Hair follicle primordium and manufacturing method thereof

Abstract

The present invention provides a hair follicle primordium that can easily and quickly form a hair shaft-like structure in vitro. The method for producing the hair follicle primordium comprises the following steps: seeding epithelial cells and mesenchymal cells; maintaining the epithelial cells and mesenchymal cells in a culture medium containing (a) laminin and entactin, and / or (b) type IV collagen; and co-culturing the epithelial cells and mesenchymal cells in the culture medium.

Description

Technical Field

[0001] The present invention relates to a hair follicle primordium and a manufacturing method thereof. Background Art

[0002] Non-Patent Document 1 describes the formation of skin organoids in vitro by seeding artificial pluripotent stem cells (iPSCs) prepared from mouse fetal fibroblasts in a 96-well plate and culturing them.

[0003] Patent Document 1 describes a method for producing an aggregate of regenerated hair follicle primordia, characterized by comprising the steps of seeding mesenchymal cells and epithelial cells on a microconcave plate composed of regularly arranged microconcave portions, and performing mixed culture while supplying oxygen to form hair follicle primordia.

[0004] Patent document 2 describes a method for producing full-thickness skin with skin appendages, characterized in that the "full-thickness skin with skin appendages" comprises at least the following (1) to (3): (1) skin comprising an epidermal layer and a dermal layer, (2) at least one skin appendage, and (3) subcutaneous tissue; the method comprises the following steps: (a) a step of stimulating an embryoid body with a physiologically active substance capable of activating the Wnt pathway, (b) a step of preparing a combination comprising the following (A) and (B), (A) all or a portion of the embryoid body stimulated in step (a), and (B) a scaffold material, (c) a step of transplanting the combination prepared in step (b) into an animal, and (d) a step of producing full-thickness skin derived from the combination in the animal.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2017 / 073625

[0008] Patent Document 2: International Publication No. 2016 / 039279

[0009] Non-patent literature

[0010] Non-patent literature 1: Jiyoon Lee et al. (2018). Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids. Cell Reports 22, 242-254 Summary of the Invention

[0011] Problems to be solved by the invention

[0012] However, the method described in Non-Patent Document 1 uses artificial pluripotent stem cells, so the operation is complicated and a relatively long culture period is required.

[0013] The present invention has been made in view of the above-mentioned problems, and one of its objects is to provide a hair follicle primordium that can form a hair shaft-like structure in vitro in a simple and short period of time.

[0014] Means for solving problems

[0015] One aspect of a method for solving the above-mentioned problems according to one embodiment of the present invention relates to a method for producing a hair follicle primordium, comprising the steps of: seeding epithelial cells and mesenchymal cells; maintaining the epithelial cells and mesenchymal cells in a culture medium in which (a) laminin and entactin, and / or (b) type IV collagen are dispersed; and co-culturing the epithelial cells and mesenchymal cells in the culture medium. According to the present invention, a method for producing a hair follicle primordium that can easily and quickly form a hair shaft-like structure in vitro is provided.

[0016] In the above method, the epithelial cells and the mesenchymal cells may be maintained in a culture medium in which the (a) laminin and entactin are dispersed. In the above method, the epithelial cells and the mesenchymal cells may be maintained in a culture medium in which the (b) type IV collagen is dispersed.

[0017] The method may also include the step of allowing the inoculated epithelial cells and mesenchymal cells to settle on a culture substrate in a culture solution, and maintaining the epithelial cells and mesenchymal cells settled on the culture substrate in a culture solution dispersed with (a) and / or (b). The method may also include the step of: after maintaining the epithelial cells and mesenchymal cells in a culture solution dispersed with (a) and / or (b), co-culturing the epithelial cells and mesenchymal cells in a culture solution having a concentration of (a) and / or (b) lower than the concentration during the maintenance. In the method, the co-culturing may also form the hair follicle primordium having a hair shaft-like structure.

[0018] Another aspect of the method of one embodiment of the present invention for solving the above-mentioned problems relates to a method for promoting the formation of a hair shaft-like structure in a hair follicle primordium, wherein the method comprises maintaining the epithelial cells and mesenchymal cells in a culture medium in which (a) laminin and entactin, and / or (b) type IV collagen are dispersed during a cell culture process comprising seeding epithelial cells and mesenchymal cells and co-culturing the epithelial cells and mesenchymal cells to form the hair follicle primordium, thereby promoting the formation of the hair shaft-like structure in the hair follicle primordium. According to the present invention, a method for promoting the formation of a hair shaft-like structure in a hair follicle primordium in vitro is provided, which is simple and short-term.

[0019] Another aspect of the method of one embodiment of the present invention for solving the above-mentioned problems relates to a method for promoting the formation of a hair shaft-like structure in a hair follicle primordium, comprising: using (a) laminin and entactin, and / or (b) type IV collagen, during a cell culture process comprising seeding epithelial cells and mesenchymal cells and co-culturing the epithelial cells and mesenchymal cells to form a hair follicle primordium. According to the present invention, a method using (a) laminin and entactin, and / or (b) type IV collagen, is provided for promoting the formation of a hair shaft-like structure in a hair follicle primordium in vitro in a simple and short-term manner.

[0020] One embodiment of the present invention, designed to address the aforementioned issues, provides a hair follicle primordium comprising epithelial and mesenchymal cells, having a hair shaft-like structure, lacking arrector pili muscle structures and / or sebaceous gland structures, and not being transplanted into a living body. The present invention provides a hair follicle primordium having a hair shaft-like structure that can be easily and quickly formed in vitro.

[0021] Effects of the Invention

[0022] According to the present invention, a hair follicle primordium capable of forming a hair shaft-like structure in vitro in a simple and short period of time and a method for producing the same are provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 1-1 of the present embodiment.

[0024] Figure 1B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 1-1 of the present embodiment.

[0025] Figure 1C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the sixth day of culture in Example 1-1 of the present embodiment.

[0026] Figure 1D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 1-2 of the present embodiment.

[0027] Figure 1E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 1-2 of the present embodiment.

[0028] Figure 1F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the sixth day of culture in Example 1-2 of the present embodiment.

[0029] Figure 2A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the fourth day of culture in Example 1-1 of the present embodiment.

[0030] Figure 2B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the fifth day of culture in Example 1-1 of the present embodiment.

[0031] Figure 2C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the sixth day of culture in Example 1-1 of the present embodiment.

[0032] Figure 3 This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 12th day of culture in Example 1-1 of the present embodiment.

[0033] Figure 4A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 2-1 of the present embodiment.

[0034] Figure 4B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 2-1 of the present embodiment.

[0035] Figure 4C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the seventh day of culture in Example 2-1 of the present embodiment.

[0036] Figure 4D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 2-2 of the present embodiment.

[0037] Figure 4E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 2-2 of the present embodiment.

[0038] Figure 4FThis is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 7th day of culture in Example 2-2 of this embodiment.

[0039] Figure 4G This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 2-3 of the present embodiment.

[0040] Figure 4H This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 2-3 of the present embodiment.

[0041] Figure 4I This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 7th day of culture in Example 2-3 of the present embodiment.

[0042] Figure 5A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-1 of the present embodiment.

[0043] Figure 5B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-2 of this embodiment.

[0044] Figure 5C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-3 of the present embodiment.

[0045] Figure 5D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-4 of the present embodiment.

[0046] Figure 5E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-5 of the present embodiment.

[0047] Figure 5F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-6 of the present embodiment.

[0048] Figure 5G This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-7 of the present embodiment.

[0049] Figure 5H This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 3-8 of the present embodiment.

[0050] Figure 6A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-1 of the present embodiment.

[0051] Figure 6B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-2 of this embodiment.

[0052] Figure 6C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-3 of the present embodiment.

[0053] Figure 6D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-4 of the present embodiment.

[0054] Figure 6E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-5 of the present embodiment.

[0055] Figure 6F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-6 of the present embodiment.

[0056] Figure 6G This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-7 of the present embodiment.

[0057] Figure 6H This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 3-8 of the present embodiment.

[0058] Figure 7A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 4 of the present embodiment.

[0059] Figure 7B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 4 of the present embodiment.

[0060] Figure 7C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 7th day of culture in Example 4 of this embodiment.

[0061] Figure 8A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 5-1 of the present embodiment.

[0062] Figure 8B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 5-2 of this embodiment.

[0063] Figure 8CThis is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 5-3 of the present embodiment.

[0064] Figure 9A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 6 of the present embodiment.

[0065] Figure 9B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 6 of the present embodiment.

[0066] Figure 10A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 7-1 of the present embodiment.

[0067] Figure 10B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 7-2 of this embodiment.

[0068] Figure 10C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 7-3 of this embodiment.

[0069] Figure 10D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 7-4 of the present embodiment.

[0070] Figure 10E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 7-5 of the present embodiment.

[0071] Figure 10F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 7-6 of the present embodiment.

[0072] Figure 11A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 7-1 of this embodiment.

[0073] Figure 11B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 7-2 of this embodiment.

[0074] Figure 11C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 7-3 of this embodiment.

[0075] Figure 11D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 7-4 of this embodiment.

[0076] Figure 11E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 7-5 of the present embodiment.

[0077] Figure 11F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 7-6 of the present embodiment.

[0078] Figure 12A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 7-1 of the present embodiment.

[0079] Figure 12B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 7-2 of this embodiment.

[0080] Figure 12C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 7-3 of this embodiment.

[0081] Figure 12D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 7-4 of the present embodiment.

[0082] Figure 12E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 7-5 of the present embodiment.

[0083] Figure 12F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 7-6 of the present embodiment.

[0084] Figure 13A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 8-1 of the present embodiment.

[0085] Figure 13B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 8-2 of this embodiment.

[0086] Figure 13C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 8-3 of the present embodiment.

[0087] Figure 13D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 8-4 of the present embodiment.

[0088] Figure 13EThis is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 8-5 of the present embodiment.

[0089] Figure 13F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example 8-6 of the present embodiment.

[0090] Figure 14A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 8-1 of the present embodiment.

[0091] Figure 14B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 8-2 of this embodiment.

[0092] Figure 14C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 8-3 of this embodiment.

[0093] Figure 14D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 8-4 of the present embodiment.

[0094] Figure 14E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 8-5 of this embodiment.

[0095] Figure 14F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 8-6 of the present embodiment.

[0096] Figure 15A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 8-1 of the present embodiment.

[0097] Figure 15B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 8-2 of this embodiment.

[0098] Figure 15C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 8-3 of this embodiment.

[0099] Figure 15D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 8-4 of the present embodiment.

[0100] Figure 15E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 8-5 of the present embodiment.

[0101] Figure 15F This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 8-6 of the present embodiment.

[0102] Figure 16A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the second day of culture in Example 9-1 of this embodiment.

[0103] Figure 16B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the second day of culture in Example 9-2 of this embodiment.

[0104] Figure 16C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the second day of culture in Example 9-3 of this embodiment.

[0105] Figure 17A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 9-1 of this embodiment.

[0106] Figure 17B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 9-2 of this embodiment.

[0107] Figure 17C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 9-3 of this embodiment.

[0108] Figure 18A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example C1-1 of this embodiment.

[0109] Figure 18B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example C1-1 of this embodiment.

[0110] Figure 18C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example C1-1 of this embodiment.

[0111] Figure 18D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the first day of culture in Example C1-2 of this embodiment.

[0112] Figure 18E This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example C1-2 of this embodiment.

[0113] Figure 18FThis is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example C1-2 of this embodiment.

[0114] Figure 19A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the second day of culture in Example C2 of this embodiment.

[0115] Figure 19B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example C2 of this embodiment.

[0116] Figure 20A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 12th day of culture in Example C3-1 of this embodiment.

[0117] Figure 20B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 12th day of culture in Example C3-2 of this embodiment.

[0118] Figure 20C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 12th day of culture in Example C3-3 of this embodiment.

[0119] Figure 20D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 12th day of culture in Example C3-4 of this embodiment.

[0120] Figure 21 This is an explanatory diagram showing an example of hair regeneration from hair follicle primordia transplanted on the back of a nude mouse in Example 10 of this embodiment.

[0121] Figure 22A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the third day of culture in Example 11 of the present embodiment.

[0122] Figure 22B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the 14th day of culture in Example 11 of the present embodiment.

[0123] Figure 23 This is an explanatory diagram showing an example of the results of measuring the length of the hair shaft-like structure formed in the hair follicle primordium during co-culture in Example 11 of the present embodiment.

[0124] Figure 24A This is an explanatory diagram showing an example of a transmission microscope photograph of a cross section of a hair shaft-like structure formed in a hair follicle primordium on the 14th day of culture in Example 11 of the present embodiment.

[0125] Figure 24B Is the enlarged display Figure 24AThe four areas enclosed by white lines are shown in the figure.

[0126] Figure 24C Is the enlarged display Figure 24B The four areas enclosed by white lines are shown in the figure.

[0127] Figure 24D Is the enlarged display Figure 24C The four areas enclosed by white lines are shown in the figure.

[0128] Figure 25A This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 12-C1 of this embodiment.

[0129] Figure 25B This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 12-1 of the present embodiment.

[0130] Figure 25C This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 12-2 of the present embodiment.

[0131] Figure 25D This is an explanatory diagram showing an example of a phase contrast microscope photograph taken on the eighth day of culture in Example 12-3 of the present embodiment. DETAILED DESCRIPTION

[0132] Hereinafter, an embodiment of the present invention will be described. However, it should be noted that the present invention is not limited to this embodiment.

[0133] The method of the present embodiment (hereinafter referred to as "the present method") includes, as one aspect thereof, a method for producing a hair follicle primordium, which comprises forming a hair follicle primordium by the following steps: inoculating epithelial cells and mesenchymal cells; maintaining the epithelial cells and mesenchymal cells in a culture medium in which (a) laminin and entactin, and / or (b) type IV collagen are dispersed; and co-culturing the epithelial cells and mesenchymal cells in the culture medium.

[0134] Specifically, the inventors of the present invention have conducted intensive research on technical means for producing hair follicle primordia in vitro and unexpectedly discovered that hair follicle primordia capable of forming a hair shaft-like structure in vitro can be produced simply and quickly by contacting epithelial cells and mesenchymal cells in a culture medium with (a) laminin and entactin, and / or (b) type IV collagen dispersed in the culture medium. This has led to the completion of the present invention.

[0135] Therefore, as another aspect, the present method includes the following method: during a cell culture comprising seeding epithelial cells and mesenchymal cells and co-culturing the epithelial cells and mesenchymal cells to form a hair follicle primordium, the epithelial cells and mesenchymal cells are maintained in a culture medium in which (a) laminin and entactin, and / or (b) type IV collagen are dispersed, thereby promoting the formation of a hair shaft-like structure in the hair follicle primordium.

[0136] In addition, as another aspect, the present method includes the following method: during a cell culture comprising inoculating epithelial cells and mesenchymal cells and co-culturing the epithelial cells and mesenchymal cells to form a hair follicle primordium, (a) laminin and entactin, and / or (b) type IV collagen are used to promote the formation of a hair shaft-like structure in the hair follicle primordium.

[0137] That is, this embodiment includes the use of (a) laminin and entactin, and / or (b) type IV collagen as a culture medium additive (more specifically, a component dispersed in the culture medium) for promoting the formation of a hair shaft-like structure in a hair follicle primordium.

[0138] The epithelial cells used in this method are not particularly limited as long as they are epithelial cells that form hair follicle primordium by co-culture with mesenchymal cells. For example, they are preferably one or more selected from epithelial cells derived from hair follicle tissue, epithelial cells derived from skin tissue, and hair follicle epithelial cells induced from stem cells in a culture system.

[0139] The epithelial cells derived from hair follicle tissue can be, for example, one or more selected from epithelial cells derived from the bulge region of hair follicle tissue (e.g., the outermost cells of the outer root sheath), and epithelial cells derived from the matrix of hair follicle tissue. The epithelial cells derived from skin tissue can be, for example, one or more selected from epidermal keratinocytes and skin epithelial cells in the formation stage. The hair follicle epithelial cells induced from stem cells in the culture system can be, for example, hair follicle epithelial cells induced from iPS (induced Pluripotent Stem) cells, ES (Embryonic Stem) cells, or EG (Embryonic Germ) cells. The epithelial cells can be epithelial stem cells.

[0140] Epithelial cells, for example, express genes related to hair growth. Specifically, epithelial cells are, for example, cells that express cytokeratin. When the epithelial cells are epithelial stem cells, the epithelial stem cells are, for example, cells that express one or more of cytokeratin 15 and CD34. Epithelial cells can be primary cells collected from an organism or pre-cultured cells (e.g., cells that have been subcultured and / or cells that have formed a cell line).

[0141] The mesenchymal cells used in this method are not particularly limited as long as they are mesenchymal cells that form hair follicle primordium by co-culture with epithelial cells. For example, they are preferably one or more selected from mesenchymal cells derived from hair follicle tissue, mesenchymal cells derived from skin tissue, and mesenchymal cells induced from stem cells in a culture system.

[0142] Mesenchymal cells derived from hair follicle tissue may be, for example, one or more selected from dermal papilla cells and hair bulb root sheath cells. Mesenchymal cells derived from skin tissue may be, for example, one or more selected from dermal root sheath cells and pubertal skin mesenchymal cells. Mesenchymal cells induced from stem cells in culture may be, for example, hair follicle mesenchymal cells induced from iPS cells, ES cells, or EG cells.

[0143] Mesenchymal cells, for example, express genes related to growth. Specifically, mesenchymal cells are defined as cells expressing one or more of the following: Versican and ALP (alkaline phosphatase). Mesenchymal cells may be primary cells collected from an organism or pre-cultured cells (e.g., subcultured cells and / or cells forming a cell line).

[0144] It should be noted that, in the present method, co-culture of only epithelial cells and mesenchymal cells can be performed, or co-culture further comprising other cells can be performed. In this case, the other cells are not particularly limited as long as the effects of the present invention can be obtained. For example, they can be one or more of pigment stem cells selected from pigment cells, pigment precursor cells, pigment stem cells, and pluripotent stem cells (e.g., iPS cells, ES cells, or Muse (Multilineage-differentiating stress-enduring, multilineage differentiation sustained stress) cells). In addition, the timing of inoculating other cells is not particularly limited as long as it is within the range of forming a hair follicle primordium comprising epithelial cells, mesenchymal cells, and the other cells.

[0145] In this method, epithelial cells and mesenchymal cells are first seeded. Regarding this point, the method described in Non-Patent Document 1 uses artificial pluripotent stem cells. Therefore, complex procedures are required to differentiate the artificial pluripotent stem cells. In contrast, this method does not require the use of pluripotent stem cells. Therefore, this method may not include the step of seeding pluripotent stem cells. Furthermore, this method may not include the step of differentiating pluripotent stem cells. Furthermore, this method may not include the step of culturing pluripotent stem cells.

[0146] The inoculation of epithelial cells and mesenchymal cells is carried out by adding the epithelial cells and mesenchymal cells to a culture vessel. Specifically, the culture fluid (cell suspension) comprising the epithelial cells and mesenchymal cells is added to the culture vessel. In addition, since the culture vessel comprises a culture substrate (for example, the bottom of the culture vessel or a culture substrate separate from the culture vessel configured in the culture vessel) to which the epithelial cells and mesenchymal cells are precipitated, it can be said that the epithelial cells and mesenchymal cells are inoculated on the culture substrate.

[0147] In the inoculation of epithelial cells and mesenchymal cells, the epithelial cells and mesenchymal cells can be inoculated simultaneously, or one of the epithelial cells and mesenchymal cells can be inoculated first, followed by inoculation of the other cell. That is, the first one of the epithelial cells and mesenchymal cells can be inoculated without contact with the other cell, and then the other cell can be inoculated to contact the one cell with the other cell. In this case, it is preferred to first inoculate the mesenchymal cells, followed by inoculation of the epithelial cells.

[0148] Specifically, for example, a cell suspension containing mesenchymal cells and not containing epithelial cells is first added to a culture vessel (e.g., a well of a 96-well plate), and the mesenchymal cells are inoculated. Subsequently, a cell suspension containing epithelial cells is added to the culture vessel containing the mesenchymal cells, and the epithelial cells are inoculated. In this case, the cell suspension containing epithelial cells may not contain mesenchymal cells.

[0149] Alternatively, one of the epithelial cells and mesenchymal cells may be seeded first, then cultured, and then the other cell may be seeded. In other words, in this case, the first of the epithelial cells and mesenchymal cells is seeded and cultured without contact with the other cell, and then the other cell is seeded to initiate co-culture of the first and second cells.

[0150] In the case where one of the epithelial cells and mesenchymal cells is inoculated first and then the other cell is inoculated, the time from inoculation of the one cell to inoculation of the other cell (i.e., for example, the time from inoculation of the one cell to the start of co-culture of the one cell and the other cell) is not particularly limited as long as it is within the range that can obtain the effect of the present invention. For example, it can be 96 hours or less, preferably 72 hours or less, more preferably 48 hours or less, and particularly preferably 24 hours or less.

[0151] The inoculated epithelial cells and mesenchymal cells are preferably dispersed in a culture solution. In this case, the epithelial cells and mesenchymal cells are mixed and dispersed in the culture solution. Each cell dispersed in the culture solution is not bound to other cells, or even if it is attached to other cells, it can be easily separated from the other cells by making the culture solution flow by operations such as pipetting. It should be noted that the culture solution used in this method is not particularly limited as long as it is a solution that can maintain the survival of epithelial cells and mesenchymal cells within the scope of the effect of the present invention.

[0152] When one of the epithelial cells and mesenchymal cells is seeded first and then the other is seeded, it is preferred that the one cell dispersed in the culture medium is seeded first, followed by the other cell dispersed in the culture medium. Specifically, for example, a cell suspension containing no epithelial cells but dispersed mesenchymal cells is first added to a culture vessel, and the mesenchymal cells are seeded. Subsequently, a cell suspension containing dispersed epithelial cells is added to the culture vessel containing the mesenchymal cells, and the epithelial cells are seeded.

[0153] The density of the seeded epithelial cells and mesenchymal cells is not particularly limited as long as it is within the range that can form hair follicle primordium in the subsequent co-culture. For example, it is preferably a density to the extent that each cell can contact adjacent cells in the culture container (especially when settled on the culture substrate).

[0154] In this method, seeded epithelial cells and mesenchymal cells are co-cultured in a culture medium to form a hair follicle primordium comprising the epithelial cells and mesenchymal cells. Specifically, by co-culturing the epithelial cells and mesenchymal cells in a culture medium, the epithelial cells and mesenchymal cells aggregate over time to form a hair follicle primordium.

[0155] More specifically, the inoculated epithelial cells and mesenchymal cells are mixed in the culture medium and maintained in a dispersed state. Afterwards, as the culture time passes, the formation of epithelial cells binding to each other, the formation of mesenchymal cells binding to each other, and the formation of the combination of epithelial cells and mesenchymal cells proceed. As a result, the epithelial cells condense to form an epithelial cell aggregate, and the mesenchymal cells condense to form a mesenchymal cell aggregate. In addition, in parallel with the formation of the epithelial cell aggregate and the mesenchymal cell aggregate, the combination of the epithelial cell aggregate and the mesenchymal cell aggregate is also formed. In this way, a hair follicle primordium comprising an epithelial cell aggregate and a mesenchymal cell aggregate is finally formed.

[0156] By first inoculating one type of cell (for example, mesenchymal cells) among epithelial cells and mesenchymal cells, and then inoculating another type of cell (for example, epithelial cells), a hair follicle primordium can be efficiently formed, which includes a first cell aggregate formed by the aggregation of the one type of cell and a second cell aggregate formed by the aggregation of the other type of cell and connected to the first cell aggregate.

[0157] In the present method, epithelial cells and mesenchymal cells need to be coagulated in order to form the hair follicle primordium. Therefore, the co-culture of epithelial cells and mesenchymal cells for forming the hair follicle primordium is performed in a fluid culture medium.

[0158] The hair follicle primordium is a cell aggregate that forms hair when transplanted into an animal. The hair follicle primordium produced in this method can be a hair follicle primordium spheroid. A hair follicle primordium spheroid is a roughly spherical cell aggregate.

[0159] The ratio of the number of epithelial cells and mesenchymal cells to the total number of cells constituting the hair follicle primordium is not particularly limited as long as the effects of the present invention can be achieved, and may be, for example, 50% or more, 70% or more, or 90% or more.

[0160] In this case, the ratio of the number of epithelial cells and mesenchymal cells to the total number of cells seeded to produce the hair follicle primordium may be, for example, 50% or more, 70% or more, or 90% or more.

[0161] Similarly, the ratio of the number of epithelial cells and mesenchymal cells to the total number of cells co-cultured to form the hair follicle primordium may be, for example, 50% or more, 70% or more, or 90% or more.

[0162] In this method, epithelial cells and mesenchymal cells can also be co-cultured on a non-adhesive culture medium. In this case, during co-culture, the epithelial cells and mesenchymal cells float in the culture medium without adhering to the culture substrate, or adhere to the culture substrate to the extent that the culture medium can be easily detached from the culture substrate by pipetting or other operations. The shape of the epithelial cells and mesenchymal cells cultured on the non-adhesive culture substrate is maintained in a roughly spherical shape.

[0163] By co-culturing cells on a non-adhesive culture substrate, non-adhesive hair follicle primordia are formed. The non-adhesive hair follicle primordia float in the culture medium or adhere to the culture substrate to such an extent that the culture medium can be easily detached by flowing the culture medium using a pipette or other operation.

[0164] The culture vessel for co-culturing epithelial cells and mesenchymal cells is not particularly limited as long as the epithelial cells and mesenchymal cells can form hair follicle primordium. For example, a relatively small pore is preferably used.

[0165] That is, the bottom area of one well used as a culture container may be, for example, 1000 mm 2 Below, preferably 100mm 2 Less than, more preferably 50mm 2 Below, particularly preferably 20mm 2 the following.

[0166] The bottom area of the hole can be, for example, 100 μm 2 Above, preferably 1000 μm 2 More than 10000 μm 2 More than, particularly preferably 100000 μm 2 above.

[0167] The area of the bottom surface of the hole can also be determined by arbitrarily combining one of the above lower limits and one of the above upper limits. Specifically, the area of the bottom surface of the hole can be, for example, 100 μm 2 Above and 1000mm 2 Below, preferably 1000 μm 2 Above and 100mm 2 Below, more preferably 10000 μm 2 Above and 50mm 2 Below, particularly preferably 100000 μm 2 Above and 20mm 2 the following.

[0168] In this method, one hair follicle primordium can also be formed in each culture vessel (e.g., each well) by co-culturing epithelial cells and mesenchymal cells. In this case, the epithelial cells and mesenchymal cells seeded in each culture vessel aggregate within the culture vessel to form a single hair follicle primordium.

[0169] Furthermore, in this method, after the epithelial cells and mesenchymal cells are seeded, the epithelial cells and mesenchymal cells are maintained in a culture medium in which one or more selected from (a) laminin and entactin, and (b) type IV collagen are dispersed.

[0170] That is, for example, epithelial cells and mesenchymal cells are maintained in a culture medium in which (a) laminin and nidogen are dispersed. In this case, laminin and nidogen preferably include a complex of laminin and nidogen.

[0171] Alternatively, for example, epithelial cells and mesenchymal cells may be maintained in a culture medium containing (b) type IV collagen. Alternatively, for example, epithelial cells and mesenchymal cells may be maintained in a culture medium containing (a) laminin and entactin, and (b) type IV collagen.

[0172] The culture solution containing the dispersed (a) and / or (b) is prepared by adding a previously prepared composition containing (a) and / or a previously prepared composition containing (b) to the culture solution.

[0173] In this method, epithelial cells and mesenchymal cells are maintained in a culture medium in which (a) and / or (b) are dispersed, and the epithelial cells and mesenchymal cells are brought into contact with (a) and / or (b) in the culture medium.

[0174] That is, in a fluid culture medium, the epithelial cells and mesenchymal cells are contacted with (a) and / or (b) dispersed in the culture medium. Specifically, in the present method, (a) and / or (b) in contact with the epithelial cells and mesenchymal cells are (a) and / or (b) dispersed in a fluid culture medium, for example, they are not (a) and / or (b) constituting a hydrogel in which the epithelial cells and mesenchymal cells are embedded, and when the epithelial cells and mesenchymal cells are retained on the surface of the hydrogel, they are not (a) and / or (b) constituting the hydrogel, nor are they (a) and / or (b) pre-immobilized on a culture substrate on which the epithelial cells and mesenchymal cells are retained.

[0175] The present method may include a process of contacting the epithelial cells and mesenchymal cells maintained on the surface of a hydrogel (e.g., a hydrogel consisting of a component containing (a) and / or (b), or a hydrogel not containing the (a) and / or (b)) with (a) and / or (b) dispersed in a culture solution. However, the present method may not include a process of contacting the epithelial cells and mesenchymal cells maintained on the surface of a hydrogel consisting of a component containing (a) and / or (b) with (a) and / or (b) dispersed in a culture solution. In addition, the present method may not include a process of contacting the epithelial cells and mesenchymal cells maintained on the surface of a hydrogel not containing (a) and / or (b) with (a) and / or (b) dispersed in a culture solution. In addition, the present method may not include a process of contacting the epithelial cells and mesenchymal cells maintained on the surface of a hydrogel with (a) and / or (b) dispersed in a culture solution.

[0176] This method may include the step of contacting the epithelial cells and mesenchymal cells held on the culture substrate on which (a) and / or (b) are immobilized with (a) and / or (b) dispersed in the culture medium. However, this method may not include the step of contacting the epithelial cells and mesenchymal cells held on the culture substrate on which (a) and / or (b) are immobilized with (a) and / or (b) dispersed in the culture medium.

[0177] Furthermore, the present method may not include the step of embedding epithelial cells and mesenchymal cells in a hydrogel comprising components (a) and / or (b) and culturing them before forming the hair follicle primordium. Furthermore, the present method may not include the step of embedding epithelial cells and mesenchymal cells in a hydrogel not containing components (a) and / or (b) and culturing them before forming the hair follicle primordium. Furthermore, the present method may not include the step of embedding epithelial cells and mesenchymal cells in a hydrogel and culturing them before forming the hair follicle primordium.

[0178] It should be noted that the cell suspension containing the epithelial cells and mesenchymal cells used in the inoculation may contain (a) and / or (b) or may not contain (a) and / or (b). When the cell suspension at the time of inoculation does not contain (a) and / or (b), after inoculation, the epithelial cells and mesenchymal cells are brought into contact with (a) and / or (b) dispersed in the culture solution by adding the (a) and / or (b) to the culture solution.

[0179] That is, when epithelial cells and mesenchymal cells are inoculated simultaneously, a cell suspension containing the epithelial cells and mesenchymal cells and (a) and / or (b) can be added to a culture container for inoculation, or a cell suspension containing the epithelial cells and mesenchymal cells but not containing (a) and / or (b) can be first added to a culture container for inoculation, and then (a) and / or (b) can be added to the culture container.

[0180] In addition, when one of epithelial cells and mesenchymal cells (e.g., mesenchymal cells) is first inoculated and then the other cell (e.g., epithelial cells) is inoculated, in each of the inoculation of the one cell and the other cell, a cell suspension containing the one cell or the other cell and (a) and / or (b) can be added to the culture container for inoculation, or a cell suspension containing the one cell or the other cell but not containing (a) and / or (b) can be first added to the culture container for inoculation, and then (a) and / or (b) can be added to the culture container.

[0181] The concentrations of (a) laminin and entactin in the culture medium for maintaining epithelial cells and mesenchymal cells are not particularly limited as long as they are within the range that can achieve the effects of the present invention. For example, they can be 1 μg / mL or more, preferably 3 μg / mL or more, and particularly preferably 5 μg / mL or more.

[0182] The concentrations of (a) laminin and entactin in the culture medium may be, for example, 3000 μg / mL or less, preferably 2500 μg / mL or less, and particularly preferably 2000 μg / mL or less.

[0183] The concentrations of (a) laminin and entactin in the culture medium can be determined by arbitrarily combining any one of the above lower limits with any one of the above upper limits. Specifically, the concentrations of (a) laminin and entactin in the culture medium for maintaining epithelial cells and mesenchymal cells can be, for example, 1 μg / mL or more and 3000 μg / mL or less, preferably 3 μg / mL or more and 2500 μg / mL or less, and particularly preferably 5 μg / mL or more and 2000 μg / mL or less.

[0184] The concentration of (b) type IV collagen in the culture medium for maintaining epithelial cells and mesenchymal cells is not particularly limited as long as it is within the range that can achieve the effects of the present invention. For example, it can be 1 μg / mL or more, preferably 3 μg / mL or more, and particularly preferably 5 μg / mL or more.

[0185] The concentration of (b) type IV collagen in the culture medium may be, for example, 1000 μg / mL or less, preferably 700 μg / mL or less, and particularly preferably 400 μg / mL or less.

[0186] The concentration of (b) type IV collagen in the culture medium can be determined by any combination of any of the above lower limits and any of the above upper limits. Specifically, the concentration of (b) type IV collagen in the culture medium for maintaining epithelial cells and mesenchymal cells can be, for example, 1 μg / mL to 1000 μg / mL, preferably 3 μg / mL to 700 μg / mL, and particularly preferably 5 μg / mL to 400 μg / mL.

[0187] In this method, the purpose of dispersing (a) and / or (b) in the culture medium is not to gel the culture medium. Therefore, the concentration of (a) and / or (b) in the culture medium can be lower than the concentration typically used for gelation. In other words, the concentration of (a) and / or (b) in the culture medium for maintaining epithelial cells and mesenchymal cells can be lower than the concentration that causes gelation of the entire culture medium.

[0188] The temperature for maintaining epithelial cells and mesenchymal cells in the culture medium in which (a) and / or (b) are dispersed is not particularly limited as long as it is within the range that can achieve the effects of the present invention. For example, it is preferably a temperature suitable for co-culture of the epithelial cells and mesenchymal cells. Specifically, for example, it is preferably a temperature of 30°C or higher and 45°C or lower, and particularly preferably a temperature of 35°C or higher and 40°C or lower.

[0189] The time for maintaining the epithelial cells and mesenchymal cells in the culture medium in which (a) and / or (b) are dispersed (the time for maintaining the epithelial cells and mesenchymal cells in the culture medium in which (a) and / or (b) are dispersed at a temperature suitable for co-culture) is not particularly limited as long as it is within the range that can achieve the effects of the present invention. For example, it can be 30 minutes or more, preferably 40 minutes or more, more preferably 50 minutes or more, and particularly preferably 60 minutes or more.

[0190] The timing of maintaining epithelial cells and mesenchymal cells in the culture medium in which (a) and / or (b) are dispersed is not particularly limited as long as the effects of the present invention can be obtained, but is preferably started before the formation of the hair follicle primordium.

[0191] Specifically, the maintenance of epithelial cells and mesenchymal cells in a culture medium in which (a) and / or (b) are dispersed can be, for example, started from the time point when the co-culture of the epithelial cells and mesenchymal cells is started (i.e., the time point when the epithelial cells and mesenchymal cells are maintained at a temperature suitable for co-culture (for example, preferably 35°C or higher and 39°C or lower, particularly preferably 36°C or higher and 38°C or lower)) to 28 hours ago, preferably started before 24 hours have passed, more preferably started before 20 hours have passed, further preferably started before 15 hours have passed, and particularly preferably started before 10 hours have passed.

[0192] That is, in the case where epithelial cells and mesenchymal cells are inoculated at the same time, and in any case where one of the epithelial cells and mesenchymal cells (e.g., mesenchymal cells) is first inoculated and then the other cell (e.g., epithelial cells) is inoculated, the maintenance of the epithelial cells and mesenchymal cells in the culture medium in which (a) and / or (b) are dispersed can be started from the time point of starting the co-culture of the epithelial cells and mesenchymal cells until the lapse of any of the above-mentioned threshold times (from the start of the co-culture at the same time as or after the start of the co-culture to the lapse of any of the above-mentioned threshold times).

[0193] In addition, when one of epithelial cells and mesenchymal cells (for example, mesenchymal cells) is first inoculated to start the culture of the one cell, and then the other cell (for example, epithelial cells) is inoculated to start co-culture, the one cell can be maintained in a culture medium in which (a) and / or (b) is dispersed, from the time point when the culture of the one cell is started (that is, the time point when the one cell is started to be maintained at a temperature suitable for culture (for example, preferably above 35°C and below 39°C, particularly preferably above 36°C and below 38°C)) until 28 hours have passed (from the start of the culture of the one cell at the same time as or after the start of the culture of the one cell to the 28 hours before the start).

[0194] In this case, the maintenance of one type of cell (the cell seeded first among the epithelial cells and mesenchymal cells) in the culture medium in which (a) and / or (b) is dispersed is preferably started before 24 hours have passed from the start of the culture of the one type of cell, more preferably before 18 hours have passed, further preferably before 12 hours have passed, and particularly preferably simultaneously with the start of the culture of the one type of cell.

[0195] The method may further comprise the step of allowing the seeded epithelial cells and mesenchymal cells to settle on a culture substrate in a culture medium, and maintaining the epithelial cells and mesenchymal cells settled on the culture substrate in a culture medium in which (a) and / or (b) are dispersed.

[0196] That is, when epithelial cells and mesenchymal cells are seeded on a culture substrate in a culture solution to which (a) and / or (b) are not added (particularly in a culture solution to which (a) is not added), for example, the seeded epithelial cells and mesenchymal cells are first allowed to settle on the culture substrate in the culture solution, and then (a) and / or (b) are added to the culture solution, and the epithelial cells and mesenchymal cells are maintained in the culture solution containing the dispersed (a) and / or (b).

[0197] In addition, when the epithelial cells and mesenchymal cells are seeded on a culture substrate in a culture solution to which (a) and / or (b) is added (particularly in a culture solution to which (a) is added), for example, the epithelial cells and mesenchymal cells are first allowed to settle on a culture substrate at a temperature lower than a temperature suitable for co-culture (for example, preferably 10°C or less (specifically, for example, more than 0°C and 10°C or less), more preferably 7°C or less, and particularly preferably 5°C or less), and then the epithelial cells and mesenchymal cells are maintained in a culture solution containing the dispersed (a) and / or (b) at a temperature suitable for the co-culture.

[0198] The method for causing the epithelial cells and mesenchymal cells to settle on the culture substrate is not particularly limited. For example, by allowing the culture container containing the culture substrate to stand still and / or by centrifuging the culture container containing the culture substrate, the epithelial cells and mesenchymal cells can be caused to settle on the culture substrate in the culture solution in the culture container.

[0199] In this method, as described above, epithelial cells and mesenchymal cells are maintained in a culture medium in which (a) and / or (b) are dispersed, and then the epithelial cells and mesenchymal cells are co-cultured in the culture medium to form a hair follicle primordium containing the epithelial cells and mesenchymal cells.

[0200] In the co-culture, a culture medium supplemented with (a) and / or (b) or a culture medium not supplemented with (a) and / or (b) may be used. Specifically, the method may include the following steps: after maintaining the epithelial cells and mesenchymal cells in a culture medium in which (a) and / or (b) are dispersed, the epithelial cells and mesenchymal cells are co-cultured in a culture medium in which the concentration of (a) and / or (b) is lower than that during the maintenance.

[0201] In this case, epithelial cells and mesenchymal cells are maintained in a culture medium containing dispersed (a) and / or (b) at a first concentration, and then the epithelial cells and mesenchymal cells are co-cultured in a culture medium in which the concentration of (a) and / or (b) is a second concentration lower than the first concentration.

[0202] The second concentration is not particularly limited as long as it is smaller than the first concentration and is within the range that can achieve the effects of the present invention. For example, it can be less than 1 / 5 of the first concentration, less than 1 / 10, less than 1 / 15, less than 1 / 20, less than 1 / 25, or less than 1 / 30.

[0203] The second concentration of (a) can be, for example, less than 1 μg / mL, less than 0.2 μg / mL, or less than 0.1 μg / mL. The second concentration of (b) can be, for example, less than 1 μg / mL, less than 0.2 μg / mL, or less than 0.1 μg / mL.

[0204] When the present method includes the step of co-culturing at the second concentration, the co-culture may be performed in a culture medium in which the concentration of (a) and / or (b) is the second concentration throughout the co-culture period until the hair follicle primordium is formed, or the co-culture may be performed in a culture medium in which the concentration of (a) and / or (b) is the second concentration only during a portion of the co-culture period.

[0205] By reducing the concentration of (a) and / or (b) in the culture medium used for co-culture, for example, the production cost of hair follicle primordium can be effectively reduced, and the operability can be improved by reducing complicated operations.

[0206] In this method, by bringing (a) and / or (b) dispersed in a culture medium into contact with epithelial cells and mesenchymal cells, and then co-culturing the epithelial cells and mesenchymal cells, a hair follicle primordium capable of forming a hair shaft-like structure can be produced in vitro simply and quickly. In other words, by further culturing the hair follicle primordium produced by this method, a hair shaft-like structure can be formed in the hair follicle primordium.

[0207] Therefore, in the present method, epithelial cells and mesenchymal cells can be maintained in a culture medium in which (a) and / or (b) are dispersed, and then the epithelial cells and mesenchymal cells are co-cultured to form a hair follicle primordium having a hair shaft-like structure.

[0208] Specifically, in this case, even after epithelial cells and mesenchymal cells aggregate to form a hair follicle primordium, co-culture of the epithelial cells and mesenchymal cells (culturing the hair follicle primordium) is continued until a hair shaft-like structure forms within the hair follicle primordium. As a result, a hair follicle primordium having a hair shaft-like structure can be produced in vitro simply and in a short period of time.

[0209] Specifically, in the present method, for example, a hair follicle primordium having a hair shaft-like structure can be formed before 480 hours have passed, preferably before 360 hours have passed, more preferably before 240 hours have passed, and particularly preferably before 170 hours have passed from the start of co-culture of epithelial cells and mesenchymal cells.

[0210] The hair shaft structure of the hair follicle primordium is a filamentous structure formed within the follicle primordium. The hair shaft structure may contain keratin. Furthermore, the hair shaft structure may contain melanin. Furthermore, the hair shaft structure may sprout from the follicle primordium. Furthermore, the hair shaft structure may also have a hair cuticle structure.

[0211] The length of the hair shaft-like structure of the hair follicle primordium is not particularly limited, and may be, for example, 30 μm or longer, 50 μm or longer, or 100 μm or longer.

[0212] The hair follicle primordium of this embodiment (hereinafter referred to as the "present hair follicle primordium") is a hair follicle primordium composed of epithelial cells and mesenchymal cells, having a hair shaft-like structure, lacking arrector pili muscle structures and / or sebaceous gland structures, and not transplanted into a living body. The present hair follicle primordium is preferably produced by the present method described above.

[0213] Although this hair follicle primordium has not yet been transplanted into a living body, it has a hair shaft-like structure. In this regard, it has been possible to manufacture hair follicle primordia that can form a hair shaft-like structure in a living body after being transplanted into the living body. However, it is difficult to form a hair shaft-like structure in a hair follicle primordium outside the living body before transplantation.

[0214] In the method described in Non-Patent Document 1, the skin tissue formed by culturing artificial pluripotent stem cells has a complex structure including arrector pili muscles and sebaceous glands, etc. In contrast, the hair follicle primordium has a relatively simple structure.

[0215] That is, the hair follicle primordium does not include the arrector pili muscle structure and / or the sebaceous gland structure described in the aforementioned Non-Patent Document 1. Specifically, the hair follicle primordium may not include the arrector pili muscle structure, may not include the sebaceous gland structure, or may not include both the arrector pili muscle structure and the sebaceous gland structure.

[0216] The use of the hair follicle primordium is not particularly limited. For example, the hair follicle primordium can be used for medical treatment such as transplantation into a patient, or can be used for research related to hair formation. The organism to which the hair follicle primordium is transplanted can be a human or a non-human animal, but is preferably a human. The transplantation of the hair follicle primordium into the organism is preferably transplanted into the skin of the organism.

[0217] Transplantation of hair follicle primordia into an organism can be for medical or research purposes. For example, transplantation of hair follicle primordia into an organism can be used to treat or prevent a disease associated with hair loss in a human patient suffering from or at risk of suffering from the disease.

[0218] The disease associated with hair loss is not particularly limited, and may be, for example, one or more selected from androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, obesity-related alopecia, seborrheic alopecia, pityriasis, traction alopecia, metabolic alopecia, compression alopecia, alopecia areata, neurotic alopecia, trichotillomania, alopecia universalis, and symptomatic alopecia (alopecia symptomatica).

[0219] Hair follicle primordium can be used, for example, to search for substances that can be used to treat or prevent diseases associated with hair loss, to search for substances related to the diseases, and to study the mechanisms of the diseases.

[0220] Next, specific examples of this embodiment will be described.

[0221] Example 1

[0222] [Collection of epithelial and mesenchymal cells]

[0223] Skin tissue from the back of 18-day-old C57BL / 6 mouse fetuses was collected and, using a modified method from Nakao et al. (Koh-ei Toyoshima et al. Nature Communications, 3, 784, 2012), treated with dispase for 1 hour at 4°C with shaking at 30 rpm to separate the epithelial and interstitial layers of the skin tissue. The epithelial layer was then treated with 100 U / mL collagenase for 1 hour and 20 minutes, followed by trypsin treatment for 10 minutes to isolate epithelial cells. Separately, the interstitial layer was treated with 100 U / mL collagenase for 1 hour and 20 minutes to isolate mesenchymal cells.

[0224] [nourish]

[0225] In Example 1-1, epithelial cells and mesenchymal cells were co-cultured using a culture medium containing laminin, entactin, and type IV collagen. First, a DMEM / F12 medium (Advanced Dulbecco's Modified Eagle Medium / Ham's F-12, GIBCO (registered trademark)) containing 1% GultaMax Supplement (GIBCO (registered trademark)) and 0.2% Normocin (InvivoGen) was prepared as the culture medium.

[0226] Then, the cells were suspended in the culture medium to a density of 5 × 10 3 cells / mL (total cell density reaches 1×10 4 cells / mL) of epithelial cells and mesenchymal cells, and further added with Matrigel (registered trademark) Basement Membrane Matrix, CORNING (registered trademark) in an amount to give a concentration of 1 v / v% to prepare a cell suspension.

[0227] The matrix gel preparation used contained 10.6 mg / mL (protein mass measured by the Lowry method) of soluble basement membrane matrix extracted from EHS (Engelbreth-Holm-Swarm) mouse tumors. The composition ratio of the basement membrane matrix was: 56% laminin, 8% entactin, and 31% type IV collagen.

[0228] Based on this, it was calculated that the cell suspension to which 1 v / v% of the matrix gel preparation was added contained 59 μg / mL of laminin, 8 μg / mL of entactin, and 33 μg / mL of type IV collagen.

[0229] Epithelial cells and mesenchymal cells were inoculated by adding 100 μL of the above cell suspension to each well of a 96-well plate (Prime surface (registered trademark), Sumitomo Bakelite). After inoculation, the 96-well plate was immediately moved to a refrigerator at 4°C and allowed to stand for 20 minutes. By standing still, in the refrigerator, in the cooled culture medium, the epithelial cells and mesenchymal cells settled to the bottom surface of the well in a state where they could contact each other. Then, the 96-well plate was moved to an incubator at 37°C, and co-culture of epithelial cells and mesenchymal cells was started in the incubator. Co-culture was carried out for 12 days.

[0230] During co-culture, the culture medium was replaced every two days. The culture medium was replaced by first removing almost all of the culture medium from each well. Next, 100 μL of DMEM / F12 medium containing no matrix gel, 1% GultaMax Supplement, and 0.2% Normocin was added to each well as a fresh culture medium.

[0231] In Comparative Example 1-2, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1, except that no matrix gel was added to the cell suspension during seeding (ie, only a culture medium containing no matrix gel was used).

[0232] [result]

[0233] Figure 1A 、 Figure 1B and Figure 1C Phase contrast microscopic photographs of the co-culture in one well of Example 1-1 were taken on the first, third, and sixth days of culture. Figure 1D 、 Figure 1E and Figure 1F Phase contrast microscopic photographs of the co-culture in one well of Example 1-2 were taken on the first, third, and sixth days of culture. Figures 1A to 1F In the figure, the scale bar represents 100 μm.

[0234] like Figure 1D to Figure 1F As shown in Example 1-2, no hair follicle primordium with a hair shaft-like structure is formed. Figures 1A to 1C As shown in Example 1-1, a hair follicle primordium having a hair shaft-like structure is formed. Figure 1C As indicated by the middle arrow, a hair shaft-like structure was formed in the hair follicle primordium on the 6th day of culture.

[0235] in addition, Figure 2A 、 Figure 2B and Figure 2CPhase contrast microscopic photographs taken on the 4th, 5th, and 6th day of culture, respectively, of the co-culture system in another well of Example 1-1 are shown. Figures 2A to 2C In the figure, the scale bar represents 100 μm. Figures 2A to 2C As indicated by the middle arrow, in Example 1-1, the formation of a hair shaft-like structure was confirmed on the 4th day of culture, and thereafter, the hair shaft-like structure extended as the culture time passed.

[0236] Furthermore, the efficiency of hair shaft formation in hair follicle primordia on culture day 10 was evaluated. Specifically, the hair shaft formation rate (%) was calculated by dividing the number of hair follicle primordia with hair shaft formation on culture day 10 by the total number of hair follicle primordia and multiplying the result by 100.

[0237] As a result, in Example 1-2, no hair shaft-like structure formation was observed in any of the 96 hair follicle primordia, and the hair shaft-like structure formation rate was 0 (zero)%. In contrast, in Example 1-1, hair shaft-like structure formation was observed in 88 of the 96 hair follicle primordia, and the hair shaft-like structure formation rate was 92%.

[0238] Figure 3 This is a scanning microscope photograph showing the hair shaft-like structure formed in the hair follicle primordium on the 12th day of culture in Example 1-1. Figure 3 In the figure, the scale bar represents 30 μm. Figure 3 As shown, the hair shaft-like structure formed in the hair follicle primordium has a cuticle structure, which is a characteristic structure of the hair shaft of an organism.

[0239] Example 2

[0240] [Collection of epithelial and mesenchymal cells]

[0241] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0242] [nourish]

[0243] In Example 2-1, epithelial cells and mesenchymal cells were seeded in a culture medium containing laminin, entactin, and type IV collagen in the same manner as in Example 1-1 of Example 1. Specifically, a DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin was prepared as the culture medium.

[0244] Then, the cells were suspended in the culture medium to a density of 5 × 10 3 cells / mL (total cell density reaches 1×10 4Epithelial cells and mesenchymal cells in an amount of 10 cells / mL) were added, and matrix gel was further added in an amount to give a concentration of 1 v / v% to prepare a cell suspension.

[0245] Epithelial and mesenchymal cells were seeded by adding 100 μL of the above cell suspension to each well of a 96-well plate. Immediately after seeding, the 96-well plate was transferred to a 4°C refrigerator and allowed to stand for 20 minutes to allow the epithelial and mesenchymal cells to settle in each well. The 96-well plate was then transferred to a 37°C incubator to initiate co-culture of the epithelial and mesenchymal cells.

[0246] After one day of culture, 100 μL of DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin and not containing Matrigel was added to each well, bringing the volume of the culture medium in each well to approximately 200 μL.

[0247] The culture medium was then replaced every two days by first removing 100 μL of the culture medium from each well and then adding 100 μL of DMEM / F12 medium containing no matrix gel, 1% GultaMax Supplement, and 0.2% Normocin to each well.

[0248] In Example 2-2, epithelial cells and mesenchymal cells were seeded in a culture medium that did not contain laminin, entactin, and type IV collagen. Then, on the first day of culture, laminin, entactin, and type IV collagen were added to the culture medium.

[0249] That is, first, each cell was suspended in DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin to a density of 5×10 3 cells / mL (total cell density reaches 1×10 4 cells / mL) of epithelial cells and mesenchymal cells to prepare a cell suspension.

[0250] Next, 100 μL of the cell suspension was added to each well of a 96-well plate to seed the epithelial and mesenchymal cells. Immediately after seeding, the 96-well plate was moved to a 37°C incubator to initiate co-culture of the epithelial and mesenchymal cells.

[0251] After one day of culture (specifically, approximately 22 hours from the start of co-culture (when the 96-well plate was moved to a 37°C incubator)), 100 μL of DMEM / F12 medium containing 1% GultaMax Supplement, 0.2% Normocin, and 2 v / v% Matrigel was added to each well. As a result, the volume of culture medium in each well reached approximately 200 μL, and the Matrigel concentration in the culture medium reached approximately 1 v / v%.

[0252] Immediately after adding the matrix gel, the 96-well plate was placed in a 4°C refrigerator and allowed to stand for 20 minutes. The 96-well plate was then moved to a 37°C incubator where co-culture of epithelial and mesenchymal cells continued. The culture medium was replaced as in Example 2-1 above.

[0253] In Example 2-3, epithelial cells and mesenchymal cells were seeded in a culture medium without laminin, entactin, and type IV collagen, and then, on day 3 of culture, laminin, entactin, and type IV collagen were added to the culture medium.

[0254] That is, similarly to Example 2-2, 100 μL of a cell suspension containing epithelial cells and mesenchymal cells was added to each well of a 96-well plate, and the epithelial cells and mesenchymal cells were seeded and co-culture was initiated in a 37°C incubator.

[0255] After culturing for one day, 100 μL of DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin was added to each well, so that the volume of the culture medium in each well reached approximately 200 μL.

[0256] After 3 days of culture (specifically, approximately 69 hours after the start of co-culture), 100 μL of the culture medium was removed from each well. Next, 100 μL of DMEM / F12 medium containing 1% GultaMax Supplement, 0.2% Normocin, and 2 v / v% Matrigel was added. As a result, the Matrigel concentration in the culture medium in each well was approximately 1 v / v%.

[0257] Immediately after adding the matrix gel, the 96-well plate was placed in a 4°C refrigerator and allowed to stand for 20 minutes. The 96-well plate was then moved to a 37°C incubator where co-culture of epithelial and mesenchymal cells continued. The culture medium was replaced as in Example 2-1 above.

[0258] [result]

[0259] Figure 4A 、 Figure 4B and Figure 4C Phase contrast microscopic photographs taken on the first, third, and seventh days of culture in the co-culture of Example 2-1 are shown. Figure 4D 、 Figure 4E and Figure 4F Phase contrast microscopic photographs taken on the first, third, and seventh days of culture in the co-culture of Example 2-2 are shown. Figure 4G 、 Figure 4H and Figure 4I Phase contrast microscopic images taken on the first, third, and seventh days of culture in the co-culture of Example 2-3 are shown. Figures 4A to 4I In the figure, the scale bar represents 200 μm.

[0260] like Figures 4A to 4C As shown in FIG2-1, a hair follicle primordium having a hair shaft-like structure is formed. Figure 4C As indicated by the arrow in the figure, a hair shaft-like structure was formed in the hair follicle primordium on the 7th day of culture. Figures 4D to 4I As shown, in Examples 2-2 and 2-3, no hair shaft-like structure was formed in the hair follicle primordium on the 7th day of culture.

[0261] Example 3

[0262] [Collection of epithelial and mesenchymal cells]

[0263] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0264] [nourish]

[0265] In Example 3-1, epithelial cells and mesenchymal cells were first seeded in a culture medium containing no laminin, entactin, or type IV collagen, and then laminin, entactin, and type IV collagen were added to the culture medium immediately after seeding (0 hours after seeding).

[0266] That is, first, each cell was suspended in DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin to a density of 5×10 3 cells / mL (total cell density reaches 1×10 4 cells / mL) of epithelial cells and mesenchymal cells to prepare a cell suspension.

[0267] Next, 100 μL of the cell suspension was added to each well of a 96-well plate to seed the epithelial and mesenchymal cells. Immediately after seeding, 100 μL of DMEM / F12 culture medium containing 1% GultaMax Supplement, 0.2% Normocin, and 2% v / v Matrigel was added to each well. This brought the volume of culture medium in each well to 200 μL, and the Matrigel concentration in the culture medium to 1% v / v.

[0268] Note that, immediately after seeding and before addition of the culture medium containing matrix gel, a portion of the epithelial cells and mesenchymal cells in each well had not settled on the bottom surface of the well but were floating in the culture medium.

[0269] Then, the 96-well plate was moved to an incubator at 37° C., and co-culture of epithelial cells and mesenchymal cells was started in the incubator. The culture medium was replaced in the same manner as in Example 2-1 of Example 2 above.

[0270] In Example 3-2, epithelial cells and mesenchymal cells were first seeded into a culture medium containing no laminin, entactin, or type IV collagen to initiate co-culture, and then laminin, entactin, and type IV collagen were added to the culture medium 0.5 hours after the start of the co-culture.

[0271] That is, each cell was suspended in DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin to a density of 5×10 3 cells / mL (total cell density reaches 1×10 4 cells / mL) of epithelial cells and mesenchymal cells to prepare a cell suspension.

[0272] Epithelial and mesenchymal cells were seeded by adding 100 μL of the cell suspension to each well of a 96-well plate. Immediately after seeding, the 96-well plate was moved to a 37°C incubator to initiate co-culture of epithelial and mesenchymal cells.

[0273] At 0.5 hours after the start of co-culture, 100 μL of DMEM / F12 culture medium containing 1% GultaMax Supplement, 0.2% Normocin, and 2 v / v% Matrigel was added to each well. This resulted in a culture volume of approximately 200 μL per well, with a Matrigel concentration of 1 v / v%.

[0274] Note that, at the time point 0.5 hours had passed since the start of co-culture immediately before the addition of the culture solution containing matrix gel, the epithelial cells and mesenchymal cells in each well had already settled to the bottom surface of the well.

[0275] Then, the co-culture of epithelial cells and mesenchymal cells was continued, and the culture medium was replaced in the same manner as in Example 2-1 of Example 2 above.

[0276] In Example 3-3, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 3-2 above, except that laminin, entactin, and type IV collagen were added to the culture medium 1 hour after the start of co-culture.

[0277] In Example 3-4, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 3-2 above, except that laminin, entactin, and type IV collagen were added to the culture medium 2 hours after the start of co-culture.

[0278] In Example 3-5, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 3-2 above, except that laminin, entactin, and type IV collagen were added to the culture medium 3 hours after the start of co-culture.

[0279] In Example 3-6, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 3-2 above, except that laminin, entactin, and type IV collagen were added to the culture medium 6 hours after the start of co-culture.

[0280] In Example 3-7, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 3-2 above, except that laminin, entactin, and type IV collagen were added to the culture medium 15 hours after the start of co-culture.

[0281] In Example 3-8, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 3-2 above, except that laminin, entactin, and type IV collagen were added to the culture medium 22 hours after the start of co-culture.

[0282] [result]

[0283] Figure 5A 、 Figure 5B 、 Figure 5C 、 Figure 5D 、 Figure 5E 、 Figure 5F 、 Figure 5G and Figure 5HPhase contrast microscopic photographs taken on the third day of culture in the co-culture of Examples 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, 3-7, and 3-8 are shown. Figure 6A 、 Figure 6B 、 Figure 6C 、 Figure 6D 、 Figure 6E 、 Figure 6F 、 Figure 6G and Figure 6H Phase contrast microscope photos taken on the 8th day of culture in the co-culture of Example 3-1, Example 3-2, Example 3-3, Example 3-4, Example 3-5, Example 3-6, Example 3-7 and Example 3-8 are shown respectively. Figures 5A to 5H and Figures 6A to 6H In the figure, the scale bar represents 200 μm.

[0284] like Figures 5A to 5H and Figures 6A to 6H As shown, from 0 to 22 hours after the start of co-culture, in all cases (Examples 3-1 to 3-8) where laminin, entactin, and type IV collagen were added to the culture medium, hair follicle primordium with a hair shaft-like structure was formed. Figures 6A to 6H As shown, in all cases, hair shaft-like structures were formed in the hair follicle primordium on day 8 of culture.

[0285] In addition, the formation rate of hair shaft-like structures was 22% in Example 3-1 (2 out of 9 hair follicle primordia), 89% in Example 3-2 (8 out of 9 hair follicle primordia), 78% in Example 3-3 (7 out of 9 hair follicle primordia), 100% in Example 3-4 (9 out of 9 hair follicle primordia), 89% in Example 3-5 (8 out of 9 hair follicle primordia), 78% in Example 3-6 (7 out of 9 hair follicle primordia), 44% in Example 3-7 (4 out of 9 hair follicle primordia), and 22% in Example 3-8 (2 out of 9 hair follicle primordia).

[0286] The reason why the hair shaft-like structure formation efficiency in Examples 3-7 and 3-8, in which the matrix gel was added 15 hours or 22 hours after the start of co-culture, was lower than that in Examples 3-2 to 3-6, in which the matrix gel was added 0.5 hours to 6 hours, is that, for example, the aggregation of epithelial cells and mesenchymal cells had already progressed by the time the matrix gel was added.

[0287] Furthermore, the reason why the hair shaft-like structure formation efficiency was low in Example 3-1, in which the matrix gel was added shortly after the start of co-culture, is that, for example, a portion of the epithelial cells and mesenchymal cells had not settled to the well bottom and were still floating at the time of addition of the matrix gel.

[0288] It should be noted that in Example 2-2 of Example 2, similar to Example 3-6, although laminin, entactin, and type IV collagen were added to the culture medium approximately 22 hours after the start of co-culture, no hair shaft-like structure was observed in the hair follicle primordium on day 6 of culture. This is believed to be due to individual differences in the mice from which the cells were collected.

[0289] Example 4

[0290] [Collection of epithelial and mesenchymal cells]

[0291] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0292] [nourish]

[0293] In Example 4, epithelial cells and mesenchymal cells were co-cultured using a culture medium containing laminin and nestin. First, a DMEM / F12 culture medium containing 1% GultaMax Supplement and 0.2% Normocin was prepared as a culture medium.

[0294] Then, the cells were suspended in the culture medium to a density of 5 × 10 3 cells / mL (total cell density reaches 1×10 4 cells / mL) of epithelial cells and mesenchymal cells, and further added with a high concentration laminin / entactin complex (HIGH CONCENTRATION LAMININ / ENTACTIN COMPLEX, CORNING (registered trademark)) in an amount to give a concentration of 1 v / v% to prepare a cell suspension.

[0295] The high-concentration laminin / nidogen complex preparation used contained 15.2 mg / mL of soluble basement membrane matrix extracted from EHS mouse tumors, and contained a laminin / nidogen complex with a purity of more than 90% as determined by SDS-PAGE, with laminin and nidogen contained in an equimolar ratio.

[0296] Based on this, it was calculated that the cell suspension to which 1 v / v% of the high-concentration laminin / nidogen complex preparation was added contained 137-152 μg / mL of laminin / nidogen complex (i.e., 68-76 μg / mL of laminin and nidogen, respectively).

[0297] That is, in the cell suspension of Example 4, the content of laminin was 115% to 129% of the corresponding content in the cell suspension of Example 1-1 of the above-mentioned Example 1, and the total content of laminin and nestin was calculated to be 204% to 227% of the corresponding content in the cell suspension of Example 1-1 of the above-mentioned Example 1.

[0298] Epithelial cells and mesenchymal cells were inoculated by adding 100 μL of the above cell suspension to each well of a 96-well plate. After inoculation, the 96-well plate was immediately moved to a 4°C refrigerator and allowed to stand for 20 minutes, thereby allowing the epithelial cells and mesenchymal cells to settle to the bottom of the wells in the cooled culture medium in the refrigerator. The 96-well plate was then moved to a 37°C incubator, and co-culture of epithelial cells and mesenchymal cells was started in the incubator. The replacement of the culture medium was carried out in the same manner as in Example 1-1 of Example 1 above.

[0299] [result]

[0300] Figure 7A 、 Figure 7B and Figure 7C Phase contrast microscopic images taken on the first, third, and seventh days of culture in the co-culture of Example 4 are shown. Figures 7A to 7C In the figure, the scale bar represents 200 μm.

[0301] like Figures 7A to 7C As shown in Figure 2, a hair follicle primordium with a hair shaft-like structure was formed by using a culture medium containing laminin and nestin. Figure 7C In the figure, as indicated by the arrow, a hair shaft-like structure was formed in the hair follicle primordium on the 7th day of culture.

[0302] Example 5

[0303] [Collection of epithelial and mesenchymal cells]

[0304] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0305] [nourish]

[0306] In Example 5-1, epithelial cells and mesenchymal cells were co-cultured using a culture medium containing laminin. Specifically, a DMEM / F12 culture medium containing 1% GultaMax Supplement and 0.2% Normocin was prepared as a culture medium.

[0307] Then, the cells were suspended in the culture medium to a density of 5 × 10 3 cells / mL (total cell density reaches 1×10 4cells / mL) of epithelial cells and mesenchymal cells, and further added with high-purity laminin (LAMININ-ULTRAPURE, MOUSE, CORNING (registered trademark)) in an amount to give a concentration of 1 v / v% to prepare a cell suspension.

[0308] The high-purity laminin preparation used contained 0.82 mg / mL of soluble basement membrane matrix extracted from EHS mouse tumors and had a laminin purity of more than 95% as determined by SDS-PAGE.

[0309] Based on this, it was calculated that the cell suspension supplemented with 1 v / v% of the high-purity laminin preparation contained approximately 8 μg / mL of laminin. In other words, the laminin content in the cell suspension of Example 5-1 was calculated to be 13% to 14% of the corresponding content in the cell suspension of Example 1-1 of Example 1.

[0310] Epithelial and mesenchymal cells were seeded by adding 100 μL of the cell suspension to each well of a 96-well plate. Immediately after seeding, the 96-well plate was moved to a 4°C refrigerator and allowed to stand for 20 minutes. This allowed the epithelial and mesenchymal cells to settle to the bottom of the wells in the cooled culture medium.

[0311] Then, the 96-well plate was moved to an incubator at 37° C., and co-culture of epithelial cells and mesenchymal cells was started in the incubator. The culture medium was replaced in the same manner as in Example 1-1 of Example 1 above.

[0312] In Example 5-2, similarly to Example 4 of the above-mentioned Example 4, co-culture of epithelial cells and mesenchymal cells was performed using a culture medium containing laminin and entactin.

[0313] In Example 5-3, epithelial cells and mesenchymal cells were co-cultured using a culture medium containing type IV collagen. Specifically, a DMEM / F12 culture medium containing 1% GultaMax Supplement and 0.2% Normocin was prepared as a culture medium.

[0314] Then, the cells were suspended in the culture medium to a density of 5 × 10 3 cells / mL (total cell density reaches 1×10 4 Cells / mL) of epithelial cells and mesenchymal cells were added, and type IV collagen (COLLAGEN IV, MOUSE, CORNING (registered trademark)) was further added in an amount to give a concentration of 1 v / v% to prepare a cell suspension.

[0315] The type IV collagen preparation used contained 1.25 mg / mL of soluble basement membrane matrix extracted from EHS mouse tumors and contained type IV collagen with a purity of more than 90% as determined by SDS-PAGE.

[0316] Based on this, it was calculated that the cell suspension supplemented with 1 v / v% of the type IV collagen preparation contained 11-13 μg / mL of type IV collagen. In other words, the type IV collagen content in the cell suspension of Example 5-3 was 34%-38% of the corresponding content in the cell suspension of Example 1-1 of Example 1.

[0317] Epithelial and mesenchymal cells were seeded by adding 100 μL of the cell suspension to each well of a 96-well plate. Immediately after seeding, the 96-well plate was moved to a 4°C refrigerator and allowed to stand for 20 minutes. This allowed the cells to settle to the bottom of the wells in the cooled culture medium.

[0318] Then, the 96-well plate was moved to an incubator at 37° C., and co-culture of epithelial cells and mesenchymal cells was started in the incubator. The culture medium was replaced in the same manner as in Example 1-1 of Example 1 above.

[0319] [result]

[0320] Figure 8A 、 Figure 8B and Figure 8C The phase contrast microscope photos taken on the 8th day of culture in Example 5-1, Example 5-2 and Example 5-3 are shown respectively. Figures 8A to 8C In the figure, the scale bar represents 200 μm.

[0321] like Figure 8A As shown in FIG5-1, in which a culture medium containing laminin was used, no hair shaft-like structure was formed in the hair follicle primordium on the 8th day of culture. Figure 8B In the example 5-2 in which the culture medium containing laminin and nestin was used, as indicated by the arrow, a hair shaft-like structure was formed in the hair follicle primordium on the 8th day of culture. Figure 8C As indicated by the middle arrow, in Example 5-3 using the culture medium containing type IV collagen, a hair shaft-like structure was also formed in the hair follicle primordium on the eighth day of culture.

[0322] Example 6

[0323] [Collection of epithelial and mesenchymal cells]

[0324] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0325] [nourish]

[0326] In Example 6, instead of the matrix gel preparation used in Example 1-1 of the above-mentioned Example 1, a matrix gel preparation with reduced growth factor content (Matrigel (registered trademark) Basement Membrane Matrix (Growth Factor Reduced), CORNING (registered trademark)) was used in an amount such that the concentration in the culture medium reached 1 v / v%. Except for this, co-culture of epithelial cells and mesenchymal cells was carried out for 8 days in the same manner as in Example 1-1 of the above-mentioned Example 1.

[0327] The matrix gel preparation (GFR) used contained 8-12 mg / mL (protein mass measured by the Lowry method) of soluble basement membrane matrix extracted from EHS (Engelbreth-Holm-Swarm) mouse tumors. The composition ratio of the basement membrane matrix was: 61% laminin, 7% entactin, and 30% type IV collagen.

[0328] [result]

[0329] Figure 9A and Figure 9B The phase contrast microscope images taken on the first and eighth days of culture in the co-culture of Example 6 are shown. Figure 9A and Figure 9B As shown in FIG6 , in Example 6, a hair follicle primordium having a hair shaft-like structure was formed. Figure 9B As shown, hair shaft-like structures were formed in the hair follicle primordium on day 8 of culture.

[0330] Furthermore, formation of a hair shaft-like structure was confirmed in 85 of the 96 hair follicle primordia, with a hair shaft-like structure formation rate of 89%. This indicates that, in Example 6, hair follicle primordia having a hair shaft-like structure were formed in the same manner as in Example 1-1, and the hair shaft-like structure formation rate was also comparable to that in Example 1-1.

[0331] Example 7

[0332] [Collection of epithelial and mesenchymal cells]

[0333] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0334] [nourish]

[0335] In Example 7-1, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set to 0.1 v / v% instead of 1 v / v%. The cell suspension to which 0.1 v / v% matrix gel was added was calculated to contain 6 μg / mL of laminin, 1 μg / mL of entactin, and 3 μg / mL of type IV collagen.

[0336] In Example 7-2, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 0.2 v / v% instead of 1 v / v%. The cell suspension to which 0.2 v / v% matrix gel was added was calculated to contain 12 μg / mL of laminin, 2 μg / mL of entactin, and 7 μg / mL of type IV collagen.

[0337] In Example 7-3, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 0.3 v / v% instead of 1 v / v%. The cell suspension to which 0.3 v / v% matrix gel was added was calculated to contain 18 μg / mL of laminin, 3 μg / mL of entactin, and 10 μg / mL of type IV collagen.

[0338] In Example 7-4, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 0.4 v / v% instead of 1 v / v%. The cell suspension to which 0.4 v / v% matrix gel was added was calculated to contain 24 μg / mL of laminin, 3 μg / mL of entactin, and 13 μg / mL of type IV collagen.

[0339] In Example 7-5, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set to 0.5 v / v% instead of 1 v / v%. The cell suspension to which 0.5 v / v% matrix gel was added was calculated to contain 30 μg / mL of laminin, 4 μg / mL of entactin, and 16 μg / mL of type IV collagen.

[0340] In Example 7-6, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 0.6 v / v% instead of 1 v / v%. The cell suspension to which 0.5 v / v% matrix gel was added was calculated to contain 36 μg / mL of laminin, 5 μg / mL of entactin, and 20 μg / mL of type IV collagen.

[0341] [result]

[0342] Figure 10A 、 Figure 10B 、 Figure 10C 、 Figure 10D 、 Figure 10E and Figure 10FPhase contrast microscopic photographs taken on the first day of culture in the co-culture of Example 7-1, Example 7-2, Example 7-3, Example 7-4, Example 7-5, and Example 7-6 are shown. Figure 11A 、 Figure 11B 、 Figure 11C 、 Figure 11D 、 Figure 11E and Figure 11F Phase contrast microscopic photographs taken on the third day of culture in the co-culture of Example 7-1, Example 7-2, Example 7-3, Example 7-4, Example 7-5, and Example 7-6 are shown. Figure 12A 、 Figure 12B 、 Figure 12C 、 Figure 12D 、 Figure 12E and Figure 12F Phase contrast microscope photos taken on day 8 of the co-culture of Example 7-1, Example 7-2, Example 7-3, Example 7-4, Example 7-5, and Example 7-6 are shown. Figures 10A to 10F 、 Figures 11A to 11F ,and Figures 12A to 12F In the figure, the scale bar represents 200 μm.

[0343] like Figures 10A to 10F 、 Figures 11A to 11F ,and Figures 12A to 12F As shown in FIG, in all the examples (Example 7-1 to Example 7-6), a hair follicle primordium having a hair shaft-like structure was formed. Figures 12A to 12F As shown, in all cases, hair shaft-like structures were formed in the hair follicle primordium cultured for 8 days.

[0344] In addition, the formation rate of hair shaft-like structures on the 8th day of culture was 25% in Example 7-1 (3 out of 12 hair follicle primordia), 58% in Example 7-2 (7 out of 12 hair follicle primordia), 92% in Example 7-3 (11 out of 12 hair follicle primordia), and 100% in Examples 7-4, 7-5, and 7-6 (12 out of 12 hair follicle primordia).

[0345] Example 8

[0346] [Collection of epithelial and mesenchymal cells]

[0347] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0348] [nourish]

[0349] In Example 8-1, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 2.0 v / v% instead of 1 v / v%. The cell suspension to which 2.0 v / v% matrix gel was added was calculated to contain 119 μg / mL of laminin, 17 μg / mL of entactin, and 66 μg / mL of type IV collagen.

[0350] In Example 8-2, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 2.5 v / v% instead of 1 v / v%. The cell suspension to which the 2.5 v / v% matrix gel was added was calculated to contain 148 μg / mL of laminin, 21 μg / mL of entactin, and 82 μg / mL of type IV collagen.

[0351] In Example 8-3, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set to 3.0 v / v% instead of 1 v / v%. The cell suspension to which the matrix gel was added was calculated to contain 178 μg / mL of laminin, 25 μg / mL of entactin, and 99 μg / mL of type IV collagen.

[0352] In Example 8-4, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set to 3.5 v / v% instead of 1 v / v%. The cell suspension to which the 3.5 v / v% matrix gel was added was calculated to contain 208 μg / mL of laminin, 30 μg / mL of entactin, and 115 μg / mL of type IV collagen.

[0353] In Example 8-5, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 4.0 v / v% instead of 1 v / v%. The cell suspension to which the matrix gel was added was calculated to contain 237 μg / mL of laminin, 34 μg / mL of entactin, and 131 μg / mL of type IV collagen.

[0354] In Example 8-6, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1-1 of Example 1, except that the concentration of the matrix gel added to the culture medium was set at 4.5 v / v% instead of 1 v / v%. The cell suspension to which the matrix gel was added was calculated to contain 267 μg / mL of laminin, 38 μg / mL of entactin, and 148 μg / mL of type IV collagen.

[0355] [result]

[0356] Figure 13A 、 Figure 13B 、 Figure 13C 、 Figure 13D 、 Figure 13E and Figure 13F Phase contrast microscopic photographs taken on the first day of culture in the co-culture of Example 8-1, Example 8-2, Example 8-3, Example 8-4, Example 8-5, and Example 8-6 are shown. Figure 14A 、 Figure 14B 、 Figure 14C 、 Figure 14D 、 Figure 14E and Figure 14F Phase contrast microscopic photographs taken on the third day of culture in the co-culture of Example 8-1, Example 8-2, Example 8-3, Example 8-4, Example 8-5, and Example 8-6 are shown. Figure 15A 、 Figure 15B 、 Figure 15C 、 Figure 15D 、 Figure 15E and Figure 15F Phase contrast microscope photos taken on the 8th day of culture in the co-culture of Example 8-1, Example 8-2, Example 8-3, Example 8-4, Example 8-5 and Example 8-6 are shown respectively. Figures 13A to 13F 、 Figures 14A to 14F ,and Figures 15A to 15F In the figure, the scale bar represents 200 μm.

[0357] like Figures 13A to 13F 、 Figures 14A to 14F ,and Figures 15A to 15F As shown in FIG, in all the examples (Example 8-1 to Example 8-6), a hair follicle primordium having a hair shaft-like structure was formed. Figures 15A to 15F As shown, in all cases, hair shaft-like structures were formed in the hair follicle primordium cultured for 8 days.

[0358] In addition, the formation rate of hair shaft-like structures on the 8th day of culture was 100% in Examples 8-1, 8-2 and 8-3 (6 out of 6 hair follicle primordia), and was also 100% in Examples 8-4, 8-5 and 8-6 (12 out of 12 hair follicle primordia).

[0359] Example 9

[0360] [Collection of epithelial and mesenchymal cells]

[0361] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0362] [nourish]

[0363] In Example 9-1, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 4 of Example 4 above, except that the concentration of the high-concentration laminin / nidogen complex preparation added to the culture medium was set to 0.5 v / v% instead of 1 v / v%. The cell suspension to which the high-concentration laminin / nidogen complex preparation was added was calculated to contain 68 to 76 μg / mL of the laminin / nidogen complex (i.e., 34 to 38 μg / mL of laminin and nidogen, respectively).

[0364] In Example 9-2, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 4 of Example 4 above, except that the concentration of the high-concentration laminin / nidogen complex preparation added to the culture medium was set to 5.0 v / v% instead of 1 v / v%. The cell suspension to which the high-concentration laminin / nidogen complex preparation was added was calculated to contain 684 to 760 μg / mL of the laminin / nidogen complex (i.e., 342 to 380 μg / mL of laminin and nidogen, respectively).

[0365] In Example 9-3, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 4 of Example 4 above, except that the concentration of the high-concentration laminin / nidogen complex preparation added to the culture medium was set at 10.0 v / v% instead of 1 v / v%. The cell suspension to which the high-concentration laminin / nidogen complex preparation was added was calculated to contain 1368 to 1520 μg / mL of the laminin / nidogen complex (i.e., 684 to 760 μg / mL of laminin and nidogen, respectively).

[0366] [result]

[0367] Figure 16A 、 Figure 16B and Figure 16C Phase contrast microscopic photographs taken on the second day of culture in the co-culture of Example 9-1, Example 9-2, and Example 9-3 are shown respectively. Figure 17A 、 Figure 17B and Figure 17C Phase contrast microscope photos taken on the 8th day of culture in the co-culture of Example 9-1, Example 9-2 and Example 9-3 are shown respectively. Figures 16A to 16C ,and Figures 17A to 17CIn the figure, the scale bar represents 200 μm.

[0368] like Figures 16A to 16C ,and Figures 17A to 17C As shown in FIG, in all the examples (Example 9-1 to Example 9-3), a hair follicle primordium having a hair shaft-like structure was formed. Figures 17A to 17C As shown, in all cases, hair shaft-like structures were formed in the hair follicle primordium cultured for 8 days.

[0369] Reference Example 1

[0370] [Collection of epithelial and mesenchymal cells]

[0371] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0372] [nourish]

[0373] In Example C1-1, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 4 of Example 4 above, except that the concentration of the high-purity laminin product added to the culture medium was set to 5 v / v% instead of 1 v / v%. The cell suspension containing 5 v / v% of the high-purity laminin product was calculated to contain 39 to 41 μg / mL of laminin.

[0374] In Example C1-2, co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 4 of Example 4 above, except that the concentration of the high-purity laminin product added to the culture medium was set to 10 v / v% instead of 1 v / v%. The cell suspension containing 10 v / v% of the high-purity laminin product was calculated to contain 78 to 82 μg / mL of laminin.

[0375] [result]

[0376] Figure 18A 、 Figure 18B and Figure 18C Phase contrast microscopic photographs taken on the first, third, and eighth day of culture in the co-culture of Example C1-1 are shown. Figure 18D 、 Figure 18E and Figure 18F Phase contrast microscopy images taken on the first, third, and eighth day of culture, respectively, in the co-culture of Example C1-2 are shown. Figures 18A to 18F In the figure, the scale bar represents 200 μm.

[0377] like Figures 18A to 18F As shown, in neither Example C1-1 nor Example C1-2, a hair follicle primordium having a hair shaft-like structure was formed.

[0378] Reference Example 2

[0379] [Collection of epithelial and mesenchymal cells]

[0380] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0381] [nourish]

[0382] In Example C2, epithelial and mesenchymal cells were co-cultured in a culture medium without type IV collagen on the surface of a culture dish previously coated with type IV collagen. First, the type IV collagen product used in Example 5-3 of Example 5 was diluted to a concentration of 100 μg / mL with 0.05 M HCl solution according to the coating instructions in the product manual to prepare a coating solution. Next, 1 mL of this coating solution was added to a 35 mm diameter culture dish and incubated at room temperature for 1 hour. The coating solution was then discarded from the dish, and the dish surface was rinsed with purified water.

[0383] On the other hand, each cell was suspended in DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normоcin to a density of 5 × 10 3 cells / mL (total cell density reaches 1×10 4 cells / mL) of epithelial cells and mesenchymal cells to prepare a cell suspension.

[0384] 2 mL of the cell suspension was added to a culture dish pre-coated with type IV collagen as described above, and epithelial cells and mesenchymal cells were seeded to initiate co-culture.

[0385] [result]

[0386] Figure 19A and Figure 19B The phase contrast microscope images of the co-culture of Example C2 were taken on the 2nd and 8th day of culture. Figure 19A and Figure 19B In the figure, the scale bar represents 200 μm.

[0387] like Figure 19A and Figure 19B As shown, even when epithelial cells and mesenchymal cells were co-cultured in a culture medium without type IV collagen on a culture dish surface coated with type IV collagen, hair follicle primordium having a hair shaft-like structure was not formed.

[0388] Reference Example 3

[0389] [Collection of epithelial and mesenchymal cells]

[0390] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0391] [nourish]

[0392] In Example C3-1, epithelial cells and mesenchymal cells were co-cultured on a gel containing laminin, entactin, and type IV collagen in a culture medium that did not contain laminin, entactin, or type IV collagen.

[0393] Specifically, 50 μL of the matrix gel preparation used in Example 1-1 of Example 1 was first dripped onto the bottom of each well of a 96-well flat-bottom culture plate and incubated at 37°C for 15 minutes to form a gel. Epithelial cells and mesenchymal cells suspended in a culture medium not containing the matrix gel preparation were then seeded onto the gel in each well and co-cultured on the gel.

[0394] In Example C3-2, epithelial and mesenchymal cells were co-cultured in a gel containing laminin, entactin, and type IV collagen.

[0395] Specifically, a matrix gel preparation was first mixed with epithelial and mesenchymal cells, and 50 μL of the resulting cell suspension was dripped onto the bottom of each well of a 96-well flat-bottom culture plate and incubated at 37°C for 15 minutes to allow gelation. Subsequently, a culture medium without the matrix gel preparation was injected onto the gel containing the dispersed epithelial and mesenchymal cells in each well, and co-culture was performed within the gel.

[0396] In Example C3-3, the hair follicle primordium formed by co-culturing epithelial cells and mesenchymal cells in a culture medium without laminin, entactin and type IV collagen was cultured on a gel containing laminin, entactin and type IV collagen in a culture medium without laminin, entactin and type IV collagen.

[0397] Specifically, first, similarly to Example 1-2 of Example 1, epithelial cells and mesenchymal cells were co-cultured for 3 days in a culture medium lacking laminin, entactin, and type IV collagen to form hair follicle primordia containing these epithelial cells and mesenchymal cells.

[0398] On the other hand, a matrix gel was formed in the same manner as in Example C3-1. Then, the hair follicle primordia formed as described above were seeded onto the gel in a culture medium without matrix gel, and the hair follicle primordia were cultured on the gel for 9 days.

[0399] In Example C3-4, hair follicle primordia formed by co-culturing epithelial cells and mesenchymal cells in a culture medium without laminin, entactin, and type IV collagen were cultured in a gel containing laminin, entactin, and type IV collagen.

[0400] Specifically, as in Example 1-2 of Example 1 above, epithelial cells and mesenchymal cells were co-cultured for 3 days in a culture medium lacking laminin, entactin, and type IV collagen to form hair follicle primordia comprising these epithelial and mesenchymal cells. Subsequently, as in Example C3-2 above, the hair follicle primordia were embedded in a matrix gel and cultured in the gel for 9 days.

[0401] [result]

[0402] Figure 20A 、 Figure 20B 、 Figure 20C and Figure 20D The phase contrast microscope images taken on the 12th day of culture are shown in Example C3-1, Example C3-2, Example C3-3 and Example C3-4 respectively. Figures 20A to 20D In the figure, the scale bar represents 200 μm.

[0403] like Figures 20A to 20D As shown, in any of Examples C3-1 to C3-4, no hair follicle primordium having a hair shaft-like structure was formed.

[0404] Example 10

[0405] [Collection of epithelial and mesenchymal cells]

[0406] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1 above.

[0407] [nourish]

[0408] In Example 10, epithelial cells and mesenchymal cells were co-cultured using a culture medium containing laminin, entactin, and type IV collagen. First, a DMEM / F12 culture medium containing 1% GultaMax Supplement and 0.2% Normocin was prepared as a culture medium.

[0409] Then, the cells were suspended in the culture medium to a density of 5 × 10 3 cells / 200 μL (total cell density reaches 1×10 4 cells / 200 μL) of epithelial cells and mesenchymal cells, and further added with matrix gel in an amount to give a concentration of 1 v / v% to prepare a cell suspension.

[0410] Epithelial and mesenchymal cells were seeded by adding 200 μL of the cell suspension to each well of a 96-well plate. Immediately after seeding, the 96-well plate was moved to a 4°C refrigerator and allowed to rest for 20 minutes. This resting allowed the epithelial and mesenchymal cells to settle to the bottom of the wells in the cooled culture medium in the refrigerator, allowing them to contact each other. The 96-well plate was then moved to a 37°C incubator, where co-culture of the epithelial and mesenchymal cells began. Co-culture was continued for 6 days.

[0411] During co-culture, the culture medium was replaced every two days. First, 100 μL of culture medium was removed from each well. Then, 100 μL of DMEM / F12 medium containing no matrix gel, 1% GultaMax Supplement, and 0.2% Normocin was added to each well as a fresh culture medium.

[0412] [Transplantation of hair follicle primordium]

[0413] The hair follicle primordia formed after 6 days of co-culture were observed under a phase contrast microscope to confirm whether a hair shaft-like structure had formed in each hair follicle primordium. Then, only the hair follicle primordia with a hair shaft-like structure were selectively recovered and transplanted subcutaneously into 5-week-old ICR nude mice (Oriental Yeast Industry Co., Ltd.) under anesthesia using an isoflurane anesthesia device for small animals (BioRearch Center Co., Ltd.). Specifically, a transplant hole was created in the back of the nude mouse using an ophthalmic V-lance (20G, Alcon Co., Ltd. in Japan), and 21 hair follicle primordia, each with a hair shaft-like structure, were inserted into the transplant hole using a pipette.

[0414] [result]

[0415] Figure 21 An example of a photograph showing the back of a nude mouse on day 22 after transplantation. Figure 21 As shown, hair was formed in the area of the nude mouse's back where the hair follicle primordium was transplanted. Specifically, hair formation was confirmed in 18 of the 21 transplanted hair follicle primordia. That is, the ratio of the number of transplanted hair follicle primordia that formed hair on the nude mouse's back to the total number of transplanted hair follicle primordia (hair regeneration efficiency) was 85.7% (= 18 / 21×100). It should be noted that this hair regeneration efficiency was higher than the case where hair follicle primordia without a hair shaft-like structure were transplanted using a culture medium that did not contain laminin, nestin, and type IV collagen (results not shown). In this way, by transplanting the hair follicle primordium produced by the above-mentioned co-culture into a living organism, it was confirmed that hair regenerated efficiently.

[0416] Example 11

[0417] [Collection of epithelial and mesenchymal cells]

[0418] Epithelial cells and mesenchymal cells were prepared in the same manner as in Example 1. However, first, on the day of inoculation of mesenchymal cells alone, mesenchymal cells were collected from a first mouse individual. Then, on the day of inoculation of epithelial cells after culturing these mesenchymal cells for one day, epithelial cells were collected from a second mouse individual different from the first mouse individual.

[0419] [nourish]

[0420] In Example 11, first, only mesenchymal cells were cultured using a culture medium containing laminin, entactin, and type IV collagen. Then, epithelial cells were added and co-cultured with epithelial and mesenchymal cells using a culture medium containing laminin, entactin, and type IV collagen.

[0421] As a culture medium, prepare DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normоcin. Suspend the cells in this culture medium to a density of 5×10 4 cells / mL, and further added with matrix gel in an amount to give a concentration of 1 v / v% to prepare a cell suspension of the mesenchymal cells.

[0422] 100 μL of mesenchymal cell suspension was added to each well of a 96-well plate and mesenchymal cells (cell density of mesenchymal cells: 5×10 3 cells / well). Immediately after seeding, the 96-well plate was placed in a 4°C refrigerator for 20 minutes to allow the mesenchymal cells to settle to the bottom of the wells. The 96-well plate was then placed in a 37°C incubator and cultured for 1 day.

[0423] One day after the start of mesenchymal cell culture, the cells were suspended in a culture medium (DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normоcin) to a density of 5×10 4 cells / mL, and further added with matrix gel in an amount to give a concentration of 1 v / v% to prepare a suspension of the epithelial cells.

[0424] On the other hand, the 96-well plate containing the mesenchymal cells cultured for one day as described above was moved to a 4°C refrigerator and allowed to stand for 20 minutes. Then, 100 μL of the epithelial cell suspension was added to each well containing mesenchymal cells, and epithelial cells (cell density of epithelial cells: 5×10 3 cells / well).

[0425] After seeding the epithelial cells, the 96-well plate containing the epithelial and mesenchymal cells was moved to a 4°C refrigerator and allowed to stand for 20 minutes. The 96-well plate was then moved to a 37°C incubator, where co-culture of the epithelial and mesenchymal cells began. The co-culture was continued for 14 days. That is, co-culture of the epithelial and mesenchymal cells was continued for 14 days starting from the day the epithelial cells were seeded in each well containing the mesenchymal cells. The culture medium was replaced in the same manner as in Example 10 above.

[0426] [result]

[0427] Figure 22A and Figure 22B Phase contrast microscopic photographs taken on the 3rd and 14th days from the start of co-culture in Example 11 are shown. Figure 22A and Figure 22B In the figure, the scale bar represents 200 μm.

[0428] like Figure 22A As shown in FIG, the hair follicle primordium formed on the third day of culture mostly contains mesenchymal cell aggregates formed by the aggregation of mesenchymal cells and epithelial cell aggregates connected in series with the mesenchymal cell aggregates formed by the aggregation of epithelial cells. Figure 22B As shown in FIG, it was also confirmed that on the 14th day of culture, a long hair shaft-like structure was formed from one hair follicle primordium.

[0429] Figure 23 The results show the measurement of the length of the hair shaft-like structure formed in the hair follicle primordium in co-culture. Figure 23 In the figure, the horizontal axis represents the number of days of co-culture, and the vertical axis represents the length of the hair shaft-like structure formed in the hair follicle primordium.

[0430] like Figure 23 As shown, hair shaft-like structures began to be observed 4 days after the start of co-culture, and then grew and lengthened as the culture time passed.

[0431] Figure 24A This is a photograph obtained by observing a cross section of a hair shaft-like structure formed in the hair follicle primordium on the 14th day of culture using a transmission microscope. Figure 24A In the figure, the scale bar represents 5 μm. Figure 24B Enlarged representation Figure 24A The quadrilateral area enclosed by the white line is shown. Figure 24B In the figure, the scale bar represents 2 μm. Figure 24C Enlarged representation Figure 24B The quadrilateral area enclosed by the white line is shown. Figure 24C In the figure, the scale bar represents 500 nm. Figure 24D Enlarged representation Figure 24C The quadrilateral area enclosed by the white line is shown. Figure 24D In the figure, the scale bar represents 200 nm.

[0432] like Figures 24A to 24D As shown, it was confirmed that the hair shaft-like structure formed in the hair follicle primordium has a structure similar to that of biological hair, such as melanin and hair cortex.

[0433] Example 12

[0434] [Collection of epithelial and mesenchymal cells]

[0435] Skin tissue from the back of 18-day-old Bulb / c mouse fetuses was collected and, using a modified method from Nakao et al. (Koh-ei Toyoshima et al. Nature Communications, 3, 784, 2012), treated with dispase for 1 hour at 4°C with shaking at 30 rpm to separate the epithelial and interstitial layers of the skin tissue. The epithelial layer was then treated with 100 U / mL collagenase for 1 hour and 20 minutes, followed by trypsin treatment for 10 minutes to isolate epithelial cells. Furthermore, mesenchymal cells were isolated by treating the interstitial layer with 100 U / mL collagenase for 1 hour and 20 minutes.

[0436] [nourish]

[0437] In Example 12-1, epithelial cells, mesenchymal cells, and melanocytes were co-cultured using a culture medium containing laminin, entactin, and type IV collagen. First, a DMEM / F12 medium containing 1% GultaMax Supplement and 0.2% Normocin was prepared as a culture medium.

[0438] Then, cells were suspended in the culture medium (total cell density: 1.125×10 4 cells / 200 μL) to a cell density of 5 × 10 3 cells / 200 μL of epithelial and mesenchymal cells, and a cell density of 1.25×10 3 Melanocytes (normal human (black) epidermal melanocytes) were added in an amount of 1 v / v% of cells / 200 μL, and a cell suspension was prepared by further adding Matrigel in an amount to give a concentration of 1 v / v%.

[0439] Epithelial cells, mesenchymal cells and melanocytes were inoculated by adding 200 μL of the above-mentioned cell suspension to each well of a 96-well plate. After inoculation, the 96-well plate was immediately moved to a refrigerator at 4°C and allowed to stand for 20 minutes. By standing still, the epithelial cells, mesenchymal cells and melanocytes settled to the bottom surface of the well in a state where they could contact each other. Then, the 96-well plate was moved to an incubator at 37°C, and co-culture of epithelial cells, mesenchymal cells and melanocytes was started in the incubator. Co-culture was performed for 8 days. The culture medium was replaced in the same manner as in Example 10 above.

[0440] In Example 12-2, in addition to using a cell density of 2.5 × 10 3 cells / 200 μL of melanocytes (total cell density: 1.25×10 4 Co-culture of epithelial cells, mesenchymal cells, and melanocytes was performed in the same manner as in Example 12-1, except that the concentration of the cells was 1:1.

[0441] In Example 12-3, in addition to using a cell density of 5 × 10 3 cells / 200 μL of melanocytes (total cell density: 1.5×10 4 Co-culture of epithelial cells, mesenchymal cells, and melanocytes was performed in the same manner as in Example 12-1, except that the concentration of the cells was 1:1.

[0442] In Example 12-C1, except that no melanocytes were used (total cell density: 1×10 4 Co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 12-1 except that the concentration of the cells was 1:1 (cells / 200 μL).

[0443] That is, the ratio of the inoculation numbers of the three types of cells (epithelial cells: mesenchymal cells: melanocytes) is "1:1:0" in Example 12-C1, "4:4:1" in Example 12-1, "2:2:1" in Example 12-2, and "1:1:1" in Example 12-3.

[0444] [result]

[0445] Figure 25A 、 Figure 25B 、 Figure 25C and Figure 25D Phase contrast microscopic photographs taken on the 8th day of culture in the co-culture of Example 12-C1, Example 12-1, Example 12-2, and Example 12-3 are shown respectively.

[0446] like Figure 25A As shown in Figure 2, co-culture of epithelial cells and mesenchymal cells from white-haired Bulb / c mice resulted in the formation of hair follicle primordia with white hair shaft-like structures. Figures 25B to 25D As shown, in all of Examples 12-1 to 12-3, by further adding melanocytes to epithelial cells and mesenchymal cells derived from Bulb / c mice having white hair and co-culturing them, hair follicle primordia having a black hair shaft-like structure were formed.

Claims

1. A method for producing a hair follicle primordium, comprising forming the hair follicle primordium by the following steps: seeding epithelial and mesenchymal cells; Prior to formation of the hair follicle primordium, the epithelial cells and the mesenchymal cells are brought into contact with (a) and / or (b) in a fluid culture medium in which (a) laminin and entactin are dispersed at a total concentration of 1 μg / mL or higher, and / or (b) type IV collagen at a concentration of 1 μg / mL or higher is / are maintained at a temperature of 30°C to 45°C; and Co-culturing the epithelial cells and the mesenchymal cells in a culture medium, in, The maintenance of the epithelial cells and the mesenchymal cells in the fluid culture medium in which (a) and / or (b) are dispersed at a temperature of 30°C to 45°C begins from the time of starting the co-culture of the epithelial cells and the mesenchymal cells until 20 hours have passed.

2. The method for producing a hair follicle primordium according to claim 1, wherein: The epithelial cells and the mesenchymal cells are maintained in a culture medium in which the (a) laminin and entactin are dispersed.

3. The method for producing a hair follicle primordium according to claim 1 or 2, wherein: The epithelial cells and the mesenchymal cells are maintained in a culture medium in which the (b) type IV collagen is dispersed.

4. The method for producing a hair follicle primordium according to claim 1, wherein: comprising the step of allowing the seeded epithelial cells and mesenchymal cells to settle on a culture substrate in a culture medium, Furthermore, the epithelial cells and the mesenchymal cells settled on the culture substrate are maintained in the culture solution in which (a) and / or (b) are dispersed.

5. The method for producing a hair follicle primordium according to claim 1, wherein: The following steps are included: After the epithelial cells and the mesenchymal cells are maintained in a culture medium in which the (a) and / or (b) are dispersed, the epithelial cells and the mesenchymal cells are co-cultured in a culture medium in which the concentration of the (a) and / or (b) is lower than the concentration during the maintenance.

6. The method for producing a hair follicle primordium according to claim 1, wherein: By performing the co-culture, the hair follicle primordium having a hair shaft-like structure is formed.

7. A method for promoting the formation of a hair shaft-like structure in a hair follicle primordium, comprising: seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to form a cell culture of the hair follicle primordium; Prior to the formation of the hair follicle primordium, the epithelial cells and the mesenchymal cells are maintained in a fluid culture medium in which (a) laminin and entactin are dispersed at a total concentration of 1 μg / mL or higher, and / or (b) type IV collagen is dispersed at a concentration of 1 μg / mL or higher, at a temperature of 30°C to 45°C, thereby bringing the epithelial cells and the mesenchymal cells into contact with (a) and / or (b), thereby promoting the formation of a hair shaft-like structure in the hair follicle primordium. in, The maintenance of the epithelial cells and the mesenchymal cells in the fluid culture medium in which (a) and / or (b) are dispersed at a temperature of 30°C to 45°C begins from the time of starting the co-culture of the epithelial cells and the mesenchymal cells until 20 hours have passed.

8. Use of (a) laminin and entactin, and / or (b) type IV collagen for promoting the formation of hair shaft-like structures in hair follicle primordium, wherein: In the cell culture process comprising seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to form the hair follicle primordium, Prior to the formation of the hair follicle primordium, the epithelial cells and the mesenchymal cells are brought into contact with (a) and / or (b) in a fluid culture medium in which (a) laminin and entactin are dispersed at a total concentration of 1 μg / mL or more, and / or (b) type IV collagen at a concentration of 1 μg / mL or more, and the culture medium is maintained at a temperature of 30°C to 45°C. The epithelial cells and the mesenchymal cells are maintained in a fluid culture medium in which (a) and / or (b) are dispersed at a temperature of 30°C to 45°C from the time when the co-culture of the epithelial cells and the mesenchymal cells is started until 20 hours have passed.

Images