WO2023058429A1

WO2023058429A1 - Method for producing cell aggregate having hair regeneration ability, and method related thereto

Info

Publication number: WO2023058429A1
Application number: PCT/JP2022/034628
Authority: WO
Inventors: 淳二福田; 達斗景山; 理樹穴竃; 龍彦肥高
Original assignee: 国立大学法人横浜国立大学; 地方独立行政法人神奈川県立産業技術総合研究所
Priority date: 2021-10-08
Filing date: 2022-09-15
Publication date: 2023-04-13
Also published as: JP2023056591A

Abstract

This method for producing a cell aggregate comprises: plating epithelial cells and mesenchymal cells; and co-culturing the epithelial cells and the mesenchymal cells to form a cell aggregate having a hair regeneration ability. The co-culturing includes a matrix process for keeping the epithelial cells and the mesenchymal cells in a culture solution having dispersed therein type-I collagen or fibronectin.

Description

METHOD FOR MANUFACTURING CELL AGGREGATE HAVING HAIR REGENERATION ABILITY AND RELATED METHOD

The present invention relates to a method for producing cell aggregates having hair regeneration ability and methods related thereto.

Non-Patent Document 1 describes the formation of 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

On the other hand, the inventors of the present invention have studied technical means for obtaining cell aggregates with excellent hair regeneration ability.

The present invention has been made in view of the above problems, and one of its objects is to provide a method for producing cell aggregates having excellent hair regeneration ability and methods related thereto.

One aspect of one embodiment of the present invention for solving the above problems is seeding epithelial cells and mesenchymal cells, and co-cultivating the epithelial cells and the mesenchymal cells to obtain hair. Forming a cell aggregate having regenerative ability, wherein the co-culturing includes a matrix treatment that retains the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, A method for producing a cell aggregate. INDUSTRIAL APPLICABILITY According to the present invention, a method for effectively producing cell aggregates having excellent hair regeneration ability is provided.

In addition, the culture solution in which the type I collagen or fibronectin is dispersed may contain type I collagen or fibronectin at a concentration within a range in which the culture solution as a whole maintains fluidity in the co-culture.

In addition, in the matrix treatment, the epithelial cells and the mesenchymal cells may be retained in the culture solution in which type I collagen is dispersed. In this case, in the matrix treatment, the epithelial cells and the mesenchymal cells may be maintained in the culture medium in which type I collagen is dispersed at a concentration of less than 480 μg/mL. Moreover, in the matrix treatment, the epithelial cells and the mesenchymal cells may be held in the culture medium in which fibronectin is dispersed.

Further, in the method, the co-cultivation may be performed to form the cell aggregate having the ability to form a hairy tissue on its surface. Further, in the method, the co-cultivation may be performed to form the cell aggregate having a hair-like tissue on its surface.

In addition, the method may further include collecting the cell aggregates formed by the co-culture for transplantation into a living body. In this case, the cell aggregate formed by the co-culture, which has the ability to form a hairy tissue on its surface and has no hairy tissue formed on its surface, is used for transplantation into a living body. It may be collected.

Further, in the method, the same conditions are used except that the co-culture is performed and a culture medium in which type I collagen or fibronectin is not dispersed is used instead of the culture medium in which type I collagen or fibronectin is dispersed. A cell aggregate may be formed in which the expression level of one or more hair growth-related genes is at least twice as large as that of the cell aggregate formed in .

Further, in the method, the co-culturing includes performing suspension culture of the epithelial cells and the mesenchymal cells to form cell aggregates, and hydrolyzing the cell aggregates formed by the suspension culture. embedding in a gel and further culturing.

Another aspect of one embodiment of the present invention for solving the above problems is seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and the mesenchymal cells. A matrix that retains the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed in the production of the cell aggregate, which includes forming a cell aggregate having hair regeneration ability. This is a method for improving the hair regeneration ability of the cell aggregate by performing the treatment. INDUSTRIAL APPLICABILITY According to the present invention, a method for effectively improving the hair regeneration ability of cell aggregates is provided.

Still another aspect of one embodiment of the present invention for solving the above problems is to seed epithelial cells and mesenchymal cells, and to co-culture the epithelial cells and the mesenchymal cells. holding the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, A use for improving the hair regeneration ability of said cell aggregates of type I collagen or fibronectin, comprising performing a matrix treatment. INDUSTRIAL APPLICABILITY According to the present invention, effective use of type I collagen or fibronectin for improving the hair regeneration ability of cell aggregates is provided.

According to the present invention, a method for producing a cell aggregate having excellent hair regeneration ability and a method related thereto 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.

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

One aspect of the method according to the present embodiment (hereinafter sometimes referred to as "this method") is to seed epithelial cells and mesenchymal cells, and to seed the epithelial cells and mesenchymal cells. to form a cell aggregate having hair regeneration ability, wherein the co-culturing retains the epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed It includes a method for producing cell aggregates, including a matrix treatment to perform the treatment.

That is, the inventors of the present invention have extensively studied technical means for producing in vitro cell aggregates having hair regeneration ability, and unexpectedly found that co-culture of epithelial cells and mesenchymal cells In the above, by performing a matrix treatment to retain the epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, it is possible to effectively improve the hair regeneration ability of the cell aggregates. The present inventors have independently discovered what they can do, and have completed the present invention.

Therefore, in another aspect, the present method includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain a cell aggregate having hair regeneration ability. In the production of cell aggregates, including forming A method for enhancing the hair regrowth ability of clumps is included.

In addition, in still another aspect, the present method includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain a cell aggregate having hair regeneration ability. In the production of cell aggregates, which comprises forming The use of collagen or fibronectin for improving the hair regeneration ability of the cell aggregates is included.

In the production of cell aggregates, epithelial cells and mesenchymal cells are seeded, and the seeded epithelial cells and mesenchymal cells are co-cultured to form cell aggregates having hair regeneration ability. .

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. Undifferentiated cells used for inducing differentiation of hair follicle epithelial cells in vitro are not particularly limited as long as they are cells capable of differentiating into the hair follicle epithelial cells in vitro. (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 are cells capable of differentiating 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., derived from adipose tissue 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, the epithelial cells and mesenchymal cells are seeded first. In this regard, in the method described in Non-Patent Document 1, induced pluripotent stem cells are seeded. Therefore, a long culture time and complicated operations for differentiating induced pluripotent stem cells are essential in culture after seeding. 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.

On the other hand, 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 addition, in 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 with 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 (that is, 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.

By co-culturing epithelial cells and mesenchymal cells, cell aggregates with hair regeneration ability can be obtained. 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.

In this regard, in the present method, co-culturing is performed by co-cultivating epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin (hereinafter collectively referred to as "matrix") is dispersed. By including the matrix treatment for retention, the hair regeneration ability of the cell aggregates formed by the co-culture is effectively improved.

A 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.

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) or solubilized fibronectin (non-insolubilized fibronectin) as a dispersed matrix.

The basal culture medium is not particularly limited as long as the effects of the present invention can be obtained, but for example, 1% 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 be added with another matrix, or may be added with no other matrix.

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, the fibronectin-dispersed matrix treatment culture medium may further contain type I collagen, laminin, entactin, or type IV collagen, or type I collagen, laminin, entactin, or type IV collagen. may not be added.

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-processing culture medium 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.

The matrix treatment culture medium contains matrix at a concentration within the range in which the culture 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 matrix that is brought into contact with epithelial cells and mesenchymal cells in 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.

It is preferable to start matrix treatment at the same time as seeding epithelial cells and mesenchymal cells (the time interval from seeding epithelial cells and mesenchymal cells to starting matrix treatment is 0 hours). 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 culture 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.

The sedimentation of epithelial cells and mesenchymal cells is preferably performed before starting matrix treatment at a temperature at which the epithelial cells and mesenchymal cells are co-cultured. 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 cells 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, it is preferable to continuously perform seeding of epithelial cells and mesenchymal cells, matrix treatment, and formation of cell aggregates 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 this method, cell aggregates with excellent hair regeneration ability can be produced. That is, in the present method, co-cultivation including the above-described matrix treatment is performed, and a culture medium in which the matrix is not dispersed (that is, a culture medium to which the matrix is not added) is used instead of the culture medium for matrix treatment. A cell aggregate with improved hair regrowth ability is produced as compared with a cell aggregate formed under the same conditions (hereinafter sometimes referred to as "control cell aggregate") except for the addition. Specifically, for example, in this method, a cell aggregate is formed 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 performing co-culturing including the matrix treatment described above, for example, it is possible to form cell aggregates that have the ability to form hair-like tissue on their surface in vitro. Thus, for example, co-cultivation involving matrix treatment can be performed to form cell aggregates having hair-like tissue on their surface.

In addition, for example, by co-cultivating with matrix treatment, cell aggregates that have the ability to form hairy tissue on their surface in vitro but do not have hairy tissue formed on their surface (i.e., By continuing the co-cultivation, it is also possible to produce a cell aggregate that has the ability to form a hairy tissue on its surface but has no hairy tissue formed on its surface yet.

For this reason, in still another aspect, the present embodiment includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain cells having hair regeneration ability. In the production of cell aggregates, including forming aggregates, by performing a matrix treatment that retains the epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, A method of promoting the formation of ciliary tissue in cell clumps is included.

In addition, as yet another aspect, this embodiment includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain cell aggregates having hair regeneration ability. In the production of cell aggregates, including forming clumps, I The use of type collagen or fibronectin to promote the formation of hair-like tissue in the cell aggregates is included.

The hair-like tissue of cell aggregates contains epithelial cells and mesenchymal cells, and is formed as a structure protruding from the surface of the cell aggregates. 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.

In addition, 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.

In addition, 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, but may be, for example, 30 μm or more, preferably 50 μm or more, and 100 μm or more. is more preferable, 500 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 a hairy 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.

In addition, unlike the tissue formed in Non-Patent Document 1, the cell aggregates formed by co-culture including matrix treatment do not contain piloerection muscle structures and/or sebaceous gland structures.

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, the cell aggregates formed by floating 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 type I collagen or fibronectin. Moreover, the hydrogel in which the cell aggregates are embedded preferably contains type I collagen or fibronectin.

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

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. In addition, 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.

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, cell aggregates may be formed by co-culturing including matrix treatment using type I collagen or fibronectin, and then the cell aggregates may be embedded in a hydrogel containing type I collagen or fibronectin and cultured. .

Also, cell aggregates are formed by co-culturing including matrix treatment using type I collagen or fibronectin, and then one or more matrices used in the matrix treatment selected from the group consisting of type I collagen and fibronectin. The cell aggregate may be embedded in a hydrogel containing 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, for example, by adding a culture medium onto the hydrogel in a culture vessel and culturing the cell aggregates inside the hydrogel.

By embedding and culturing a cell aggregate that does not have hairy tissue on its surface in a hydrogel, it is possible to effectively form a hairy tissue 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.

The use of the cell aggregates obtained in this embodiment is not particularly limited, but the cell aggregates have excellent hair regeneration ability and are useful as grafts to living organisms. Therefore, the method for producing cell aggregates may further include collecting the cell aggregates formed by co-culturing including matrix treatment for transplantation into a living body. In this case, a cell aggregate having a hairy tissue on its surface may be used as a graft to a living body, but it is preferable to use a cell aggregate on which no hairy tissue has yet been formed.

That is, a cell aggregate having the ability to form a hairy tissue on its surface and having no hairy tissue formed on its surface formed by co-culture including matrix treatment is used for transplantation into a living body. It is preferable to collect at

In this case, the cell aggregates are collected before the hair-like tissue is formed on the surface. Specifically, collection of cell aggregates for transplantation into a living body is preferably performed within 168 hours, more preferably within 144 hours, from the seeding of epithelial cells and mesenchymal cells, for example. It is even more preferable to carry out within 120 hours, and particularly preferably within 96 hours.

Furthermore, collection of cell aggregates for transplantation into a living body is preferably performed, for example, within 84 hours, more preferably within 72 hours, and 60 hours after seeding epithelial cells and mesenchymal cells. It is more preferable to carry out within 48 hours, and particularly preferably within 48 hours.

In addition, collection of cell aggregates for transplantation into a living body may be performed, for example, after 12 hours, preferably after 15 hours, and preferably after 18 hours from the seeding of epithelial cells and mesenchymal cells. It is more preferable to carry out after 21 hours, even more preferably after 24 hours, and particularly preferably after 24 hours. The timing of collecting cell aggregates for transplantation into a living body may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.

In addition, a cell aggregate that does not have a hair-like tissue on its surface formed by co-culturing may not have a cyst-like structure inside. In this regard, in this method, it is preferable to collect cell aggregates for transplantation into a living body before cyst-like structures are formed therein.

The collection of cell aggregates is performed by taking out the cell aggregates from the culture vessel in which the cell aggregates were cultured. The collected cell aggregates may be transferred to a container other than the culture container.

The method may further comprise cold storage of the cell aggregate formed by co-culturing including matrix treatment for transplantation into a living body. In this case, for example, cell aggregates formed by co-cultivation are collected, and the collected cell aggregates are stored in a cold state until they are transplanted into a living body.

Specifically, for example, the cell aggregate collected from the culture vessel is transferred to a container separate from the culture vessel and stored in a cold state. Alternatively, for example, cell aggregates formed by co-cultivation may be stored in a cold state without being collected. That is, in this case, for example, the cell aggregates in the culture vessel are stored by cooling as they are.

The temperature for cold storage of cell aggregates 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 lower (more than 0 ° C. and 10 ° C. or lower), and 7 ° C. or lower. is more preferable, and 5° C. or less is particularly preferable.

As one aspect, this method includes a hair regeneration method including transplanting the cell aggregate collected as described above into a living body. A living body into which the cell aggregates are transplanted may be a human or a non-human animal, but is preferably a human. Transplantation of the cell aggregates into the living body is preferably to the skin of the living body, and particularly preferably to the scalp of the living body.

The transplantation of cell aggregates into a living body may be for medical or research purposes. Transplantation of cell aggregates into a living body is preferably for the treatment or prevention of diseases associated with hair loss. That is, transplantation of cell aggregates into a living body is preferably transplantation into a human patient suffering from or likely to suffer 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 accompanied by 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, the cell aggregates obtained in this embodiment 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. It may be possible to

Next, a specific example according to this embodiment will be described.

[Collection of epithelial cells and mesenchymal cells]
Back skin tissue was collected from a C57BL/6 mouse fetus of 18 days of gestational age, and dispase treatment was performed at 4° C. for 1 hour under shaking conditions of 20 to 30 rpm to separate the epithelial layer and 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 left 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 2 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, epithelial cells and mesenchymal cells began to aggregate after the start of 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. 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 the 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 the 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 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. 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 aggregate formed by co-culturing epithelial cells and mesenchymal cells in this example had a cyst-like structure in its central part. 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.

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 day 4 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. After that, 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 the cell aggregates were embedded and cultured in the hydrogel for another 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 regressed 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.

During the 8-day co-cultivation period, the culture solution 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, after the start of 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. 13 shows 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 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 medium 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. .

Immediately after seeding (0 hours after seeding), 100 μL of a culture solution containing type I collagen at a concentration of 240 μg/mL and cooled to 4° C. was added to the wells of Example 4-1. 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 left 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 adding 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 a micrograph of cell aggregates on day 8 of culture in a culture system to 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 an enlarged photograph 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, FIG. 21 shows a group of genes significantly (Fold change>2) increased in the cell aggregates of Example 5-1 compared to the cell aggregates of Example 5-2 (i.e., the cell aggregates 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.

As Example 6-2, instead of type I collagen, a culture solution was used in which Matrigel undiluted solution (Matrigel (registered trademark) Basement Membrane Matrix, CORNING (registered trademark)) was added at a final concentration of 2 v/v%. 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 conditions using a culture medium 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.

Claims

Seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to form a cell aggregate having hair regeneration ability,
The co-cultivation includes a matrix treatment that retains the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed,
A method for producing cell aggregates.
The culture medium in which the type I collagen or fibronectin is dispersed contains type I collagen or fibronectin at a concentration within a range in which the culture medium as a whole maintains fluidity in the co-culture.
The method for producing the cell aggregate according to claim 1.
In the matrix treatment, the epithelial cells and the mesenchymal cells are retained in the culture medium in which type I collagen is dispersed.
The method for producing a cell aggregate according to claim 1 or 2.
In the matrix treatment, the epithelial cells and the mesenchymal cells are retained in the culture medium in which type I collagen is dispersed at a concentration of less than 480 μg/mL.
The method for producing a cell aggregate according to claim 3.
In the matrix treatment, the epithelial cells and the mesenchymal cells are retained in the culture medium in which fibronectin is dispersed.
3. A method according to claim 1 or 2.
performing the co-culturing to form the cell clumps having the ability to form hairy tissue on their surface;
The method for producing a cell aggregate according to any one of claims 1 to 5.
performing the co-culturing to form the cell aggregate having a hairy tissue on its surface;
The method for producing a cell aggregate according to any one of claims 1 to 6.
further comprising collecting the cell aggregates formed by the co-culture for transplantation into a living body;
The method for producing a cell aggregate according to any one of claims 1 to 7.
The cell aggregate formed by the co-cultivation, which has the ability to form a hairy tissue on its surface and has no hairy tissue formed on its surface, is collected for transplantation into a living body.
The method for producing a cell aggregate according to claim 8.
Cell aggregates formed under the same conditions except that the co-culture is performed and a culture medium in which type I collagen or fibronectin is not dispersed is used instead of the culture medium in which type I collagen or fibronectin is dispersed. Forming a cell aggregate in which the expression level of one or more hair growth-related genes is two or more times greater than that of
The method for producing a cell aggregate according to any one of claims 1 to 9.
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 1 to 10, comprising
Said cell aggregate, comprising seeding epithelial cells and mesenchymal cells, and co-culturing said epithelial cells and said mesenchymal cells to form a cell aggregate having hair regeneration ability. A method for improving the hair regeneration ability of the cell aggregate by performing a matrix treatment to retain the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed in the production of .
Said cell aggregate, comprising seeding epithelial cells and mesenchymal cells, and co-culturing said epithelial cells and said mesenchymal cells to form a cell aggregate having hair regeneration ability. In the production of the cell aggregates of type I collagen or fibronectin, comprising performing a matrix treatment that retains the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed. Use for improving hair regrowth.