JP2023056592A - Hair-like tissue for transplant and method related thereto - Google Patents

Abstract

To provide a hair-like tissue for transplant for hair restoration and a method related thereto.SOLUTION: A method for producing a hair-like tissue for transplant includes culturing cells to give a cell aggregate having hair-like tissue on its surface, and cutting the hair-like tissue from the cell aggregate and recovering it for transplant to an organism.SELECTED DRAWING: None

Description

The present invention relates to hair-like tissue for transplantation and related methods.

Non-Patent Document 1 describes forming skin organoids in vitro by seeding and culturing induced pluripotent stem cells (iPSCs) made from fetal mouse fibroblasts in a 96-well plate. It is stated that

Patent Document 1 discloses a process of seeding mesenchymal cells and epithelial cells on a micro-intaglio plate consisting of regularly arranged micro-concavities and culturing them while supplying oxygen to form a hair follicle primordium. A method for producing aggregates of regenerated follicle primordia is described, comprising:

Patent Document 2 discloses a method for producing full-thickness skin having skin appendages, wherein the "full-thickness skin having skin appendages" includes at least the following (1) to (3); (1) epidermal layer (2) at least one type of skin appendage; and (3) subcutaneous tissue, the method comprising the steps of: stimulating with an active substance, (b): preparing a conjugate comprising (A) and (B); (A) all or part of the embryoid body stimulated in step (a) ( B) a scaffolding material, (c) implanting said conjugate prepared in step (b) into an animal, and (d) producing full thickness skin from said conjugate in said animal. A method is described comprising:

Seeding epithelial cells and mesenchymal cells in Patent Document 3; holding the mesenchymal cells; and forming a hair follicle primordium by co-culturing the epithelial cells and the mesenchymal cells in a culture medium. A method for making the base is described.

US Pat. No. 5,300,003 discloses a method of making mammalian hair follicles comprising the steps of: (a) providing at least one de novo papilla; (b) fibroblasts, keratinocytes and/or providing at least one other cell population selected from the group of melanocytes; and (c) co-culturing said de novo papillae with said at least one other cell population under non-adherent culture conditions. wherein step (a) comprises (1) providing at least one dermal papilla (DP) derived from at least one mammalian hair follicle; (3) isolating the DPF from the DP by fixing the dermal papilla fibroblasts (DPF) to the basal lamina and allowing the dermal papilla fibroblasts (DPF) to flow out; (4) condensing the expanded DPF into cell aggregates exhibiting physiological DP size and shape to obtain de novo papillae; (5) coating the cell aggregates with an extracellular matrix protein, wherein the DPF is differentiated in a non-adherent culture vessel at a cell concentration per culture vessel surface of 1000-100000 DFP/cm ² ; A method of making a mammalian hair follicle is described, comprising the steps of:

In Patent Document 5, in a method for producing spheroids, a carrier population composed of a plurality of carriers having cell adhesiveness and adhesion-dependent cells are mixed under mixed conditions in which the carrier population does not aggregate together with the cells. A method for producing spheroids is described, which includes a mixing step of mixing and a culturing step of culturing the mixture for a predetermined period.

WO2017/073625 WO2016/039279 WO2020/225934 Japanese translation of PCT publication No. 2011-515095 Japanese Patent Application Laid-Open No. 2003-219865

Jiyoon Lee et al. (2018). Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids. Cell Reports 22, 242-254

On the other hand, the inventors of the present invention have studied technical means for hair regenerative medicine.

The present invention has been made in view of the above problems, and one of its objects is to provide a hair-like tissue for transplantation for hair regeneration and a method related thereto.

A method for producing a hairy tissue for transplantation according to an embodiment of the present invention for solving the above-mentioned problems comprises performing cell culture to obtain a cell aggregate having a hairy tissue formed on its surface; cutting and recovering the ciliary tissue from the cell clump for transplantation into a living body. According to the present invention, a method for producing hair-like tissue for transplantation for hair regeneration is provided.

Also, the hair-like tissue may have a hair shaft-like structure. Moreover, the hair-like tissue of the cell aggregate may have a dermal papilla-like structure at the tip portion, which is a free end. Also, the ciliary tissue may not contain capillaries. Also, the cell aggregate may not contain a pili structure and/or a sebaceous gland structure. Moreover, the ciliary tissue cut and recovered from the cell aggregate may have one tip portion that does not have a cut surface and the other tip portion that has a cut surface.

Further, the cell culture may be co-culturing of the epithelial cells and the mesenchymal cells, including seeding the epithelial cells and the mesenchymal cells. In this case, the co-cultivation includes a matrix treatment in which the epithelial cells and the mesenchymal cells are maintained in a culture medium in which type I collagen, fibronectin, laminin and entactin, or type IV collagen is dispersed. good too. Furthermore, in this case, in the matrix treatment, the epithelial cells and the mesenchymal cells may be maintained in a culture solution in which type I collagen is dispersed.

In addition, the co-cultivation includes performing suspension culture of the epithelial cells and the mesenchymal cells to form cell aggregates, and enveloping the cell aggregates formed by the suspension culture in a hydrogel. burying and further culturing.

A hair-like tissue for transplantation according to one embodiment of the present invention for solving the above problems has one tip portion and the other tip portion which are free ends, and contains epithelial cells and mesenchymal cells, Does not contain capillaries. According to the present invention, there is provided a hair-like tissue transplant for hair regeneration.

Moreover, the hair-like tissue for transplantation may have a hair shaft-like structure. Moreover, the hair-like tissue for transplantation may have a dermal papilla-like structure at the one tip portion. Also, the hair-like tissue for transplantation may have the one tip portion without a cut surface and the other tip portion with a cut surface.

A hair regeneration method according to an embodiment of the present invention for solving the above problems includes transplanting any of the hairy tissues described above into a living body. According to the present invention, an effective hair regrowth method is provided.

In accordance with the present invention, hair-like tissue transplants and related methods for hair regeneration are provided.

FIG. 3 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 1 according to the present embodiment. FIG. 2 is an explanatory diagram showing the relationship between the concentration of type I collagen in the culture solution at the time of seeding and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture in Example 1 according to the present embodiment. Explanatory drawing showing the relationship between the type I collagen concentration in the culture medium at the time of seeding and the number of hairy tissues formed per cell aggregate on day 8 of culture in Example 1 according to the present embodiment. be. FIG. 2 is an explanatory view showing a micrograph of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 μg/mL in Example 1 according to the present embodiment. . In Example 1 according to the present embodiment, the results of HE staining of frozen sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 μg / mL are shown. It is an explanatory diagram showing. Versican was fluorescently stained in cryosections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 μg/mL in Example 1 according to the present embodiment. It is explanatory drawing which shows a result. In Example 1 according to the present embodiment, CD34 was fluorescently stained in frozen sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 μg/mL. It is explanatory drawing which shows a result. FIG. 2 is an explanatory diagram schematically showing a cell aggregate having a hair-like tissue obtained in Example 1 according to the present embodiment; Photomicrographs taken on the 8th day, 12th day, 18th day, 22nd day and 27th day of culture for one cell aggregate embedded in the hydrogel in Example 2 according to the present embodiment It is an explanatory view showing . FIG. 10 is an explanatory view showing an enlarged portion (ciliary tissue) surrounded by a dotted square in each of the five photographs included in FIG. 9; FIG. 10 is an explanatory diagram showing changes over time in the length of hair-like tissue formed in Example 2 according to the present embodiment; FIG. 10 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 3 according to the present embodiment. FIG. 10 is an explanatory diagram showing the correspondence between the fibronectin concentration in the culture solution at the time of seeding and the formation efficiency of hairy tissue in the cell aggregates on the eighth day of culture in Example 3 according to the present embodiment. FIG. 10 is an explanatory diagram showing the relationship between the fibronectin concentration in the culture medium at the time of seeding and the number of hairy tissues formed per cell aggregate on day 8 of culture in Example 3 according to the present embodiment. Explanation showing the results of HE staining of frozen sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of fibronectin in the culture solution at the time of seeding was 100 μg/mL in Example 3 according to the present embodiment It is a diagram. FIG. 10 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 4 according to the present embodiment. FIG. 10 is an explanatory diagram showing the relationship between the addition timing of type I collagen and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture in Example 4 according to the present embodiment. FIG. 4 is an explanatory diagram showing the relationship between the timing of addition of type I collagen and the number of hairy tissues formed per cell aggregate on day 8 of culture in Example 4 according to the present embodiment. FIG. 10 is an explanatory view showing a micrograph of cell aggregates on day 8 of culture formed in a culture system in which type I collagen was added immediately after cell seeding in Example 4 according to the present embodiment. FIG. 4 is an explanatory view showing an enlarged photograph of a hairy tissue extending from a cell aggregate on day 8 of culture formed in a culture system in which type I collagen was added immediately after cell seeding in Example 4 according to the present embodiment. . In Example 5 according to the present embodiment, microarray analysis was performed on cell aggregates formed using a culture medium containing type I collagen and cell aggregates formed using a culture medium containing no type I collagen. is an explanatory diagram showing a part of the result of performing. FIG. 10 is an explanatory diagram showing the result of visually counting the number of regenerated hairs 4 weeks after the transplantation of the cell aggregates in Example 6 according to the present embodiment. FIG. 11 is an explanatory diagram showing a micrograph of hair-like tissue recovered from a cell aggregate formed in Example 7 according to the present embodiment. FIG. 10 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 8 according to the present embodiment. FIG. 10 is an explanatory diagram showing the result of fluorescently staining the nuclei of cells contained in cell aggregates formed using a culture solution containing Matrigel in Example 8 according to the present embodiment and observing them with a confocal microscope. FIG. 10 is an explanatory diagram showing observation results of cell aggregates formed using a culture solution containing Matrigel in Example 8 according to the present embodiment, by HE staining and fluorescent staining. FIG. 10 is an explanatory diagram showing the results of observing the hair-like tissue of cell aggregates formed using a culture solution containing Matrigel in Example 8 according to the present embodiment. FIG. 10 is an explanatory diagram showing the results of observation with a transmission microscope of hair-like tissue of cell aggregates formed using a culture medium containing Matrigel and body hair of a mouse in Example 8 according to the present embodiment. FIG. 10 is an explanatory diagram showing a micrograph of hairy tissue collected from a cell aggregate on day 14 of culture in Example 9-1 of Example 9 according to the present embodiment. FIG. 10 is an explanatory view showing micrographs of hairy tissue collected from cell aggregates on days 12 and 22 of culture in Example 9-2 of Example 9 according to the present embodiment. FIG. 10 is an explanatory diagram showing a micrograph of cell aggregates on day 23 of culture in Example 9-2 of Example 9 according to the present embodiment. FIG. 10 is an explanatory diagram showing a micrograph of ciliary tissue recovered from a cell aggregate in Example 10 according to the present embodiment. FIG. 11 is an explanatory diagram showing a micrograph of a hydrogel-embedded and cultured hair-like graft in Example 11 according to the present embodiment.

An embodiment of the present invention will be described below. Note that the present invention is not limited to this embodiment.

As one aspect of this embodiment, cell culture is performed to obtain a cell aggregate having a hairy tissue formed on its surface, and the hairy tissue is transplanted into a living body. cutting and recovering from the clump.

In addition, this embodiment includes, as another aspect, a hair regeneration method including transplanting into a living body hairy tissue containing epithelial cells and mesenchymal cells and not containing capillaries. That is, this embodiment includes the use of hairy tissue containing epithelial cells and mesenchymal cells and not containing capillaries for transplantation into a living body. In addition, as still another aspect, the present embodiment provides a hair for transplantation that has one tip portion and the other tip portion that are free ends, contains epithelial cells and mesenchymal cells, and does not contain capillaries. contains similar tissue.

In the production of hairy tissue for transplantation, first, cell culture is performed to obtain a cell aggregate having a hairy tissue formed on its surface. Cell culture for obtaining a cell aggregate having a hairy tissue on its surface is not particularly limited as long as the effect of the present invention can be obtained. For example, seeding epithelial cells and mesenchymal cells includes Co-culture of the epithelial cells and mesenchymal cells is preferred. Specifically, epithelial cells and mesenchymal cells are seeded, and the seeded epithelial cells and mesenchymal cells are co-cultured to form a cell aggregate having a hairy tissue formed on its surface. is preferred.

The epithelial cells used to form the cell aggregates are not particularly limited as long as the effects of the present invention are obtained. For example, one or more selected from the group consisting of hair follicle epithelial cells and their precursor cells. is preferred. Hair follicle epithelial cells are epithelial cells that contribute to hair growth (more specifically, for example, epithelial cells that contribute to hair growth in cooperation with hair follicle mesenchymal cells).

The hair follicle epithelial cells may be those collected from hair follicles in vivo, or may be those induced to differentiate from undifferentiated cells in vitro. The undifferentiated cells used for inducing differentiation of hair follicle epithelial cells in vitro are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle epithelial cells in vitro. For example, pluripotent stem cells (e.g., iPS (induced pluripotent stem) cells, ES (embryonic stem) cells, Muse (multilineage-differentiating stress-ending) cells or EG (embryonic germ) cells), and stem cells other than the pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells).

Specifically, the hair follicle epithelial lineage cells are selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, outer root sheath cells, and inner root sheath cells. is preferably one or more, and particularly preferably one or more selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, and outer root sheath cells.

The progenitor cells of hair follicle epithelial cells are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle epithelial cells in vitro. epithelial cells derived from the epidermal layer of neonatal skin), and stem cells other than pluripotent stem cells that have the ability to differentiate into the hair follicle epithelial cells in vitro. is preferred.

The mesenchymal cells used for forming the cell aggregates are not particularly limited as long as the effects of the present invention can be obtained. For example, one or more selected from the group consisting of hair follicle mesenchymal cells and their progenitor cells. Preferably. Hair follicle mesenchymal cells are mesenchymal cells that contribute to hair growth (more specifically, for example, mesenchymal cells that contribute to hair growth in cooperation with hair follicle epithelial cells).

The hair follicle mesenchymal cells may be those collected from hair follicles in vivo, or may be those induced to differentiate from undifferentiated cells in vitro. The undifferentiated cells used for inducing differentiation of hair follicle mesenchymal cells in vitro are not particularly limited as long as they have the ability to differentiate into the hair follicle mesenchymal cells in vitro. stem cells (e.g., iPS cells, ES cells, Muse cells or EG cells), and stem cells other than the pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells, and mesenchymal stem cells (e.g., , adipose tissue-derived mesenchymal stem cells)).

Specifically, the hair follicle mesenchymal cells are preferably one or more selected from the group consisting of dermal papilla cells and dermal sheath cup cells, and are dermal papilla cells. is particularly preferred.

Progenitor cells of hair follicle mesenchymal cells are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle mesenchymal cells in vitro. , mesenchymal cells derived from the dermal layer of fetal or neonatal skin), and stem cells other than pluripotent stem cells that have the ability to differentiate into the hair follicle mesenchymal cells in vitro (e.g., adipose tissue-derived mesenchymal stem cells). The adipose tissue-derived mesenchymal stem cells are not particularly limited as long as the effects of the present invention can be obtained, but are, for example, collected from adipose tissue (subcutaneous adipose tissue and/or other adipose tissue) in a living body.

In co-culturing, only epithelial cells and mesenchymal cells may be co-cultured to form cell aggregates, but other cells may be added to co-culture to form cell aggregates. good. Other cells are not particularly limited as long as the effects of the present invention can be obtained, but are preferably one or more selected from the group consisting of melanocyte, melanocyte precursor cell, and melanocyte stem cell, for example.

Other cells may be those collected from hair follicles in vivo, or those induced to differentiate from undifferentiated cells in vitro. Undifferentiated cells used for inducing differentiation of other cells in vitro are not particularly limited as long as they are cells that have the ability to differentiate into the other cells in vitro. cells, ES cells, Muse cells, or EG cells), and stem cells other than the pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells). .

The cells used for co-culture are not particularly limited as long as they are derived from animals having hair follicles, and may be cells derived from humans or non-human animals (non-human animals, e.g., primates (e.g., , monkeys), rodents (e.g. mice, rats, hamsters, guinea pigs, rabbits), carnivorous animals (e.g. dogs, cats), and ungulates (e.g. pigs, cows, horses, goats, sheep), etc. non-human mammals))). However, human cells are used for transplantation into humans.

The cells used for co-culture are preferably derived from the individual to whom the cells are to be transplanted, but may be derived from an individual other than the individual to which the cells are to be transplanted. For example, the human cells used for co-culture are preferably derived from a human patient to whom the human cells are to be transplanted, but are derived from a human other than the patient (e.g., multiple cells derived from a human other than the patient). Potential stem cells (for example, cells induced to differentiate in vitro from iPS cells, ES cells, Muse cells or EG cells stored in cell banks) may be used.

In the co-culture of epithelial cells and mesenchymal cells, first, the epithelial cells and mesenchymal cells are seeded. In this regard, as a cell culture for obtaining a cell aggregate having a hairy tissue formed on its surface, as described in Non-Patent Document 1, cells having the hairy tissue are obtained by inducing the differentiation of pluripotent stem cells. A method of forming agglomerates may also be employed. However, methods involving induction of differentiation of pluripotent stem cells require long culture times and complicated operations. Also, in methods involving seeding of pluripotent stem cells, it is necessary to add special components such as Matrigel (registered trademark) to the culture solution. Tissues other than skin tissue may also be formed in methods involving seeding of pluripotent stem cells.

In contrast, when seeding epithelial cells and mesenchymal cells, it is not necessary to use pluripotent stem cells. Therefore, the method according to this embodiment may not include seeding pluripotent stem cells. Also, the method according to this embodiment may not include differentiating pluripotent stem cells. Also, the method according to this embodiment may not include culturing pluripotent stem cells.

Seeding of epithelial cells and mesenchymal cells is performed by placing the epithelial cells and mesenchymal cells in a culture vessel (eg, well for cell culture). The culture vessel for co-cultivating epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained. A relatively small volume culture vessel suitable for forming a single cell aggregate from cells including mesenchymal cells is preferably used.

Specifically, the area of the bottom surface of the culture vessel (for example, the bottom surface of one well) may be, for example, 1000 mm ² or less, preferably 500 mm ² or less, and more preferably 100 mm ² or less. , 50 mm ² or less, and particularly preferably 20 mm ² or less.

In addition, the area of the bottom surface of the culture vessel may be, for example, 0.01 mm ² or more, preferably 0.10 mm ² or more, more preferably 0.30 mm ² or more, and 0.50 mm ^{2 or more} . It is more preferably 0.70 mm 2 or more, and particularly preferably 0.70 mm ² or more. The area of the bottom surface of the culture vessel may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.

In the seeding of epithelial cells and mesenchymal cells, it is preferable to seed the epithelial cells and the mesenchymal cells at the same time. , the other may be sown further.

The time interval between the seeding of one of epithelial cells and mesenchymal cells and the seeding of the other (that is, the time from the seeding of one cell to the completion of seeding of both cells) is the effect of the present invention. However, for example, it may be 48 hours or less (0 hours or more and 48 hours or less). That is, in this case, within 48 hours after seeding one of epithelial cells and mesenchymal cells, the other is also seeded to complete seeding of both the epithelial cells and mesenchymal cells.

The time interval between the seeding of one of epithelial cells and mesenchymal cells and the other is, for example, preferably 45 hours or less, more preferably 36 hours or less, and 30 hours or less. More preferably, it is particularly preferably 24 hours or less.

Furthermore, the time interval between the seeding of one of the epithelial cells and the mesenchymal cells and the seeding of the other is, for example, preferably 18 hours or less, more preferably 15 hours or less, and 12 hours or less. is even more preferable, and 9 hours or less is particularly preferable.

Furthermore, the time interval between seeding one of epithelial cells and mesenchymal cells and seeding the other is, for example, preferably 6 hours or less, more preferably 3 hours or less, and 1 hour or less. is even more preferable, and 0 hours (that is, seeding epithelial cells and mesenchymal cells at the same time) is particularly preferable.

In the seeding of epithelial cells and mesenchymal cells, it is preferable to seed the dispersed epithelial cells and the dispersed mesenchymal cells. That is, when epithelial cells and mesenchymal cells are seeded at the same time, a cell suspension in which the epithelial cells and mesenchymal cells are dispersed is placed in a culture vessel. When one of the epithelial cells and mesenchymal cells is first seeded and then the other is further seeded, the cell suspension in which the one of the cells is dispersed is first placed in a culture vessel, and then the cell suspension is placed in a culture vessel. A cell suspension in which the other cells are dispersed is additionally put into the culture vessel.

The epithelial cells and mesenchymal cells seeded using the cell suspension as described above are dispersed and mixed in the culture medium in the culture vessel. Individual cells dispersed in the culture medium are not substantially bound to other cells, or adhere to other cells, but the culture medium can be fluidized by an operation such as pipetting. It is easily separated from the other cells.

The seeding density of epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained. Density is preferred.

Specifically, the seeding density of epithelial cells and mesenchymal cells (the total number of epithelial cells and mesenchymal cells seeded per 1 cm ² of the bottom surface of the culture vessel) is, for example, 0.1×10 ⁴ cells/cm. The number may be ² or more, preferably 0.5×10 ⁴ pieces/cm ² or more, more preferably 1.0×10 ⁴ pieces/cm ² or more, and 2.5×10 ⁴ pieces/cm 2 or more. /cm ² or more, and particularly preferably 5.0×10 ⁴ /cm ² or more. In addition, the seeding density of epithelial cells and mesenchymal cells may be, for example, 1000×10 ⁴ cells/cm ² or less, preferably 700×10 ⁴ cells/cm ² or less, and 500×10 cells/cm 2 or less. It is more preferably ⁴ pieces/cm ² or less, even more preferably 400×10 ⁴ pieces/cm ² or less, and particularly preferably 300×10 ⁴ pieces/cm ² or less. The seeding density of epithelial cells and mesenchymal cells may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.

The ratio of the total number of epithelial cells and mesenchymal cells to the total number of cells seeded in coculture is not particularly limited as long as the effect of the present invention can be obtained, but for example, it is 50% or more. 60% or more is preferable, 70% or more is more preferable, 80% or more is even more preferable, and 90% or more is particularly preferable.

Similarly, the ratio of the total number of epithelial cells and mesenchymal cells to the total number of cells composing the cell aggregates formed by co-culturing is particularly limited as long as the effects of the present invention are obtained. However, for example, it may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and 90% or more. is particularly preferred.

In addition, the ratio of the number of seeded epithelial cells and the number of seeded mesenchymal cells in co-culture (mesenchymal: epithelial seeding ratio) is not particularly limited as long as the effects of the present invention can be obtained. For example, it may be in the range of 1:10 to 10:1, preferably in the range of 1:9 to 9:1, and preferably in the range of 1:8 to 8:1. It is more preferably in the range of 1:7 to 7:1, even more preferably in the range of 1:6 to 6:1.

Furthermore, the mesenchymal: epithelial seeding ratio is, for example, preferably in the range of 1:5 to 5:1, more preferably in the range of 1:4 to 4:1, 1:3 to 3 :1 is more preferred, and 1:2 to 2:1 is particularly preferred.

In co-cultivation, epithelial cells and mesenchymal cells are mixed and cultured in a culture solution to aggregate the epithelial cells and mesenchymal cells to form cell aggregates. More specifically, in co-cultivation, epithelial cells and mesenchymal cells are seeded in a culture solution in a dispersed and mixed state, and the epithelial cells aggregate with the passage of culture time. causing the mesenchymal cells to aggregate and form intercellular connections between some epithelial cells and some mesenchymal cells. As a result, epithelial cell aggregates formed by aggregation of epithelial cells, mesenchymal cell aggregates formed by aggregation of mesenchymal cells, and between some epithelial cells and some Cell clumps containing cell-to-cell junctions with leaf lineage cells are obtained.

In order to form cell aggregates, it is necessary to aggregate epithelial cells and mesenchymal cells in a culture medium, so co-epithelial cells and mesenchymal cells are required to form the cell aggregates. Cultivation is performed in a fluid culture medium as a whole.

In the co-culturing, it is preferable to perform suspension culture of epithelial cells and mesenchymal cells to form cell aggregates. In suspension culture, epithelial cells and mesenchymal cells are cultured in a non-adhesive state to form non-adherent cell aggregates.

A culture vessel having a non-cell-adhesive bottom surface is preferably used for suspension culture. In this case, the epithelial cells and mesenchymal cells are cultured on the non-cell-adhesive bottom surface without substantially adhering to the bottom surface (that is, in a non-adherent state). That is, for example, epithelial cells and mesenchymal cells sedimented on a non-cell-adhesive bottom surface do not adhere to the bottom surface in the culture solution, or the culture solution is made to flow by an operation such as pipetting. It adheres to the bottom surface weakly enough to be easily detached from the bottom surface. The shape of epithelial cells and mesenchymal cells cultured on the non-cell-adhesive bottom surface is maintained approximately spherical.

In addition, cell aggregates are formed on a non-cell-adhesive bottom surface without substantially adhering to the bottom surface (ie, in a non-adhesive state). That is, for example, cell aggregates formed on a non-adhesive bottom surface do not adhere to the bottom surface in the culture medium, or are easily removed from the bottom surface by flowing the culture medium by pipetting or the like. It adheres to the bottom surface so weakly that it detaches from the bottom surface.

In co-culture, in one culture vessel (e.g., one well), one cell aggregate can be formed from cells including epithelial cells and mesenchymal cells seeded in the culture vessel. preferable.

Co-culturing of epithelial cells and mesenchymal cells for producing a cell aggregate having a hairy tissue is not particularly limited as long as the effects of the present invention can be obtained. It is preferable to include a matrix treatment in which cells are retained in a culture medium in which type I collagen, fibronectin, laminin and entactin, or type IV collagen (hereinafter collectively referred to as "matrix") are dispersed. .

Matrix treatment is, for example, a treatment for retaining epithelial cells and mesenchymal cells in a culture solution in which type I collagen is dispersed as a matrix. Matrix treatment is, for example, a treatment for retaining epithelial cells and mesenchymal cells in a culture solution in which fibronectin is dispersed as a matrix.

Matrix treatment is, for example, a treatment for retaining epithelial cells and mesenchymal cells in a culture solution in which laminin and entactin (for example, laminin/entactin complex) are dispersed as a matrix. Matrix treatment is, for example, a treatment of retaining epithelial cells and mesenchymal cells in a culture solution in which type IV collagen is dispersed as a matrix.

The matrix-dispersed culture medium used for matrix treatment (hereinafter sometimes referred to as "matrix treatment culture medium") is a basal culture medium (e.g., used for co-cultivating epithelial cells and mesenchymal cells). possible culture medium) by adding the matrix. That is, the matrix treatment medium contains an exogenously added matrix.

The matrix treatment culture medium contains the added matrix in a solubilized state (not insolubilized state). That is, the matrix treatment culture medium contains solubilized type I collagen (non-insolubilized type I collagen), solubilized fibronectin (non-insolubilized fibronectin), and solubilized laminin as dispersed matrices. and entactin (non-insolubilized laminin and entactin) or solubilized type IV collagen (non-insolubilized type IV collagen).

The basic culture medium is not particularly limited as long as the effect of the present invention is obtained, but for example, 1% in DMEM/F12 medium (Advanced Dulbecco's Modified Eagle Medium/Ham's F-12, GIBCO (registered trademark)) A culture solution prepared by adding GultaMax Supplement (GIBCO (registered trademark)) and 0.2% Normocin (InvivoGen) is preferably used.

The matrix added in the preparation of the matrix treatment culture medium is not particularly limited as long as the effects of the present invention can be obtained. For example, commercially available matrix products such as those used in the examples below is preferably used.

In addition, the matrix to be added may be derived from animals (human or non-human animals), may be derived from cultured cells, or may be synthesized using genetic recombination technology. may be

In addition, the matrix to be added may be treated to reduce antigenicity. Thus, for example, the collagen may be atelocollagen with the telopeptide portion removed.

The matrix-processing culture medium in which a specific matrix is dispersed may or may not contain other matrices.

That is, for example, the matrix treatment culture medium in which type I collagen is dispersed may be further added with fibronectin, laminin, entactin or type IV collagen, or fibronectin, laminin, entactin or type IV collagen may be added. It may be assumed that it is not.

Further, for example, fibronectin-dispersed matrix treatment culture medium may be further added with fibronectin, laminin, entactin or type IV collagen, or fibronectin, laminin, entactin or type IV collagen may be added. It is also possible not to do so.

Further, for example, the matrix treatment culture medium in which laminin and entactin are dispersed may be further added with type I collagen, fibronectin or type IV collagen, or type I collagen, fibronectin or type IV collagen may be added. It may be assumed that it is not.

Further, for example, the matrix treatment culture medium in which type IV collagen is dispersed may be further added with type I collagen, fibronectin, laminin or entactin, or type I collagen, fibronectin, laminin or entactin may be added. It may be assumed that it is not.

As the matrix treatment culture medium, a matrix treatment culture medium in which type I collagen is dispersed is particularly preferable. Moreover, it is preferable that the matrix treatment culture medium mainly contains type I collagen as a matrix. That is, with respect to the sum of the content of type I collagen, the content of fibronectin, the content of laminin, the content of entactin, and the content of type IV collagen in the matrix treatment medium in which type I collagen is dispersed, The weight percentage of the type I collagen content may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and 80% by weight or more. is even more preferable, and 90% by weight or more is particularly preferable.

In addition, the matrix treatment culture solution in which fibronectin is dispersed may mainly contain fibronectin as a matrix. That is, the fibronectin content relative to the sum of the type I collagen content, the fibronectin content, the laminin content, the entactin content, and the type IV collagen content in the matrix treatment medium in which the fibronectin is dispersed. The weight percentage of the content may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and even more preferably 80% by weight or more. It is preferably 90% by weight or more, and particularly preferably 90% by weight or more.

In addition, the matrix treatment culture solution in which laminin and entactin are dispersed may mainly contain laminin and entactin as the matrix. That is, the total amount of type I collagen content, fibronectin content, laminin content, entactin content, and type IV collagen content in the matrix treatment medium in which laminin and entactin are dispersed, The total weight ratio of the laminin content and the entactin content may be, for example, 50% by weight or more, preferably 60% by weight or more, and more preferably 70% by weight or more. , more preferably 80% by weight or more, and particularly preferably 90% by weight or more.

Moreover, the matrix treatment culture solution in which type IV collagen is dispersed may mainly contain type IV collagen as a matrix. That is, the total amount of type I collagen content, fibronectin content, laminin content, entactin content, and type IV collagen content in the matrix treatment culture medium in which type IV collagen is dispersed, The weight ratio of the type IV collagen content may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and 80% by weight or more. is even more preferable, and 90% by weight or more is particularly preferable.

The matrix treatment medium contains a matrix at a concentration within a range in which the medium as a whole maintains fluidity in the co-culture of epithelial cells and mesenchymal cells. In this regard, for example, in general, when cells are embedded in a matrix hydrogel and cultured, the cells and gelation are first allowed to occur under conditions that do not cause gelation of the matrix (for example, a temperature that does not cause gelation). A suspension containing the matrix at a suitable concentration is prepared, and then a suspension containing the cells and the matrix in a culture vessel under conditions suitable for gelation of the matrix (e.g., temperature at which gelation occurs). to gel the whole. As a result, an immobile hydrogel filling the culture vessel is formed.

On the other hand, the matrix treatment culture medium does not allow co-cultivation of epithelial cells and mesenchymal cells in the culture vessel (for example, well). The matrix is contained at a concentration within a range in which the treatment medium as a whole maintains fluidity (that is, in a concentration within which a non-fluid hydrogel filling the culture vessel is not formed). The matrix-processing culture medium that maintains fluidity as a whole is, for example, a gel-like liquid floating in the culture medium that is formed due to local unevenness in the concentration of the dispersed matrix in the culture medium. may contain objects. However, the gel-like material in this case is not, for example, intentionally formed hydrogel beads (eg, hydrogel beads in which cells are embedded), and does not have a specific shape such as a spherical shape.

Since the purpose of dispersing the matrix in the matrix treatment culture medium in the matrix treatment is not gelation of the entire culture medium, the concentration of the matrix in the culture medium is generally Low compared to the concentrations used.

The concentration of the type I collagen in the matrix treatment medium in which the type I collagen is dispersed is not particularly limited as long as the effects of the present invention can be obtained. It is more preferably 460 μg/mL or less, even more preferably 420 μg/mL or less, and particularly preferably 400 μg/mL or less.

Further, the concentration of type I collagen in the matrix treatment culture medium is, for example, preferably 380 μg/mL or less, more preferably 350 μg/mL or less, and even more preferably 300 μg/mL or less. Preferably, it is particularly preferably 260 μg/mL or less.

Further, the concentration of type I collagen in the matrix treatment culture medium is, for example, preferably 3 μg/mL or higher, more preferably 5 μg/mL or higher, and even more preferably 10 μg/mL or higher. 12 μg/mL or more is particularly preferred.

Further, the concentration of type I collagen in the matrix treatment culture medium is, for example, preferably 14 μg/mL or more, more preferably 16 μg/mL or more, and even more preferably 18 μg/mL or more. Preferably, it is particularly preferably 20 μg/mL or more. The concentration of type I collagen in the matrix-treatment culture medium may be specified by arbitrarily combining one of the above upper limits and one of the above lower limits.

The concentration of fibronectin in the matrix treatment culture medium in which fibronectin is dispersed is not particularly limited as long as the effects of the present invention can be obtained, but for example, it is preferably 450 μg/mL or less, and 400 μg/mL or less. is more preferably 350 μg/mL or less, and particularly preferably 300 μg/mL or less.

Further, the concentration of fibronectin in the matrix treatment culture medium is preferably, for example, 250 μg/mL or less, more preferably 200 μg/mL or less, and even more preferably 150 μg/mL or less, 120 μg/mL or less is particularly preferred.

In addition, the concentration of fibronectin in the matrix treatment culture medium is, for example, preferably 1 μg/mL or more, more preferably 2 μg/mL or more, even more preferably 3 μg/mL or more, and 4 μg/mL. mL or more is particularly preferred. The concentration of fibronectin in the matrix treatment culture medium may be specified by any combination of one of the above upper limits and one of the above lower limits.

The concentration of laminin and entactin in the matrix treatment culture medium in which laminin and entactin are dispersed (sum of laminin concentration and entactin concentration) is not particularly limited as long as the effects of the present invention can be obtained. It is preferably 1500 μg/mL or less, more preferably 1000 μg/mL or less, and particularly preferably 500 μg/mL or less.

In addition, the concentration of laminin and entactin in the matrix treatment culture medium is, for example, preferably 1 μg/mL or more, more preferably 3 μg/mL or more, and particularly preferably 5 μg/mL or more. The concentrations of laminin and entactin in the matrix treatment medium may be specified by any combination of one of the above upper limits and one of the above lower limits.

The concentration of type IV collagen in the matrix treatment culture medium in which type IV collagen is dispersed is not particularly limited as long as the effects of the present invention can be obtained. It is more preferably 300 μg/mL or less, particularly preferably 300 μg/mL or less.

In addition, the concentration of type IV collagen in the matrix treatment culture medium is, for example, preferably 1 μg/mL or more, more preferably 3 μg/mL or more, and particularly preferably 5 μg/mL or more. The concentration of type IV collagen in the matrix-treatment culture medium may be specified by arbitrarily combining one of the above upper limits and one of the above lower limits.

The matrix that is brought into contact with epithelial cells and mesenchymal cells in the matrix treatment is a solubilized matrix that is dispersed in a matrix treatment culture medium that has fluidity as a whole. That is, the dispersed matrix that is brought into contact with epithelial cells and mesenchymal cells in matrix treatment is, for example, a hydrogel that does not have fluidity as a whole (for example, a solution containing a hydrogel polymer is poured into a culture vessel, and the culture is When epithelial cells and mesenchymal cells are embedded in a hydrogel formed by gelling the entire solution in a container, it is not a matrix that constitutes the hydrogel. In addition, the dispersed matrix that is brought into contact with the epithelial cells and mesenchymal cells in the matrix treatment is, for example, the case where the epithelial cells and mesenchymal cells are held on the surface of a hydrogel that does not have fluidity as a whole. is not the matrix that constitutes the hydrogel. In addition, the dispersed matrix that is brought into contact with the epithelial cells and mesenchymal cells in the matrix treatment is, for example, a matrix that is preliminarily immobilized on the culture surface on which the epithelial cells and mesenchymal cells are held (e.g., pre-coated matrix on the bottom of culture vessels such as wells).

In matrix treatment, epithelial cells and mesenchymal cells retained on the surface of a hydrogel that does not have fluidity as a whole (hydrogel containing a matrix or hydrogel that does not contain a matrix) are treated as a whole. It may be held in a fluid matrix treatment culture medium and brought into contact with the matrix dispersed in the culture medium.

However, in the matrix treatment, the epithelial cells and mesenchymal cells retained on the surface of the hydrogel containing the matrix and having no fluidity as a whole are retained in the matrix treatment culture medium, may not include contacting with the matrix dispersed in the

In the matrix treatment, the epithelial cells and mesenchymal cells held on the surface of the hydrogel that does not contain a matrix and have no fluidity as a whole are held in a culture medium for matrix treatment, and the culture is performed. It may not include contacting with a matrix dispersed in a liquid.

In matrix treatment, epithelial cells and mesenchymal cells retained on the surface of a hydrogel that does not have fluidity as a whole (regardless of whether the hydrogel contains a matrix or not) are used for matrix treatment. It may not include being held in a culture medium and brought into contact with a matrix dispersed in the culture medium.

In the matrix treatment, the epithelial cells and mesenchymal cells retained on the culture surface on which the matrix has been immobilized in advance are retained in a culture medium for matrix treatment, and the cells dispersed in the culture medium are treated. Contacting with a matrix may be included.

However, in the matrix treatment, the epithelial cells and mesenchymal cells held on the culture surface on which the matrix is previously immobilized are held in the matrix treatment culture medium and dispersed in the culture medium. It is good also as not including contacting with the said matrix.

Matrix treatment may not include embedding and culturing epithelial cells and mesenchymal cells in a hydrogel containing a matrix before cell aggregates are formed. In addition, the matrix treatment may not include embedding and culturing the epithelial cells and mesenchymal cells in a matrix-free hydrogel before the cell aggregates are formed. In addition, matrix treatment involves embedding and culturing epithelial cells and mesenchymal cells in a hydrogel (regardless of whether the hydrogel contains a matrix) before cell aggregates are formed. may not be included.

In the matrix treatment, the temperature at which the epithelial cells and mesenchymal cells are maintained in the matrix treatment medium is not particularly limited as long as the effects of the present invention can be obtained. (e.g., 30 ° C. or higher and 45 ° C. or lower, preferably 33 ° C. or higher and 41 ° C. or lower, more preferably 34 ° C. or higher and 40 ° C. or lower, still more preferably 35 ° C. Above, the temperature is preferably 39° C. or less, particularly preferably 36° C. or more and 38° C. or less.

The time for which the epithelial cells and mesenchymal cells are retained in the matrix treatment culture medium in the matrix treatment is not particularly limited as long as the effects of the present invention can be obtained. It is preferably 6 hours or longer, more preferably 9 hours or longer, and particularly preferably 12 hours or longer.

Furthermore, in the matrix treatment, the epithelial cells and mesenchymal cells are preferably retained in the matrix treatment medium for 15 hours or longer, more preferably 18 hours or longer, and more preferably 21 hours or longer. More preferably, holding for 24 hours or more is particularly preferable.

The timing of starting the matrix treatment is not particularly limited as long as the effects of the present invention can be obtained. is preferred.

Furthermore, the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, more preferably 66 hours or less, even more preferably 60 hours or less, and 54 hours. The following are particularly preferred.

Furthermore, the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 48 hours or less, more preferably 45 hours or less, and 42 hours or less. is even more preferable, and 39 hours or less is particularly preferable.

Furthermore, the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 36 hours or less, more preferably 33 hours or less, and 30 hours or less. is even more preferable, and 27 hours or less is particularly preferable.

Furthermore, the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 24 hours or less, more preferably 21 hours or less, and 18 hours or less. is even more preferable, and 15 hours or less is particularly preferable.

Furthermore, the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 12 hours or less, more preferably 5 hours or less, and 3 hours or less. is even more preferable, and one hour or less is particularly preferable.

Matrix treatment is preferably started simultaneously with seeding of epithelial cells and mesenchymal cells (time interval from seeding of epithelial cells and mesenchymal cells to start of matrix treatment is 0 hour). That is, in this case, for example, by placing a cell suspension prepared by dispersing epithelial cells and mesenchymal cells in a matrix treatment culture medium into a culture vessel, the epithelial cells and mesenchymal cells Start matrix treatment at the same time as seeding.

On the other hand, when matrix treatment is started after seeding epithelial cells and mesenchymal cells, for example, epithelial cells and mesenchymal cells are first seeded in a culture medium to which no matrix is added, and then the seeding is performed. to the above-mentioned time interval, the amount of matrix required for matrix treatment (for example, the concentration in the culture medium after addition is one of the above-mentioned upper limits and / or the above-mentioned A quantity of matrix that provides a concentration within the range specified by one of the lower limits specified above) is added to the culture solution containing the epithelial cells and mesenchymal cells to initiate matrix treatment.

In the matrix treatment, it is preferable to retain the sedimented epithelial cells and mesenchymal cells in the matrix treatment medium. That is, in this case, co-culturing involves sedimenting the seeded epithelial cells and mesenchymal cells in a culture medium, and placing the sedimented epithelial cells and mesenchymal cells in a culture medium for matrix treatment. Including matrix processing that holds in .

More specifically, for example, first, the dispersed epithelial cells and mesenchymal cells are allowed to settle in the culture solution and deposited on the bottom surface of the culture vessel. Then, the epithelial cells and mesenchymal cells deposited on the bottom surface are held in a matrix treatment culture medium having fluidity as a whole, and the epithelial cells and mesenchymal cells are dispersed in the culture medium. contact with the matrix.

The method for sedimenting epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained. and/or a method of centrifuging the culture vessel is preferably used.

Sedimentation of epithelial cells and mesenchymal cells is preferably performed at a temperature at which the epithelial cells and mesenchymal cells are co-cultured before starting matrix treatment. That is, for example, the culture for matrix treatment is first performed at a temperature lower than the co-culturing temperature (e.g., preferably 10°C or lower (more than 0°C, 10°C or lower), more preferably 7°C or lower, particularly preferably 5°C or lower). A temperature at which epithelial cells and mesenchymal cells are precipitated in a liquid and then co-cultured (for example, a temperature of 30 ° C. or higher and 45 ° C. or lower, preferably a temperature of 33 ° C. or higher and 41 ° C. or lower, more preferably The matrix treatment may be performed at a temperature of 34° C. or higher and 40° C. or lower, more preferably 35° C. or higher and 39° C. or lower, particularly preferably 36° C. or higher and 38° C. or lower.

Alternatively, for example, epithelial cells and mesenchymal cells are first precipitated in a culture medium to which no matrix is added, and then the amount of matrix required for matrix treatment is added to the culture medium (for example, The concentration of matrix is added in an amount specified by one of the upper limit values and/or one of the lower limit values described above), and matrix treatment is performed at a temperature suitable for co-cultivation. may be In this case, the addition of the matrix to the sedimented epithelial cells and mesenchymal cells is carried out at a temperature lower than the co-culturing temperature (e.g., preferably 10°C or lower (greater than 0°C, 10°C or lower), more preferably 7 C. or lower, particularly preferably 5.degree.

Matrix treatment is performed as part or all of the co-culture of epithelial and mesenchymal cells. That is, the co-cultivation may be performed entirely in the matrix treatment medium. Alternatively, co-cultivation may be performed in a matrix treatment medium until cell aggregates are formed. Also, the co-cultivation may be carried out partly in a matrix treatment culture medium and the other part in a culture medium having a lower matrix concentration than the matrix treatment culture medium.

More specifically, for example, co-culturing includes performing matrix treatment of epithelial cells and mesenchymal cells in a matrix treatment culture medium containing a matrix at a first concentration, Continuing the co-culturing of the epithelial and mesenchymal cells in a medium containing the matrix at a second concentration that is less than the one concentration.

In this case, the second concentration is not particularly limited as long as the effect of the present invention is obtained, but the ratio of the second concentration to the first concentration may be, for example, 90% or less, or 70%. % or less, 50% or less, 30% or less, or 10% or less. Also, two or more different densities may be employed as the second density. That is, the second concentration may change (for example, the second concentration may decrease) as the culture time elapses.

The method for reducing the matrix concentration in the culture medium during co-cultivation to be lower than that in the matrix-treatment culture medium is not particularly limited as long as the effects of the present invention can be obtained. After the passage of time, part of the matrix-treatment culture medium in the culture vessel is removed, and instead a culture medium with a matrix concentration lower than that of the matrix-treatment culture medium (for example, a culture medium to which the matrix is not added) is added. liquid) may be added.

In co-culturing, seeding of epithelial cells and mesenchymal cells, matrix treatment, and formation of cell aggregates are preferably carried out continuously in the same culture vessel (for example, the same well). That is, once the epithelial cells and mesenchymal cells are seeded in the culture vessel, matrix treatment and cell aggregate formation are performed in the culture vessel without removing the epithelial cells and mesenchymal cells from the culture vessel. Forming is preferred.

According to co-cultivation including matrix treatment, cells were formed under the same conditions except that a culture medium in which the matrix was not dispersed (that is, a culture medium to which the matrix was not added) was used instead of the culture medium for matrix treatment. It is possible to obtain a cell aggregate in which the expression level of one or more hair growth-related genes is amplified as compared with the cell aggregate (hereinafter sometimes referred to as "control cell aggregate"). Specifically, for example, a cell aggregate is obtained in which the expression level of one or more hair growth-related genes is at least twice as high as that of the control cell aggregate.

The hair growth-related gene expressed by the cell aggregates is not particularly limited as long as it is a gene related to hair growth. , Edaradd, Pdgfa, and Lgr4).

That is, the cell aggregates obtained by co-culturing including matrix treatment may have twice or more the expression level of the Tgfb2 gene and two times or more the expression level of the Sox21 gene as compared with the control cell aggregates. The expression level of the Lgr5 gene may be 2 times or more, the expression level of the Lhx2 gene may be 2 times or more, and the Edaradd gene expression level may be 2 times or more, The expression level of the Pdgfa gene may be doubled or more, or the expression level of the Lgr4 gene may be doubled or more.

By adopting co-culturing of the epithelial cells and mesenchymal cells, including seeding the epithelial cells and mesenchymal cells, in vitro, a cell aggregate having a hairy tissue formed on its surface can be manufactured effectively.

In addition, cell culture for obtaining a cell aggregate with a hairy tissue formed on its surface is, for example, differentiation-inducing culture of pluripotent stem cells such as iPS cells as described in Non-Patent Document 1 above. There may be.

In this respect, Non-Patent Document 1 described above describes the following differentiation-inducing culture method. First, iPS cells were suspended in an ectoderm differentiation medium and seeded at a density of 3×10 ³ cells/well (100 μL) in a well (96-well plate) having a U-shaped bottom with low cell adhesion. do.

On day 1 of culture, remove half the volume (50 μL) of the culture medium in the wells and add 4% (v/v) Matrigel (registered trademark) (final concentration after addition is 2% (v/v)). Add 50 μL of fresh ectodermal differentiation medium.

On day 3 of culture, add 25 μL of fresh ectodermal differentiation medium without Matrigel containing 50 ng/mL to 250 ng/mL BMP-4 and 5 μM SB431542 (an ALK inhibitor) to the wells (BMP-4 after addition). 4 concentration is 10 ng/mL to 50 ng/mL, SB431542 concentration is 1 μM, culture volume 125 μL/well).

On day 4 of culture, 25 μL of fresh ectodermal differentiation medium containing 6 μM LDN (ALK inhibitor) and 150 ng/mL FGF-2 without Matrigel is added to the wells (LDN concentration after addition is 1 μM, FGF-2 -2 concentration is 25 ng/mL, culture volume is 150 μL/well).

On day 8 of culture, the cell aggregates in the wells are transferred to wells (24-well plate) containing 500 μL of maturation medium containing 1% (v/v) matrigel.

From the 10th day of culture, the medium is replaced once every two days until the 30th day of culture by removing half the culture medium (250 μL) in the well and adding 250 μL of fresh maturation medium without Matrigel.

However, the method of forming hair-like tissue in vitro using differentiation induction of pluripotent stem cells such as iPS cells is complicated in operation, and the hair-like tissue formed on the surface of the cell aggregates is not formed. Care must be taken to ensure safety when transplanting the cells that produce cells into a living body.

In contrast, epithelial cells and mesenchymal cells are seeded without using pluripotent stem cells, and the epithelial cells and mesenchymal cells are co-cultured to form cell aggregates having hairy tissue. The method is easy to operate, and it is easy to ensure safety for transplanting the hairy tissue into a living body.

The hairy tissue of the cell aggregate contains epithelial cells and mesenchymal cells, and is formed as a structure protruding from the surface of the cell aggregate. The hair-like tissue formed on the surface of the cell aggregate has, for example, a dermal papilla-like structure at its free end, the tip portion. The hair-like tissue formed on the surface of the cell aggregate does not have a dermal papilla-like structure at its root portion (the end opposite to the tip portion, which is a free end).

The dermal papilla-like structure of the hair-like tissue has a structure similar to the dermal papilla in the hair follicle of the living body. That is, the dermal papilla-like structure has a spherical shape. Dermal papilla-like structures also contain mesenchymal cells (eg, dermal papilla cells). That is, dermal papilla-like structures are identified, for example, as aggregates of Versican-positive cells.

Specifically, the ratio of the number of mesenchymal cells (for example, dermal papilla cells) contained in the dermal papilla-like structure to the total number of cells constituting the dermal papilla-like structure is, for example, 50% or more. Well, it is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

Also, the hair-like tissue has, for example, a hair shaft-like structure. The hair shaft-like structure of the hair-like tissue has a structure similar to the hair shaft in the hair follicle of the body. That is, the hair shaft-like structure contains keratin. The hair shaft-like structure also has a cuticle structure. Moreover, the hair shaft-like structure of the hair-like tissue extends from the vicinity of the dermal papilla-like structure included in the tip portion of the hair-like tissue toward the root portion of the hair-like tissue. The shaft-like structure preferably contains melanin.

Also, ciliary tissue does not contain capillaries. That is, for example, a dermal papilla-like structure contained in a hair-like tissue does not contain capillaries, unlike a dermal papilla contained in a hair follicle collected from a living body.

The length of the hairy tissue protruding from the surface of the cell aggregate changes with the passage of culture time. is more preferable, 800 μm or more is even more preferable, and 1000 μm or more is particularly preferable.

Furthermore, the length of the hairy tissue protruding from the surface of the cell aggregate is, for example, preferably 2 mm (2000 μm) or more, more preferably 4 mm or more, and even more preferably 6 mm or more, 8 mm or more is particularly preferred. Moreover, the length of the hair-like tissue protruding from the surface of the cell aggregate may be, for example, 100 mm or less.

A cell aggregate having hair-like tissue may have a cyst-like structure inside. This cyst-like structure is identified as a non-nucleated structure surrounded by cells with a nucleus. Specifically, the cyst-like structure is, for example, in the central part of the HE (hematoxylin-eosin)-stained cross-section of the cell aggregate, the periphery is covered with cells having cell nuclei, and is colored pink. Observed as a stained structure.

Further, for example, when a cell aggregate is formed without using pluripotent stem cells, the cell aggregate does not contain a pili muscle structure and/or a sebaceous gland structure, unlike the tissue formed in Non-Patent Document 1 above. You can do it. Moreover, the cell aggregates preferably have hair regeneration ability. The hair regeneration ability of a cell aggregate is the ability to induce hair growth at the site where the cell aggregate is transplanted when the cell aggregate is transplanted into a living body.

Co-cultivation is performed by performing suspension culture of epithelial cells and mesenchymal cells to form cell aggregates, and embedding the cell aggregates formed by the suspension culture in hydrogel and further culturing may include doing.

In this case, first, in suspension culture, cell aggregates that do not have hairy tissue on their surface (for example, have the ability to form hairy tissue on their surface, but have not yet formed hairy tissue on their Cell aggregates) may be formed, or cell aggregates having hair-like tissue on their surface may be formed.

Next, cell aggregates formed by suspension culture are embedded in hydrogel and cultured. That is, first, in a culture vessel, cell aggregates are held in a solution for forming hydrogel (hereinafter sometimes referred to as "hydrogel-forming solution"), and then the solution in the culture vessel By gelling the entire cell aggregate, the cell aggregate is embedded to form a hydrogel that does not have fluidity as a whole.

The hydrogel-forming solution is not particularly limited as long as the effects of the present invention can be obtained, but preferably contains a matrix, for example. That is, the hydrogel-forming solution preferably contains one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen. More preferably, it contains one or more selected from the group consisting of, and particularly preferably contains type I collagen.

In addition, the hydrogel in which the cell aggregates are embedded preferably contains one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, type I collagen, fibronectin, and , laminin and entactin, and particularly preferably type I collagen.

The matrix-containing hydrogel-forming solution and the matrix-containing hydrogel may or may not have other matrices added thereto.

That is, for example, the hydrogel-forming solution containing type I collagen and the hydrogel containing type I collagen may further contain fibronectin, laminin, entactin, or type IV collagen. , entactin or type IV collagen may not be added.

The hydrogel-forming solution containing the matrix may be a basic solution (e.g., a culture medium that can be used for co-culturing epithelial cells and mesenchymal cells), similar to the matrix-processing culture medium. and an aqueous solution capable of sustaining the survival of mesenchymal cells), by adding the matrix.

The concentration of the matrix contained in the hydrogel-forming solution is not particularly limited as long as the effects of the present invention can be obtained. is preferred.

Specifically, when forming a hydrogel containing type I collagen, the concentration of type I collagen in the hydrogel-forming solution and the concentration of type I collagen in the hydrogel obtained by gelling the entire solution are, for example, It is preferably 500 μg/mL or more, more preferably 1000 μg/mL or more, even more preferably 1500 μg/mL or more, and particularly preferably 2000 μg/mL or more. Also, the type I collagen concentration in the hydrogel-forming solution and the type I collagen concentration in the hydrogel obtained by gelling the entire solution may be, for example, 3500 μg/mL or less.

Further, when forming a hydrogel containing fibronectin, the fibronectin concentration in the hydrogel-forming solution and the fibronectin concentration in the hydrogel obtained by gelling the entire solution are, for example, 500 μg/mL or more. It is preferably 1000 μg/mL or more, even more preferably 1500 μg/mL or more, and particularly preferably 2000 μg/mL or more. Also, the fibronectin concentration in the hydrogel-forming solution and the fibronectin concentration in the hydrogel obtained by gelling the entire solution may be, for example, 3500 μg/mL or less.

In addition, when forming a hydrogel containing laminin and entactin, the concentration of laminin and entactin in the hydrogel-forming solution (the sum of the concentration of laminin and the concentration of entactin), and the hydrogel obtained by gelling the entire solution The concentrations of laminin and entactin in the gel are, for example, preferably 1800 μg/mL or higher, more preferably 2000 μg/mL or higher, and particularly preferably 2200 μg/mL or higher. Further, the concentrations of laminin and entactin in the hydrogel-forming solution and the concentrations of laminin and entactin in the hydrogel obtained by gelling the entire solution may be, for example, 3500 μg/mL or less.

The type of matrix contained in the hydrogel that embeds the cell aggregates may be the same as or different from the type of matrix used in the matrix treatment in the co-culture for forming the cell aggregates. good too.

That is, a cell aggregate is formed by co-cultivation including matrix treatment using one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, and then type I collagen, fibronectin, The cell aggregate may be embedded in a hydrogel containing one or more selected from the group consisting of laminin, entactin, and type IV collagen and cultured.

Also, a cell aggregate is formed by co-culturing including matrix treatment using one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, and then type I collagen, fibronectin, The cell aggregates may be embedded in a hydrogel containing one or more matrices selected from the group consisting of laminin, entactin, and type IV collagen used in the matrix treatment and cultured.

From the viewpoint of cost and safety, cell aggregates are formed by co-cultivation including matrix treatment using type I collagen, and then the cell aggregates are embedded in a hydrogel containing type I collagen and cultured. is particularly preferred.

Hydrogel-embedded culture of cell aggregates is preferably carried out by, for example, adding a culture solution onto the hydrogel in a culture vessel and culturing the cell aggregates inside the hydrogel.

By embedding and culturing a cell aggregate having no hairy tissue on its surface in a hydrogel, a hairy tissue can be effectively formed on the surface of the cell aggregate within the hydrogel. In addition, by embedding and culturing the cell aggregate with the hairy tissue formed on the surface in the hydrogel, the hairy tissue can be effectively grown in the hydrogel (for example, the hairy tissue (effectively increasing the length of ). In this way, co-cultivation including hydrogel-embedded culture of cell aggregates can effectively promote the formation of a hair-like structure in the cell aggregates.

Then, in the production of the hair-like tissue for transplantation, the hair-like tissue formed on the surface of the cell aggregate as described above is cut from the cell aggregate and collected for transplantation into a living body. The method of cutting the hairy tissue from the cell aggregate is not particularly limited as long as the effect of the present invention can be obtained, but for example, it is preferable to cut the root portion of the hairy tissue using a cutting instrument such as scissors. .

The hair-like tissue cut and collected from the cell aggregate (i.e., hair-like tissue for transplantation) is a graft manufactured for transplantation into a living body, and contains epithelial cells and interstitial cells independent of the cell aggregate. It is an organization containing leaf cells.

A hair-like tissue for transplantation that has not yet been transplanted into a living body (hereinafter sometimes referred to as a "hair-like graft") has one tip portion that is a free end and the other tip portion that is a free end. . More specifically, the hair-like graft is, for example, one tip portion that does not have a cut surface due to being separated from the cell aggregate (hairy tissue formed on the surface of the cell aggregate, A tip portion that was a free end) and the other tip portion having a cut surface.

A hair-like graft, for example, has a dermal papilla-like structure on one tip portion (eg, the tip portion without a cut surface). The dermal papilla-like structure of the hair-like graft has a structure similar to the dermal papilla in the hair follicle of the living body. That is, the dermal papilla-like structure has a spherical shape. Dermal papilla-like structures also contain mesenchymal cells (eg, dermal papilla cells). That is, the dermal papilla-like structure is mainly composed of mesenchymal cells (for example, dermal papilla cells).

Specifically, the ratio of the number of mesenchymal cells (for example, dermal papilla cells) contained in the dermal papilla-like structure to the total number of cells constituting the dermal papilla-like structure is, for example, 50% or more. Well, it is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. In addition, the hair-like graft does not have a dermal papilla-like structure at the other tip portion (for example, the tip portion having the cut surface).

Hair-like grafts also have, for example, hair-shaft-like structures. The hair shaft-like structure of the hair-like graft has a structure similar to the hair shaft within the hair follicle of the body. That is, the hair shaft-like structure contains keratin. The hair shaft-like structure also has a cuticle structure. Also, the hair shaft-like structure of the hair-like graft extends from the vicinity of the dermal papilla-like structure included in one tip portion of the hair-like graft toward the other tip portion of the hair-like graft. Also, the hair shaft-like structure preferably contains melanin.

Also, ciliary tissue does not contain capillaries. That is, for example, a dermal papilla-like structure contained in a hair-like tissue does not contain capillaries, unlike a dermal papilla contained in a hair follicle collected from a living body.

The length of the hair-like graft is not particularly limited as long as the effects of the present invention can be obtained. , more preferably 800 μm or more, and particularly preferably 1000 μm or more.

Furthermore, the length of the hair-like graft is, for example, preferably 2 mm or longer, more preferably 4 mm or longer, even more preferably 6 mm or longer, and particularly preferably 8 m or longer.

Also, the length of the hair-like graft may be, for example, 100 mm or less, preferably 50 mm or less, more preferably 30 mm or less, and particularly preferably 20 mm or less. The length of the hair-like graft may be specified by any combination of one of the above upper limits and one of the above lower limits.

A method according to this embodiment may include cold storage of the harvested hair-like graft for implantation into the living body. In this case, for example, the hair-like graft is stored in a cold state until it is transplanted into a living body. Specifically, for example, the hair-like graft collected from the culture container is transferred to a container different from the culture container and stored in a cold state.

The temperature for cold storage of the hair-like graft is not particularly limited as long as the effects of the present invention can be obtained, but for example, it is preferably 10°C or less (greater than 0°C and 10°C or less), and is 7°C or less. is more preferable, and 5° C. or less is particularly preferable.

The hair regeneration method according to this embodiment includes transplanting the hair-like graft produced in vitro as described above into a living body. The hair-like graft is transplanted into the living body so that the structure of the hair-like graft after transplantation resembles the structure of the hair follicle in the living body.

That is, in the hair follicle of a living body, the dermal papilla is arranged inside the living body, and the free end of the hair shaft is arranged outside the living body. For this reason, the hair-like graft is transplanted into the living body such that one tip portion including the dermal papilla-like structure of the hair-like graft after transplantation is on the inside of the living body. Moreover, it is preferable to transplant the hair-like graft into the living body so that the other tip portion of the hair-like graft that does not contain the dermal papilla-like structure becomes a free end in the living body after transplantation.

The living body to which the hair-like graft is to be transplanted may be a human or a non-human animal, but is preferably a human. The hair-like graft is transplanted into the living body preferably into the skin of the living body, and particularly preferably into the scalp of the living body.

Implantation of the hair-like graft into a living body may be for medical or research purposes. Transplantation of the hair-like graft into a living body is preferably for the treatment or prevention of a disease associated with hair loss. That is, transplantation of the hair-like graft into a living body is preferably transplantation into a human patient suffering from or potentially suffering from a disease associated with hair loss. Therefore, the hair regeneration method according to this embodiment is preferably a method for treating or preventing a disease associated with hair loss.

Diseases associated with hair loss are not particularly limited, but for example, androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrhea Composed of alopecia areata, alopecia pityriasis, traction alopecia, metabolic alopecia, alopecia areata compressive, alopecia areata, alopecia areata, alopecia nervosa, trichotillomania, alopecia generalis, and symptomatic alopecia It may be one or more selected from the group.

In addition, in vitro-produced hair-like grafts can be used, for example, to search for substances that can be used for the treatment or prevention of diseases associated with hair loss, to search for substances involved in the diseases, and to study the mechanisms of the diseases. may be used.

Next, specific examples according to this embodiment will be described.

[Collection of epithelial cells and mesenchymal cells]
A dorsal skin tissue was collected from a C57BL/6 mouse embryo of 18 days of embryonic age, and subjected to dispase treatment at 4° C. for 1 hour under shaking conditions of 20 to 30 rpm to separate the epithelial layer and the mesenchymal layer of the skin tissue. . Thereafter, the epithelial layer was treated with 100 U/mL collagenase for 80 minutes and then treated with trypsin for 10 minutes to isolate epithelial cells. In addition, mesenchymal cells were isolated by treating the mesenchymal layer with 100 U/mL collagenase for 80 minutes.

[Production of cell aggregates]
First, 1% GultaMax Supplement (GIBCO (registered trademark)) and 0.2% Normocin (InvivoGen) were added to DMEM/F12 medium (Advanced Dulbecco's Modified Eagle Medium/Ham's F-12, GIBCO (registered trademark)). to prepare the basal medium.

Next, type I collagen undiluted solution (Cellmatrix (registered trademark) Type IA, type I collagen concentration 2.4 mg/mL, Nitta Gelatin Co., Ltd.) cooled to 4°C was added to the basal medium cooled to 4°C. It was added to concentrations of 2.4 μg/mL, 24 μg/mL, 120 μg/mL, 240 μg/mL, 360 μg/mL, or 480 μg/mL to prepare six culture solutions with different concentrations of type I collagen.

Then, to each culture solution at 4°C, the amount of epithelial cells and mesenchymal cells that each cell density becomes 5 × 10 ⁴ cells / mL (the total cell density becomes 1 × 10 ⁵ cells / mL) was suspended and a cell suspension (i.e., epithelial and mesenchymal cells dispersed and 2.4 μg/mL, 24 μg/mL, 120 μg/mL, 240 μg/mL, 360 μg/mL, or 480 μg/mL A culture medium for matrix treatment in which type I collagen was dispersed at a concentration of mL was prepared. Then, 200 μL of the cell suspension at 4° C. was poured into each well of a 96-well plate to seed 1×10 ⁴ cells/well of epithelial cells and 1×10 ⁴ cells/well of mesenchymal cells. .

After seeding, the 96-well plate was placed in a refrigerator at 4° C. for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).

During the co-cultivation period of 8 days, the culture solution was exchanged once every two days. The culture medium was exchanged by removing half of the culture medium (100 μL) from each well and then adding 100 μL of the culture medium to which type I collagen was not added (basic medium). Under all conditions, the culture medium in the wells maintained fluidity as a whole throughout the culture period.

[result]
FIG. 1 shows micrographs on day 8 of culture. As shown in FIG. 1, epithelial cells and mesenchymal cells aggregated in all six culture systems with different concentrations of type I collagen in the culture medium at the time of seeding (that is, the concentration of type I collagen in the matrix treatment). , cell clumps were formed, one in each well.

In addition, in four culture systems in which the concentration of type I collagen in the culture medium at the time of seeding was 24 μg/mL, 120 μg/mL, 240 μg/mL or 360 μg/mL, hairy tissue (four photographs included in FIG. In each case, the formation of cell aggregates having a portion indicated by an arrow) was confirmed.

That is, in these four culture systems, after starting suspension culture, epithelial cells and mesenchymal cells began to aggregate, and cell aggregates were formed on the first day of culture. Thereafter, on the 4th to 6th days of culture, hairy tissue began to form on the surface of the cell aggregates. The hair-like tissue of cell aggregates elongated with the passage of culture time.

FIG. 2 shows the relationship between the concentration of type I collagen in the culture solution at the time of seeding (at the time of matrix treatment) and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture. As shown in FIG. 2, in culture systems where the type I collagen concentration was 24 μg/mL, 120 μg/mL or 240 μg/mL, hairy tissue was formed in all cell aggregates (hairy tissue formation efficiency 100%). In addition, in the culture system in which the type I collagen concentration was 360 μg/mL, hairy tissue was formed in 3 out of 6 cell aggregates (hairy tissue formation efficiency: 50%). On the other hand, in culture systems with type I collagen concentrations of 2.4 μg/mL and 480 μg/mL, no hairy tissue was formed in any of the cell aggregates (hairy tissue formation efficiency 0%).

FIG. 3 shows the relationship between the type I collagen concentration in the culture solution at the time of seeding and the number of hair-like tissues formed per cell aggregate on day 8 of culture. That is, in FIG. 3, the horizontal axis indicates the type I collagen concentration in the culture medium at the time of seeding, and the vertical axis indicates the total number of hairy tissue formed in the cell aggregates divided by the number of the cell aggregates. The arithmetic mean value is shown.

As shown in FIG. 3, in culture systems with type I collagen concentrations of 24 μg/mL, 120 μg/mL, or 240 μg/mL, an average of 3 to 5 hair-like tissues were formed in one cell aggregate. rice field.

FIG. 4 shows a micrograph of cell aggregates on day 8 of culture in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 μg/mL. Photograph (ii) in FIG. 4 shows an enlarged portion of the square frame shown in photograph (i).

As indicated by arrows in photograph (i) of FIG. 4, it was confirmed that at least four hair-like tissues were formed in one cell aggregate. In addition, as indicated by arrowheads in photograph (ii) of FIG. 4, a bulging structure was observed at the tip, which is the free end of the hairy tissue extending from the cell aggregate.

FIG. 5 shows the results of HE staining of sections of cell aggregates on day 8 of culture in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 μg/mL. Photograph (ii) in FIG. 5 shows an enlarged portion of the square frame shown in photograph (i).

As indicated by black arrows in photograph (i) of FIG. 5, two hairy tissues were confirmed in this section. Furthermore, as indicated by the white arrow in photograph (ii), at the tip of the hair-like tissue, there is a dermal papilla-like structure similar to the dermal papilla of the living body, and a dermal papilla-like structure formed in the vicinity of the dermal papilla-like structure. A part of a black hair shaft-like structure similar to

In addition, as indicated by the white arrow in photograph (i), a pink-stained cyst-like structure was confirmed in the central portion of the cell aggregate. This cyst-like structure was a structure without a cell nucleus. In addition, the periphery of the cyst-like structure was covered by cells with cell nuclei.

FIG. 6 shows the results of fluorescent staining of Versican on sections of cell aggregates formed on day 8 of culture in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 μg/mL. Photograph (ii) in FIG. 6 shows an enlarged portion of the square frame shown in photograph (i). The part enclosed by the white dotted line in photograph (ii) shows the hair shaft-like structure.

As shown in FIG. 6, the hair-like tissue formed in the cell aggregates has Versican-positive cell aggregates, ie, dermal papilla cell aggregates (dermal papilla-like structure) at its tip. confirmed.

FIG. 7 shows the results of fluorescent staining of CD34 on sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 μg/mL. In FIG. 7, the portion surrounded by the white dotted line indicates the tip portion of the ciliary tissue.

As shown in FIG. 7, it was confirmed that CD34-positive cells, i.e., hair follicle epithelial stem cells, were contained in the periphery of the hair shaft-like structure close to the dermal papilla-like structure at the tip of the hair-like tissue. .

FIG. 8 schematically shows a cell aggregate having a hairy tissue obtained in this example based on the observation results described above. Note that FIG. 8 is only a schematic diagram, and the size and arrangement of the cell aggregates, the hairy tissue, and the cells and structures contained therein do not limit the cell aggregates according to the present invention.

As shown in FIG. 8, the cell aggregates formed by co-culturing epithelial cells and mesenchymal cells in this Example had a cyst-like structure in the center. Also, the cell clumps had one or more hair-like tissue on their surface. This hair-like tissue consists of a dermal papilla-like structure (agglomerate of Versican-positive cells) formed at the tip, which is the free end, and hair extending from the vicinity of the dermal papilla-like structure to the root of the hair-like tissue. It had a stem-like structure. In addition, the hair-like tissue contained hair follicle epithelial stem cells (CD34-positive cells) around the periphery of the portion of the hair shaft-like structure close to the dermal papilla-like structure.

[Production of cell aggregates]
1×10 ⁴ cells/well were added to each well of a 96-well plate in the same manner as in Example 1 above, except that the culture solution to which type I collagen was added to a final concentration of 12 μg/mL was used. Epithelial cells and 1×10 ⁴ cells/well of mesenchymal cells were seeded.

After seeding, the 96-well plate was placed in a refrigerator at 4° C. for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).

During the 4-day co-cultivation period, the culture solution was exchanged in the same manner as in Example 1 above. On day 4 of culture, one cell aggregate was formed in each well. However, formation of hairy tissue was not confirmed in the cell aggregates on day 4 of culture.

Next, 7 cell aggregates without hairy tissue on the 4th day of culture were collected in a centrifuge tube with a volume of 15 mL, and the culture medium was removed from the centrifuge tube as much as possible. Then, a type I collagen stock solution (Cellmatrix (registered trademark) Type IA, type I collagen concentration: 2.4 mg/mL, manufactured by Nitta Gelatin) was added to the centrifuge tube, and cell aggregates were added to the type I collagen stock solution. Suspended.

Thereafter, 0.4 mL of a hydrogel-forming solution containing cell aggregates and type I collagen was dropped into one well of a 6-well plate and held in an incubator at 37°C for 20 minutes to gel type I collagen. . As a result, the entire solution in the well gelled to form a hydrogel having no fluidity. Moreover, in the hydrogel, the seven cell aggregates were three-dimensionally dispersed and embedded separately from each other.

After that, 2 mL of basal medium was added onto the hydrogel in the well, and cell aggregates were embedded and cultured in the hydrogel for an additional 23 days. That is, a total of 27 days of co-culture was performed.

[result]
FIG. 9 shows one cell aggregate embedded in the hydrogel on day 8 of culture (day 4 after starting hydrogel embedding culture), day 12, day 18, and day 22. Photomicrographs taken on day 27 and day 27 are shown. FIG. 10 shows an enlarged portion (ciliary tissue) surrounded by a dotted box in each of the five photographs included in FIG. FIG. 11 shows changes over time in the length of the hair-like tissue shown in FIGS. In FIG. 11, the horizontal axis indicates the number of culture days (days), and the vertical axis indicates the length of the hairy tissue (μm).

9 to 11, in the hydrogel-embedded culture from the 8th day of the culture to the 22nd day of the culture, the hair-like tissue on the surface of the cell aggregates elongated with the passage of the culture time. increased. The length of the ciliary tissue reached approximately 2 mm (2000 μm) on day 22 of culture.

On the other hand, from the 22nd day to the 27th day of culture, the hairy tissue retracted and its length decreased. Such changes in ciliary tissue were consistent with the first hair growth cycle (3 to 4 weeks) in mice. In the hydrogel-embedded culture, on the second day of the hydrogel-embedded culture (six days after cell seeding), hairy tissue began to form on the surface of the cell aggregates.

[Production of cell aggregates]
Instead of type I collagen, fibronectin undiluted solution (fibronectin solution (derived from human plasma), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with a final concentration of 6 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL or 100 μg / In each well of a 96-well plate, 1×10 ⁴ cells/well of epithelial cells were added in the same manner as in Example 1 above, except that the culture solution prepared by adding to the basal medium was used so as to make the volume of mL. and 1×10 ⁴ cells/well of mesenchymal cells were seeded.

After seeding, the 96-well plate was placed in a refrigerator at 4° C. for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).

During the 8-day co-cultivation period, the medium was exchanged in the same manner as in Example 1 above. Under all conditions, the culture medium in the wells maintained fluidity as a whole throughout the culture period.

[result]
FIG. 12 shows micrographs on day 8 of culture. As shown in FIG. 12, epithelial cells and mesenchymal cells were aggregated in all five culture systems with different fibronectin concentrations in the culture medium at the time of seeding, and one cell aggregate was formed in each well. rice field.

In addition, in all five culture systems in which the fibronectin concentration in the culture medium at the time of seeding (at the time of matrix treatment) was 6 μg/mL, 12 μg/mL, 25 μg/mL, 50 μg/mL or 100 μg/mL, hairy tissue Formation of cell aggregates having (indicated by arrows in each of the five photographs included in FIG. 12) was confirmed.

That is, in these five culture systems, epithelial cells and mesenchymal cells began to aggregate after starting suspension culture, and cell aggregates were formed on the first day of culture. Thereafter, on the 4th to 6th days of culture, hairy tissue began to form on the surface of the cell aggregates. The hair-like tissue of cell aggregates elongated with the passage of culture time.

FIG. 13 shows the relationship between the fibronectin concentration in the culture solution at the time of seeding and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture. As shown in FIG. 13, the formation efficiency of hair-like tissue was 40% to 70%.

FIG. 14 shows the relationship between the fibronectin concentration in the culture solution at the time of seeding and the number of hairy tissues formed per cell aggregate on day 8 of culture. As shown in FIG. 14, 1 to 3 hair-like tissues were formed on average per cell aggregate.

FIG. 15 shows the results of HE staining of sections of cell aggregates on day 8 of culture in a culture system in which fibronectin in the culture medium at the time of seeding was 100 μg/mL. As indicated by arrows in FIG. 15, two strands of hairy tissue were identified in this section. In addition, a dermal papilla-like structure was confirmed at the tip of the hair-like tissue. On the other hand, a cyst-like structure was confirmed in the central portion of the cell aggregate.

[Production of cell aggregates]
Six culture systems with different timings of adding type I collagen (timing of adding type I collagen: 0 hours (Example 4-1), 6 hours (Example 4-2), 12 hours (Example 4-2) after cell seeding 3), after 24 hours (Example 4-4), 36 hours (Example 4-5) or 48 hours (Example 4-6)), in the same manner as in Example 1 above, between epithelial cells and Co-culture with leaf lineage cells was performed.

First, a basal medium to which type I collagen was not added was prepared in the same manner as in Example 1 described above. Then, in a basal medium cooled to 4°C, epithelial cells and mesenchymal cells were added in an amount that each gave a cell density of 1 x 10 ⁵ cells/mL (an amount that gave a total cell density of 2 x 10 ⁵ cells/mL). Cells were suspended to prepare a cell suspension. On the other hand, a culture solution containing type I collagen at a concentration of 240 μg/mL was prepared by adding type I collagen undiluted solution cooled to 4° C. to a basal medium cooled to 4° C.

Then, 100 μL of the cell suspension at 4° C. was poured into each well of a 96-well plate to seed 1×10 ⁴ cells/well of epithelial cells and 1×10 ⁴ cells/well of mesenchymal cells. .

To the wells of Example 4-1, 100 μL of a culture solution containing type I collagen at a concentration of 240 μg/mL and cooled to 4° C. was added immediately after seeding (0 hours after seeding). As a result, in the wells of Example 4-1, epithelial cells and mesenchymal cells were retained in the matrix treatment culture medium in which type I collagen was dispersed at a concentration of 120 μg/mL.

The 96-well plate was then placed in a refrigerator at 4° C. for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).

When 5 hours and 40 minutes had passed since the cells were seeded, the 96-well plate was transferred to a refrigerator at 4° C. and allowed to stand for 20 minutes to cool the culture medium containing the cells in the wells. After cooling for 20 minutes, that is, when 6 hours had passed since seeding the cells, 100 μL of the culture medium containing type I collagen at a concentration of 240 μg/mL and cooled to 4° C. was added to the wells of Example 4-2. As a result, in the wells of Example 4-2, epithelial cells and mesenchymal cells were retained in the matrix treatment medium in which type I collagen was dispersed at a concentration of 120 μg/mL.

After addition of type I collagen to the wells of Example 4-2, the 96-well plate was shaken in a refrigerator at 4°C for 20 minutes. After that, the 96-well plate was transferred to a 37° C. incubator to continue co-cultivation (suspension culture).

Similarly, at 12 hours, 24 hours, 36 hours, and 48 hours after seeding the cells, Example 4-3, Example 4-4, and Example Matrix treatment was initiated by adding type I collagen to the wells of Examples 4-5 and 4-6. In addition, in all the wells, the culture solution was changed for the first time on the third day of culture, and thereafter, the culture solution was changed once every two days. Exchange of the culture solution was performed in the same manner as in Example 1 above.

[result]
FIG. 16 shows micrographs on day 8 of culture. As shown in FIG. 16, epithelial cells and mesenchymal cells aggregated to form one cell aggregate in each well in all of the six culture systems with different addition timings of type I collagen.

In addition, in all of the six culture systems in which type I collagen was added at different timings, cell aggregates with hair-like tissue formed on the surface (indicated by arrows in each of the six photographs included in FIG. 16) were obtained. was taken.

FIG. 17 shows the timing of addition of type I collagen (time from seeding of cells to addition of type I collagen) in correspondence with the formation efficiency of hairy tissue in cell aggregates on day 8 of culture. As shown in FIG. 17, in the culture system to which type I collagen was added immediately after seeding the cells (time 0), hairy tissue was formed in 9 out of 12 cell aggregates (hair formation efficiency of like tissue 75%). In addition, at 6 hours, 12 hours, 24 hours, 36 hours, and 48 hours after seeding the cells, the formation efficiencies of hairy tissue in the culture system to which type I collagen was added were 58%, 58%, and 58%, respectively. 50%, 33% and 25%. That is, it was confirmed that the efficiency of hair-like tissue formation tends to decrease as the timing of adding type I collagen is delayed.

FIG. 18 shows the relationship between the timing of addition of type I collagen and the number of hair-like tissues formed per cell aggregate on day 8 of culture. As shown in FIG. 18, it was confirmed that as the timing of adding type I collagen was delayed, the number of hair-like tissues formed in one cell aggregate tended to decrease.

FIG. 19 shows micrographs of cell aggregates on day 8 of culture in a culture system in which type I collagen was added immediately after cell seeding. Photograph (ii) in FIG. 19 shows an enlarged portion of the square frame shown in photograph (i). As shown in FIG. 19, a swollen structure was observed at the tip, which is the free end of the hair-like tissue protruding from the surface of the cell aggregate.

FIG. 20 shows enlarged photographs of hairy tissue formed on the surface of cell aggregates on day 8 of culture in a culture system in which type I collagen was added immediately after cell seeding. The portion surrounded by the white dotted line in FIG. 20 is the ciliary tissue. As shown in FIG. 20, a bulging structure is observed at the tip portion, which is the free end of the hairy tissue, and a black hair shaft extending from the bulging structure toward the root of the hairy tissue. A similar structure was observed.

[Production of cell aggregates]
As Example 5-1, a culture medium to which type I collagen was added to a final concentration of 120 μg/mL was used, and in the same manner as in Example 1 above, epithelial cells and mesenchymal cells were co-cultured (suspended culture) was started.

In addition, as Example 5-2, co-culture of epithelial cells and mesenchymal cells ( Floating culture) was started.

[result]
In both Examples 5-1 and 5-2, cell aggregates without hairy tissue were formed on the second day of culture. RNA was extracted from the cell aggregates collected on day 2 of culture using RNeasy mini-kit (QIAGEN) and subjected to microarray analysis using Mouse Genome 430 2.0 Array (Applied Biosystems).

FIG. 21 shows cell aggregates formed using a culture solution in which type I collagen is dispersed in Example 5-1, and cell aggregates formed using a culture solution in which type I collagen is not dispersed in Example 5-2. The results of GO (Gene Ontology) analysis using a microarray are shown for the cell aggregates obtained. That is, in FIG. 21, in the cell aggregate of Example 5-1, the gene group significantly increased (Fold change>2) compared to the cell aggregate of Example 5-2 (i.e., the cell aggregate of Example 5-1 Gene groups whose expression levels in clumps were at least twice that of cell clumps in Example 5-2).

As shown in FIG. 21, five GO terms of hair growth-related genes were extracted from the top 10 gene groups that increased significantly in the cell aggregates of Example 5-1 (hair cycle, skin development, epidermis development , hair follicle development and hair cycle process).

That is, in the cell aggregates of Example 5-1 formed by co-culture including matrix treatment using type I collagen, the cell aggregates of Example 5-2 formed by co-culture without the matrix treatment In comparison, it was confirmed that the expression levels of hair growth-related genes were significantly increased.

Specifically, for example, in the cell aggregates of Example 5-1, the expression levels of Tgfb2, Sox21, Lgr5, Lhx2, Edaradd, Pdgfa, and Lgr4, which are marker genes related to hair follicle development, are all It was more than double that of 2 cell clumps.

Therefore, the cell aggregates of Example 5-1 formed by co-culture including the matrix treatment using type I collagen were compared to the cell aggregates of Example 5-2 formed by co-culture without the matrix treatment. Therefore, it was considered that the hair regrowth ability was remarkably improved.

[Production of cell aggregates]
As Example 6-1, a culture medium to which type I collagen was added to a final concentration of 120 μg/mL was used, and 1×10 ⁴ cells were added to each well of a 96-well plate in the same manner as in Example 1 above. Epithelial cells/well and 1×10 ⁴ cells/well of mesenchymal cells were seeded.

After seeding, the 96-well plate was placed in a refrigerator at 4° C. for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).

As Example 6-2, instead of type I collagen, a culture medium containing a Matrigel undiluted solution (Matrigel (registered trademark) Basement Membrane Matrix, CORNING (registered trademark)) at a final concentration of 2 v/v% was used. Co-culture (suspension culture) of epithelial cells and mesenchymal cells was started in the same manner as in Example 6-1 except for the above.

The Matrigel undiluted solution contains 10.6 mg/mL (protein amount measured by the Lowry method) of soluble basement membrane matrix extracted from EHS (Engelbreth-Holm-Swarm) mouse tumor, and the composition ratio in the basement membrane matrix is , 56% laminin, 8% entactin, and 31% type IV collagen.

Therefore, it was calculated that the culture solution to which the Matrigel stock solution was added in an amount to give a final concentration of 2 v/v% contained 118 μg/mL laminin, 16 μg/mL entactin, and 66 μg/mL type IV collagen. be.

As Example 6-3, epithelial cells and mesenchymal cells were obtained in the same manner as in Example 6-1 above, except that a culture medium (basic medium) to which neither type I collagen nor Matrigel was added was used. Co-culture (suspension culture) was started.

[Transplantation of cell aggregates]
In any of the culture systems of Examples 6-1, 6-2 and 6-3, cell aggregates without hairy tissue were formed on day 3 of culture. From each of the culture systems of Examples 6-1, 6-2, and 6-3, cell aggregates on day 3 of culture were collected, and the cell aggregates were applied to 5-week-old SCID- by a patch method. Implanted on the skin of nu mice. Specifically, 10 cell aggregates of Example 6-1, 50 cell aggregates of Example 6-2, and 50 cell aggregates of Example 6-2 were placed in three separate transplant sites formed near the dermis or fascia of mouse skin. and 50 cell aggregates of Example 6-3, respectively.

[result]
FIG. 22 shows the results of visually counting the number of regenerated hairs at each transplantation site of the mouse 4 weeks after transplantation. As shown in FIG. 22, at the transplantation site of 50 cell aggregates formed under conditions using a culture solution (basal medium) in which neither type I collagen nor Matrigel was dispersed (“no matrix” in the figure). 128 hairs were regenerated. That is, the number of regenerated hairs per transplanted cell aggregate was 2.6.

In addition, 285 hairs were regenerated at the transplanted site of 50 cell aggregates formed under the conditions using the culture solution in which Matrigel was dispersed ("Matrigel" in the figure). That is, the number of regenerated hairs per transplanted cell aggregate was 5.7. The number of regenerated hairs per cell aggregate formed using Matrigel was 2.2 times that of the cell aggregate formed without type I collagen and Matrigel.

In addition, 208 hairs were regenerated at the transplanted site of 10 cell aggregates formed under the conditions using the culture solution in which type I collagen was dispersed (“collagen” in the figure). That is, the number of regenerated hairs per transplanted cell aggregate was 20.8.

That is, the number of regenerated hairs per cell aggregate formed using type I collagen was 8 times that of cell aggregates formed without using type I collagen and Matrigel, and the number of regenerated hairs using Matrigel was 8 times that of cell aggregates formed without using type I collagen and Matrigel. 3.6 times that of the formed cell clumps.

[Production of cell aggregates]
In the same manner as in Example 2 above, first, coculture (suspension culture) of epithelial cells and mesenchymal cells was carried out for 4 days using a culture solution to which type I collagen was added so that the final concentration was 12 μg/mL. gone. Next, cell aggregates without hairy tissue on day 4 of culture were collected and embedded in type I collagen hydrogel, and hydrogel embedding culture of the cell aggregates was performed.

[Recovery of ciliary tissue]
On the 14th day of culture (10th day of hydrogel-embedded culture), it was confirmed that a hairy tissue was formed on the surface of the cell aggregate. The root portion of the hairy tissue of the cell aggregate embedded in the hydrogel on the 14th day of culture was cut with scissors, and the cut hairy tissue was recovered.

[result]
FIG. 23 shows a photomicrograph of the ciliary tissue cut and collected from the cell aggregates as described above. For each of the three hairy tissues shown in FIG. 23, the white arrowhead indicates one tip portion that was free before being cut from the cell clump, and the black arrowhead was formed by the cutting. The other tip portion (the root portion that was bound to the cell clump before the cutting) is shown. As shown in FIG. 23, the hair-like tissue formed on the surface of the cell aggregate could be separated from the cell aggregate and collected.

[Production of cell aggregates]
As Example 8-1, in place of type I collagen, the final concentration is 2 v / v% (laminin final concentration is 118 μg / mL, entactin final concentration is 16 μg / mL, and type IV collagen final concentration is 66 μg / Epithelial cells and mesenchymal cells were co-cultured (suspension culture) in the same manner as in Example 1 above, except that the culture medium to which Matrigel undiluted solution was added was used.

As Example 8-2, in place of type I collagen, Matrigel undiluted solution (Matrigel (registered trademark) Basement Membrane Matrix (Growth Factor Reduced), CORNING (registered trademark)) with reduced growth factor content was added to a final concentration of Co-culture (suspension culture) of epithelial cells and mesenchymal cells was carried out in the same manner as in Example 1 above, except that the culture solution added in an amount of 1 v/v % was used.

The matrigel undiluted solution (GFR) with reduced growth factor content contains 8 to 12 mg/mL (protein amount measured by the Lowry method) of soluble basement membrane matrix extracted from EHS mouse tumor, and the basement membrane The composition ratio in the matrix was 61% laminin, 7% entactin, and 30% type IV collagen. Therefore, the culture medium to which Matrigel stock solution (GFR) was added in an amount to give a final concentration of 1 v / v% was 49-73 μg / mL laminin, 6-8 μg / mL entactin, and 24-36 μg / mL IV It is calculated that it contained type collagen.

As Example 8-3, instead of type I collagen, a culture supplemented with a high-concentration laminin/entactin complex undiluted solution (HIGH CONCENTRATION LAMININ/ENTACTIN COMPLEX, CORNING (registered trademark)) at a final concentration of 1 v/v%. Co-culture (suspension culture) of epithelial cells and mesenchymal cells was carried out in the same manner as in Example 1 above, except that the liquid was used.

The high-concentration laminin/entactin complex stock solution contains 15.2 mg/mL of soluble basement membrane matrix extracted from EHS mouse tumor, contains laminin/entactin complex with a purity of 90% or more by SDS-PAGE, and contains laminin and Entactin was included in an equimolar ratio. Therefore, the culture medium to which the high-concentration laminin/entactin complex stock solution was added at a final concentration of 1 v/v% contained 137 to 152 μg/mL laminin/entactin complex (that is, 68 to 76 μg/mL, respectively). laminin and entactin).

As Example 8-4, instead of type I collagen, a culture medium containing type IV collagen undiluted solution (COLLAGEN IV, MOUSE, CORNING (registered trademark)) at a final concentration of 1 v/v% was used. Epithelial cells and mesenchymal cells were co-cultured (suspension culture) in the same manner as in Example 1 described above.

The type IV collagen stock solution contained 1.25 mg/mL of the soluble basement membrane matrix extracted from the EHS mouse tumor, and contained type IV collagen with a purity of 90% or higher by SDS-PAGE. Therefore, it is calculated that the culture medium to which the type IV collagen stock solution was added in such an amount that the final concentration was 1 v/v %, contained 11 to 13 μg/mL of type IV collagen.

As Example 8-5, instead of type I collagen, a high-concentration laminin/entactin complex stock solution with a final concentration of 1 v/v% and a type IV collagen stock solution with a final concentration of 1 v/v%. Co-culture (suspension culture) of epithelial cells and mesenchymal cells was carried out in the same manner as in Example 1 above, except that a culture solution containing was used. Under all conditions, the culture medium in the wells maintained fluidity as a whole throughout the culture period.

[result]
FIG. 24 shows micrographs of cell aggregates on day 8 of culture. As shown in FIG. 24, Example 8-1 using a culture medium in which Matrigel was dispersed, Example 8-2 using a culture medium in which Matrigel with a reduced growth factor content was dispersed, and laminin/entactin complex Example 8-3 using a culture medium in which body was dispersed, Example 8-4 using a culture medium in which type IV collagen was dispersed, and culture medium in which a laminin/entactin complex and type IV collagen were dispersed were used. In all of the culture systems of Example 8-5 used, one cell aggregate with hairy tissue was formed in each well after 8 days of culture. In addition, in each of the five photographs included in FIG. 24, the arrows indicate the ciliary tissue formed on the surface of the cell aggregates.

FIG. 25 shows the results of fluorescent staining of the nuclei of cells contained in cell aggregates formed using the culture medium in which Matrigel was dispersed and observation with a confocal microscope. As shown in FIG. 25, the entire cell aggregate including multiple hair-like tissues formed on the surface of the cell aggregate was stained.

FIG. 26 shows the results of observation by HE staining and fluorescent staining of cell aggregates formed using the culture medium in which Matrigel was dispersed. In FIG. 26, photograph (i) shows an HE-stained image, and photograph (ii) shows an enlarged portion surrounded by a dotted line in the photograph (i). As shown in photographs (i) and (ii) of FIG. 26, the hair-like tissue formed in the cell aggregate consists of a dermal papilla-like structure formed at the free end of the dermal papilla-like structure and the dermal papilla-like structure. It had a hair shaft-like structure extending from the vicinity of the hair tissue to the root of the hair tissue.

In FIG. 26, photograph (iii) shows a fluorescence microscope image of a cell aggregate fluorescently stained with CD34, and photograph (iv) shows an enlarged portion surrounded by a dotted line in the photograph (iii). As shown in photographs (iii) and (iv) of FIG. 26, CD34-positive cells (ie, hair follicle epithelial stem cells) were observed along the periphery of the hair shaft-like structure of the cell aggregates.

In FIG. 26, photograph (v) shows a fluorescence microscope image of cell aggregates fluorescently stained with Versican, a marker for dermal papilla cells, and photograph (vi) shows an enlarged portion surrounded by a dotted line in the photograph (v). shown as As shown in photographs (v) and (vi) of FIG. 26, at the tip, which is the free end of the hair-like tissue of the cell aggregate, there is an aggregate (dermal papilla-like structure) of Versican-positive cells (that is, dermal papilla cells). ) was observed.

In FIG. 26, photograph (vii) shows a fluorescent microscopic image of cell aggregates fluorescently stained with Gp100, a pigment cell marker, and photograph (viii) is an enlarged portion surrounded by a dotted line in photograph (vii). is shown. As shown in photographs (vii) and (viii) in FIG. 26, Gp100-positive cells (i.e., pigment cells) were observed around the dermal papilla-like structure at the tip, which is the free end of the hair-like tissue of the cell aggregate. was done.

FIG. 27 shows the results of observing the hair-like tissue of cell aggregates formed using the culture medium in which Matrigel was dispersed. Photograph (i) in FIG. 27 is an image of the root portion of the hair-like tissue formed on the surface of the cell aggregate, taken with a scanning electron microscope (SEM). As shown in this photograph (i), it was confirmed that the hair shaft-like structure contained in the hair-like tissue has a cuticle structure.

Photograph (ii) of Photograph 27 is an image of the tip portion, which is the free end of the hairy tissue formed on the surface of the cell aggregate, taken in a bright field of a microscope. As shown in this photograph (ii), the hair-like tissue has a dermal papilla-like structure at its tip, and from the vicinity of the dermal papilla-like structure to the root portion of the hair-like tissue, there is a hair shaft-like structure. It was confirmed that the structure was stretched. Since epithelial cells take up melanin, they are observed as black cells under a microscope, but dermal papilla cells do not take up melanin, so they are identified as non-black cells under a microscope.

FIG. 28 shows the results of observation by a transmission microscope (TEM) of hair-like tissue of cell aggregates formed using the culture solution in which Matrigel was dispersed and mouse body hair. As shown in FIG. 28, the hair-like tissue formed in the cell aggregates was confirmed to have a hair shaft-like structure similar to the hair shaft of mouse hair, and a melanosome and cuticle structure similar to mouse hair. .

[Production of cell aggregates]
As Example 9-1, in place of type I collagen, the final concentration is 2 v / v% (laminin final concentration is 118 μg / mL, entactin final concentration is 16 μg / mL, and type IV collagen final concentration is 66 μg / Epithelial cells and mesenchymal cells were co-cultured (suspension culture) in the same manner as in Example 1 above, except that the culture medium to which Matrigel undiluted solution was added was used. In the suspension culture of Example 9-1, hairy tissue began to form on the surface of the cell aggregates from day 4 to day 6 of culture. After that, the length of the ciliary tissue of the cell clumps increased over time.

In Example 9-2, co-culture (suspension culture) of epithelial cells and mesenchymal cells was first started in the same manner as in Example 9-1 above. Next, the cell aggregates without hairy tissue on the first day of culture were collected in a centrifuge tube, and as much of the culture medium as possible was removed from the centrifuge tube. After that, Matrigel undiluted solution was added to the centrifuge tube, and cell aggregates were suspended in the Matrigel undiluted solution.

After that, 0.4 mL of a solution containing cell aggregates and Matrigel was dropped into wells of a 6-well plate and held in an incubator at 37°C for 30 minutes to gel Matrigel. As a result, the entire solution in the well gelled to form a hydrogel having no fluidity. Moreover, in the hydrogel, the cell aggregates were three-dimensionally dispersed and embedded in a state separated from each other.

After that, 2 mL of basal medium was added onto the hydrogel in the well to initiate hydrogel-embedded culture of cell aggregates. In the hydrogel-embedded culture of Example 9-2, hairy tissue was formed on the surface of the cell aggregates on the 4th to 6th days of the culture (3rd to 5th days from the start of the hydrogel-embedded culture). began to be After that, the length of the ciliary tissue of the cell clumps increased over time.

[Recovery of ciliary tissue]
In Example 9-1, the root portion of the hairy tissue of the cell aggregates floating in the culture medium on day 14 of culture was cut with scissors, and the cut hairy tissue was collected. In Example 9-2, the root portion of the hairy tissue of the cell aggregates on day 12 of culture (day 11 of hydrogel-embedded culture) and day 22 of culture (day 21 of hydrogel-embedded culture) was cut with scissors. The cut hairy tissue was collected.

[result]
FIG. 29 shows a photomicrograph of the ciliary tissue cut and recovered from the cell aggregate on day 14 of culture in Example 9-1. For each of the two hair-like tissues shown in photograph (i) and the two hair-like tissues shown in photograph (ii) of FIG. 29, the white arrowheads were free ends before being cut from the cell clump. One tip portion is shown, and the black arrowhead indicates the other tip portion formed by the cutting (the root portion that was bound to the cell aggregate before the cutting).

FIG. 30 shows micrographs of ciliary tissues collected from cell aggregates on day 12 (photograph (i)) and day 22 (photograph (ii)) of culture in Example 9-2. For each of the one hairy tissue shown in photograph (i) of FIG. 30 and the three hairy tissues shown in photograph (ii), the white arrowheads were free ends before being cut from the cell clumps. One tip portion is shown, and the black arrowhead indicates the other tip portion formed by the cutting (the root portion that was bound to the cell aggregate before the cutting). As shown in FIGS. 29 and 30, the hairy tissue formed on the surface of the cell aggregates could be separated from the cell aggregates and recovered.

FIG. 31 shows a micrograph of cell aggregates on day 23 of culture (day 22 of hydrogel-embedded culture) in Example 9-2. As shown in FIG. 31, one of the hair-like tissues formed on the surface of the cell aggregate reached about 4 cm in length. This hairy tissue exceeded 5 cm in length on day 30 of culture.

In FIG. 31, a partial photograph (i) shows an enlarged tip portion (portion surrounded by a black square), which is the free end of the ciliary tissue. As shown in this partial photograph (i), the hair-like tissue had a swollen structure at the tip and a shaft-like structure extending from the tip to the root.

In FIG. 31, partial photograph (ii) shows an enlarged view of small projections (parts surrounded by white squares) formed on the surface of the cell aggregates. As shown in this partial photograph (ii), in addition to the two elongated hair-like tissues, a new hair-like tissue was being formed on the surface of the cell aggregate.

[Production of cell aggregates]
In the same manner as in Example 9-2 above, cell aggregates were embedded and cultured in hydrogels containing Matrigel. Formation of hairy tissue was confirmed in the cell aggregates on day 6 of culture (fifth day from the start of hydrogel-embedded culture). After that, the length of the ciliary tissue of the cell clumps increased over time.

[Recovery of ciliary tissue]
The root portion of the hair-like tissue formed on the surface of the cell aggregate embedded in the hydrogel on the 15th day of the culture (the 14th day of the hydrogel-embedded culture) was cut with scissors, and the cut hair was obtained. Similar tissues were collected.

[result]
FIG. 32 shows micrographs of ciliary tissue recovered from cell clumps. Photograph (i) of FIG. 32 is a bright field micrograph of the recovered ciliary tissue. Photograph (ii) in FIG. 32 shows the result of Calcein-AM staining of the hairy tissue shown in photograph (i) and observation with a fluorescence microscope. In each of the two photographs (i) and (ii) in FIG. 32 , the white arrowhead indicates one tip portion that was free before being cut from the cell aggregate, and the black arrowhead indicates the The other tip portion that was formed (the root portion that was bound to the cell clump before the cutting) is shown.

As shown in this photograph (ii), the cells composing the collected hairy tissue (hairy graft) were stained with Calcein-AM and confirmed to be viable.

[Production of cell aggregates]
Instead of type I collagen, the final concentration is 2 v / v% (laminin final concentration is 118 μg / mL, entactin final concentration is 16 μg / mL, and type IV collagen final concentration is 66 μg / mL). Co-culture (suspension culture) of epithelial cells and mesenchymal cells was carried out in the same manner as in Example 1 above, except that the culture medium to which Matrigel undiluted solution was added was used.

Next, the cell aggregates without hairy tissue on the first day of culture were collected in a centrifuge tube, and as much of the culture medium as possible was removed from the centrifuge tube. After that, Matrigel undiluted solution was added to the centrifuge tube, and cell aggregates were suspended in the Matrigel undiluted solution.

After that, 0.4 mL of a solution containing cell aggregates and Matrigel was dropped into wells of a 6-well plate and held in an incubator at 37°C for 30 minutes to gel Matrigel. As a result, the entire solution in the well gelled to form a hydrogel having no fluidity. Moreover, in the hydrogel, the cell aggregates were three-dimensionally dispersed and embedded in a state separated from each other.

After that, 2 mL of basal medium was added onto the hydrogel in the well to initiate hydrogel-embedded culture of cell aggregates. In the hydrogel-embedded culture, hairy tissue began to form on the surface of the cell aggregates on the 4th to 6th days of the culture (3rd to 5th days from the start of the hydrogel-embedded culture). After that, the length of the ciliary tissue of the cell clumps increased over time.

[Recovery of ciliary tissue]
The root portion of the hairy tissue of the cell aggregate on day 14 of culture (day 10 of hydrogel-embedded culture) was cut with scissors, and the cut hairy tissue was collected.

[Hydrogel-embedded culture of hair-like grafts]
The hairy tissue (hairy graft) collected as described above was added to the Matrigel undiluted solution, 0.4 mL of the solution containing the hairy graft and Matrigel was added dropwise to the wells of a 6-well plate, and heated at 37°C. Matrigel was gelled by holding in an incubator for 30 minutes. As a result, the entire solution in the well gelled to form a hydrogel having no fluidity. Thereafter, 2 mL of basal medium was added onto the hydrogel in the well to initiate hydrogel embedding culture of the hair-like explant. The hydrogel in which the hair-like graft was embedded was used as an artificial tissue simulating the tissue of a living body into which the hair-like graft was to be implanted.

[result]
FIG. 33 shows micrographs of hydrogel-embedded cultured hair-like explants. In the photograph (i) of FIG. 33, immediately after the start of the hydrogel embedding culture (d0), in the photograph (ii), the first day of the hydrogel embedding culture (d1), in the photograph (iii), the hydrogel embedding culture On day 2 (d2), in photo (iv) on day 3 of hydrogel embedding culture (d3), in photo (v) on day 5 of hydrogel embedding culture (d5), in photo (vi) Photo (vii) shows hairy explants on day 7 of gel-embedded culture (d7) and on day 8 of hydrogel-embedded culture (d8), respectively.

In FIG. 33, the white arrowhead indicates one tip portion that was free before being cut from the cell aggregate, and the black arrowhead indicates the other tip portion formed by the cutting (before cutting The root portion that was associated with cell clumps) is shown.

As shown in FIG. 33, the hair graft survives after being cut from the cell clump, and the length of the hair graft (especially the length of the shaft-like structure contained in the hair graft) ) increased over time. This result supported the usefulness of hair-like tissue cut from cell clumps as a hair-like graft.

Claims (15)

performing cell culture to obtain a cell aggregate having a hair-like tissue formed on its surface; and
cutting and recovering the ciliary tissue from the cell aggregate for transplantation into a living body;
A method for producing hairy tissue for transplantation, comprising: The hair-like tissue has a hair shaft-like structure,
The method for producing a hair-like tissue for transplantation according to claim 1. The hair-like tissue of the cell aggregate has a dermal papilla-like structure at the tip, which is a free end,
The method for producing a hair-like tissue for transplantation according to claim 1 or 2. the ciliary tissue does not contain capillaries;
A method for producing a hair-like tissue for transplantation according to any one of claims 1 to 3. The cell aggregate does not contain arrector pili structure and/or sebaceous gland structure,
A method for producing a hair-like tissue for transplantation according to any one of claims 1 to 4. The ciliary tissue cut and collected from the cell aggregate has one tip portion that does not have a cut surface and the other tip portion that has a cut surface,
A method for producing a hair-like tissue for transplantation according to any one of claims 1 to 5. The cell culture is a co-culture of the epithelial cells and the mesenchymal cells, comprising seeding the epithelial cells and the mesenchymal cells.
A method for producing a hair-like tissue for transplantation according to any one of claims 1 to 6. The co-culturing includes matrix treatment in which the epithelial cells and the mesenchymal cells are maintained in a culture medium in which type I collagen, fibronectin, laminin and entactin, or type IV collagen is dispersed,
The method for producing a hair-like tissue for transplantation according to claim 7. In the matrix treatment, the epithelial cells and the mesenchymal cells are held in a culture medium in which type I collagen is dispersed.
The method for producing a hair-like tissue for transplantation according to claim 8. The co-culture is
performing suspension culture of the epithelial cells and the mesenchymal cells to form cell aggregates;
Embedding the cell aggregate formed by the suspension culture in a hydrogel and further culturing it;
The method for producing a cell aggregate according to any one of claims 7 to 9, comprising Having one tip portion and the other tip portion that are free ends,
including epithelial cells and mesenchymal cells,
does not contain capillaries,
Hairy tissue for transplantation. having a hair shaft-like structure,
Hair-like tissue for transplantation according to claim 11. Having a dermal papilla-like structure at the one tip portion,
Hair-like tissue for transplantation according to claim 11 or 12. Having said one tip portion without a cutting surface and said other tip portion having a cutting surface,
Hair-like tissue for transplantation according to any one of claims 11 to 13. Transplanting the hairy tissue according to any one of claims 11 to 14 into a living body,
Hair regrowth method.

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