K.E., et al., Nature Communications, 3: 784 (2012) Veraitch, O., et al., J Invest Dermatol, 133: 1479-1488, (2013)

  The object of the present invention is to facilitate or suppress the formation of hair follicle tissues or the like, a kit for use in such a method, or the formation of hair follicle tissues in vitro (in vitro). An object of the present invention is to provide a method for simply evaluating the above or a kit used for such a method.

  The inventors of the present invention have been intensively researched to solve the above problems. In the process, it was considered that hair follicle tissue and the like could be produced if the in vivo form of hair follicle tissue and the like could be simulated in vitro. Therefore, an approximately spherical gel (pluripotent stem cell gel) that embeds pluripotent stem cells covers an approximately spherical gel (cell growth factor gel) that contains cell growth factor and embeds epidermal cells. A three-layer gel formed by coating a spherical gel (epidermal cell gel) with the pluripotent stem cell gel is further prepared and cultured in a cell growth medium containing hair papilla cell growth factor. When culturing was carried out instead of the cell growth culture solution, hair follicle tissue and the like were found, and the present invention was completed.

That is, the present invention is as follows.
[1] A kit for evaluating hair follicle tissue or surrounding tissue formation, comprising the following (p) to (s):
(P) a gel material for embedding mammalian epidermal cells in a substantially spherical gel;
(Q) a gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel;
(R) a gel material for containing a cell growth factor in a substantially spherical gel;
(S) A substantially spherical gel that embeds mammalian pluripotent stem cells is coated on a substantially spherical gel containing a cell growth factor, and the substantially spherical gel that embeds mammalian epidermis cells Package insert describing coating of a substantially spherical gel embedding pluripotent stem cells;
[2] (p) a gel material for embedding mammalian epidermal cells in a substantially spherical gel, and (q) a gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel. The evaluation kit according to [1] above, wherein the gel material for containing the collagen (r) cell growth factor in a substantially spherical gel is alginic acid or a salt thereof.
[3] The evaluation kit according to [1] or [2] above, wherein the cell growth factor is fibroblast growth factor (FGF) -2.
[4] A method for evaluating formation of a hair follicle tissue or its surrounding tissue, comprising the following steps (A) to (C), wherein the basic culture solution for mammalian cell culture in step (A): And the said evaluation method by which a test chemical | medical agent or a test substance is contained in any one or both of the basic culture solutions for mammalian cell cultures in a process (B).
(A) a substantially spherical gel that embeds mammalian epidermal cells;
A substantially spherical gel for embedding mammalian pluripotent stem cells, coated with a substantially spherical gel for embedding the mammalian epidermal cells;
A three-layered gel comprising a substantially spherical gel containing a cell growth factor coated with a substantially spherical gel embedding the pluripotent stem cells, and one or more hair papilla cell growth factors Culturing in a basal culture medium for mammalian cell culture, containing serum or serum replacement;
(B) After culturing in step (A), culturing the three-layer gel in a basal culture medium for mammalian cell culture containing serum or a serum substitute;
(C) After the culture in step (B), a step of detecting an increase or decrease in the formation efficiency of the hair follicle tissue or the surrounding tissue in the three-layer gel;
[5] The evaluation method according to [4], wherein the culture in the step (A) is performed for 4 to 13 days.
[6] A substantially spherical gel that embeds mammalian epidermal cells, and a substantially spherical gel that embeds mammalian pluripotent stem cells are collagen gels, and a substantially spherical gel containing a cell growth factor is The evaluation method according to [4] or [5] above, which is an alginate gel with a divalent or higher cation.
[7] The above-mentioned [4] to [6], wherein the two or more dermal papilla cell growth factors include at least bovine pituitary extract (BPE) and cyproterone acetate (Cyp). The evaluation method described.
[8] The evaluation method according to any one of [4] to [7], wherein the cell growth factor is fibroblast growth factor (FGF) -2.
[9] A kit for preparing a hair follicle tissue or its surrounding tissue, comprising the following (p) to (s).
(P) a gel material for embedding mammalian epidermal cells in a substantially spherical gel;
(Q) a gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel;
(R) a gel material for containing a cell growth factor in a substantially spherical gel;
(S) A substantially spherical gel that embeds mammalian pluripotent stem cells is coated on a substantially spherical gel containing a cell growth factor, and the substantially spherical gel that embeds mammalian epidermis cells Package insert describing coating of a substantially spherical gel embedding pluripotent stem cells;
[10] (p) a gel material for embedding mammalian epidermal cells in a substantially spherical gel, and (q) a gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel. The preparation kit according to [9] above, wherein the gel material for containing (r) the cell growth factor in a substantially spherical gel is alginic acid or a salt thereof.
[11] The preparation kit according to [9] or [10] above, wherein the cell growth factor is fibroblast growth factor (FGF) -2.
[12] A method for producing an in vitro hair follicle tissue or its surrounding tissue comprising the following steps (a) and (b):
(A) a substantially spherical gel that embeds mammalian epidermal cells;
A substantially spherical gel for embedding mammalian pluripotent stem cells, coated with a substantially spherical gel for embedding the mammalian epidermal cells;
A three-layered gel comprising a substantially spherical gel containing a cell growth factor coated with a substantially spherical gel embedding the pluripotent stem cells, and one or more hair papilla cell growth factors Culturing in a basal culture medium for mammalian cell culture, containing serum or serum replacement;
(B) a step of culturing the three-layer gel after culturing in the step (a) in a basic culture solution for mammalian cell culture containing serum or a serum substitute;
[13] The production method according to [12] above, wherein the culture in the step (a) is performed for 4 to 13 days.
[14] A substantially spherical gel that embeds mammalian epidermal cells and a substantially spherical gel that embeds mammalian pluripotent stem cells are collagen gels, and a substantially spherical gel containing a cell growth factor is The method according to [12] or [13] above, which is an alginate gel with a divalent or higher cation.
[15] The above [12] to [14], wherein the two or more dermal papilla cell growth factors include at least bovine pituitary extract (BPE) and cyproterone acetate (Cyp). The manufacturing method as described.
[16] The production method according to any one of [12] to [15] above, wherein the cell growth factor is fibroblast growth factor (FGF) -2.

  Further, as another embodiment of the present invention, a screening method for a prophylactic or therapeutic agent for alopecia or alopecia or hirsutism characterized by comprising the above steps (A) to (C), A screening method for a skin irritant substance that affects the increase or decrease of hair, any one of a basic culture solution for mammalian cell culture in step (A) and a basic culture solution for mammalian cell culture in step (B) Examples of the screening method include one or both of a test drug or a test substance (hereinafter, these may be collectively referred to as “the present screening method”).

  Further, as another embodiment of the present invention, a screening kit for a prophylactic or therapeutic agent for alopecia or alopecia or hirsutism characterized by comprising the above (p) to (s), A screening kit for skin irritants that affects the increase or decrease in hair (hereinafter, these may be collectively referred to as “the screening kit”). This screening kit relates to a use invention relating to a kit for screening a preventive or therapeutic agent for alopecia or alopecia, or hirsutism, and a kit for screening a skin irritant substance that affects the increase or decrease of hair. It is a use invention, and as such a kit, there are generally used components of this type of kit, such as a carrier, a pH buffer, a stabilizer, a cross-linking agent for gelling the gel material, one type or two or more types of hair. It may contain papillary cell growth factor, serum, serum replacement, basic culture medium for mammalian cell culture, and instruction manual.

  When the production method or production kit of the present invention is used, hair follicle tissue and the like can be produced relatively easily in vitro. The produced hair follicle tissue or the like is useful for treatment of patients with alopecia (alopecia areata, male pattern alopecia, etc.) or alopecia (congenital alopecia, acquired alopecia, etc.). In addition, when the evaluation method or the evaluation kit of the present invention is used, the test drug or the test substance has an action of promoting the formation of hair follicle tissue or the like, or the formation suppression (inhibition) of hair follicle tissue or the like. Hair cycle caused by causes such as alopecia; alopecia; hirsutism; nutritional deficiencies, damage to the scalp and hair, lack of blood flow, decreased female hormones, stress, etc. Useful for screening for prophylactic or therapeutic agents for diseases related to hair follicle tissues such as abnormalities (states in which hair is removed in a thin and short state), or screening for skin irritants that affect the increase or decrease in hair It is.

A substantially spherical calcium alginate gel layer (first layer) containing fibroblast growth factor (FGF) -2 (also referred to as basic fibroblast growth factor: bFGF), ES It is a conceptual diagram of a substantially spherical three-layer gel comprising a type I collagen gel layer (second layer) embedding cells and a type I collagen gel layer (third layer) embedding epidermal cells. It is a figure which shows a phase-contrast microscope image when the three-layer gel of FIG. 1 is observed from the upper part of a 3rd layer. A portion surrounded by a broken line indicates a first layer, a second layer, and a third layer in order from the inside. It is a figure which shows the hematoxylin eosin (HE) dyeing | staining image in the Combination group of the 28th day after culture | cultivating the 3 layer gel of FIG. Enlarged regions (a and b) surrounded by broken lines in the upper image are shown as a and b in the lower image. Moreover, the scale bar in the figure indicates 200 μm. The figure which shows the HE staining image (left figure a) of the hair follicle tissue in a mouse fetus, and the HE staining image (right figure b) of the hair follicle tissue in the Combination group on the 48th day after culturing the three-layer gel of FIG. It is. The arrow in the figure indicates the hair follicle tissue. It is a figure which shows the HE dyeing | staining image (upper part), cytokeratin immunostaining image (middle part), and smooth muscle actin immunostaining image (lower part) in the Combination group of 28-49 days after culture | cultivating the 3 layer gel of FIG. . The arrow in the first figure from the left indicates the hair follicle tissue, and an enlarged view thereof is shown in the second figure from the left. Moreover, the scale bar in the figure indicates 100 μm. A two-layer gel consisting of a type I collagen gel layer (second layer) embedding ES cells and a type I collagen gel layer (third layer) embedding epidermal cells was observed from the top of the third layer. It is a phase contrast microscope image. Portions surrounded by broken lines indicate the second layer and the third layer in order from the inside. It is a phase-contrast microscope image when the monolayer gel of the type I collagen gel layer which embeds ES cells and epidermal cells is observed from above. A substantially spherical calcium alginate gel layer (first layer) containing FGF-2, a type I collagen gel layer (second layer) embedding ES cells, and a type I collagen gel layer (first layer) embedding epidermal cells It is a phase-contrast microscope image when the sheet-like 3 layer gel which consists of 3 layers) is observed from the upper part of the 3rd layer. It is a figure which shows the HE dyeing | staining image in the Combination group of the 28th day after culture | cultivating the bilayer gel of FIG. It is a figure which shows the phase-contrast microscope image in the Combination group of the 18th day after culture | cultivating the sheet-like three-layer gel of FIG.

  The production method of the present invention includes a substantially spherical gel (hereinafter sometimes referred to as “epidermal cell gel”) embedding mammalian-derived epidermal cells, and a mammalian-derived coating coated on the epidermal cell gel. A substantially spherical gel that embeds pluripotent stem cells (hereinafter sometimes referred to as “pluripotent stem cell gel”), and a substantially spherical gel containing a cell growth factor coated on the pluripotent stem cell gel. (Hereinafter referred to as “cell growth factor gel”) is a three-layer gel (hereinafter sometimes referred to as “the present three-layer gel”), one or more dermal papilla cell growth factor, and serum or serum. A step (a) of culturing in a basic culture solution for mammalian cell culture (hereinafter sometimes referred to as “the culture medium for differentiation induction”) containing the alternative, and the three layers after the culture in step (a) Basic culture for mammalian cell culture containing gel, serum or serum replacement (Hereinafter referred to as “the present basic culture solution”) and the step (b) of culturing in vitro (in vitro) a method for producing a hair follicle tissue or its surrounding tissue (hereinafter referred to as “the present production method”) Is not particularly limited.

  In addition, the preparation kit of the present invention includes a gel material (p) for embedding mammalian-derived epidermal cells in a substantially spherical gel, and a pluripotent stem cell derived from a mammal. A gel material (q) for embedding in a cell, a gel material (r) for containing a cell growth factor in a substantially spherical gel, and a pluripotent stem cell gel coated on the cell growth factor gel, A kit for preparing a hair follicle tissue or a surrounding tissue thereof (hereinafter referred to as “the present preparation kit”), comprising a package insert (s) described to coat the gel with the pluripotent stem cell gel. The kit for producing the present invention is a use invention relating to a kit for producing a hair follicle tissue or its surrounding tissue, and such a kit is generally used for this kind of kit. Ingredients such as carriers, H buffer, stabilizer, cross-linking agent for gelling the gel material, one or more dermal papilla cell growth factor, serum, serum substitute, basic culture solution for mammalian cell culture, instruction manual May be included.

  As the evaluation method of the present invention, the step (A) of culturing the three-layer gel in the differentiation-inducing culture medium, and the culture in the step (A), the three-layer gel in the basic culture solution A follicular tissue comprising: a step (B) for culturing; and a step (C) for detecting an increase or decrease in the formation efficiency of the hair follicle tissue or the surrounding tissue in the three-layer gel after the culture in the step (B). A method for evaluating formation or peripheral tissue formation of hair follicle tissue, wherein the test drug is used in one or both of the differentiation-inducing culture medium in step (A) and the basic culture medium in step (B). Or if it is the said evaluation method (henceforth "this evaluation method") containing a test substance, it will not restrict | limit in particular.

  Moreover, as an evaluation kit of this invention, evaluation of hair follicle tissue formation provided with the said gel material (p)-(r) and the said package insert (s), or evaluation of surrounding tissue formation of a hair follicle tissue. The kit is not particularly limited as long as it is a kit for use in the present invention (hereinafter sometimes referred to as “the present evaluation kit”), and the present evaluation kit is used to evaluate the formation of hair follicle tissue or the surrounding tissue of the hair follicle tissue. The kit is a use invention relating to the kit, and as such a kit, components such as a carrier, a pH buffer, a stabilizer, a cross-linking agent for gelling the gel material, one type or two types are generally used. It may contain the above-described dermal papilla cell growth factor, serum, serum substitute, basic culture solution for mammalian cell culture, and instruction manual.

  In the present invention, the “hair follicle tissue” is composed of a root sheath (inner root sheath and outer root sheath) that is an epithelial component on the inside, and a connective tissue hair follicle that is a connective tissue component on the outside. Means a double-layered structure. In the present invention, the “peripheral tissue of the hair follicle tissue” means a tissue attached to the hair follicle tissue and present in the periphery thereof (for example, napped muscles, hair bulges, sebaceous glands, etc.).

  In the present invention, “alopecia or alopecia” means a state in which hair is reduced due to genetic (congenital) factors or acquired factors such as aging, stress, administration of drugs such as anticancer agents, or hair The alopecia includes alopecia areata, androgenetic alopecia, diffuse alopecia, and alopecia due to administration of drugs such as anticancer agents. In the present invention, “hyperhirsia” means dermatomyositis, systemic diseases (such as advanced HIV infection), endocrine diseases such as hypothyroidism, malnutrition, corticosteroids, immunosuppressants, etc. This means that male hair is overproduced or the reactivity to male hormone is improved, resulting in a state where soft hair has changed to hard hair.

  In this preparation method, this evaluation method, or this screening method, the formation of hair follicle tissue or the like is caused by the hematoxylin Eosin (HE) staining method or the hair follicle tissue marker (cytokeratin, Versican [Vcan ], An immunohistochemical staining method using an antibody against ALP [Alkaline phosphatase], CD200, CD34, etc.) or an antibody against a marker around the hair follicle (smooth muscle actin [αSMA], calponin, K15, CD200, etc.) It can be confirmed by a phase contrast microscope.

  Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, rabbits such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, cats such as dogs and cats, humans, Primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc. can be exemplified, and when the prepared hair follicle tissue etc. are used for screening therapeutics for these diseases, mice or humans are preferred When using a hair follicle tissue or the like for regenerative medicine, a human is preferable.

  In the present invention, “substantially spherical” as a whole has a shape of a ball such as a true sphere or an ellipsoid (a shape solidified like a ball), and has fine irregularities (wrinkles, protrusions, depressions, etc.) on the surface. It means an almost spherical shape. The “substantially spherical” includes a substantially polyhedral shape (substantially tetrahedron, substantially pentahedron, substantially hexahedron, etc.) and a substantially cylindrical shape for convenience. As the “polyhedron” in the “substantially polyhedral shape” described above, “convex polyhedron” is preferably mentioned.

  In the present invention, “X is coated with Y” means that at least a part of X may be covered with Y as long as the effects of the present invention can be obtained. Of these, the proportion covered with Y is usually within the range of 40 to 100%, preferably 45 to 100%, more preferably 50 to 100%, still more preferably 55 to 100%, and even more preferably. Is 60% to 100%, particularly preferably 65 to 100%, particularly preferably 70 to 100%, particularly preferably 75 to 100%, particularly more preferably 80 to 100%, and even more preferably 85 to 100%. %, Most preferably 90% to 100%. In addition, this ratio may be the same for each layer, or may be different.

  As the gel in the above epidermal cell gel or pluripotent stem cell gel, when gel material (solution) containing epidermal cells or pluripotent stem cells is gelled, these cells can be embedded in the gel, There is no adverse effect on the culture of these cells, and the gel state may be maintained at the culture temperature (usually 30 to 40 ° C.) of the present three-layer gel. When a gel material (solution) containing a growth factor is gelled, the cell growth factor contained in the gel can be gradually released (sustained release), and the gel state is maintained at the culture temperature of the present three-layer gel. Specifically, alginate gel with divalent or higher cation (for example, calcium ion, divalent or trivalent iron ion, aluminum ion, etc.); gelatin gel; collagen Agarose gel; carrageenan gel; cellulose gel and the like. In the case of epidermal cell gel and pluripotent stem cell gel, collagen gel is preferable, and in the case of cell growth factor gel, 2 Alginate gels with higher valence cations are preferred.

  The collagen gel may be a fibrillar collagen gel capable of forming fibrils. Specifically, type I, type II, type III, type V, type XI collagen gels, type I, type II, III Examples thereof include a gel made of a combination of two or more selected from type, type V, and type XI collagen, and a type I collagen gel can be suitably exemplified.

  Moreover, as said gel material (p) and gel material (q), when gel material (solution) containing an epidermal cell or a pluripotent stem cell is gelatinized, these cells may be embedded in a gel. It is sufficient that the culture of these cells is not adversely affected and the gel state is maintained at the culture temperature of the present three-layer gel, and the gel material (r) contains a cell growth factor. When the gel material (solution) is gelled, the cell growth factor contained in the gel can be gradually released (sustained release), and the gel state is maintained at the culture temperature of the three-layer gel. Specific examples thereof include alginic acid or a salt thereof, gelatin, collagen, agar or a purified product thereof (agarose), carrageenan, cellulose and the like. Examples of gel material (p) and gel material (q) If collagen Le is preferred if the gel material (r), alginic acid or a salt thereof.

  Examples of the salt of alginic acid include alginates with divalent or higher cations (for example, calcium ions, divalent or trivalent iron ions, aluminum ions, etc.) and monovalent cations (sodium ions, potassium ions, ammonium ions). Etc.) and alginates.

  The collagen may be any fibrous collagen capable of forming fibrosis, specifically, type I, type II, type III, type V, type XI collagen, and combinations of two or more selected from these. Can be mentioned, and type I collagen can be preferably exemplified.

  The epidermal cells may be those prepared from mammals each time (prepared at the time of use) as long as they are cells derived from epidermal tissues, or those established by in vitro culture. Examples of such established epidermal cells include epidermal cells derived from adult humans (HaCaT cells), epidermal cells derived from newborn mice (Balb / MK cells), epidermal cells derived from rat fetuses (FRSK cells), and the like. it can.

  The pluripotent stem cell (pluripotent stem ce11) is not particularly limited as long as it can be induced to differentiate into hair follicle tissue and the like, embryonic stem cells (embryonic stem cells: ES cells) isolated from early embryos, Isolated from embryonic germ cells (EG cells) isolated from fetal primordial germ cells (see, for example, Proc Natl Acad Sci US A. 1998, 95: 13726-31) and testis immediately after birth Germline stem cells (GS cells) (for example, see Nature. 2008, 456: 344-9) and cells with pluripotency obtained by selecting from mesenchymal tissues such as skin and bone marrow (Muse cells [Multi-lineage differentiating Stress Enduring cells]) and by introducing multiple genes into somatic cells such as skin cells, it induces dedifferentiation of the subject's own somatic cells and is as multipotent as ES cells Induced pluripotent stem cells (iPS; induced plur) ipotent stemcell) and hair follicle stem cells obtained by isolating nestin-expressing cells (Japanese Patent Publication No. 2005-502377), among which ES cells and iPS cells are preferable.

  ES cells are cultured in the above serum-containing or serum-free medium on feeder cells such as primary fibroblasts [MEF], STO cells, SNL cells derived from mouse embryos treated with mitomycin C. Can be manufactured. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718 and the like. EG cells can be produced by culturing primordial germ cells in the above serum-containing or serum-free medium containing mSCF, LIF and FGF-2 (see, for example, Ce11. 1992, 70: 841-847). . Muse cells select stress-resistant cells by applying stress such as trypsin or hypoxia treatment to mesenchymal tissues such as skin and bone marrow, or SSEA-3, which is a surface antigen of pluripotent stem cells. It can be produced by selecting cells using expression as an indicator, and further isolating the cells by overlapping suspension culture in a single cell state (Japanese Patent No. 5185443, Proc Natl Acad Sci US A. 2010). , 107: 8639-8643). iPS cells are produced by introducing reprogramming factors such as Oct3 / 4, Sox2 and Klf4 (c-Myc or n-Myc as necessary) into somatic cells (eg, fibroblasts, skin cells, etc.). (Eg, Ce11. 2006, 126: 663-676, Nature. 2007, 448: 313-317, Nat Biotechno 1. 2008, 26; 101-106, Cel1. 2007, 131: 861-872, Science. 2007 , 318: 1917-1920, Ce11 Stem Cells. 2007, 1: 55-70, Nat Biotechnol. 2007, 25: 1177-1181, Nature. 2007, 448: 318-324, Cell Stem Cells. 2008, 2: 10- 12, Nature. 2008, 451: 141-146, Science. 2007, 318: 1917-1920). Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (eg, Nature. 1997, 385: 810-813, Science. 1998, 280 : 1256-1258, Nature Biotechnology. 1999, 17: 456-461, Nature. 1998, 394: 369-374, Nature Genetics. 1999, 22: 127-128, Proc. Natl. Acad. Sci. USA. 1999, 96 : 14984-14989, Nature Genetics. 2000, 24: 109-110). Specific examples of pluripotent stem cells include human ES cell H9 strain (WA09), human ES cell H1 (WA01) strain (National Stem Cell bank, WISC Bank), KhES-1, KhES-2 and KhES-3. (Estimated stem cell medicine research center attached to Kyoto University Research Institute for Regeneration) Human ES cells such as mouse ES cells (EB3, EB5, etc.) (RIKEN Cell Bank), HES3, HES4, and HES6 (National Stem Cell bank, Monash University) Or iPS cells (RIKEN BioResource Center, Kyoto University) obtained by introducing Oct3 / 4 gene, Klf4 gene, C-Myc gene and Sox2 gene, Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky ( JCRB1329 strain), Toe (JCRB1338 strain), Lollipop ( CRB1336 strain) (National Center for Child Health and Development, Research Institute for Intractable Diseases and Diseases, JCRB Cell Bank), UTA-1 strain and UTA-1-SF-2-2 strain (both from the University of Tokyo), Oct3 Examples include iPS cells such as iPS cells (Nat Biotechnol. 2008, 26: 101-106) obtained by introducing / 4 gene, Klf4 gene and Sox2 gene.

  The cell growth factor may be any substance (compound, cytokine, extract, etc.) that can promote cell growth of mammalian cells. Specifically, bovine pituitary extract (BPE), Cypro Cyproterone acetate (Cyp), Growth Blocking Peptide, Minoxidil, Polyamine (ORYZA POLYAMINE, Putrescine, Spermidine, Spermine, etc.), Insulin, Transferrin , Triiodothyronine (TIT), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (Insulin-like Growth Factor-1) IGF-1), Wnt10, Sonic Hedgehog ( Sonic hedgehog (SHH), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), FGF-2, FGF-7, FGF-9, bone morphogenetic protein 4 [Bone Morphogenetic Protein 4: BMP4], BMP5, Activin A, Stem Cell Factor (SCF), Dkk1 (Dickkopf-1), Wnt inhibitors (XAV, IWP4, etc.), Wnt activators (CHIR, Kenpaullone, etc.), leukemia Inhibitory factors (Leukemia Inhibitory Factor: LIF), Wnt, TGF-β, and combinations thereof can be mentioned, and among these, FGF-2 is preferred.

  Specific examples of the serum include fetal bovine serum (FBS) and calf bovine serum (CS). The serum substitute is a component (component) used for culturing and proliferating cells instead of serum, and may have any effect similar to that of serum. Specifically, commercially available B27 A supplement (-insulin) (made by Life Technologies), N2 supplement (made by Life Technologies), B27 supplement (made by Life Technologies), Knockout Serum Replacement (made by Invitrogen) etc. can be mentioned.

  Examples of the basic culture solution for mammalian cell culture include nutrient sources necessary for the growth of mammalian cells, such as amino acids (glutamine, alanine, asparagine, serine, aspartic acid, cysteine, glutamic acid, glycine, proline, tyrosine, arginine, Histidine, leucine, isoleucine, methionine, lysine, ornithine, phenylalanine, taurine, threonine, tryptophan, valine, etc.), vitamins (choline chloride, pantothenate, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid, Biotin, inositol, etc.) and sugars (glucose, glucosamine, etc.) may be used. Specifically, GMEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), M-199 (M edium 199), EMEM (Eagle's Minimum Essential Medium), αMEM, DMEM (Dulbecco's modified Eagle's Medium), Ham's F12, RPMI 1640, Fischer's, AIM-V, PCGM (TMTPGM-250, manufactured by Toyobo Co., Ltd.) be able to. The basic culture solution for mammalian cell culture includes an iron source (transferrin, lactoferrin, etc.), minerals (sodium selenite, cobalt carbonate, calcium iodate, etc.), steroids (progesterone, β- Estradiol, etc.), antibiotics (eg, penicillin, streptomycin, etc.), buffers (TRIS [Tris (hydroxymethyl) aminomethane], HEPES [4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid], PIPES [Piperazine-1,4 Additives such as -bis (2-ethanesulfonic acid], MOPS [3-Morpholinopropanesulfonic acid], etc.) may be appropriately supplemented.

  One or more types of dermal papilla cell growth factor may be any one that can promote cell proliferation of dermal papilla cells (compound, cytokine, extract, etc.), and specifically, BPE, Cyp, JP, Growth inhibitory peptide, minoxidil, polyamine (ORYZA POLYAMINE, putrescine, spermidine, spermine, etc.), FGF-2, FGF-7, insulin, transferrin, TIT, VEGF, HGF, IGF described in JP-A-2006-45098 -1, Wnt10, SHH, and combinations thereof, and those containing at least BPE and Cyp are preferred.

  The present three-layer gel can be prepared, for example, according to the following procedure. First, a cell growth factor is mixed with the gel material (r) in the form of a solution to prepare a cell growth factor-containing solution, and an incubator using a minute amount dropping (discharge) means (microdispenser, microdispenser, etc.) After dropping on a glass or plastic multi-well plate, culture dish [petri dish, dish], etc., gelation treatment is performed to prepare a cell growth factor gel. The concentration of the cell growth factor in the gel solution is appropriately determined according to the volume and / or surface area of the gel, the type of cell growth factor to be used, etc., but is usually in the range of 0.1 ng / mL to 100 μg / mL. It is preferably 1 ng / mL to 10 μg / mL, more preferably 1 ng / mL to 3 μg / mL, still more preferably 1 to 1000 ng / mL, and most preferably 5 to 500 ng / mL. Moreover, as the volume of the cell growth factor-containing gel solution to be dropped, the gel solution dropped on the incubator may be substantially spherical, and is usually within a range of 0.1 μL to 10 mL, preferably 0.1 to 1000 μL. More preferably, it is 0.3-300 microliters, More preferably, it is 0.3-100 microliters, Still more preferably, it is 0.3-30 microliters, Most preferably, it is 0.5-5 microliters.

Subsequently, a mammalian pluripotent stem cell is suspended in the gel material (q) in a solution to prepare a mammalian pluripotent stem cell suspension, and a cell growth factor is prepared using the minute amount dropping means. After dropping on the gel, gelation treatment is performed to prepare a cell growth factor gel (substantially spherical two-layer gel) coated with a pluripotent stem cell gel. The concentration of mammalian pluripotent stem cells in the suspension is appropriately determined according to the volume and / or surface area of the gel, the type of mammalian pluripotent stem cells to be used, etc., but usually 1 × 10 to 1 × 10 ⁷ cells / μL, preferably 1 × 10 ² to 1 × 10 ⁶ cells / μL, more preferably 1 × 10 ² to 1 × 10 ⁵ cells / μL, even more preferable. Is 1 × 10 ³ to 1 × 10 ⁴ cells / μL. In addition, the volume of the mammalian pluripotent stem cell suspension to be dropped may be approximately spherical when the cell suspension dropped on the incubator is usually in a range of 0.1 μL to 10 mL, preferably It is 0.1-1000 microliters, More preferably, it is 0.5-300 microliters, More preferably, it is 1-100 microliters, Still more preferably, it is 1-30 microliters, Most preferably, it is 1-10 microliters.

Next, mammalian epidermis cells are suspended in the gel material (p) in a solution state to prepare a mammalian epidermis cell suspension, which is dropped onto the pluripotent stem cell gel using the minute amount dropping means. Then, a gelation treatment is performed to prepare a cell growth factor gel (this three-layer gel) sequentially coated with an epidermal cell gel and a pluripotent stem cell gel. The concentration of mammalian epidermal cells in the suspension is appropriately determined according to the volume and / or surface area of the gel, the type of mammalian epidermal cells used, etc., but is usually 1 × 10 to 1 × 10 ⁷ cells. / ΜL, preferably 1 × 10 ² to 1 × 10 ⁶ cells / μL, more preferably 1 × 10 ² to 1 × 10 ⁵ cells / μL, and even more preferably 1 × 10 6 ³ to 1 × 10 ⁴ cells / μL. Moreover, the volume of the mammalian epidermal cell suspension to be dripped is not particularly limited as long as the cell suspension dripped onto the incubator is substantially spherical, and is usually in the range of 0.1 μL to 10 mL, preferably 0.8. It is 1-1000 microliters, More preferably, it is 0.5-500 microliters, More preferably, it is 1-200 microliters, More preferably, it is 1-50 microliters, Most preferably, it is 1-20 microliters.

  The present three-layer gel is formed by covering a cell growth factor gel with a pluripotent stem cell gel and further covering the pluripotent stem cell gel with an epidermal cell gel. For this reason, the volume of each gel is usually appropriately increased in the order of cell growth factor gel, pluripotent stem cell gel, and epidermal cell gel (1: 2: 3, 1: 2: 4, 1: 3). : 5, 1: 3: 4, 1: 3: 6, etc.).

  When using alginic acid as the gel material, considering its characteristics (insoluble in water), in the alginate solution with monovalent cations to which monovalent cations (sodium ion, potassium ion, ammonium ion, etc.) are added, Cell growth factors are mixed, or mammalian pluripotent stem cells or epidermal cells are suspended. In addition, when an alginate with a divalent or higher cation is used as the gel material, in consideration of its characteristics (ion crosslinking [gelation occurs by a divalent or higher cation]), a chelating agent (EDTA [Ethylene Diamine Tetraacetic Acid] EGTA [Ethylene Glycol Tetraacetic Acid]), a cell growth factor is mixed in a solution of alginate with a divalent or higher cation, and mammalian pluripotent stem cells or epidermal cells are suspended. In addition, when collagen is used as the gel material, it is usually adjusted to neutrality with an alkali agent (potassium hydroxide or sodium, potassium carbonate or sodium, etc.) in consideration of its characteristics (acidity and coagulation temperature around 15 ° C.). A cell growth factor is mixed in a collagen solution at a low temperature (usually 0 to 20 ° C.) or mammalian pluripotent stem cells or epidermal cells are suspended. In addition, when agar, agarose, or carrageenan is used as the gel material, cell growth factors are mixed in 30-40 ° C agar, agarose, or carrageenan solution, considering their characteristics (coagulation temperature around 35 ° C). Or suspending mammalian pluripotent stem cells or epidermal cells.

  In the gelation treatment, a crosslinking agent, temperature conditions, and the like can be appropriately selected depending on the type of gel material. For example, when the gel material is alginic acid or a salt thereof, the above-mentioned divalent or higher cation is added as a cross-linking agent to cause gelation by causing ionic cross-linking with the divalent or higher cation. When the gel material is collagen, warm a low-temperature (usually 0 to 20 ° C.) cell growth factor-containing solution, pluripotent stem cell or epidermal cell suspension to a range of 25 to 40 ° C. To gel. When the gel material is agar, agarose, or carrageenan, the cell growth factor-containing solution at 30 to 40 ° C. or the pluripotent stem cell or epidermal cell suspension is cooled to a range of 0 to 28 ° C. To gel.

  As long as the effect of this invention is acquired, this 3 layer gel may contain arbitrary structures other than 3 layer gel, and gel layers other than 3 layer gel are mentioned as this arbitrary structure. The present three-layer gel includes one having one or two or more arbitrary gel layers other than the three-layer gel, for example, two or more gel layers corresponding to the first layer in the present three-layer gel. And those having two or more gel layers corresponding to the second layer in the present three-layer gel, those having two or more gel layers corresponding to the third layer in the present three-layer gel, and the like. For example, when two gel layers corresponding to the first layer are provided, the two layers may have the same composition, or the types of cell growth factors may be different.

  In the step (a) of the production method and the step (A) of the evaluation method or the screening method, the three-layer gel is placed in the differentiation-inducing culture medium, that is, in the basic culture liquid containing hair papilla cell growth factor. When cultured in, the mammalian pluripotent stem cells in the present three-layer gel are induced to differentiate into hair follicle tissue or the like. On the other hand, if such culture is excessive, the ratio of inducing differentiation into tissues (nerve tissue, myocardial tissue, etc.) other than the hair follicle tissue increases, so after the step (a) or step (A), the present three layers The gel is cultured in the basic culture medium instead of the differentiation-inducing culture medium, that is, in the basic culture medium containing little or no dermal papilla cell growth factor.

  As the culture period in the step (a) of the production method and the culture period in the step (A) of the evaluation method or the screening method, mammalian pluripotent stem cells in the three-layer gel are used as a hair follicle tissue or the like. It is a period sufficient for inducing differentiation and a period sufficient for not inducing differentiation into tissues other than hair follicle tissue or the like, and is usually within a range of 12 hours to 20 days, for example, 12 hours -18 days, 12 hours to 16 days, 12 hours to 14 days, 12 hours to 13 days, 12 hours to 12 days, 12 hours to 11 days, 12 hours to 10 days, 1 to 20 days, 1 to 18 days, 1-16 days, 1-14 days, 1-13 days, 1-12 days, 1-11 days, 1-10 days, 3-20 days, 3-18 days, 3-16 days, 3-14 days, 3-13 days, 3-12 days, 3-11 days, 3-10 days, 4-20 days, 4-18 days, 4 16 days, 4-14 days, 4-13 days, 4-12 days, 4-11 days, 4-10 days, 5-20 days, 5-18 days, 5-16 days, 5-14 days, 5- 13 days, 5-12 days, 5-11 days, 5-10 days, 6-20 days, 6-18 days, 6-16 days, 6-14 days, 6-13 days, 6-12 days, 6- 11 days, 6-10 days, 7-20 days, 7-18 days, 7-16 days, 7-14 days, 7-13 days, 7-12 days, 7-11 days, 7-10 days However, 4 to 13 days is preferable.

  In the step (b) of the present production method and the step (B) of the present evaluation method or the present screening method, after the cultivation of the step (a) or the step (A), the present three-layer gel contains serum or a serum substitute. Culturing in a basic culture for mammalian cell culture. When culturing in the step (b) or the step (B), the culture medium for differentiation induction is performed using a culture solution removing means (aspirator, pipette, pipetman, etc.) after the culture in the step (a) or the step (A). It is preferable to add a new basic culture solution. The culture period in the step (b) of the present preparation method and the step (B) of the present screening method may be a period sufficient for the formation of hair follicle tissue and the like, and is usually within a range of 5 to 60 days. It is preferably 10 to 56 days, more preferably 20 to 53 days, still more preferably 30 to 50 days, and even more preferably 36 to 46 days.

In the steps (a) and (b) of the production method and the steps (A) and (B) of the evaluation method or the screening method, the culture of the three-layer gel is performed on mammalian pluripotent stem cells or epidermal cells. It can be carried out under conditions suitable for culture. For example, the differentiation-inducing culture medium or the basic culture medium is added to the incubator so that the entire three-layer gel prepared on the incubator is subsurface, and the appropriate temperature (usually about 30 to 40 ° C.) is added. , Preferably 36-38 ° C., CO ₂ concentration (usually in the range of 1-10%, preferably 4-6%), and humidity (usually in the range of 70-100%, preferably 95-100%). In addition, the culture medium for differentiation induction and the basic culture medium for differentiation of the present invention may be appropriately replaced with new ones as needed during the culture period.

  In the step (C) of the present evaluation method or the present screening method, the formation efficiency (%) of the hair follicle tissue or the like in the present three-layer gel is determined by the aforementioned HE staining method, the antibody against the hair follicle tissue marker, or the hair follicle periphery. An immunohistochemical staining method using an antibody against a tissue marker was performed, and the number of the three-layer gels in which the hair follicle tissue was formed by a phase contrast microscope and the total number of the analyzed three-layer gels were calculated. It can be calculated by inputting [(number of the present three-layer gel in which the hair follicle tissue or the like is formed / the total number of the present three-layer gel) × 100].

  In the step (C) of the evaluation method, when an increase in the formation efficiency of the hair follicle tissue or the like in the three-layer gel is detected, the test drug or the test substance has an action of promoting the formation of the hair follicle tissue or the like. Can be evaluated. In the step (C) of the present evaluation method, when a decrease in the formation efficiency of the hair follicle tissue or the like in the three-layer gel is detected, the test drug or the test substance acts to inhibit (inhibit) formation of the hair follicle tissue or the like Can be evaluated.

  When an increase in the formation efficiency of hair follicle tissue or the like in the present three-layer gel is detected in step (C) of the present screening method, the test drug or test substance is used as a prophylactic or therapeutic agent for alopecia or alopecia Can be selected as a candidate drug or substance, or can be selected as a skin irritating substance that affects the increase in hair. In the step (C) of the present screening method, when a decrease in the formation efficiency of the hair follicle tissue or the like in the present three-layer gel is detected, the test drug or test substance is selected as a candidate drug for the prevention or treatment of hirsutism It can be selected as a substance or a skin irritant substance that affects hair loss.

  In the step (C) of the present evaluation method and the step (C) of the present screening method, the comparison target for determining the increase or decrease in the formation efficiency of hair follicle tissue is usually the absence of the test drug or test substance This is the formation efficiency of hair follicle tissue and the like when the present three-layer gel is cultured (preferably cultured under the same conditions). The formation efficiency of the comparison target may be calculated each time the screening method is performed or may be calculated in advance.

  The artificial operation in steps (a) and (b) of the production method is preferably performed aseptically using a clean bench or the like in order to avoid contamination (contamination) of dust, bacteria, and the like.

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, cell culture was performed in an incubator (37 ° C., 5% CO ₂ ).

1. Preparation of hair follicle tissue and surrounding tissue using layer gel It was considered that hair follicle tissue and surrounding tissue could be prepared if the in vivo morphology of hair follicle tissue could be simulated in vitro. Therefore, a three-layer gel (example sample) shown in FIG. 1 was prepared and tried to be cultured in vitro.

1-1 Method [ES cell preparation]
Mouse embryonic stem cells (Embryonic Stem Cell: ES cells, EB3 clone, RIKEN Cell Bank) were frozen and thawed, and then a 25 cm ² cell culture flask (BioLite 25 cm ² Flask, Vented, ThermoScientific) coated with gelatin with 0.1% gelatin solution 10% Fetal Bovine Serum (FBS), 1% Antibiotic-antifungal (Antimycotic-Antibiotic: Invitrogen), 0.1 mM MEM non-essential amino acid (NEAA, Nacalai) Tesque), 1 mM sodium pyruvate (100 mmol / L Sodiun Pyruvate Solution [× 100], Wako Pure Chemical Industries), 0.1 mM 2-mercaptoethanol (2-Mercaptoethanol, Merck), 1000 units / mL leukemia inhibitory factor (Leukemia Inhibitory Factor) : LIF, Wako Pure Chemical Industries) 100 μL, and Blasticidin S Solutio The cells were cultured in GMEM (Glasgow Modified Minimum Essential Medium, Sigma Aldrich) containing n) and grown to the number of cells required for the experiment.

[Preparation of epidermal cells]
A pregnant mouse on the 17th day of gestation was anesthetized, and the fetal mouse was removed from the uterus, and then a skin piece was collected, immersed in a 0.25% trypsin solution, and subjected to enzyme treatment. 10% FBS, 100 units / mL penicillin (Meiji Seika), 100 units / mL kanamycin (Meiji Seika), 10 ng / mL cholera toxin (Sigma Aldrich), 10 ng / mL EGF (Wako Pure Chemical Industries), Dulbecco's Modified Eagle Minimal Essential Medium (Dulbecco's Modified) containing 0.5 μg / mL hydrocortisone (Pfizer), 5 μg / mL insulin (Japan Eli Lilly), and 2 × 10 ⁻⁹ M triiodothyronine (Takeda Pharmaceutical) After stirring in Eagle Medium (DMEM) to disperse the cells, the skin residue was removed using a cell strainer (Becton Dickinson, Franklin Lakes, NJ). The obtained cell suspension was centrifuged and the supernatant was removed to obtain cells (epidermal cells) (Iffect of Teprenone on Cultured Normal Human Keratinocytes) Journal of Japan Society of Plastic and Reconstructive Surgery 31: 224-230 (2011)).

[Method for producing three-layer gel]
First, a calcium alginate gel containing cell growth factor (FGF-2) was prepared as the first layer. Specifically, FGF-2 (BioVision) is mixed in a 0.2% sodium alginate solution so that the final concentration is 40 ng / mL, and 1 μL is dropped on a cell culture dish having a diameter of 60 mm with a microdispenser. did. Then, 5 mL of 102 mM calcium chloride aqueous solution was added to the said culture dish, the sodium alginate solution was gelatinized, and the calcium alginate gel was prepared. In addition, the excess calcium chloride aqueous solution was removed after gelatinization.

Subsequently, the ES cells and epidermal cells prepared according to the methods described in the above-mentioned [Preparation of ES cells] and [Preparation of epidermis cells] were each 2.4 mg / mL type I collagen neutral solution (I-AC , Koken) was dispersed so that the number of cells was 1.4 to 1.8 × 10 ⁴ cells / 3 μL beads and 1.4 to 1.8 × 10 ⁴ cells / 5 μL beads. The prepared ES cell-containing collagen solution and epidermal cell-containing collagen solution were respectively 3 μL (1.4 to 1.8 × 10 ⁴ cells) and 5 μL (1.4 to 1.8 × 10 ⁴ ) using a microdispenser. was added dropwise in this order on the cell) by alginate gel beads, an incubator (37 ° C., allowed to gel at 5% CO ₂₎ in, to prepare a three-layer gel (Figure 2).
In the three-layer gel, the first layer is covered with about 90 to 100% of the total surface area by the second layer (type I collagen gel layer embedding ES cells), and the second layer is Approximately 90-100% of its total surface area was covered by a third layer (type I collagen gel layer embedding epidermal cells).

[Trilayer gel culture and immunohistochemical staining]
A three-layer gel (Example sample) prepared according to the method described in [Method for preparing a three-layer gel] above was prepared by using 1% FCS (FBS) and dermal papilla cell growth factor (0.5% ITT [insulin transferrin]. Triiodothyronine mixed solution] Papilla group cultured in PCGM culture solution (TMTPGM-250, manufactured by Toyobo Co., Ltd.) containing 1% BPE and 0.5% Cyp) for 28 to 49 days, in such PCGM culture solution Combination group cultured for additional 21-39 days in GMEM medium containing 10% FBS, 1% antibiotic-antimycotic, 0.1 mM NEAA, and 1 mM sodium pyruvate after culturing for 7-10 days, and such The culture was divided into 3 groups of GMEM group cultured for 28-49 days in GMEM culture solution. The culture medium was changed once every 2-3 days.
The cultured three-layer gel was fixed with 10% neutral buffered formalin, embedded in paraffin, paraffin sections were prepared according to a conventional method, and cells were stained using the HE staining method. In addition, an anti-cytokeratin antibody (AE1 / AE3 clone, DAKO) is used to stain the entire hair follicle tissue, and an anti-smooth muscle actin (αSMA) antibody (DAKO) is used to stain the surrounding tissue of the hair follicle tissue (napped muscle). ) And DAB (3,3′-diaminobenzidine) staining according to a conventional method. As a control, hair follicle tissue in the mouse fetal skin tissue was similarly stained.

1-2 Results and discussion
In the Combination group, 20 three-layer gels (example samples) were analyzed by the HE staining method, and 9 (45%) of them were smoothed around the concentric hair follicle-like tissue (FIG. 3b) and the surrounding area. It was shown that muscle-like tissue (FIG. 3a) was formed. This hair follicle-like tissue was shown to have the same structure as mouse fetal hair follicle tissue (Fig. 4a) and to be positive for cytokeratin (upper and middle rows in Fig. 5). Furthermore, the smooth muscle-like tissue was shown to be positive for smooth muscle actin (lower part of FIG. 5). These results indicate that when the three-layer gel is cultured in a basic culture solution and then cultured in a basic culture solution containing hair papilla cell growth factor, a hair follicle tissue and its surrounding tissue are formed.
On the other hand, in the Papilla group, the hair follicle tissue and its surrounding tissues are formed only at a low rate of 3% (1/30), and in the GMEM group, the hair follicle tissue and its surrounding tissues are completely (0 / 20) not formed. From this result, it is shown that the culture in the basic culture solution of the three-layer gel and the subsequent culture in the basic culture solution containing the dermal papilla cell growth factor are necessary for the formation of the hair follicle tissue and surrounding tissues. ing.
In the above three groups, the three-layer gel contracted with the passage of culture time, and tissue regeneration was observed. In particular, the combination group and the Papilla group had a large contraction rate and remarkable tissue regeneration.

2. Evaluation of the three-layer gel When culturing the three-layer gel (Example sample), it was shown that the hair follicle tissue and the surrounding tissue were formed. It was analyzed whether a follicular tissue and its surrounding tissue were formed when the sheet was formed into a sheet rather than a substantially spherical shape. Specifically, a two-layer gel (comparative sample 1) consisting of a second layer (ES cell) and a third layer (epidermal cell) without a calcium alginate gel layer (first layer), ES cell and epidermal cell A single (1) layer gel (comparative sample 2) consisting of a mixture of the above and a sheet-like three-layer gel (comparative sample 3) instead of a substantially spherical shape, according to the method described in "2-1 Method" below. It was prepared and cultured according to the method described in [Culture of three-layer gel and immunohistochemical staining], and immunohistochemical staining was performed.

2-1 Method [Production Method of Comparative Example Sample 1]
The ES cells and epidermal cells prepared according to the methods described in the above [Preparation of ES cells] and [Preparation of epidermis cells] were each 2.4 mg / mL type I collagen neutral solution (I-AC, Koken). ) Was dispersed so that the number of cells became 1.4 to 1.8 × 10 ⁴ cells / 3 μL beads and 1.4 to 1.8 × 10 ⁴ cells / 5 μL beads. The prepared ES cell-containing collagen solution and epidermal cell-containing collagen solution were respectively 3 μL (1.4 to 1.8 × 10 ⁴ cells) and 5 μL (1.4 to 1.8 × 10 ⁴ ) using a microdispenser. The cells were dropped on a cell culture dish having a diameter of 60 mm. Thereafter, an incubator _{(37 ℃, 5% CO 2} ) is gelled in, to prepare a comparative sample 1 (Fig. 6).

[Production Method of Comparative Sample 2]
The ES cells and epidermal cells prepared according to the methods described in the above [Preparation of ES cells] and [Preparation of epidermal cells] were mixed with a 2.4 mg / mL type I collagen neutral solution (I-AC, Koken). The cells were mixed and dispersed so that the number of cells became 1.4 to 1.8 × 10 ⁴ cells / 8 μL beads. 8 μL of the prepared ES cells and epidermis cell-containing collagen solution (ES cells 1.4 to 1.8 × 10 ⁴ cells and epidermis cells 1.4 to 1.8 × 10 ⁴ cells) were grown using a microdispenser. After dropping in order on the alginate gel beads not containing the factor, gelation was performed in an incubator (37 ° C., 5% CO ₂ ) to produce Comparative Example Sample 2 (FIG. 7).

[Production Method of Comparative Sample 3]
According to the method described in the above item [Method for producing a three-layer gel], 100 μL of a calcium alginate gel containing a cell growth factor (FGF-2) is prepared on a cell culture dish having a diameter of 60 mm. A calcium alginate gel was prepared. Subsequently, the ES cells and epidermal cells prepared according to the methods described in the above-mentioned [Preparation of ES cells] and [Preparation of epidermis cells] were each 2.4 mg / mL type I collagen neutral solution (I-AC , Koken) was dispersed so that the number of cells became 1.4-1.8 × 10 ⁶ cells / 300 μL gel sheet and 1.4-1.8 × 10 ⁶ cells / 500 μL beads. Prepared ES cell-containing collagen solution, and the epidermal cells containing collagen solution, respectively 300μL (1.4~1.8 × 10 ⁶ cells) using a micro dispenser, and 500μL (1.4~1.8 × 10 ⁶ Cells) were dropped on the sheet-like calcium alginate gel. Thereafter, an incubator _{(37 ℃, 5% CO 2} ) is gelled in, to prepare a comparative sample 3 (Fig. 8).

2-2 Results and Discussion When Comparative Sample 1 was used, in the Combination group and Papilla group, the gel contraction rate increased with the lapse of culture time, and tissue regeneration was observed, but the hair follicles also included the GMEM group. Tissue and surrounding tissues (0/20) were not formed at all (FIG. 9). For Comparative Samples 2 and 3, in all three groups (Combination group, Papilla group, and GMEM group), the gel contraction rate is small, and tissue regeneration is not recognized so much. Was not formed at all (0/15) (FIG. 10). From these results, pluripotent stem cells and epidermal cells are arranged so that the three-dimensional positional relationship is controlled, and cell growth factors are gradually released from the early stage of culture in pluripotent stem cells. It has been shown that culturing under conditions is necessary for the formation of hair follicle tissue and surrounding tissue in vitro. Furthermore, based on the results in Example 1, it is necessary to carry out the above culture for about 7 to 10 days in a cell growth medium containing dermal papilla cell growth factor, and then perform the culture instead of the normal cell growth medium. It was also shown that there was.

  When the production method or production kit of the present invention is used, hair follicle tissue and the like can be produced relatively easily in vitro. Therefore, the present invention contributes to the treatment of alopecia or alopecia. In addition, when the evaluation method or the evaluation kit of the present invention is used, the test drug or the test substance has an action of promoting the formation of hair follicle tissue or the like, or the formation suppression (inhibition) of hair follicle tissue or the like. Hair cycle caused by causes such as alopecia; alopecia; hirsutism; nutritional deficiencies, damage to the scalp and hair, lack of blood flow, decreased female hormones, stress, etc. Useful for screening for prophylactic or therapeutic agents for diseases related to hair follicle tissues such as abnormalities (states in which hair is removed in a thin and short state), or screening for skin irritants that affect the increase or decrease in hair It is. Therefore, the present invention contributes to the development of a preventive or therapeutic agent for alopecia or alopecia or hirsutism.

Claims (16)

A kit for evaluating hair follicle tissue or surrounding tissue formation, comprising the following (p) to (s).
(P) a gel material for embedding mammalian epidermal cells in a substantially spherical gel;
(Q) a gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel;
(R) a gel material for containing a cell growth factor in a substantially spherical gel;
(S) A substantially spherical gel that embeds mammalian pluripotent stem cells is coated on a substantially spherical gel containing a cell growth factor, and the substantially spherical gel that embeds mammalian epidermis cells Package insert describing coating of a substantially spherical gel embedding pluripotent stem cells;   (P) Gel material for embedding mammalian epidermal cells in a substantially spherical gel, and (q) Gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel are collagen. The evaluation kit according to claim 1, wherein the gel material for containing (r) the cell growth factor in a substantially spherical gel is alginic acid or a salt thereof. The evaluation kit according to claim 1 or 2, wherein the cell growth factor is fibroblast growth factor (FGF) -2. A method for evaluating the formation of a hair follicle tissue or its surrounding tissue, comprising the following steps (A) to (C), the basic culture solution for mammalian cell culture in step (A), and the step (A): The evaluation method, wherein a test drug or a test substance is contained in one or both of the basic culture solutions for mammalian cell culture in B).
(A) a substantially spherical gel that embeds mammalian epidermal cells;
A substantially spherical gel for embedding mammalian pluripotent stem cells, coated with a substantially spherical gel for embedding the mammalian epidermal cells;
A three-layered gel comprising a substantially spherical gel containing a cell growth factor coated with a substantially spherical gel embedding the pluripotent stem cells, and one or more hair papilla cell growth factors Culturing in a basal culture medium for mammalian cell culture, containing serum or serum replacement;
(B) After culturing in step (A), culturing the three-layer gel in a basal culture medium for mammalian cell culture containing serum or a serum substitute;
(C) After the culture in step (B), a step of detecting an increase or decrease in the formation efficiency of the hair follicle tissue or the surrounding tissue in the three-layer gel;   The evaluation method according to claim 4, wherein the culture in the step (A) is performed for 4 to 13 days.   A substantially spherical gel that embeds mammalian epidermal cells and a substantially spherical gel that embeds mammalian pluripotent stem cells are collagen gels, and a substantially spherical gel containing a cell growth factor is bivalent or more. The evaluation method according to claim 4, wherein the gel is an alginate gel with cations.   The evaluation method according to any one of claims 4 to 6, wherein the two or more hair papilla cell growth factors contain at least bovine pituitary extract (BPE) and cyproterone acetate (Cyp).   The evaluation method according to any one of claims 4 to 7, wherein the cell growth factor is fibroblast growth factor (FGF) -2. A kit for preparing a hair follicle tissue or its surrounding tissue, comprising the following (p) to (s).
(P) a gel material for embedding mammalian epidermal cells in a substantially spherical gel;
(Q) a gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel;
(R) a gel material for containing a cell growth factor in a substantially spherical gel;
(S) A substantially spherical gel that embeds mammalian pluripotent stem cells is coated on a substantially spherical gel containing a cell growth factor, and the substantially spherical gel that embeds mammalian epidermis cells Package insert describing coating of a substantially spherical gel embedding pluripotent stem cells;   (P) Gel material for embedding mammalian epidermal cells in a substantially spherical gel, and (q) Gel material for embedding mammalian pluripotent stem cells in a substantially spherical gel are collagen. The preparation kit according to claim 9, wherein the gel material for containing (r) the cell growth factor in a substantially spherical gel is alginic acid or a salt thereof.   The preparation kit according to claim 9 or 10, wherein the cell growth factor is fibroblast growth factor (FGF) -2. A method for producing an in vitro hair follicle tissue or its surrounding tissue comprising the following steps (a) and (b):
(A) a substantially spherical gel that embeds mammalian epidermal cells;
A substantially spherical gel for embedding mammalian pluripotent stem cells, coated with a substantially spherical gel for embedding the mammalian epidermal cells;
A three-layered gel comprising a substantially spherical gel containing a cell growth factor coated with a substantially spherical gel embedding the pluripotent stem cells, and one or more hair papilla cell growth factors Culturing in a basal culture medium for mammalian cell culture, containing serum or serum replacement;
(B) a step of culturing the three-layer gel after culturing in the step (a) in a basic culture solution for mammalian cell culture containing serum or a serum substitute;   The production method according to claim 12, wherein the culture in the step (a) is performed for 4 to 13 days.   A substantially spherical gel that embeds mammalian epidermal cells and a substantially spherical gel that embeds mammalian pluripotent stem cells are collagen gels, and a substantially spherical gel containing a cell growth factor is bivalent or more. The preparation method according to claim 12, wherein the gel is an alginate gel with cations.   The production method according to any one of claims 12 to 14, wherein the two or more types of hair papilla cell growth factor contain at least bovine pituitary extract (BPE) and cyproterone acetate (Cyp).   The production method according to any one of claims 12 to 15, wherein the cell growth factor is fibroblast growth factor (FGF) -2.