Hair-like tissue for transplantation and related methods

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to hair-like tissue for transplantation and related methods. [Background technology]

[0002] Non-patent document 1 describes how induced pluripotent stem cells (iPSCs) derived from mouse fetal fibroblasts can be seeded in a 96-well plate and cultured to form skin organoids in vitro.

[0003] Patent Document 1 describes a method for producing an aggregate of regenerated hair follicle primordia, characterized by comprising the step of seeding mesenchymal cells and epithelial cells onto a micro-indentation plate consisting of regularly arranged minute recesses, and then forming hair follicle primordia by mixed culture while supplying oxygen.

[0004] Patent Document 2 describes 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) skin including the epidermal layer and the dermis layer, (2) at least one type of skin appendage, and (3) subcutaneous tissue, and the method is characterized by comprising the following steps: (a) stimulating an embryoid body with a physiologically active substance capable of activating the Wnt pathway, (b) preparing a conjugate comprising the following (A) and (B): (A) all or part of the embryoid body stimulated in step (a), (B) scaffold material, (c) transplanting the conjugate prepared in step (b) into an animal, and (d) producing full-thickness skin derived from the conjugate in the animal.

[0005] Patent Document 3 describes a method for producing hair follicle primordia, comprising: seeding epithelial cells and mesenchymal cells; holding the epithelial cells and mesenchymal cells in a culture medium in which laminin and enterin and / or type IV collagen are dispersed; and forming hair follicle primordia by co-culturing the epithelial cells and mesenchymal cells in the culture medium.

[0006] Patent Document 4 describes a method for producing mammalian hair follicles, comprising the following steps: (a) providing at least one de novo papilla; (b) providing at least one other cell population selected from the group of fibroblasts, keratinocytes and / or melanocytes; and (c) co-culturing the de novo papilla together with the at least one other cell population under non-adherent culture conditions, wherein step (a) is a step of providing at least one dermal papilla (DP) derived from at least one mammalian hair follicle. (2) A step of isolating dermal dermal papilla fibroblasts (DPF) from the DP by mechanically fixing the DP to the surface of a cell culture vessel, thereby creating holes in the basement plate and allowing the DPF to flow out; (3) A step of growing the isolated DPF in monolayer culture without collagen coating and passing the DPF at least once; (4) A step of obtaining de novo papillae by condensing the grown DPF into cell aggregates exhibiting the size and shape of physiological DP, wherein the DPF is 1,000 to 1,000,000 DFP / cm 2 A method for producing mammalian hair follicles is described, characterized by comprising the steps of (5) differentiating cells in a non-adherent culture vessel at a cell concentration per surface of the culture vessel, and (6) coating the cell aggregates with extracellular matrix proteins.

[0007] Patent Document 5 describes a method for producing spheroids, which includes a mixing step of mixing a carrier group composed of multiple carriers having cell adhesion properties with adhesion-dependent cells under mixing conditions in which the carrier group does not aggregate integrally with the cells, and a culturing step of culturing the mixture for a predetermined period of time. [Prior art documents]

Patent Document

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Document

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] On the other hand, the inventors of the present invention have studied technical means for hair regeneration medicine.

[0011] The present invention has been made in view of the above problems, and one of its objects is to provide a hair-like tissue for transplantation for hair regeneration and a method related thereto.

Means for Solving the Problems

[0012] A method for producing hair-like tissue for transplantation according to one embodiment of the present invention, which solves the above problems, includes performing cell culture to obtain a cell aggregate on which hair-like tissue is formed on its surface, and cutting and recovering the hair-like tissue from the cell aggregate for transplantation into a living organism. According to the present invention, a method for producing hair-like tissue for transplantation for hair regeneration is provided.

[0013] Furthermore, the hair-like tissue may have a hair shaft-like structure. Also, the hair-like tissue of the cell aggregate may have a dermal papilla-like structure at its free end. Furthermore, the hair-like tissue may not contain capillaries. Furthermore, the cell aggregate may not contain arrector pili muscle structures and / or sebaceous gland structures. Furthermore, the hair-like tissue that is cut and recovered from the cell aggregate may have one end portion without a cut surface and the other end portion with a cut surface.

[0014] Furthermore, the cell culture may be a co-culture of epithelial cells and mesenchymal cells, which includes seeding of epithelial cells and mesenchymal cells. In this case, the co-culture may include a matrix treatment in which the epithelial cells and mesenchymal cells are held in a culture medium dispersed with type I collagen, fibronectin, laminin and entactin, or type IV collagen. In this case, the matrix treatment may also involve holding the epithelial cells and mesenchymal cells in a culture medium dispersed with type I collagen.

[0015] Furthermore, the co-culture may include performing suspension culture of the epithelial cells and the mesenchymal cells to form cell aggregates, and embedding the cell aggregates formed by the suspension culture in a hydrogel and culturing them further.

[0016] To solve the above problems, a transplantable hair-like tissue according to one embodiment of the present invention has one tip portion that is a free end and the other tip portion, contains epithelial cells and mesenchymal cells, and does not contain capillaries. According to the present invention, a transplantable hair-like tissue for hair regeneration is provided.

[0017] Furthermore, the transplantable hair-like tissue may have a hair shaft-like structure. Also, the transplantable hair-like tissue may have a hair papilla-like structure at one of its tip portions. Furthermore, the transplantable hair-like tissue may have one tip portion without a cut surface and the other tip portion with a cut surface.

[0018] A hair regeneration method according to one embodiment of the present invention for solving the above problems includes transplanting any of the aforementioned hair-like tissues into a living organism. According to the present invention, an effective hair regeneration method is provided. [Effects of the Invention]

[0019] According to the present invention, hair-like tissue for transplantation for hair regeneration and related methods are provided. [Brief explanation of the drawing]

[0020] [Figure 1] This is an explanatory diagram showing a micrograph of a cell aggregate formed on day 8 of culture in Example 1 according to this embodiment. [Figure 2] This diagram illustrates the correspondence between the concentration of type I collagen in the culture medium at the time of seeding in Example 1 of this embodiment and the efficiency of ciliary tissue formation in the cell aggregates on day 8 of culture. [Figure 3] This diagram illustrates the relationship between the concentration of type I collagen in the culture medium at the time of seeding in Example 1 of this embodiment and the number of hair-like tissues formed per cell aggregate on day 8 of culture. [Figure 4] This is an explanatory diagram showing a micrograph of a cell aggregate formed on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL, according to Example 1 of this embodiment. [Figure 5] This is an explanatory diagram showing the results of HE staining of frozen sections of cell aggregates formed on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL in Example 1 of this embodiment. [Figure 6] This is an explanatory diagram showing the results of fluorescent staining with Versican on frozen sections of cell aggregates formed on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL, according to Example 1 of this embodiment. [Figure 7] This is an explanatory diagram showing the results of fluorescent staining of CD34 in frozen sections of cell aggregates formed on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL, according to Example 1 of this embodiment. [Figure 8] This is a schematic diagram illustrating a cell aggregate having hair-like tissue obtained in Example 1 of this embodiment. [Figure 9] This diagram shows micrographs of a single cell aggregate embedded in the hydrogel in Example 2 of this embodiment, taken on days 8, 12, 18, 22, and 27 of culture. [Figure 10] Figure 9 is an explanatory diagram showing a magnified view of the area enclosed by a dotted rectangle (ciliary tissue) in each of the five photographs included in the figure. [Figure 11] This is an explanatory diagram showing the change in the length of the ciliary tissue formed in Example 2 of this embodiment over time. [Figure 12] This is an explanatory diagram showing a micrograph of a cell aggregate formed on day 8 of culture in Example 3 according to this embodiment. [Figure 13] This diagram illustrates the correspondence between the fibronectin concentration in the culture medium at the time of seeding in Example 3 of this embodiment and the efficiency of ciliary tissue formation in the cell aggregates on day 8 of culture. [Figure 14] This diagram illustrates the relationship between the fibronectin concentration in the culture medium at the time of seeding in Example 3 of this embodiment and the number of hair-like tissues formed per cell aggregate on day 8 of culture. [Figure 15] This is an explanatory diagram showing the results of HE staining of frozen sections of cell aggregates formed on day 8 of culture in a culture system where the fibronectin concentration in the culture medium at the time of seeding was 100 μg / mL in Example 3 of this embodiment. [Figure 16] This is an explanatory diagram showing a micrograph of a cell aggregate formed on day 8 of culture in Example 4 according to this embodiment. [Figure 17] This diagram illustrates the correspondence between the timing of type I collagen addition in Example 4 of this embodiment and the efficiency of ciliary tissue formation in cell aggregates on day 8 of culture. [Figure 18] This diagram illustrates the relationship between the timing of type I collagen addition in Example 4 according to this embodiment and the number of hair-like tissue strands formed per cell aggregate on day 8 of culture. [Figure 19] This is an explanatory diagram showing a micrograph of a cell aggregate formed on day 8 of culture in a culture system in which type I collagen was added immediately after cell seeding, according to Example 4 of this embodiment. [Figure 20] This is an explanatory diagram showing a magnified photograph of ciliary tissue extending from a cell aggregate formed on day 8 of culture in a culture system in which type I collagen was added immediately after cell seeding, according to Example 4 of this embodiment. [Figure 21] This diagram shows some of the results of microarray analysis performed on cell aggregates formed using a culture medium containing type I collagen and cell aggregates formed using a culture medium that does not contain type I collagen, in Example 5 of this embodiment. [Figure 22] This is an explanatory diagram showing the results of visually counting the number of hairs that had regenerated 4 weeks after transplantation of cell aggregates in Example 6 according to this embodiment. [Figure 23] This is an explanatory diagram showing a micrograph of the ciliary tissue recovered from the cell aggregate formed in Example 7 according to this embodiment. [Figure 24] This is an explanatory diagram showing a micrograph of a cell aggregate formed on day 8 of culture in Example 8 according to this embodiment. [Figure 25] This is an explanatory diagram showing the results of fluorescently staining the nuclei of cells contained in a cell aggregate formed using a culture medium containing Matrigel in Example 8 of this embodiment and observing them with a confocal microscope. [Figure 26]This is an explanatory diagram showing the observation results of HE staining and fluorescence staining of cell aggregates formed using a culture medium containing Matrigel in Example 8 of this embodiment. [Figure 27] This is an explanatory diagram showing the results of observing the hair-like tissue of cell aggregates formed using a culture medium containing Matrigel in Example 8 of this embodiment. [Figure 28] This diagram illustrates the results of observing, using a transmission microscope, the hair-like tissue of cell aggregates formed using a culture medium containing Matrigel in Example 8 of this embodiment, and the body hair of a mouse. [Figure 29] This is an explanatory diagram showing a micrograph of ciliary tissue recovered from a cell aggregate on day 14 of culture in Example 9-1 of Example 9 according to this embodiment. [Figure 30] This is an explanatory diagram showing micrographs of ciliary tissue recovered from cell aggregates on day 12 and day 22 of culture in Example 9 of this embodiment. [Figure 31] This is an explanatory diagram showing a micrograph of a cell aggregate on day 23 of culture in Example 9-2 of Example 9 according to this embodiment. [Figure 32] This is an explanatory diagram showing a micrograph of the ciliary tissue recovered from the cell aggregate in Example 10 according to this embodiment. [Figure 33] This is an explanatory diagram showing a micrograph of a hair-like graft that was cultured in hydrogel in Example 11 of this embodiment. [Modes for carrying out the invention]

[0021] One embodiment of the present invention is described below. However, the present invention is not limited to this embodiment.

[0022] As one aspect, this embodiment includes a method for producing graftable hilar tissue, which involves performing cell culture to obtain cell aggregates on which hilar tissue is formed on the surface, and then cutting and recovering the hilar tissue from the cell aggregates for transplantation into a living organism.

[0023] Furthermore, this embodiment also includes, as another aspect, a hair regeneration method that includes transplanting hair-like tissue containing epithelial cells and mesenchymal cells but without capillaries into a living organism. That is, this embodiment includes the use of hair-like tissue containing epithelial cells and mesenchymal cells but without capillaries for transplantation into a living organism. Furthermore, as yet another aspect, this embodiment also includes transplantable hair-like tissue having one end portion that is a free end and the other end portion, containing epithelial cells and mesenchymal cells, and without capillaries.

[0024] In the production of hair-like tissue for transplantation, first, cell culture is performed to obtain cell aggregates in which hair-like tissue is formed on the surface. The cell culture for obtaining cell aggregates having hair-like tissue on the surface is not particularly limited as long as the effects of the present invention are obtained, but it is preferable to co-culture epithelial cells and mesenchymal cells, for example, by seeding epithelial cells and mesenchymal cells. That is, it is preferable to seed epithelial cells and mesenchymal cells and co-culture the seeded epithelial cells and mesenchymal cells to form cell aggregates in which hair-like tissue is formed on the surface.

[0025] The epithelial cells used in the formation of cell aggregates are not particularly limited as long as the effects of the present invention are obtained, but it is preferable that they be one or more selected from the group consisting of, for example, hair follicle epithelial cells and their precursor cells. Hair follicle epithelial cells are epithelial cells that contribute to hair growth (more specifically, epithelial cells that contribute to hair growth in cooperation with hair follicle mesenchymal cells).

[0026] Hair follicle epithelial cells may be collected from hair follicles in a living organism, or they may be differentiated from undifferentiated cells in vitro. The undifferentiated cells used to induce differentiation of hair follicle epithelial cells in vitro are not particularly limited as long as they have the ability to differentiate into hair follicle epithelial cells in vitro, but it is preferable that they be one or more selected from the group consisting of, for example, pluripotent stem cells (e.g., iPS (induced Pluripotent Stem) cells, ES (Embryonic Stem) cells, Muse (Multilineage-differentiating stress-enduring) cells, or EG (Embryonic Germ) cells) and stem cells other than pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells).

[0027] Specifically, the hair follicle epithelial cells are preferably one or more selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, outer root sheath cells, and inner root sheath cells, and are particularly preferably one or more selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, and outer root sheath cells.

[0028] The precursor cells of hair follicle epithelial cells are not particularly limited as long as they are cells that have the ability to differentiate into hair follicle epithelial cells in vitro, but it is preferable that they be one or more selected from the group consisting of, for example, fetal or neonatal cutaneous epithelial cells (e.g., epithelial cells derived from the epidermal layer of fetal or neonatal skin) and stem cells other than pluripotent stem cells that have the ability to differentiate into hair follicle epithelial cells in vitro.

[0029] The mesenchymal cells used in the formation of cell aggregates are not particularly limited as long as the effects of the present invention are obtained, but it is preferable that they be one or more selected from the group consisting of, for example, hair follicle mesenchymal cells and their precursor cells. 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).

[0030] Hair follicle mesenchymal cells may be collected from hair follicles in a living organism, or they may be differentiated from undifferentiated cells in vitro. The undifferentiated cells used to induce differentiation of hair follicle mesenchymal cells in vitro are not particularly limited as long as they have the ability to differentiate into hair follicle mesenchymal cells in vitro, but it is preferable that they be one or more selected from the group consisting of pluripotent stem cells (e.g., iPS cells, ES cells, Muse cells, or EG cells) and stem cells other than pluripotent stem cells (e.g., one or more selected from the group consisting of stem cells obtained by reprogramming differentiated cells and mesenchymal stem cells (e.g., adipose tissue-derived mesenchymal stem cells)).

[0031] 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 particularly preferably dermal papilla cells.

[0032] The precursor cells of hair follicle mesenchymal cells are not particularly limited as long as they have the ability to differentiate into hair follicle mesenchymal cells in vitro, but it is preferable that they be one or more selected from the group consisting of, for example, fetal or neonatal skin mesenchymal cells (e.g., mesenchymal cells derived from the dermis layer of fetal or neonatal skin) and stem cells other than pluripotent stem cells that have the ability to differentiate into hair follicle mesenchymal cells in vitro (e.g., adipose tissue-derived mesenchymal stem cells). The adipose tissue-derived mesenchymal stem cells are not particularly limited as long as the effects of the present invention are obtained, but for example, they are collected from the adipose tissue of a living organism (subcutaneous adipose tissue and / or other adipose tissue).

[0033] In co-culture, cell aggregates may be formed by co-culturing only epithelial cells and mesenchymal cells, or by co-culturing with other cells added. The other cells are not particularly limited as long as the effects of the present invention are obtained, but it is preferable that they be one or more selected from the group consisting of pigment cells, pigment progenitor cells, and pigment stem cells.

[0034] Other cells may be taken from hair follicles of living organisms, or they may be differentiated from undifferentiated cells in vitro. The undifferentiated cells used to induce differentiation of other cells in vitro are not particularly limited as long as they have the ability to differentiate into the other cells in vitro, but it is preferable that they be one or more selected from the group consisting of, for example, pluripotent stem cells (e.g., iPS cells, ES cells, Muse cells, or EG cells) and stem cells other than pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells).

[0035] The cells used in co-culture are not particularly limited as long as they originate from animals that have hair follicles; they may be derived from humans, or from animals other than humans (non-human animals, such as primates (e.g., monkeys), rodents (e.g., mice, rats, hamsters, guinea pigs, rabbits), carnivores (e.g., dogs, cats), and ungulates (e.g., pigs, cows, horses, goats, sheep, etc.)). However, if the purpose is transplantation into humans, human cells should be used.

[0036] The cells used in co-culture are preferably derived from the individual to whom the cells will be transplanted, but they may also be derived from an individual other than the individual to whom the cells will be transplanted. For example, the human cells used in co-culture are preferably derived from the human patient to whom the human cells will be transplanted, but they may also be derived from a human other than the patient (for example, cells differentiated in vitro from pluripotent stem cells (e.g., iPS cells, ES cells, Muse cells, or EG cells stored in a cell bank) derived from a human other than the patient).

[0037] In co-culture of epithelial cells and mesenchymal cells, the epithelial cells and mesenchymal cells are first seeded. In this regard, as a cell culture to obtain cell aggregates in which hair-like tissue is formed on the surface, a method can be employed in which cell aggregates having such hair-like tissue are formed by inducing differentiation of pluripotent stem cells, as described in Non-Patent Document 1 above. However, methods involving the induction of differentiation of pluripotent stem cells require long culture times and complicated procedures. Furthermore, methods involving the seeding of pluripotent stem cells require the addition of special components such as Matrigel (registered trademark) to the culture medium. In addition, in methods involving the seeding of pluripotent stem cells, tissues other than skin tissue may also be formed.

[0038] In contrast, when seeding epithelial cells and mesenchymal cells, it is not necessary to use pluripotent stem cells. Therefore, the method according to this embodiment may not include seeding pluripotent stem cells. Furthermore, the method according to this embodiment may not include differentiating pluripotent stem cells. Furthermore, the method according to this embodiment may not include culturing pluripotent stem cells.

[0039] Seeding of epithelial cells and mesenchymal cells is performed by placing the epithelial cells and mesenchymal cells into a culture vessel (for example, a well for cell culture). The culture vessel for co-culturing epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention are obtained, but for example, a culture vessel with a relatively small capacity that is suitable for forming a single cell aggregate from the cells including epithelial cells and mesenchymal cells seeded in the culture vessel is preferably used.

[0040] Specifically, the area of ​​the bottom surface of the culture vessel (for example, the bottom surface of one well) is, for example, 1000 mm². 2 The following are also acceptable: 500mm 2 Preferably, the following: 100 mm 2 More preferably, 50 mm 2 It is even more preferable that the following is true: 20 mm 2 The following is particularly preferable:

[0041] Also, 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, even more preferably 0.50 mm 2 or more, still more preferably 0.70 mm 2 or more, particularly preferably. The area of the bottom surface of the culture vessel may be specified by arbitrarily combining one of the above-described lower limit values and one of the above-described upper limit values.

[0042] In the seeding of epithelial cells and mesenchymal cells, it is preferable to seed the epithelial cells and the mesenchymal cells simultaneously, but one of the epithelial cells and the mesenchymal cells may be seeded first, and then the other may be seeded further.

[0043] The time interval between the seeding of one of the epithelial cells and the mesenchymal cells and the seeding of the other (that is, the time from the seeding of one type of cell to the completion of the seeding of both types of cells) is not particularly limited as long as the effects of the present invention can be obtained. 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 the epithelial cells and the mesenchymal cells, the other is also seeded to complete the seeding of both the epithelial cells and the mesenchymal cells.

[0044] The time interval between the seeding of one of the epithelial cells and the mesenchymal cells and the seeding of the other is preferably, for example, 45 hours or less, more preferably 36 hours or less, even more preferably 30 hours or less, and particularly preferably 24 hours or less.

[0045] Furthermore, the time interval between the seeding of one of the epithelial cells and the mesenchymal cells and the seeding of the other is preferably, for example, 18 hours or less, more preferably 15 hours or less, even more preferably 12 hours or less, and particularly preferably 9 hours or less.

[0046] Furthermore, the time interval between seeding one type of cell (epithelial cells or mesenchymal cells) and seeding the other is preferably, for example, 6 hours or less, more preferably 3 hours or less, even more preferably 1 hour or less, and particularly preferably 0 hours (i.e., seeding epithelial cells and mesenchymal cells simultaneously).

[0047] In seeding epithelial cells and mesenchymal cells, it is preferable to seed dispersed epithelial cells and dispersed mesenchymal cells. That is, when seeding epithelial cells and mesenchymal cells simultaneously, a cell suspension in which the epithelial cells and mesenchymal cells are dispersed is placed in the culture vessel. Also, when seeding one of the epithelial cells or mesenchymal cells first, and then seeding the other, a cell suspension in which the cells of the first type are dispersed is first placed in the culture vessel, and then a cell suspension in which the cells of the other type are dispersed is added to the culture vessel.

[0048] As described above, epithelial and mesenchymal cells seeded using a cell suspension are dispersed and mixed in the culture medium within the culture vessel. Individual cells dispersed in the culture medium are substantially not bound to other cells, or are attached to other cells but can be easily separated from them by flowing the culture medium through operations such as pipetting.

[0049] The seeding density of epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention are obtained, but it is preferable that the density is such that individual seeded cells can come into contact with adjacent cells in the culture vessel.

[0050] Specifically, the seeding density of epithelial and mesenchymal cells (1 cm from the bottom of the culture vessel) 2 The total number of epithelial and mesenchymal cells seeded per area is, for example, 0.1 × 10⁻⁶. 4 pieces / cm 2 It may be greater than or equal to 0.5 × 10 4 pieces / cm 2 Preferably, it is 1.0 × 10 4 pieces / cm 2 It is more preferable that the above be the case, 2.5 × 104 pieces / cm 2 It is even more preferable that the above is true, 5.0 × 10 4 pieces / cm 2 The above is particularly preferable. Furthermore, the seeding density of epithelial cells and mesenchymal cells should be, for example, 1000 × 10 4 pieces / cm 2 The following are also acceptable: 700 x 10 4 pieces / cm 2 Preferably, it is 500 × 10 4 pieces / cm 2 It is more preferable that the following conditions apply: 400 × 10 4 pieces / cm 2 It is even more preferable that the following conditions apply: 300 × 10 4 pieces / cm 2 The following is particularly preferable: The seeding densities of epithelial and mesenchymal cells may be determined by arbitrarily combining one of the lower limits and one of the upper limits mentioned above.

[0051] In co-culture, the ratio of the total number of epithelial cells to the total number of mesenchymal cells to the total number of cells seeded is not particularly limited as long as the effects of the present invention are obtained, but may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0052] Similarly, the ratio of the total number of epithelial cells to the total number of mesenchymal cells to the total number of cells constituting the cell aggregate formed by co-culture is not particularly limited as long as the effects of the present invention are obtained, but may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0053] Furthermore, the ratio of the number of epithelial cells seeded to the number of mesenchymal cells seeded in co-culture (mesenchymal:epithelial seeding ratio) is not particularly limited as long as the effects of the present invention are 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, more preferably in the range of 1:8 to 8:1, even more preferably in the range of 1:7 to 7:1, and particularly preferably in the range of 1:6 to 6:1.

[0054] Furthermore, the mesenchymal:epithelial seeding ratio is preferably in the range of 1:5 to 5:1, more preferably in the range of 1:4 to 4:1, even more preferably in the range of 1:3 to 3:1, and particularly preferably in the range of 1:2 to 2:1.

[0055] In co-culture, epithelial cells and mesenchymal cells are mixed and cultured in a culture medium, causing the epithelial cells and mesenchymal cells to aggregate and form cell aggregates. More specifically, in co-culture, epithelial cells and mesenchymal cells are seeded in a dispersed and mixed state in a culture medium, and as the culture time progresses, the epithelial cells aggregate with each other, the mesenchymal cells aggregate with each other, and intercellular junctions are formed between some epithelial cells and some mesenchymal cells. As a result, a cell aggregate is obtained that includes epithelial cell aggregates formed by the aggregation of epithelial cells, mesenchymal cell aggregates formed by the aggregation of mesenchymal cells, and intercellular junctions between some epithelial cells and some mesenchymal cells.

[0056] In order to form cell aggregates, epithelial cells and mesenchymal cells need to aggregate in the culture medium. Therefore, the co-culture of epithelial cells and mesenchymal cells for the formation of such cell aggregates is carried out in a culture medium that is generally fluid.

[0057] Furthermore, in co-culture, 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-adherent state to form non-adherent cell aggregates.

[0058] For suspension culture, a culture vessel with a non-cell-adherent bottom surface is preferably used. In this case, epithelial cells and mesenchymal cells are cultured on the non-cell-adherent bottom surface without substantially adhering to it (i.e., in a non-adherent state). That is, for example, epithelial cells and mesenchymal cells that have settled on the non-cell-adherent bottom surface do not adhere to the bottom surface in the culture medium, or they adhere to the bottom surface so weakly that they can be easily detached by flowing the culture medium through operations such as pipetting. The shape of epithelial cells and mesenchymal cells cultured on a non-cell-adherent bottom surface is maintained to be approximately spherical.

[0059] Furthermore, cell aggregates are formed on a non-cell-adherent bottom surface without substantially adhering to it (i.e., in a non-adherent state). That is, for example, cell aggregates formed on a non-adherent bottom surface do not adhere to the bottom surface in the culture medium, or they adhere to the bottom surface so weakly that they can be easily detached by flowing the culture medium through operations such as pipetting.

[0060] Furthermore, in co-culture, it is preferable to form a single cell aggregate within a single culture vessel (for example, a single well) from cells including epithelial cells and mesenchymal cells seeded in the culture vessel.

[0061] The co-culture of epithelial cells and mesenchymal cells for producing cell aggregates having hair-like tissue is not particularly limited as long as the effects of the present invention are obtained, but it is preferable to include a matrix treatment in which the epithelial cells and mesenchymal cells are held in a culture medium in which type I collagen, fibronectin, laminin and entactin, or type IV collagen (hereinafter these may be collectively referred to as "matrix") is dispersed.

[0062] Matrix treatment is a process that, for example, maintains epithelial cells and mesenchymal cells in a culture medium in which type I collagen is dispersed as the matrix. Alternatively, matrix treatment can also be a process that maintains epithelial cells and mesenchymal cells in a culture medium in which fibronectin is dispersed as the matrix.

[0063] Matrix treatment is a process of maintaining epithelial and mesenchymal cells in a culture medium in which laminin and enterin (e.g., laminin / entactin complex) are dispersed as the matrix. Matrix treatment is also a process of maintaining epithelial and mesenchymal cells in a culture medium in which type IV collagen is dispersed as the matrix.

[0064] A culture medium containing a dispersed matrix, used for matrix treatment (hereinafter sometimes referred to as "matrix treatment culture medium"), is prepared by adding the matrix to a basic culture medium (for example, a culture medium that can be used for co-culturing epithelial cells and mesenchymal cells). In other words, the matrix treatment culture medium contains an externally added matrix.

[0065] The culture medium for matrix treatment contains the added matrix in a solubilized (not insolubilized) state. That is, the culture medium for matrix treatment contains, as dispersed matrix, solubilized type I collagen (non-insolubilized type I collagen), solubilized fibronectin (non-insolubilized fibronectin), solubilized laminin and entactin (non-insolubilized laminin and entactin), or solubilized type IV collagen (non-insolubilized type IV collagen).

[0066] The basic culture medium is not particularly limited as long as the effects of the present invention are obtained, but for example, a culture medium prepared by adding 1% GultaMax Supplement (GIBCO®) and 0.2% Normocin (InvivoGen) to DMEM / F12 medium (Advanced Dulbecco's Modified Eagle Medium / Ham's F-12, GIBCO®) is preferably used.

[0067] The matrix added in the preparation of the culture medium for matrix treatment is not particularly limited as long as the effects of the present invention are obtained, but commercially available matrix products, such as those used in the examples described later, are preferably used.

[0068] Furthermore, the added matrix may be derived from animals (human or non-human animals), cultured cells, or synthesized using genetic engineering technology.

[0069] Furthermore, the added matrix may be treated to reduce its antigenicity. For example, the collagen may be atelocollagen from which the telopeptide portion has been removed.

[0070] A matrix-treated culture medium containing a specific matrix may or may not contain any other matrix.

[0071] In other words, for example, a matrix treatment culture medium containing dispersed type I collagen may further contain fibronectin, laminin, entactin, or type IV collagen, or it may not contain fibronectin, laminin, entactin, or type IV collagen.

[0072] Furthermore, for example, a matrix treatment culture medium containing dispersed fibronectin may also contain fibronectin, laminin, entactin, or type IV collagen, or it may not contain fibronectin, laminin, entactin, or type IV collagen.

[0073] Furthermore, for example, the matrix treatment culture medium in which laminin and entactin are dispersed may also contain type I collagen, fibronectin, or type IV collagen, or it may not contain type I collagen, fibronectin, or type IV collagen.

[0074] Furthermore, for example, a matrix treatment culture medium containing dispersed type IV collagen may also contain type I collagen, fibronectin, laminin, or entactin, or it may not contain type I collagen, fibronectin, laminin, or entactin.

[0075] As a culture medium for matrix treatment, a culture medium for matrix treatment in which type I collagen is dispersed is particularly preferred. Furthermore, it is preferable that the culture medium for matrix treatment mainly contains type I collagen as the matrix. That is, in a culture medium for matrix treatment in which type I collagen is dispersed, the weight ratio of the content of type I collagen to the total content of type I collagen, fibronectin, laminin, entactin, and type IV collagen may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and particularly preferably 90% by weight or more.

[0076] Furthermore, the matrix treatment culture medium in which fibronectin is dispersed may mainly contain fibronectin as the matrix. That is, in the matrix treatment culture medium in which fibronectin is dispersed, the weight ratio of the fibronectin content to the total content of type I collagen, fibronectin, laminin, entactin, and type IV collagen may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and particularly preferably 90% by weight or more.

[0077] Furthermore, the matrix treatment culture medium in which laminin and entactin are dispersed may mainly contain laminin and entactin as the matrix. That is, in the matrix treatment culture medium in which laminin and entactin are dispersed, the weight ratio of the total content of laminin and entactin to the total content of type I collagen, fibronectin, laminin, entactin, and type IV collagen may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and particularly preferably 90% by weight or more.

[0078] Furthermore, the matrix treatment culture medium in which type IV collagen is dispersed may mainly contain type IV collagen as the matrix. That is, in the matrix treatment culture medium in which type IV collagen is dispersed, the weight ratio of the content of type IV collagen to the total content of type I collagen, fibronectin, laminin, entactin, and type IV collagen may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, even more preferably 80% by weight or more, and particularly preferably 90% by weight or more.

[0079] The culture medium for matrix treatment contains the matrix at a concentration that allows the culture medium as a whole to maintain fluidity in the co-culture of epithelial cells and mesenchymal cells. In this regard, for example, when cells are generally cultured embedded in a hydrogel of matrix, first a suspension containing the cells and the matrix at a concentration suitable for gelation is prepared under conditions that do not cause the matrix to gel (e.g., a temperature at which gelation does not occur), and then the entire suspension containing the cells and matrix in the culture vessel is gelled under conditions suitable for the gelation of the matrix (e.g., a temperature at which gelation occurs). As a result, a non-fluid hydrogel is formed that fills the culture vessel.

[0080] In contrast, the matrix-treated culture medium contains the matrix at a concentration that allows the matrix-treated culture medium in the culture vessel (e.g., a well) to maintain overall fluidity even when epithelial cells and mesenchymal cells are co-cultured in the matrix-treated culture medium. (i.e., a concentration that does not form a non-fluid hydrogel filling the culture vessel.) The matrix-treated culture medium that maintains overall fluidity may contain, for example, gel-like substances suspended in the culture medium due to localized imbalances in the concentration of dispersed matrix. However, these gel-like substances are not, for example, intentionally formed hydrogel beads (e.g., hydrogel beads with embedded cells), and do not have a specific shape such as a sphere.

[0081] In matrix processing, the purpose of dispersing the matrix in the culture medium for matrix processing is not to gel the entire culture medium; therefore, the concentration of the matrix in the culture medium is generally lower than the concentration used to gel the entire culture medium.

[0082] The concentration of type I collagen in the matrix treatment culture medium in which type I collagen is dispersed is not particularly limited as long as the effects of the present invention are obtained, but for example, it is preferably less than 480 μg / mL, more preferably 460 μg / mL or less, even more preferably 420 μg / mL or less, and particularly preferably 400 μg / mL or less.

[0083] Furthermore, the concentration of type I collagen in the matrix treatment culture medium is preferably, for example, 380 μg / mL or less, more preferably 350 μg / mL or less, even more preferably 300 μg / mL or less, and particularly preferably 260 μg / mL or less.

[0084] Furthermore, the concentration of type I collagen in the matrix treatment culture medium is preferably, for example, 3 μg / mL or more, more preferably 5 μg / mL or more, even more preferably 10 μg / mL or more, and particularly preferably 12 μg / mL or more.

[0085] Furthermore, the concentration of type I collagen in the matrix-treated culture medium is preferably, for example, 14 μg / mL or higher, more preferably 16 μg / mL or higher, even more preferably 18 μg / mL or higher, and particularly preferably 20 μg / mL or higher. The concentration of type I collagen in the matrix-treated culture medium may be determined by arbitrarily combining one of the upper limits and one of the lower limits mentioned above.

[0086] 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 are obtained, but for example, it is preferably 450 μg / mL or less, more preferably 400 μg / mL or less, even more preferably 350 μg / mL or less, and particularly preferably 300 μg / mL or less.

[0087] Furthermore, 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, even more preferably 150 μg / mL or less, and particularly preferably 120 μg / mL or less.

[0088] Furthermore, the concentration of fibronectin in the matrix-treated culture medium is preferably, for example, 1 μg / mL or more, more preferably 2 μg / mL or more, even more preferably 3 μg / mL or more, and particularly preferably 4 μg / mL or more. The concentration of fibronectin in the matrix-treated culture medium may be determined by arbitrarily combining one of the upper limits and one of the lower limits mentioned above.

[0089] The concentrations of laminin and entactin (the sum of the laminin and entactin concentrations) in the matrix treatment culture medium in which laminin and entactin are dispersed are not particularly limited as long as the effects of the present invention are obtained, but for example, it is preferably 1500 μg / mL or less, more preferably 1000 μg / mL or less, and particularly preferably 500 μg / mL or less.

[0090] Furthermore, the concentrations of laminin and entactin in the matrix-treated culture medium are preferably, for example, 1 μg / mL or higher, more preferably 3 μg / mL or higher, and particularly preferably 5 μg / mL or higher. The concentrations of laminin and entactin in the matrix-treated culture medium may be determined by arbitrarily combining one of the upper limits and one of the lower limits mentioned above.

[0091] The concentration of type IV collagen in the matrix treatment culture medium in which type IV collagen is dispersed is not particularly limited as long as the effects of the present invention are obtained, but for example, it is preferably 1000 μg / mL or less, more preferably 500 μg / mL or less, and particularly preferably 300 μg / mL or less.

[0092] Furthermore, the concentration of type IV collagen in the matrix-treated culture medium is preferably, for example, 1 μg / mL or more, more preferably 3 μg / mL or more, and particularly preferably 5 μg / mL or more. The concentration of type IV collagen in the matrix-treated culture medium may be determined by arbitrarily combining one of the upper limits and one of the lower limits mentioned above.

[0093] The matrix that comes into contact with epithelial and mesenchymal cells during matrix treatment is a solubilized matrix dispersed in a matrix treatment culture medium that is fluid overall. In other words, the dispersed matrix that comes into contact with epithelial and mesenchymal cells during matrix treatment is not, for example, the matrix constituting the hydrogel in a case where epithelial and mesenchymal cells are embedded in a hydrogel that is not fluid overall (for example, a hydrogel formed by pouring a solution containing a hydrogel polymer into a culture vessel and gelling the entire solution within the culture vessel). Furthermore, the dispersed matrix that comes into contact with epithelial and mesenchymal cells during matrix treatment is not, for example, the matrix constituting the hydrogel in a case where epithelial and mesenchymal cells are held on the surface of a hydrogel that is not fluid overall. Furthermore, the dispersed matrix that comes into contact with epithelial and mesenchymal cells during matrix treatment is not, for example, a matrix that is pre-immobilized on the culture surface on which epithelial and mesenchymal cells are held (for example, a matrix pre-coated on the bottom surface of a culture vessel such as a well).

[0094] In matrix treatment, epithelial cells and mesenchymal cells held on the surface of a hydrogel that does not have overall fluidity (a hydrogel containing a matrix, or a hydrogel without a matrix) may be held in a matrix treatment culture medium that has overall fluidity and brought into contact with the matrix dispersed in the culture medium.

[0095] However, the matrix treatment may not include holding epithelial cells and mesenchymal cells, which are held on the surface of a hydrogel containing a matrix and not having fluidity as a whole, in a matrix treatment culture medium and bringing them into contact with the matrix dispersed in the culture medium.

[0096] Furthermore, the matrix treatment may not include holding epithelial cells and mesenchymal cells, which are held on the surface of a hydrogel that does not contain a matrix and is not fluid as a whole, in a matrix treatment culture medium and bringing them into contact with the matrix dispersed in the culture medium.

[0097] Furthermore, the matrix treatment may not include holding epithelial cells and mesenchymal cells, which are held on the surface of a hydrogel that does not have overall fluidity (whether or not the hydrogel contains a matrix), in a matrix treatment culture medium and bringing them into contact with the matrix dispersed in the culture medium.

[0098] Matrix treatment may include holding epithelial cells and mesenchymal cells, which are held on a culture surface with a matrix immobilized on it, in a matrix treatment culture medium and bringing them into contact with the matrix dispersed in the culture medium.

[0099] However, the matrix treatment may not include bringing epithelial cells and mesenchymal cells, which are held on a culture surface with a matrix already immobilized, into contact with the matrix dispersed in the culture medium for matrix treatment.

[0100] The matrix treatment may not include embedding and culturing epithelial and mesenchymal cells in a matrix-containing hydrogel before cell aggregates are formed. Alternatively, the matrix treatment may not include embedding and culturing epithelial and mesenchymal cells in a matrix-free hydrogel before cell aggregates are formed. Furthermore, the matrix treatment may not include embedding and culturing epithelial and mesenchymal cells in a hydrogel (whether or not the hydrogel contains a matrix) before cell aggregates are formed.

[0101] In matrix treatment, the temperature at which epithelial cells and mesenchymal cells are maintained in the matrix treatment culture medium is not particularly limited as long as the effects of the present invention are obtained, but it is preferable that the temperature is suitable for culturing the epithelial cells and mesenchymal cells (for example, a temperature of 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, even more preferably 35°C or higher and 39°C or lower, and particularly preferably 36°C or higher and 38°C or lower).

[0102] In matrix treatment, the time for retaining epithelial cells and mesenchymal cells in the matrix treatment culture medium is not particularly limited as long as the effects of the present invention are obtained, but for example, it may be 3 hours or more, preferably 6 hours or more, more preferably 9 hours or more, and particularly preferably 12 hours or more.

[0103] Furthermore, in the matrix treatment, it is preferable to retain epithelial cells and mesenchymal cells in the matrix treatment culture medium for 15 hours or more, more preferably for 18 hours or more, even more preferably for 21 hours or more, and particularly preferably for 24 hours or more.

[0104] The timing for initiating the matrix treatment is not particularly limited as long as the effects of the present invention are obtained, but it is preferable, for example, to start the matrix treatment within 72 hours of seeding epithelial and mesenchymal cells for co-culture.

[0105] Furthermore, the time interval from seeding of the epithelial and mesenchymal cells to the start of matrix treatment is more preferably 66 hours or less, even more preferably 60 hours or less, and particularly preferably 54 hours or less.

[0106] Furthermore, the time interval from seeding of epithelial and mesenchymal cells to the start of matrix treatment is preferably 48 hours or less, more preferably 45 hours or less, even more preferably 42 hours or less, and particularly preferably 39 hours or less.

[0107] Furthermore, the time interval from seeding of epithelial and mesenchymal cells to the start of matrix treatment is preferably 36 hours or less, more preferably 33 hours or less, even more preferably 30 hours or less, and particularly preferably 27 hours or less.

[0108] Furthermore, the time interval from seeding of epithelial and mesenchymal cells to the start of matrix treatment is preferably 24 hours or less, more preferably 21 hours or less, even more preferably 18 hours or less, and particularly preferably 15 hours or less.

[0109] Furthermore, the time interval from seeding of epithelial and mesenchymal cells to the start of matrix treatment is preferably 12 hours or less, more preferably 5 hours or less, even more preferably 3 hours or less, and particularly preferably 1 hour or less.

[0110] It is preferable to start the matrix treatment simultaneously with the seeding of epithelial and mesenchymal cells (the time interval between seeding of epithelial and mesenchymal cells and the start of the matrix treatment is 0 hours). In this case, for example, the cell suspension prepared by dispersing epithelial and mesenchymal cells in a matrix treatment culture medium is placed in a culture vessel, thereby starting the matrix treatment simultaneously with seeding the epithelial and mesenchymal cells.

[0111] On the other hand, if matrix treatment is to be started after seeding of epithelial and mesenchymal cells, for example, the epithelial and mesenchymal cells are first seeded in a culture medium without added matrix, and then, after a period of time within the time intervals described above has elapsed since seeding, the amount of matrix required for matrix treatment (for example, an amount of matrix such that the concentration in the culture medium after addition falls within the range specified by one of the upper and / or lower limits described above) is added to the culture medium containing the epithelial and mesenchymal cells to start the matrix treatment.

[0112] In matrix treatment, it is preferable to retain the epithelial and mesenchymal cells in a settled state within the matrix treatment culture medium. That is, in this case, co-culture includes matrix treatment, which involves settling the seeded epithelial and mesenchymal cells in the culture medium and retaining the settled epithelial and mesenchymal cells in the matrix treatment culture medium.

[0113] More specifically, for example, first, dispersed epithelial and mesenchymal cells are allowed to settle in the culture medium and accumulate on the bottom surface of the culture vessel. Then, the epithelial and mesenchymal cells accumulated on the bottom surface are held in a matrix-treated culture medium that is fluid overall, bringing the epithelial and mesenchymal cells into contact with the matrix dispersed in the culture medium.

[0114] The method for settling epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention are obtained, but for example, a method of letting the culture vessel in which the epithelial cells and mesenchymal cells are seeded stand still, and / or a method of centrifugation of the culture vessel are preferably used.

[0115] It is preferable to perform the sedimentation of epithelial cells and mesenchymal cells before initiating the matrix treatment at the temperature at which the epithelial cells and mesenchymal cells will be co-cultured. That is, for example, the epithelial cells and mesenchymal cells may first be sedimented in a matrix treatment culture medium at a temperature lower than the temperature at which the co-culture will be performed (for example, preferably 10°C or lower (above 0°C and below 10°C), more preferably 7°C or lower, and particularly preferably 5°C or lower), and then the matrix treatment may be performed at the temperature at which the co-culture will be performed (for example, a temperature of 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, even more preferably 35°C or higher and 39°C or lower, and particularly preferably 36°C or higher and 38°C or lower).

[0116] Alternatively, for example, epithelial cells and mesenchymal cells may first be allowed to settle in a culture medium without the addition of matrix, and then the amount of matrix necessary for matrix treatment (for example, an amount of matrix such that the concentration in the culture medium after addition is determined by one of the upper and / or lower limits mentioned above) may be added to the culture medium, and the matrix treatment may be carried out at a temperature suitable for co-culture. In this case, the matrix may be added to the settled epithelial cells and mesenchymal cells at a temperature lower than the temperature at which co-culture is performed (for example, preferably 10°C or lower (above 0°C and below 10°C), more preferably 7°C or lower, and particularly preferably 5°C or lower), and then the matrix treatment may be carried out at a temperature suitable for co-culture.

[0117] Matrix treatment is performed as part or all of a co-culture of epithelial and mesenchymal cells. That is, the entire co-culture may be performed in a matrix-treated culture medium. Alternatively, the co-culture may be performed in a matrix-treated culture medium until cell aggregates are formed. Alternatively, part of the co-culture may be performed in a matrix-treated culture medium, and the remaining part in a culture medium with a lower matrix concentration than the matrix-treated culture medium.

[0118] More specifically, for example, co-culture may include matrix treatment of epithelial cells and mesenchymal cells in a matrix treatment culture medium containing the matrix at a first concentration, and then continuing the co-culture of the epithelial cells and mesenchymal cells in a culture medium containing the matrix at a second concentration lower than the first concentration.

[0119] In this case, the second concentration is not particularly limited as long as the effects of the present invention are obtained, but the ratio of the second concentration to the first concentration may be, for example, 90% or less, 70% or less, 50% or less, 30% or less, or 10% or less. Furthermore, two or more different concentrations may be used as the second concentration. That is, the second concentration may change (for example, the second concentration may decrease) as the culture time progresses.

[0120] The method for reducing the matrix concentration in the culture medium during co-culturing to that of the matrix-treated culture medium is not particularly limited as long as the effects of the present invention are obtained. For example, after a certain amount of time has elapsed since the start of the co-culturing, a portion of the matrix-treated culture medium in the culture vessel may be removed and replaced with a culture medium in which the matrix concentration is lower than that of the matrix-treated culture medium (for example, a culture medium without the matrix added).

[0121] In co-culture, it is preferable to continuously perform seeding of epithelial cells and mesenchymal cells, matrix treatment, and cell aggregate formation within the same culture vessel (e.g., the same well). That is, once epithelial cells and mesenchymal cells are seeded in the culture vessel, it is preferable to perform matrix treatment and cell aggregate formation within the same culture vessel without removing the epithelial cells and mesenchymal cells from the vessel.

[0122] Co-culture including matrix treatment can yield cell aggregates with amplified expression levels of one or more hair growth-related genes compared to cell aggregates formed under identical conditions except that a culture medium without a dispersed matrix (i.e., a culture medium without the matrix) is used instead of a matrix-treated culture medium (hereinafter sometimes referred to as "control cell aggregates"). Specifically, for example, cell aggregates can be obtained in which the expression level of one or more hair growth-related genes is more than twice as high as that of the control cell aggregates.

[0123] The hair growth-related genes expressed by the cell aggregates are not particularly limited as long as they are genes related to hair growth, but may be, for example, one or more marker genes related to hair follicle development (e.g., one or more selected from the group consisting of Tgfb2, Sox21, Lgr5, Lhx2, Edaradd, Pdgfa, and Lgr4).

[0124] In other words, the cell aggregates obtained by co-culture including matrix treatment may have twice or more expression levels of the Tgfb2 gene, twice or more expression levels of the Sox21 gene, twice or more expression levels of the Lgr5 gene, twice or more expression levels of the Lhx2 gene, twice or more expression levels of the Edaradd gene, twice or more expression levels of the Pdgfa gene, or twice or more expression levels of the Lgr4 gene compared to the control cell aggregates.

[0125] By employing a co-culture of epithelial cells and mesenchymal cells, which includes seeding of epithelial cells and mesenchymal cells, it is possible to effectively produce cell aggregates with ciliary tissue formed on their surface in vitro.

[0126] Furthermore, the cell culture required to obtain cell aggregates with hair-like tissue formed on their surface may, for example, be a differentiation induction culture of pluripotent stem cells such as iPS cells, as described in Non-Patent Document 1 above.

[0127] In this regard, Non-Patent Document 1 described above describes the following differentiation induction culture method. First, iPS cells are suspended in ectodermal differentiation medium and placed in wells (96-well plate) with a low cell adhesion U-shaped base in 3 × 10⁶ 3 Seeds are sown at a density of one seed per well (100 μL).

[0128] On day 1 of culture, remove half (50 μL) of the culture medium from the wells and add 50 μL of fresh ectoderm differentiation medium containing 4% (v / v) Matrigel® (final concentration after addition is 2% (v / v)).

[0129] On day 3 of culture, 25 μL of fresh ectoderm differentiation medium containing 50 ng / mL to 250 ng / mL of BMP-4 and 5 μM of SB431542 (ALK inhibitor), without Matrigel, is added to each well (after addition, the BMP-4 concentration is 10 ng / mL to 50 ng / mL, the SB431542 concentration is 1 μM, and the culture volume is 125 μL / well).

[0130] On day 4 of culture, 25 μL of fresh ectoderm differentiation medium containing 6 μM LDN (ALK inhibitor) and 150 ng / mL FGF-2, without Matrigel, is added to each well (after addition, the LDN concentration is 1 μM, the FGF-2 concentration is 25 ng / mL, and the culture volume is 150 μL / well).

[0131] On day 8 of culture, the cell aggregates in the wells are transferred to wells (24-well plate) containing 500 μL of maturation medium with 1% (v / v) Matrigel.

[0132] From day 10 of culture until day 30 of culture, perform a medium change every two days by removing half (250 μL) of the culture medium in the well and adding 250 μL of fresh mature medium that does not contain Matrigel.

[0133] However, the method of forming hair-like tissue in vitro using differentiation induction of pluripotent stem cells such as iPS cells is complicated, and care must be taken to ensure the safety of the cells that make up the hair-like tissue formed on the surface of the cell aggregates when transplanting them into a living organism.

[0134] In contrast, a method that involves seeding epithelial cells and mesenchymal cells without using pluripotent stem cells, and then co-culturing these epithelial and mesenchymal cells to form cell aggregates containing hair-like tissue, is simpler to perform and also easier to ensure the safety of transplanting the hair-like tissue into a living organism.

[0135] The pilosarconiae of a cell aggregate contains epithelial and mesenchymal cells and is formed as a structure that protrudes from the surface of the cell aggregate. The pilosarconiae formed on the surface of a cell aggregate has, for example, a dermal papilla-like structure at its free end (the tip). However, the pilosarconiae formed on the surface of a cell aggregate does not have a dermal papilla-like structure at its base (the end opposite the free end).

[0136] The dermal papilla-like structures in hair-like tissue have a structure similar to the dermal papilla within a living hair follicle. That is, the dermal papilla-like structures have a spherical shape. Furthermore, the dermal papilla-like structures contain mesenchymal cells (e.g., dermal papilla cells). In other words, the dermal papilla-like structures are identified, for example, as aggregates of Versican-positive cells.

[0137] Specifically, the ratio of the number of mesenchymal cells (e.g., dermal papilla cells) contained in a dermal papilla-like structure to the total number of cells constituting the dermal papilla-like structure may be, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.

[0138] Furthermore, 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 a living hair follicle. 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 contained in the tip of the hair-like tissue toward the root of the hair-like tissue. It is preferable that the hair shaft-like structure contains melanin.

[0139] Furthermore, the pilosa tissue does not contain capillaries. In other words, for example, the dermal papilla-like structure contained in the pilosa tissue does not contain capillaries, unlike the dermal papilla contained in hair follicles taken from living organisms.

[0140] The length of the cilia protruding from the surface of the cell aggregate changes over time, but may be, for example, 200 μm or more, preferably 400 μm or more, more preferably 600 μm or more, even more preferably 800 μm or more, and particularly preferably 1000 μm or more.

[0141] Furthermore, the length of the cilia protruding from the surface of the cell aggregate is preferably, for example, 2 mm (2000 μm) or more, more preferably 4 mm or more, even more preferably 6 mm or more, and particularly preferably 8 mm or more. Alternatively, the length of the cilia protruding from the surface of the cell aggregate may be, for example, 100 mm or less.

[0142] Cell aggregates with hair-like tissue may contain cyst-like structures within them. These cyst-like structures are identified as structures without a cell nucleus, surrounded by cells containing a cell nucleus. Specifically, the cyst-like structure is observed, for example, in the central part of a cross-section of a cell aggregate stained with HE (hematoxylin-eosin), as a structure without a cell nucleus, surrounded by cells containing a cell nucleus, and stained pink.

[0143] Furthermore, for example, when forming cell aggregates without using pluripotent stem cells, the cell aggregates may differ from the tissue formed in Non-Patent Document 1 above in that they do not contain arrector pili muscle structures and / or sebaceous gland structures. It is also preferable that the cell aggregates have hair regeneration capabilities. The hair regeneration capability of the cell aggregates is their ability to cause hair growth at the site where they are transplanted into a living organism.

[0144] The co-culture may include performing suspension culture of epithelial cells and mesenchymal cells to form cell aggregates, and embedding the cell aggregates formed by the suspension culture in a hydrogel and culturing them further.

[0145] In this case, in suspension culture, cell aggregates without pithyroid tissue on their surface may be formed (for example, cell aggregates that have the ability to form pithyroid tissue on their surface but have not yet formed it), or cell aggregates with pithyroid tissue on their surface may be formed.

[0146] Next, the cell aggregates formed by suspension culture are embedded in a hydrogel and cultured. Specifically, the cell aggregates are first held in a hydrogel-forming solution (hereinafter sometimes referred to as "hydrogel-forming solution") within a culture vessel, and then the entire solution in the culture vessel is gelled, thereby embedding the cell aggregates and forming a hydrogel that does not flow as a whole.

[0147] The hydrogel-forming solution is not particularly limited as long as the effects of the present invention are obtained, but it is preferable to include a matrix, for example. That is, the hydrogel-forming solution preferably contains one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, more preferably contains one or more selected from the group consisting of type I collagen, fibronectin, and laminin and entactin, and is particularly preferable to contain type I collagen.

[0148] Furthermore, the hydrogel in which the cell aggregates are embedded preferably contains one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, more preferably contains one or more selected from the group consisting of type I collagen, fibronectin, and laminin and entactin, and particularly preferably contains type I collagen.

[0149] The hydrogel-forming solution containing the matrix, and the hydrogel containing the matrix, may or may not contain other matrices.

[0150] That is, for example, a hydrogel-forming solution containing type I collagen and a hydrogel containing type I collagen may have fibronectin, laminin, entactin, or type IV collagen added to them, or they may not have fibronectin, laminin, entactin, or type IV collagen added.

[0151] A hydrogel-forming solution containing the matrix is ​​prepared by adding the matrix to a basic solution (for example, an aqueous solution capable of maintaining the survival of epithelial and mesenchymal cells, which may be a culture medium suitable for co-culturing epithelial and mesenchymal cells), similar to a culture medium for matrix treatment.

[0152] The concentration of the matrix contained in the hydrogel-forming solution is not particularly limited as long as the effects according to the present invention are obtained, but it is preferable that the concentration is such that the entire solution in the culture vessel gels at a temperature suitable for cultivation.

[0153] 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 the gelation of the entire solution are preferably, for example, 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. Alternatively, the concentration of type I collagen in the hydrogel-forming solution and the concentration of type I collagen in the hydrogel obtained by the gelation of the entire solution may be, for example, 3500 μg / mL or less.

[0154] Furthermore, when forming a hydrogel containing fibronectin, the fibronectin concentration in the hydrogel-forming solution and the fibronectin concentration in the hydrogel obtained by the gelation of the entire solution are preferably, for example, 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. Alternatively, the fibronectin concentration in the hydrogel-forming solution and the fibronectin concentration in the hydrogel obtained by the gelation of the entire solution may be, for example, 3500 μg / mL or less.

[0155] Furthermore, when forming a hydrogel containing laminin and entactin, the concentrations of laminin and entactin in the hydrogel-forming solution (the sum of the laminin and entactin concentrations) and the concentrations of laminin and entactin in the hydrogel obtained by the gelation of the entire solution are preferably, for example, 1800 μg / mL or higher, more preferably 2000 μg / mL or higher, and particularly preferably 2200 μg / mL or higher. Alternatively, the concentrations of laminin and entactin in the hydrogel-forming solution and the concentrations of laminin and entactin in the hydrogel obtained by the gelation of the entire solution may be, for example, 3500 μg / mL or lower.

[0156] The type of matrix contained in the hydrogel embedding the cell aggregates may be the same as, or different from, the type of matrix used in the matrix treatment during the co-culture for the formation of the cell aggregates.

[0157] In other words, cell aggregates may be formed by co-culture including matrix treatment with one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, and then the cell aggregates may be embedded in a hydrogel containing one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen and cultured.

[0158] Alternatively, cell aggregates may be formed by co-culture including a matrix treatment using one or more selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen. Subsequently, the cell aggregates may be embedded in a hydrogel containing one or more of the matrix used in the matrix treatment, selected from the group consisting of type I collagen, fibronectin, laminin and entactin, and type IV collagen, and cultured.

[0159] Furthermore, from the viewpoint of cost and safety, it is particularly preferable to form cell aggregates by co-culture including matrix treatment with type I collagen, and then embed the cell aggregates in a hydrogel containing type I collagen and culture them.

[0160] Hydrogel-embedded culture of cell aggregates is preferably carried out, for example, by adding a culture medium to the hydrogel in a culture vessel and culturing the cell aggregates inside the hydrogel.

[0161] By embedding cell aggregates without ciliary tissue on their surface in a hydrogel and culturing them, ciliary tissue can be effectively formed on the surface of the cell aggregates within the hydrogel. Furthermore, by embedding cell aggregates with ciliary tissue already formed on their surface in a hydrogel and culturing them, the ciliary tissue can be effectively grown within the hydrogel (for example, its length can be effectively increased). Thus, by including hydrogel-embedded culture of cell aggregates in the co-culture, the formation of ciliary structures in the cell aggregates can be effectively promoted.

[0162] In the production of graftable hair-like tissue, the hair-like tissue formed on the surface of the cell aggregate as described above is cut and recovered from the cell aggregate for transplantation into a living organism. The method of cutting the hair-like tissue from the cell aggregate is not particularly limited as long as the effects of the present invention are obtained, but it is preferable to cut the root portion of the hair-like tissue using a cutting tool such as scissors.

[0163] The pithyroid tissue (i.e., graftable pithyroid tissue) that is cut and recovered from the cell aggregate is a graft prepared for transplantation into a living organism, and is a tissue body containing epithelial cells and mesenchymal cells, independent of the cell aggregate.

[0164] A graft of hairy tissue that has not yet been transplanted into a living organism (hereinafter sometimes referred to as a "hairy graft") has one tip that is a free end and another tip that is also a free end. More specifically, a hairy graft has, for example, one tip that does not have a cut surface (the tip that was a free end in the hairy tissue formed on the surface of the cell aggregate) and another tip that has a cut surface, due to being separated from a cell aggregate.

[0165] A hair-like graft has, for example, a dermal papilla-like structure at one of its tip portions (e.g., the tip portion without a cut surface). The dermal papilla-like structure of the hair-like graft has a structure similar to the dermal papilla in a living hair follicle. That is, the dermal papilla-like structure has a spherical shape. Furthermore, the dermal papilla-like structure contains mesenchymal cells (e.g., dermal papilla cells). In other words, the dermal papilla-like structure is mainly composed of mesenchymal cells (e.g., dermal papilla cells).

[0166] Specifically, the ratio of the number of mesenchymal cells (e.g., dermal papilla cells) contained in the dermal papilla-like structure to the total number of cells constituting the dermal papilla-like structure may be, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more. Note that the other end of the hair-like graft (e.g., the end with the cut surface) does not have a dermal papilla-like structure.

[0167] Furthermore, the hair-like graft has, for example, a hair shaft-like structure. The hair shaft-like structure of the hair-like graft has a structure similar to the hair shaft in a living hair follicle. 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 graft extends from the vicinity of the dermal papilla-like structure contained in one end portion of the hair-like graft toward the other end portion of the hair-like graft. Furthermore, it is preferable that the hair shaft-like structure contains melanin.

[0168] Furthermore, the pilosa tissue does not contain capillaries. In other words, for example, the dermal papilla-like structure contained in the pilosa tissue does not contain capillaries, unlike the dermal papilla contained in hair follicles taken from living organisms.

[0169] The length of the ciliary graft is not particularly limited as long as the effects of the present invention are obtained, but for example it may be 200 μm or more, preferably 400 μm or more, more preferably 600 μm or more, even more preferably 800 μm or more, and particularly preferably 1000 μm or more.

[0170] Furthermore, the length of the ciliary graft is preferably 2 mm or more, more preferably 4 mm or more, even more preferably 6 mm or more, and particularly preferably 8 mm or more.

[0171] Furthermore, the length of the pithyroid graft may be, for example, 100 mm or less, preferably 50 mm or less, more preferably 30 mm or less, and particularly preferably 20 mm or less. The length of the pithyroid graft may be determined by arbitrarily combining one of the upper limits and one of the lower limits mentioned above.

[0172] The method according to this embodiment may include cooling and storing the hair follicle grafts recovered for transplantation into a living organism. In this case, for example, the hair follicle grafts are cooled and stored until they are transplanted into a living organism. Specifically, for example, the hair follicle grafts recovered from the culture vessel are transferred to a container separate from the culture vessel and cooled and stored there.

[0173] The temperature at which the ciliary grafts are cooled and stored is not particularly limited as long as the effects of the present invention are obtained, but it is preferably 10°C or lower (greater than 0°C and 10°C or lower), more preferably 7°C or lower, and particularly preferably 5°C or lower.

[0174] The hair regeneration method according to this embodiment includes transplanting a hair-like graft, manufactured in vitro as described above, into a living organism. The transplantation of the hair-like graft into the living organism is carried out in such a way that the structure of the transplanted hair-like graft resembles the structure of a hair follicle in the living organism.

[0175] In other words, in a living hair follicle, the dermal papilla is located on the inside of the body, and the free end of the hair shaft is located on the outside of the body. For this reason, the hair-like graft is transplanted into the living body such that one end portion containing the dermal papilla-like structure is on the inside of the body. It is also preferable to transplant the hair-like graft into the living body such that the other end portion not containing the dermal papilla-like structure is the free end.

[0176] The organism to which the hair-like graft is transplanted may be human or a non-human animal, but it is preferable that it be human. The transplantation of the hair-like graft into the organism is preferably into the skin of the organism, and particularly preferably into the scalp of the organism.

[0177] The transplantation of hair-like grafts into living organisms may be for medical or research purposes. Preferably, the transplantation of hair-like grafts into living organisms is for the treatment or prevention of diseases involving hair loss. That is, preferably, the transplantation of hair-like grafts into living organisms is for human patients who have or are at risk of developing diseases involving hair loss. Therefore, the hair regeneration method according to this embodiment is preferably a method for the treatment or prevention of diseases involving hair loss.

[0178] Diseases involving hair loss are not particularly limited, but may be one or more selected from the group consisting of, for example, male pattern baldness (androgenetic alopecia: AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrheic alopecia, pityriasis versicolor, traction alopecia, metabolic alopecia, compression alopecia, alopecia areata, neurogenic alopecia, trichotillomania, alopecia generalis, and symptomatic alopecia.

[0179] Furthermore, hair-like grafts produced in vitro may be used, for example, to search for substances that can be used to treat or prevent diseases involving hair loss, to search for substances involved in such diseases, and to study the mechanisms of such diseases.

[0180] Next, a specific example of this embodiment will be described. [Examples]

[0181] [Collection of epithelial and mesenchymal cells] Skin tissue was collected from the back of 18-day-old C57BL / 6 mouse fetuses and treated with dispase at 4°C for 1 hour with shaking at 20-30 rpm to separate the epithelial and mesenchymal layers of the skin tissue. Subsequently, the epithelial layer was treated with 100 U / mL collagenase for 80 minutes, followed by trypsin treatment for 10 minutes to isolate epithelial cells. Similarly, the mesenchymal layer was treated with 100 U / mL collagenase for 80 minutes to isolate mesenchymal cells.

[0182] [Production of cell aggregates] First, a basic culture medium was prepared by adding 1% GultaMax Supplement (GIBCO®) and 0.2% Normocin (InvivoGen) to DMEM / F12 medium (Advanced Dulbecco's Modified Eagle Medium / Ham's F-12, GIBCO®).

[0183] Next, to a basic culture medium cooled to 4°C, type I collagen stock solution (Cellmatrix® Type IA, type I collagen concentration 2.4 mg / mL, Nitta Gelatin Co., Ltd.), cooled to 4°C, was added to prepare six different culture media with varying type I collagen concentrations: final concentrations of 2.4 μg / mL, 24 μg / mL, 120 μg / mL, 240 μg / mL, 360 μg / mL, or 480 μg / mL.

[0184] Subsequently, each culture medium at 4°C was filled to a cell density of 5 × 10⁶. 4 The amount that results in cells / mL (total cell density is 1 × 10⁻⁶) 5 Epithelial and mesenchymal cells were suspended in an amount equivalent to cells / mL to prepare a cell suspension (i.e., a matrix treatment culture medium in which epithelial and mesenchymal cells were dispersed and type I collagen was dispersed at concentrations of 2.4 μg / mL, 24 μg / mL, 120 μg / mL, 240 μg / mL, 360 μg / mL, or 480 μg / mL). Then, 200 μL of the cell suspension at 4°C was poured into each well of a 96-well plate to obtain 1 × 10⁶ cells. 4 Epithelial lineage cells and 1 × 10⁶ cells / well 4Mesenchymal cells were seeded in cells / well.

[0185] After seeding, the 96-well plate was left to stand in a 4°C refrigerator for 30 minutes to allow the cells to settle and accumulate on the bottom of the wells. Then, the 96-well plate was transferred to a 37°C incubator to start co-culture (suspension culture).

[0186] During the 8-day co-culture period, the culture medium was changed every two days. This was done by removing half (100 μL) of the culture medium from each well, followed by the addition of 100 μL of culture medium without type I collagen (basic medium). Under all conditions, the overall fluidity of the culture medium in each well was maintained throughout the culture period.

[0187] [result] Figure 1 shows a micrograph of the cells after 8 days of culture. As shown in Figure 1, in all six culture systems with different types of collagen concentrations in the culture medium at the time of seeding (i.e., types of collagen concentrations in the matrix treatment), epithelial and mesenchymal cells aggregated, and one cell aggregate was formed in each well.

[0188] Furthermore, in four culture systems where the type I collagen concentration in the culture medium at the time of seeding was 24 μg / mL, 120 μg / mL, 240 μg / mL, or 360 μg / mL, the formation of cell aggregates containing pithyroid tissue (the parts indicated by arrows in each of the four photographs included in Figure 1) was confirmed.

[0189] Specifically, in these four culture systems, epithelial and mesenchymal cells began to aggregate after the start of suspension culture, and cell aggregates were formed on day 1 of culture. Subsequently, from day 4 to day 6 of culture, cilia-like tissue began to form on the surface of the cell aggregates. The cilia-like tissue of the cell aggregates elongated as the culture time progressed.

[0190] Figure 2 shows the correlation between the type I collagen concentration in the culture medium at seeding (matrix treatment) and the efficiency of ciliary tissue formation in cell aggregates on day 8 of culture. As shown in Figure 2, in culture systems with type I collagen concentrations of 24 μg / mL, 120 μg / mL, and 240 μg / mL, ciliary tissue was formed in all cell aggregates (100% ciliary tissue formation efficiency). In addition, in a culture system with a type I collagen concentration of 360 μg / mL, ciliary tissue was formed in 3 out of 6 cell aggregates (50% ciliary tissue formation efficiency). On the other hand, in culture systems with type I collagen concentrations of 2.4 μg / mL and 480 μg / mL, ciliary tissue was not formed in any cell aggregates (0% ciliary tissue formation efficiency).

[0191] Figure 3 shows the relationship between the concentration of type I collagen in the culture medium at the time of seeding and the number of ciliary tissues formed per cell aggregate on day 8 of culture. Specifically, in Figure 3, the horizontal axis represents the concentration of type I collagen in the culture medium at the time of seeding, and the vertical axis represents the arithmetic mean obtained by dividing the total number of ciliary tissues formed in a cell aggregate by the number of such cell aggregates.

[0192] As shown in Figure 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 each cell aggregate.

[0193] Figure 4 shows a micrograph of cell aggregates on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL. Photograph (ii) in Figure 4 shows a magnified view of the area within the square frame shown in photograph (i).

[0194] As indicated by the arrows in photograph (i) of Figure 4, at least four hair-like structures were observed in a single cell aggregate. Furthermore, as indicated by the arrowheads in photograph (ii) of Figure 4, a swollen structure was observed at the free end of the hair-like structure extending from the cell aggregate.

[0195] Figure 5 shows the results of HE staining of cell aggregate sections on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at seeding was 120 μg / mL. Photograph (ii) in Figure 5 shows a magnified view of the area within the square frame shown in photograph (i).

[0196] As indicated by the black arrows in photograph (i) of Figure 5, two hair-like structures were identified in this section. Furthermore, as indicated by the white arrows in photograph (ii), a hair papilla-like structure similar to that of a living hair papilla was identified at the tip of the hair-like structure, and a portion of a black hair shaft-like structure similar to that of a living hair shaft was identified near the hair papilla-like structure.

[0197] Furthermore, as indicated by the white arrow in photograph (i), a pink-stained cyst-like structure was observed in the central part of the cell aggregate. This cyst-like structure lacked a cell nucleus. The outer periphery of the cyst-like structure was covered by cells containing a cell nucleus.

[0198] Figure 6 shows the results of fluorescent staining with Versican on a section of cell aggregates formed on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL. Photograph (ii) in Figure 6 shows a magnified view of the area within the square frame shown in photograph (i). In photograph (ii), the area enclosed by the white dotted line shows a hair shaft-like structure.

[0199] As shown in Figure 6, it was confirmed that the hair-like tissue formed in the cell aggregates had aggregates of Versican-positive cells, i.e., aggregates of dermal papilla cells (dermal papilla-like structures), at their tips.

[0200] Figure 7 shows the results of fluorescent staining of CD34 in sections of cell aggregates formed on day 8 of culture in a culture system where the type I collagen concentration in the culture medium at the time of seeding was 120 μg / mL. In Figure 7, the area enclosed by the white dotted line indicates the tip of the ciliary tissue.

[0201] As shown in Figure 7, it was confirmed that CD34-positive cells, i.e., hair follicle epithelial stem cells, are contained in the outer periphery of the hair shaft-like structure near the dermal papilla-like structure at the tip of the hair-like tissue.

[0202] Figure 8 schematically shows a cell aggregate having cilia-like tissue obtained in this embodiment, based on the observations described above. Note that Figure 8 is merely a schematic diagram, and the size and arrangement of the cell aggregate, cilia-like tissue, and the cells and structures contained therein are not in any way limited to the cell aggregate according to the present invention.

[0203] As shown in Figure 8, the cell aggregates formed by co-culturing epithelial cells and mesenchymal cells in this embodiment had a cyst-like structure in their center. Furthermore, the cell aggregates had one or more hair-like structures on their surface. These hair-like structures had a dermal papilla-like structure (an aggregate of Versican-positive cells) formed at its free end, and a hair shaft-like structure extending from the vicinity of the dermal papilla-like structure to the base of the hair-like structure. Additionally, the hair-like structures contained hair follicle epithelial stem cells (CD34-positive cells) around the outer circumference of the hair shaft-like structure near the dermal papilla-like structure. [Examples]

[0204] [Production of cell aggregates] Except for using a culture medium to which type I collagen was added to achieve a final concentration of 12 μg / mL, the procedure was the same as in Example 1 described above, and 1 × 10⁶ of collagen was added to each well of a 96-well plate. 4 Epithelial lineage cells and 1 × 10⁶ cells / well 4 Mesenchymal cells were seeded in cells / well.

[0205] After seeding, the 96-well plate was left to stand in a 4°C refrigerator for 30 minutes to allow the cells to settle and accumulate on the bottom of the wells. Then, the 96-well plate was transferred to a 37°C incubator to start co-culture (suspension culture).

[0206] During the 4-day co-culture period, the culture medium was changed in the same manner as in Example 1 described above. On the 4th day of culture, one cell aggregate was formed in each well. However, no ciliary tissue formation was observed in the cell aggregate on the 4th day of culture.

[0207] Next, seven cell aggregates lacking ciliary tissue on day 4 of culture were collected in a 15 mL centrifuge tube, and the culture medium was removed from the tube as much as possible. Then, type I collagen stock solution (Cellmatrix® Type IA, type I collagen concentration: 2.4 mg / mL, manufactured by Nitta Gelatin Co., Ltd.) was added to the centrifuge tube, and the cell aggregates were suspended in the type I collagen stock solution.

[0208] Subsequently, 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 incubated in a 37°C incubator for 20 minutes to gel the type I collagen. As a result, the entire solution in the well gelled, forming a non-fluid hydrogel. Within the hydrogel, the seven cell aggregates were dispersed three-dimensionally and embedded in a separated state.

[0209] Subsequently, 2 mL of basic culture medium was added to the hydrogel in the well, and the cell aggregates were embedded and cultured in the hydrogel for a further 23 days. In total, co-culture was performed for 27 days.

[0210] [result] Figure 9 shows micrographs of a single cell aggregate embedded in hydrogel, taken on day 8 (4 days after the start of hydrogel-embedded culture), day 12, day 18, day 22, and day 27 of culture. Figure 10 shows magnified views of the area enclosed by the dotted rectangle (ciliary tissue) in each of the five photographs included in Figure 9. Figure 11 shows the change in the length of the ciliary tissue shown in Figures 9 and 10 over time. In Figure 11, the horizontal axis represents the number of culture days, and the vertical axis represents the length of the ciliary tissue (μm).

[0211] Figures 9 to 11 show that in hydrogel-embedded cultures from day 8 to day 22, the ciliary tissue on the surface of the cell aggregates elongated and increased in length as the culture time progressed. The length of the ciliary tissue reached approximately 2 mm (2000 μm) on day 22 of culture.

[0212] On the other hand, from day 22 to day 27 of culture, the pithyroid tissue regressed and its length decreased. This change in pithyroid tissue was consistent with the mouse's first hair growth cycle (3 to 4 weeks). In hydrogel-embedded culture, pithyroid tissue began to form on the surface of the cell aggregates on day 2 of the hydrogel-embedded culture (day 6 from cell seeding). [Examples]

[0213] [Production of cell aggregates] Except for using a culture medium prepared by adding fibronectin stock solution (fibronectin solution (derived from human plasma), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to the basic medium to a final concentration of 6 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL, or 100 μg / mL, the same procedure as in Example 1 described above was followed, and 1 × 10⁶ cells were placed in each well of a 96-well plate. 4 Epithelial lineage cells and 1 × 10⁶ cells / well 4 Mesenchymal cells were seeded in cells / well.

[0214] After seeding, the 96-well plate was left to stand in a 4°C refrigerator for 30 minutes to allow the cells to settle and accumulate on the bottom of the wells. Then, the 96-well plate was transferred to a 37°C incubator to start co-culture (suspension culture).

[0215] During the 8-day co-culture period, the culture medium was replaced in the same manner as in Example 1 described above. Under all conditions, the culture medium in the wells maintained its overall fluidity throughout the culture period.

[0216] [result] Figure 12 shows a micrograph of the cells after 8 days of culture. As shown in Figure 12, in all five culture systems with different fibronectin concentrations in the culture medium at the time of seeding, epithelial and mesenchymal cells aggregated, and one cell aggregate was formed in each well.

[0217] Furthermore, in all five culture systems where the fibronectin concentration in the culture medium at the time of seeding (matrix treatment) was 6 μg / mL, 12 μg / mL, 25 μg / mL, 50 μg / mL, or 100 μg / mL, the formation of cell aggregates containing pithyroid tissue (indicated by arrows in each of the five photographs included in Figure 12) was confirmed.

[0218] Specifically, in these five culture systems, epithelial and mesenchymal cells began to aggregate after the start of suspension culture, and cell aggregates were formed on day 1 of culture. Subsequently, from day 4 to day 6 of culture, cilia-like tissue began to form on the surface of the cell aggregates. The cilia-like tissue of the cell aggregates elongated as the culture time progressed.

[0219] Figure 13 shows the relationship between the fibronectin concentration in the culture medium at the time of seeding and the efficiency of ciliary tissue formation in cell aggregates on day 8 of culture. As shown in Figure 13, the efficiency of ciliary tissue formation was 40% to 70%.

[0220] Figure 14 shows the relationship between the fibronectin concentration in the culture medium at the time of seeding and the number of hair-like tissues formed per cell aggregate on day 8 of culture. As shown in Figure 14, an average of 1 to 3 hair-like tissues were formed per cell aggregate.

[0221] Figure 15 shows the results of HE staining of a cell aggregate section on day 8 of culture in a culture system where the fibronectin content in the culture medium at seeding was 100 μg / mL. As indicated by the arrows in Figure 15, two hair-like structures were identified in this section. In addition, a hair papilla-like structure was identified at the tip of the hair-like structure. On the other hand, a cyst-like structure was identified in the central part of the cell aggregate. [Examples]

[0222] [Production of cell aggregates] In six different culture systems, each with a different timing for adding type I collagen (type I collagen addition timing: 0 hours (Example 4-1), 6 hours (Example 4-2), 12 hours (Example 4-3), 24 hours (Example 4-4), 36 hours (Example 4-5), or 48 hours (Example 4-6) after cell seeding), co-culture of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 described above.

[0223] First, a basic culture medium without type I collagen was prepared in the same manner as in Example 1 described above. Next, the basic culture medium was cooled to 4°C and each cell density was 1 × 10⁶ 5 The amount that results in cells / mL (total cell density is 2 × 10⁻⁶) 5 A cell suspension was prepared by suspending epithelial cells and mesenchymal cells (in an amount equivalent to cells / mL). Meanwhile, a culture medium containing type I collagen at a concentration of 240 μg / mL was prepared by adding type I collagen stock solution, cooled to 4°C, to a basic culture medium cooled to 4°C.

[0224] Then, by pouring 100 μL of the 4°C cell suspension into each well of a 96-well plate, 1 × 10⁶ 4 Epithelial lineage cells and 1 × 10⁶ cells / well 4 Mesenchymal cells were seeded in cells / well.

[0225] In Example 4-1, 100 μL of culture medium containing type I collagen at a concentration of 240 μg / mL and cooled to 4°C was added to the well immediately after seeding (0 hours after seeding). As a result, epithelial and mesenchymal cells were retained in the well of Example 4-1 in a matrix treatment culture medium in which type I collagen was dispersed at a concentration of 120 μg / mL.

[0226] Next, the 96-well plate was left to stand in a 4°C refrigerator for 30 minutes to allow the cells to settle and accumulate on the bottom of the wells. After that, the 96-well plate was transferred to a 37°C incubator to start co-culture (suspension culture).

[0227] Five hours and forty minutes after cell seeding, the 96-well plate was transferred to a 4°C refrigerator and left to stand for 20 minutes to cool the culture medium containing the cells in the wells. After 20 minutes of cooling, i.e., six hours after cell seeding, 100 μL of 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, epithelial and mesenchymal cells were retained in the wells of Example 4-2 in a matrix treatment culture medium dispersed with type I collagen at a concentration of 120 μg / mL.

[0228] After adding type I collagen to the wells in Example 4-2, the 96-well plate was shaken in a refrigerator at 4°C for 20 minutes. Then, the 96-well plate was transferred to a 37°C incubator and co-culture (suspension culture) was continued.

[0229] Similarly, at 12 hours, 24 hours, 36 hours, and 48 hours after cell seeding, type I collagen was added to the wells of Examples 4-3, 4-4, 4-5, and 4-6, respectively, to initiate matrix treatment. In addition, the culture medium was changed for the first time on the third day of culture in all wells, and thereafter every two days. The culture medium was changed in the same manner as in Example 1 described above.

[0230] [result] Figure 16 shows a micrograph of the cells after 8 days of culture. As shown in Figure 16, in all six culture systems with different timings of type I collagen addition, epithelial and mesenchymal cells aggregated, and one cell aggregate was formed in each well.

[0231] Furthermore, in all six culture systems with different timings for adding type I collagen, cell aggregates were obtained in which cilia-like tissue (indicated by arrows in each of the six photographs in Figure 16) was formed on the surface.

[0232] Figure 17 shows the correlation between the timing of type I collagen addition (time from cell seeding to type I collagen addition) and the efficiency of pilosarconia formation in cell aggregates on day 8 of culture. As shown in Figure 17, in the culture system in which type I collagen was added immediately after cell seeding (0 hours), pilosarconia was formed in 9 out of 12 cell aggregates (pilosarconia formation efficiency of 75%). Furthermore, the pilosarconia formation efficiency in the culture systems in which type I collagen was added at 6 hours, 12 hours, 24 hours, 36 hours, and 48 hours after cell seeding was 58%, 58%, 50%, 33%, and 25%, respectively. In other words, it was observed that the efficiency of pilosarconia formation tended to decrease as the timing of type I collagen addition became later.

[0233] Figure 18 shows the relationship between the timing of type I collagen addition and the number of hair follicles formed per cell aggregate on day 8 of culture. As shown in Figure 18, a tendency was observed for the number of hair follicles formed per cell aggregate to decrease as the timing of type I collagen addition became later.

[0234] Figure 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 Figure 19 shows a magnified view of the area within the square frame shown in photograph (i). As shown in Figure 19, a swollen structure was observed at the tip, which is the free end of the ciliary tissue protruding from the surface of the cell aggregate.

[0235] Figure 20 shows a magnified image of the ciliary tissue formed on the surface of cell aggregates on day 8 of culture in a culture system to which type I collagen was added immediately after cell seeding. In Figure 20, the area enclosed by the white dotted line is the ciliary tissue. As shown in Figure 20, a swollen structure was observed at the tip, which is the free end of the ciliary tissue, and a black hair shaft-like structure was observed extending from this swollen structure toward the base of the ciliary tissue. [Examples]

[0236] [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 co-culture (suspension culture) of epithelial cells and mesenchymal cells was started in the same manner as in Example 1 described above.

[0237] Furthermore, in Example 5-2, co-culture (suspension culture) of epithelial cells and mesenchymal cells was initiated in the same manner as in Example 1 above, except that a culture medium (basic medium) without type I collagen was used.

[0238] [result] In both Example 5-1 and Example 5-2, cell aggregates without pithyroid tissue were formed on day 2 of culture. RNA was extracted from the cell aggregates collected on day 2 of culture using the RNeasy mini-kit (QIAGEN), and microarray analysis was performed using the Mouse Genome 430 2.0 Array (Applied Biosystems).

[0239] Figure 21 shows the results of GO (Gene Ontology) analysis using microarrays on cell aggregates formed using a culture medium in which type I collagen was dispersed in Example 5-1, and on cell aggregates formed using a culture medium in which type I collagen was not dispersed in Example 5-2. Specifically, Figure 21 shows the gene groups that were significantly increased (Fold change > 2) in the cell aggregates of Example 5-1 compared to the cell aggregates of Example 5-2 (i.e., the gene groups whose expression levels were more than twice as high in the cell aggregates of Example 5-1 compared to the cell aggregates of Example 5-2).

[0240] As shown in Figure 21, five GO Term hair growth-related genes were extracted from the top 10 gene groups that were significantly increased in the cell aggregates of Example 5-1 (hair cycle, skin development, epidermis development, hair follicle development, and hair cycle process).

[0241] That is, in the cell aggregates of Example 5-1 formed by co-culture including matrix treatment using type I collagen, it was confirmed that the expression level of hair growth-related genes was significantly increased compared to the cell aggregates of Example 5-2 formed by co-culture not including the matrix treatment.

[0242] Specifically, for example, in the cell aggregates of Example 5-1, the expression levels of marker genes related to hair follicle development, namely Tgfb2, Sox21, Lgr5, Lhx2, Edaradd, Pdgfa, and Lgr4, were all more than twice those of the cell aggregates of Example 5-2.

[0243] Therefore, it was considered that the hair regeneration ability of the cell aggregates of Example 5-1 formed by co-culture including matrix treatment using type I collagen was significantly improved compared to the cell aggregates of Example 5-2 formed by co-culture not including the matrix treatment.

Example

[0244] [Production of cell aggregates] As Example 6-1, using a culture solution added with type I collagen to a final concentration of 120 μg / mL, in the same manner as in Example 1 above, 1×10 4 cells / well of epithelial cells and 1×10 4 cells / well of mesenchymal cells were seeded in each well of a 96-well plate.

[0245] After seeding, the 96-well plate was left standing in a refrigerator at 4°C for 30 minutes to allow the cells to sediment and deposit on the bottom surface of the wells. Then, the 96-well plate was transferred to an incubator at 37°C to start co-culture (suspension culture).

[0246] As Example 6-2, except that a culture solution added with a matrix gel stock solution (Matrigel (registered trademark) Basement Membrane Matrix, CORNING (registered trademark)) in an amount to a final concentration of 2 v / v% was used instead of type I collagen, co-culture (suspension culture) of epithelial cells and mesenchymal cells was started in the same manner as in Example 6-1 above.

[0247] The Matrigel stock solution contained 10.6 mg / mL (protein amount measured by the Lowry method) of soluble basement membrane matrix extracted from EHS (Engelbreth-Holm-Swarm) mouse tumors. The composition ratio of this basement membrane matrix was 56% laminin, 8% entactin, and 31% type IV collagen.

[0248] Therefore, it is calculated that the culture medium to which the Matrigel stock solution was added in an amount that resulted in a final concentration of 2 v / v% contained 118 μg / mL of laminin, 16 μg / mL of entactin, and 66 μg / mL of type IV collagen.

[0249] As Example 6-3, co-culture (suspension culture) of epithelial cells and mesenchymal cells was initiated in the same manner as in Example 6-1 above, except that a culture medium (basic medium) without type I collagen or Matrigel was used.

[0250] [Transplantation of cell aggregates] In all three culture systems of Example 6-1, Example 6-2, and Example 6-3, cell aggregates without hair-like tissue were formed on day 3 of culture. Cell aggregates were collected from each of the culture systems of Example 6-1, Example 6-2, and Example 6-3 on day 3 of culture, and these cell aggregates were transplanted into the skin of 5-week-old SCID-nu mice using a patch method. Specifically, 10 cell aggregates from Example 6-1, 50 cell aggregates from Example 6-2, and 50 cell aggregates from Example 6-3 were injected into three separate transplantation sites formed near the dermis or fascia of the mouse skin.

[0251] [result] Figure 22 shows the results of visually counting the number of hairs that had regenerated at each transplant site in mice four weeks after transplantation. As shown in Figure 22, 128 hairs regenerated at the transplant sites of 50 cell aggregates formed using a culture medium (basic medium) in which neither type I collagen nor Matrigel was dispersed ("no matrix" in the figure). In other words, the number of regenerated hairs per transplanted cell aggregate was 2.6.

[0252] Furthermore, 285 hairs were regenerated at the transplantation site of 50 cell aggregates formed under conditions using a culture medium in which Matrigel was dispersed (labeled "Matrigel" in the figure). In other words, 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 cell aggregates formed without using type I collagen or Matrigel.

[0253] Furthermore, 208 hairs were regenerated at the transplantation site of 10 cell aggregates formed under conditions using a culture medium dispersed with type I collagen (labeled "collagen" in the figure). In other words, the number of regenerated hairs per transplanted cell aggregate was 20.8.

[0254] In other words, the number of regenerated hairs per cell aggregate formed using type I collagen was eight times that of cell aggregates formed without using type I collagen or Matrigel, and 3.6 times that of cell aggregates formed using Matrigel. [Examples]

[0255] [Production of cell aggregates] Similar to Example 2 described above, first, epithelial cells and mesenchymal cells were co-cultured (suspended in water) for 4 days using a culture medium to which type I collagen was added to a final concentration of 12 μg / mL. Next, cell aggregates without pithyroid tissue were collected on day 4 of culture and embedded in a hydrogel of type I collagen, and the cell aggregates were subjected to hydrogel-embedded culture.

[0256] [Recovery of ciliary tissue] At 14 days of culture (10 days of hydrogel-embedded culture), the formation of cilia-like tissue on the surface of the cell aggregates was confirmed. The base of the cilia-like tissue of the cell aggregates embedded in the hydrogel on 14 days of culture was cut with scissors, and the cut cilia-like tissue was collected.

[0257] [result] Figure 23 shows micrographs of the ciliary tissue that was cut and recovered from the cell aggregate as described above. For each of the three ciliary tissues shown in Figure 23, the white arrowhead indicates one end that was a free end before being cut from the cell aggregate, and the black arrowhead indicates the other end formed by the cut (the base that was attached to the cell aggregate before the cut). As shown in Figure 23, the ciliary tissue formed on the surface of the cell aggregate could be separated from the cell aggregate and recovered. [Examples]

[0258] [Production of cell aggregates] As Example 8-1, co-culture (suspension culture) of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 above, except that a culture medium containing an amount of Matrigel stock solution with a final concentration of 2 v / v% (a final laminin concentration of 118 μg / mL, an entactin final concentration of 16 μg / mL, and a final type IV collagen concentration of 66 μg / mL) was used instead of type I collagen.

[0259] As Example 8-2, co-culture (suspension culture) of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 above, except that a culture medium was used to which Matrigel stock solution (Matrigel® Basement Membrane Matrix (Growth Factor Reduced), CORNING®), which has a reduced growth factor content, was added at a final concentration of 1 v / v%, instead of type I collagen.

[0260] In addition, Matrigel stock solution (GFR) with a reduced growth factor content contains 8 - 12 mg / mL (protein amount measured by the Lowry method) of a soluble basement membrane matrix extracted from EHS mouse tumors. The composition ratio in the basement membrane matrix was 61% laminin, 7% entactin, and 30% type IV collagen. Therefore, the culture medium to which an amount of Matrigel stock solution (GFR) that results in a final concentration of 1 v / v% is added was calculated to contain 49 - 73 μg / mL of laminin, 6 - 8 μg / mL of entactin, and 24 - 36 μg / mL of type IV collagen.

[0261] As Example 8 - 3, except that a culture medium to which a high - concentration laminin / entactin complex stock solution (HIGH CONCENTRATION LAMININ / ENTACTIN COMPLEX, CORNING (registered trademark)) at a final concentration of 1 v / v% was added instead of type I collagen was used, co - culture (suspension culture) of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 above.

[0262] The high - concentration laminin / entactin complex stock solution contains 15.2 mg / mL of a soluble basement membrane matrix extracted from EHS mouse tumors, contains a laminin / entactin complex with a purity of 90% or more by SDS - PAGE, and laminin and entactin are contained in an equimolar ratio. Therefore, the culture medium to which an amount of the high - concentration laminin / entactin complex stock solution that results in a final concentration of 1 v / v% is added was calculated to contain 137 - 152 μg / mL of the laminin / entactin complex (i.e., 68 - 76 μg / mL each of laminin and entactin).

[0263] As Example 8 - 4, except that a culture medium to which a type IV collagen stock solution (COLLAGEN IV, MOUSE, CORNING (registered trademark)) at a final concentration of 1 v / v% was added instead of type I collagen was used, co - culture (suspension culture) of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 above.

[0264] The type IV collagen stock solution contained 1.25 mg / mL of soluble basement membrane matrix extracted from EHS mouse tumors and contained type IV collagen with a purity of over 90% as determined by SDS-PAGE. Therefore, it can be calculated that the culture medium to which the type IV collagen stock solution was added in an amount that resulted in a final concentration of 1 v / v% contained 11-13 μg / mL of type IV collagen.

[0265] As Example 8-5, co-culture (suspension culture) of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 above, except that a culture medium containing a high-concentration laminin / entactin complex stock solution with a final concentration of 1 v / v% and a type IV collagen stock solution with a final concentration of 1 v / v% was used instead of type I collagen. Under all conditions, the culture medium in the wells maintained overall fluidity throughout the culture period.

[0266] [result] Figure 24 shows micrographs of cell aggregates on day 8 of culture. As shown in Figure 24, in all culture systems—Example 8-1 using a culture medium dispersed with Matrigel, Example 8-2 using a culture medium dispersed with Matrigel with reduced growth factor content, Example 8-3 using a culture medium dispersed with laminin / entactin complex, Example 8-4 using a culture medium dispersed with type IV collagen, and Example 8-5 using a culture medium dispersed with both laminin / entactin complex and type IV collagen—one cell aggregate with pithyroid tissue was formed in each well on day 8 of culture. In each of the five photographs included in Figure 24, the arrows indicate the pithyroid tissue formed on the surface of the cell aggregate.

[0267] Figure 25 shows the results of fluorescently staining the nuclei of cells in a cell aggregate formed using a culture medium dispersed with Matrigel, and observing them under a confocal microscope. As shown in Figure 25, the entire cell aggregate, including multiple ciliary tissues formed on its surface, was stained.

[0268] Figure 26 shows the results of HE staining and fluorescence staining observations of cell aggregates formed using a culture medium in which Matrigel was dispersed. In Figure 26, photograph (i) shows the HE stained image, and photograph (ii) shows a magnified view of the area enclosed by the dotted line in photograph (i). As shown in photographs (i) and (ii) of Figure 26, the hair-like tissue formed in the cell aggregates had a dermal papilla-like structure formed at its free end, the tip, and a hair shaft-like structure extending from the vicinity of the dermal papilla-like structure to the root of the hair-like tissue.

[0269] In Figure 26, photograph (iii) shows a fluorescence microscope image of a cell aggregate stained with CD34, and photograph (iv) shows a magnified view of the area enclosed by the dotted line in photograph (iii). As shown in photographs (iii) and (iv) of Figure 26, CD34-positive cells (i.e., hair follicle epithelial stem cells) were observed along the outer periphery of the hair shaft-like structure of the cell aggregate.

[0270] In Figure 26, photograph (v) shows a fluorescence microscope image of a cell aggregate stained with Versican, a marker for dermal papilla cells, and photograph (vi) shows a magnified view of the area enclosed by the dotted line in photograph (v). As shown in photographs (v) and (vi) of Figure 26, aggregates of Versican-positive cells (i.e., dermal papilla cells) (dermal papilla-like structures) were observed at the tip of the cell aggregate, which is the free end of the hair-like tissue.

[0271] In Figure 26, photograph (vii) shows a fluorescence microscope image of a cell aggregate stained with Gp100, a marker for pigment cells, and photograph (viii) shows a magnified view of the area enclosed by the dotted line in photograph (vii). As shown in photographs (vii) and (viii) of Figure 26, Gp100-positive cells (i.e., pigment cells) were observed around the dermal papilla-like structure at the tip of the cell aggregate, which is the free end of the hair-like tissue.

[0272] Figure 27 shows the results of observing the hair-like tissue of cell aggregates formed using a culture medium in which Matrigel was dispersed. Photograph (i) in Figure 27 is a scanning electron microscope (SEM) image of the base of the hair-like tissue formed on the surface of the cell aggregate. As shown in this photograph (i), it was confirmed that the hair shaft-like structures contained in the hair-like tissue possess a cuticle structure.

[0273] Photograph (ii) in Figure 27 is a bright-field image of the tip of the hair-like tissue, which is the free end, formed on the surface of a cell aggregate. As shown in Photograph (ii), the hair-like tissue has a dermal papilla-like structure at its tip, and it was confirmed that a hair shaft-like structure extends from the vicinity of this dermal papilla-like structure toward the base of the hair-like tissue. Epithelial cells take up melanin and are therefore observed as black cells under a microscope, but dermal papilla cells do not take up melanin and are therefore identified as non-black cells under a microscope.

[0274] Figure 28 shows the results of observing hair-like tissue from cell aggregates formed using a culture medium dispersed with Matrigel, and mouse body hair, using a transmission microscope (TEM). As shown in Figure 28, the hair-like tissue formed in the cell aggregates was found to have a hair shaft-like structure similar to that of mouse body hair, as well as melanosomes and cuticle structures similar to those of mouse body hair. [Examples]

[0275] [Production of cell aggregates] As Example 9-1, co-culture (suspension culture) of epithelial and mesenchymal cells was performed in the same manner as in Example 1 above, except that a culture medium containing an amount of Matrigel stock solution with a final concentration of 2 v / v% (a final laminin concentration of 118 μg / mL, an entactin final concentration of 16 μg / mL, and a final type IV collagen concentration of 66 μg / mL) was used instead of type I collagen. In the suspension culture of Example 9-1, pilosamic tissue began to form on the surface of cell aggregates from day 4 to day 6 of culture. Subsequently, the length of the pilosamic tissue on the cell aggregates increased over time.

[0276] In Example 9-2, first, co-culture (suspension culture) of epithelial cells and mesenchymal cells was started in the same manner as in Example 9-1 described above. Next, cell aggregates without ciliary tissue on day 1 of culture were collected in a centrifuge tube, and the culture medium was removed from the centrifuge tube as much as possible. Then, stock Matrigel was added to the centrifuge tube, and the cell aggregates were suspended in the stock Matrigel.

[0277] Subsequently, 0.4 mL of the solution containing cell aggregates and Matrigel was dropped into the wells of a 6-well plate and incubated in a 37°C incubator for 30 minutes to gel the Matrigel. As a result, the entire solution in the wells gelled, forming a non-fluid hydrogel. Within the hydrogel, the cell aggregates were dispersed three-dimensionally and embedded in a separated state.

[0278] Subsequently, 2 mL of basic culture medium was added to the hydrogel in the well, and hydrogel-embedded culture of the cell aggregates was started. In the hydrogel-embedded culture of Example 9-2, cilia-like tissue began to form on the surface of the cell aggregates from day 4 to day 6 of culture (days 3 to 5 from the start of hydrogel-embedded culture). Thereafter, the length of the cilia-like tissue on the cell aggregates increased over time.

[0279] [Recovery of ciliary tissue] In Example 9-1, the base of the hair-like tissue of cell aggregates suspended in the culture medium on day 14 of culture was cut with scissors, and the cut hair-like tissue was collected. In Example 9-2, the base of the hair-like tissue of cell aggregates on day 12 of culture (day 11 of hydrogel-embedded culture) and day 22 of culture (day 21 of hydrogel-embedded culture) was cut with scissors, and the cut hair-like tissue was collected.

[0280] [result] Figure 29 shows micrographs of the cilia tissue that was cut and recovered from the cell aggregate on day 14 of culture in Example 9-1. For each of the two cilia tissues shown in photograph (i) and photograph (ii) of Figure 29, the white arrowhead indicates one end that was a free end before being cut from the cell aggregate, and the black arrowhead indicates the other end formed by the cut (the base that was attached to the cell aggregate before the cut).

[0281] Figure 30 shows micrographs of the cilia tissue recovered from the cell aggregates of Example 9-2 on day 12 (photograph (i)) and day 22 (photograph (ii)). For each of the cilia tissues shown in photograph (i) and photograph (ii) of Figure 30, the white arrowhead indicates one end that was a free end before being cut from the cell aggregate, and the black arrowhead indicates the other end formed by the cut (the base that was attached to the cell aggregate before the cut). As shown in Figures 29 and 30, the cilia tissue formed on the surface of the cell aggregates could be separated from the cell aggregates and recovered.

[0282] Figure 31 shows a micrograph of the cell aggregate in Example 9-2 on day 23 of culture (day 22 of hydrogel-embedded culture). As shown in Figure 31, one of the cilia-like tissues formed on the surface of the cell aggregate reached a length of approximately 4 cm. By day 30 of culture, this cilia-like tissue exceeded 5 cm in length.

[0283] In Figure 31, partial photograph (i) shows a magnified view of the tip (the area enclosed by the black square), which is the free end of the ciliary tissue. As shown in this partial photograph (i), the ciliary tissue had a swollen structure at its tip and a hair shaft-like structure extending from the tip to the root.

[0284] In Figure 31, section photograph (ii) shows a magnified view of small protrusions (areas enclosed in white squares) formed on the surface of the cell aggregate. As shown in this section photograph (ii), in addition to the two elongated cilia, new cilia were being formed on the surface of the cell aggregate. [Examples]

[0285] [Production of cell aggregates] Cell aggregates were embedded and cultured in a hydrogel containing Matrigel, similar to the procedure described in Example 9-2 above. On day 6 of culture (day 5 from the start of hydrogel embedding), the formation of pithyroid tissue was observed in the cell aggregates. Subsequently, the length of the pithyroid tissue in the cell aggregates increased over time.

[0286] [Recovery of ciliary tissue] On day 15 of culture (day 14 of hydrogel-embedded culture), the base of the cilia-like tissue formed on the surface of the cell aggregate embedded in the hydrogel was cut with scissors, and the cut cilia-like tissue was collected.

[0287] [result] Figure 32 shows micrographs of ciliary tissue recovered from cell aggregates. Photograph (i) in Figure 32 is a bright-field micrograph of the recovered ciliary tissue. Photograph (ii) in Figure 32 shows the results of observing the ciliary tissue shown in photograph (i) after Calcein-AM staining using a fluorescence microscope. In both photographs (i) and (ii) in Figure 32, the white arrowhead indicates one end that was a free end before being cut from the cell aggregate, and the black arrowhead indicates the other end formed by the cut (the base portion that was attached to the cell aggregate before the cut).

[0288] As shown in this photograph (ii), the cells constituting the recovered pithyroid tissue (pithyroid graft) were stained with Calcein-AM and confirmed to be viable. [Examples]

[0289] [Production of cell aggregates] Co-culture (suspension culture) of epithelial cells and mesenchymal cells was performed in the same manner as in Example 1 above, except that a culture medium containing an amount of Matrigel stock solution with a final concentration of 2 v / v% (a final laminin concentration of 118 μg / mL, an entactin final concentration of 16 μg / mL, and a final type IV collagen concentration of 66 μg / mL) was used instead of type I collagen.

[0290] Next, cell aggregates lacking ciliary tissue on day 1 of culture were collected in a centrifuge tube, and the culture medium was removed from the centrifuge tube as much as possible. Then, Matrigel stock solution was added to the centrifuge tube, and the cell aggregates were suspended in the Matrigel stock solution.

[0291] Subsequently, 0.4 mL of the solution containing cell aggregates and Matrigel was dropped into the wells of a 6-well plate and incubated in a 37°C incubator for 30 minutes to gel the Matrigel. As a result, the entire solution in the wells gelled, forming a non-fluid hydrogel. Within the hydrogel, the cell aggregates were dispersed three-dimensionally and embedded in a separated state.

[0292] Subsequently, 2 mL of basic culture medium was added to the hydrogel in the well, and hydrogel-embedded culture of the cell aggregates was initiated. In the hydrogel-embedded culture, cilia-like tissue began to form on the surface of the cell aggregates from day 4 to day 6 of culture (from day 3 to day 5 from the start of hydrogel-embedded culture). Subsequently, the length of the cilia-like tissue on the cell aggregates increased over time.

[0293] [Recovery of ciliary tissue] On day 14 of culture (day 10 of hydrogel-embedded culture), the base of the hair-like tissue in the cell aggregate was cut with scissors, and the cut hair-like tissue was collected.

[0294] [Hydrogel-embedded culture of cilia] As described above, the recovered hair-like tissue (hair-like graft) was added to stock Matrigel, and 0.4 mL of the solution containing the hair-like graft and Matrigel was dropped into the wells of a 6-well plate. The Matrigel was then gelled by incubating at 37°C for 30 minutes. As a result, the entire solution in the well gelled, forming a non-fluid hydrogel. Subsequently, 2 mL of basic culture medium was added to the hydrogel in the well to start hydrogel-embedded culture of the hair-like graft. The hydrogel in which the hair-like graft was embedded was used as an artificial tissue that mimicked the tissue of the living organism to which the hair-like graft was transplanted.

[0295] [result] Figure 33 shows micrographs of hair-like grafts cultured in hydrogel. Photograph (i) in Figure 33 shows the hair-like graft immediately after the start of hydrogel-embedded culture (d0), photograph (ii) shows it on day 1 of hydrogel-embedded culture (d1), photograph (iii) shows it on day 2 of hydrogel-embedded culture (d2), photograph (iv) shows it on day 3 of hydrogel-embedded culture (d3), photograph (v) shows it on day 5 of hydrogel-embedded culture (d5), photograph (vi) shows it on day 7 of hydrogel-embedded culture (d7), and photograph (vii) shows it on day 8 of hydrogel-embedded culture (d8).

[0296] In Figure 33, the white arrowhead indicates one end that was a free end before being cut from the cell aggregate, and the black arrowhead indicates the other end formed by the cut (the base portion that was attached to the cell aggregate before the cut).

[0297] As shown in Figure 33, the hair-like grafts survived even after being cut from the cell aggregate, and the length of the hair-like grafts (particularly the length of the hair shaft-like structures contained within the hair-like grafts) increased over time. This result supports the usefulness of hair-like tissue cut from cell aggregates as hair-like grafts.

Claims

1. To obtain a cell aggregate of epithelial cells and mesenchymal cells, wherein a hair-like tissue protrudes from the surface of the cell aggregate formed by co-culturing epithelial cells and mesenchymal cells, the hair-like tissue comprises the epithelial cells and mesenchymal cells, includes a hair shaft-like structure containing keratin and having a cuticle structure, has a length of 200 μm or more, and includes a dermal papilla-like structure having a spherical shape at the free end portion, which is an aggregate of mesenchymal cells, and To transplant the hair-like tissue into a living organism, the root portion of the hair-like tissue is cut and the hair-like tissue is recovered from the cell aggregate. Includes, The aforementioned co-culture is Seeding the epithelial cells and the mesenchymal cells, The seeded epithelial cells and mesenchymal cells are subjected to a matrix treatment in which they are held in a culture medium dispersed with type I collagen, fibronectin, laminin and entactin, or type IV collagen, and The epithelial cells and mesenchymal cells that have undergone the matrix treatment are cultured in suspension. Includes, In the matrix treatment described above, the culture medium is (i) Type I collagen in a concentration of 10 μg / mL or higher and 420 μg / mL or lower; (ii) Fibronectin in concentrations of 3 μg / mL or higher and 150 μg / mL or lower; (iii) Laminin and entactin in a total concentration of 137 μg / mL or higher and 152 μg / mL or lower; (iv) Type IV collagen in a concentration of 11 μg / mL or higher and 13 μg / mL or lower; or (v) Laminin and entactin in a total concentration of 55 μg / mL or higher and 152 μg / mL or lower, and type IV collagen in a concentration of 11 μg / mL or higher and 66 μg / mL or lower; including, A method for producing hair-like tissue for transplantation.

2. The aforementioned ciliary tissue does not contain capillaries. A method for producing hair-like tissue for transplantation according to claim 1.

3. The cell aggregate does not contain arrector pili muscle structures and / or sebaceous gland structures. A method for producing hair-like tissue for transplantation according to claim 1 or 2.

4. The hair-like tissue that is cut and recovered from the cell aggregate has one tip portion that does not have a cut surface and the other tip portion that has a cut surface. A method for producing hair-like tissue for transplantation according to any one of claims 1 to 3.

5. In the matrix treatment, the epithelial cells and the mesenchymal cells are maintained in a culture medium in which type I collagen is dispersed at a concentration of 10 μg / mL or more and 420 μg / mL or less. A method for producing hair-like tissue for transplantation according to any one of claims 1 to 4.

6. The aforementioned co-culture is Performing the suspension culture of the epithelial cells and mesenchymal cells that have undergone the matrix treatment, thereby forming cell aggregates on which ciliary tissue protrudes from their surface, and The cell aggregates formed by the suspension culture are embedded in a hydrogel and cultured further. A method for producing hair-like tissue for transplantation according to any one of claims 1 to 5, including the method described above.

7. The aforementioned co-culture is Perform the suspension culture of the epithelial cells and mesenchymal cells that have undergone the matrix treatment to form cell aggregates on their surface in which the ciliary tissue is not formed, and The cell aggregates formed by the suspension culture, which do not have ciliary tissue on their surface, are embedded in a hydrogel containing one or more selected from the group consisting of type I collagen, fibronectin, laminin, entactin, and type IV collagen at a concentration that causes the entire culture medium to gel, and cultured further to obtain cell aggregates in which ciliary tissue protrudes from their surface. A method for producing hair-like tissue for transplantation according to any one of claims 1 to 5, including the method described above.

8. It has one end portion that is a free end and does not have a cut surface, and the other end portion that is a free end and has a cut surface. It includes epithelial cells and mesenchymal cells, It contains keratin and a hair shaft-like structure having a cuticle structure, The length is 200 μm or more, The free end portion, which has no cut surface, contains a hair papilla-like structure which is an aggregate of mesenchymal cells having a spherical shape and not containing capillaries. Hair-like tissue for transplantation.

9. The procedure includes transplanting the hair-like tissue described in claim 8 into a non-human animal. Hair regeneration methods.

Images