WO2023058429A1 - Procédé de production d'agrégat cellulaire ayant une capacité de régénération capillaire, et procédé associé - Google Patents

Procédé de production d'agrégat cellulaire ayant une capacité de régénération capillaire, et procédé associé Download PDF

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WO2023058429A1
WO2023058429A1 PCT/JP2022/034628 JP2022034628W WO2023058429A1 WO 2023058429 A1 WO2023058429 A1 WO 2023058429A1 JP 2022034628 W JP2022034628 W JP 2022034628W WO 2023058429 A1 WO2023058429 A1 WO 2023058429A1
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cells
culture
collagen
cell
epithelial cells
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PCT/JP2022/034628
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English (en)
Japanese (ja)
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淳二 福田
達斗 景山
理樹 穴竃
龍彦 肥高
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国立大学法人横浜国立大学
地方独立行政法人神奈川県立産業技術総合研究所
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Publication of WO2023058429A1 publication Critical patent/WO2023058429A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the present invention relates to a method for producing cell aggregates having hair regeneration ability and methods related thereto.
  • Patent Document 1 discloses a process of seeding mesenchymal cells and epithelial cells on a micro-intaglio plate consisting of regularly arranged micro-concavities and culturing them while supplying oxygen to form a hair follicle primordium.
  • a method for producing aggregates of regenerated follicle primordia comprising:
  • Patent Document 2 discloses a method for producing full-thickness skin having skin appendages, wherein the "full-thickness skin having skin appendages" includes at least the following (1) to (3); (1) epidermal layer (2) at least one type of skin appendage; and (3) subcutaneous tissue, the method comprising the steps of: stimulating with an active substance, (b): preparing a conjugate comprising (A) and (B); (A) all or part of the embryoid body stimulated in step (a) ( B) a scaffolding material, (c) implanting said conjugate prepared in step (b) into an animal, and (d) producing full thickness skin from said conjugate in said animal.
  • a method is described comprising:
  • Patent Document 3 Seeding epithelial cells and mesenchymal cells in Patent Document 3; holding the mesenchymal cells; and forming a hair follicle primordium by co-culturing the epithelial cells and the mesenchymal cells in a culture medium.
  • a method for making the base is described.
  • US Pat. No. 5,300,003 discloses a method of making mammalian hair follicles comprising the steps of: (a) providing at least one de novo papilla; (b) fibroblasts, keratinocytes and/or providing at least one other cell population selected from the group of melanocytes; and (c) co-culturing said de novo papillae with said at least one other cell population under non-adherent culture conditions.
  • step (a) comprises (1) providing at least one dermal papilla (DP) derived from at least one mammalian hair follicle; (3) isolating the DPF from the DP by fixing the dermal papilla fibroblasts (DPF) to the basal lamina and allowing the dermal papilla fibroblasts (DPF) to flow out; (4) condensing the expanded DPF into cell aggregates exhibiting physiological DP size and shape to obtain de novo papillae; (5) coating the cell aggregates with an extracellular matrix protein, wherein the DPF is differentiated in a non-adherent culture vessel at a cell concentration per culture vessel surface of 1000-100000 DFP/cm 2 ;
  • a method of making a mammalian hair follicle is described, comprising the steps of:
  • Patent Document 5 in a method for producing spheroids, a carrier population composed of a plurality of carriers having cell adhesiveness and adhesion-dependent cells are mixed under mixed conditions in which the carrier population does not aggregate together with the cells.
  • a method for producing spheroids is described, which includes a mixing step of mixing and a culturing step of culturing the mixture for a predetermined period.
  • the inventors of the present invention have studied technical means for obtaining cell aggregates with excellent hair regeneration ability.
  • the present invention has been made in view of the above problems, and one of its objects is to provide a method for producing cell aggregates having excellent hair regeneration ability and methods related thereto.
  • One aspect of one embodiment of the present invention for solving the above problems is seeding epithelial cells and mesenchymal cells, and co-cultivating the epithelial cells and the mesenchymal cells to obtain hair.
  • Forming a cell aggregate having regenerative ability, wherein the co-culturing includes a matrix treatment that retains the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed A method for producing a cell aggregate.
  • INDUSTRIAL APPLICABILITY According to the present invention, a method for effectively producing cell aggregates having excellent hair regeneration ability is provided.
  • the culture solution in which the type I collagen or fibronectin is dispersed may contain type I collagen or fibronectin at a concentration within a range in which the culture solution as a whole maintains fluidity in the co-culture.
  • the epithelial cells and the mesenchymal cells may be retained in the culture solution in which type I collagen is dispersed.
  • the epithelial cells and the mesenchymal cells may be maintained in the culture medium in which type I collagen is dispersed at a concentration of less than 480 ⁇ g/mL.
  • the epithelial cells and the mesenchymal cells may be held in the culture medium in which fibronectin is dispersed.
  • the co-cultivation may be performed to form the cell aggregate having the ability to form a hairy tissue on its surface. Further, in the method, the co-cultivation may be performed to form the cell aggregate having a hair-like tissue on its surface.
  • the method may further include collecting the cell aggregates formed by the co-culture for transplantation into a living body.
  • the cell aggregate formed by the co-culture which has the ability to form a hairy tissue on its surface and has no hairy tissue formed on its surface, is used for transplantation into a living body. It may be collected.
  • a cell aggregate may be formed in which the expression level of one or more hair growth-related genes is at least twice as large as that of the cell aggregate formed in .
  • the co-culturing includes performing suspension culture of the epithelial cells and the mesenchymal cells to form cell aggregates, and hydrolyzing the cell aggregates formed by the suspension culture. embedding in a gel and further culturing.
  • Another aspect of one embodiment of the present invention for solving the above problems is seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and the mesenchymal cells.
  • This is a method for improving the hair regeneration ability of the cell aggregate by performing the treatment.
  • INDUSTRIAL APPLICABILITY According to the present invention, a method for effectively improving the hair regeneration ability of cell aggregates is provided.
  • Still another aspect of one embodiment of the present invention for solving the above problems is to seed epithelial cells and mesenchymal cells, and to co-culture the epithelial cells and the mesenchymal cells. holding the epithelial cells and the mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, A use for improving the hair regeneration ability of said cell aggregates of type I collagen or fibronectin, comprising performing a matrix treatment.
  • INDUSTRIAL APPLICABILITY According to the present invention, effective use of type I collagen or fibronectin for improving the hair regeneration ability of cell aggregates is provided.
  • a method for producing a cell aggregate having excellent hair regeneration ability and a method related thereto are provided.
  • FIG. 3 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 1 according to the present embodiment.
  • FIG. 2 is an explanatory diagram showing the relationship between the concentration of type I collagen in the culture solution at the time of seeding and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture in Example 1 according to the present embodiment.
  • Explanatory drawing showing the relationship between the type I collagen concentration in the culture medium at the time of seeding and the number of hairy tissues formed per cell aggregate on day 8 of culture in Example 1 according to the present embodiment. be.
  • FIG. 1 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 1 according to the present embodiment.
  • FIG. 2 is an explanatory diagram showing the relationship between the concentration of type I collagen in the culture solution at the time of seeding and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture in Example 1 according to the present embodiment.
  • Explanatory drawing showing the relationship between the type I collagen concentration in the culture medium at
  • Example 2 is an explanatory view showing a micrograph of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 ⁇ g/mL in Example 1 according to the present embodiment.
  • Example 1 the results of HE staining of frozen sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 ⁇ g / mL are shown. It is an explanatory diagram showing.
  • Versican was fluorescently stained in cryosections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 ⁇ g/mL in Example 1 according to the present embodiment. It is explanatory drawing which shows a result.
  • CD34 was fluorescently stained in frozen sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture medium at the time of seeding was 120 ⁇ g/mL. It is explanatory drawing which shows a result.
  • FIG. 10 is an explanatory view showing an enlarged portion (ciliary tissue) surrounded by a dotted square in each of the five photographs included in FIG. 9;
  • FIG. 10 is an explanatory diagram showing changes over time in the length of hair-like tissue formed in Example 2 according to the present embodiment;
  • FIG. 10 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 3 according to the present embodiment.
  • FIG. 10 is an explanatory diagram showing the correspondence between the fibronectin concentration in the culture solution at the time of seeding and the formation efficiency of hairy tissue in the cell aggregates on the eighth day of culture in Example 3 according to the present embodiment.
  • FIG. 10 is an explanatory diagram showing the relationship between the fibronectin concentration in the culture medium at the time of seeding and the number of hairy tissues formed per cell aggregate on day 8 of culture in Example 3 according to the present embodiment.
  • Explanation showing the results of HE staining of frozen sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of fibronectin in the culture solution at the time of seeding was 100 ⁇ g/mL in Example 3 according to the present embodiment It is a diagram.
  • FIG. 10 is an explanatory diagram showing the correspondence between the fibronectin concentration in the culture solution at the time of seeding and the formation efficiency of hairy tissue in the cell aggregates on the eighth day of culture in Example 3 according to the present embodiment.
  • FIG. 10 is an explan
  • FIG. 10 is an explanatory diagram showing a micrograph of cell aggregates formed on day 8 of culture in Example 4 according to the present embodiment.
  • FIG. 10 is an explanatory diagram showing the relationship between the addition timing of type I collagen and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture in Example 4 according to the present embodiment.
  • FIG. 4 is an explanatory diagram showing the relationship between the timing of addition of type I collagen and the number of hairy tissues formed per cell aggregate on day 8 of culture in Example 4 according to the present embodiment.
  • FIG. 10 is an explanatory view showing a micrograph of cell aggregates on day 8 of culture formed in a culture system in which type I collagen was added immediately after cell seeding in Example 4 according to the present embodiment.
  • Example 4 is an explanatory view showing an enlarged photograph of a hairy tissue extending from a cell aggregate on day 8 of culture formed in a culture system in which type I collagen was added immediately after cell seeding in Example 4 according to the present embodiment.
  • Example 5 microarray analysis was performed on cell aggregates formed using a culture medium containing type I collagen and cell aggregates formed using a culture medium containing no type I collagen.
  • FIG. 10 is an explanatory diagram showing the result of visually counting the number of regenerated hairs 4 weeks after the transplantation of the cell aggregates in Example 6 according to the present embodiment.
  • This method is to seed epithelial cells and mesenchymal cells, and to seed the epithelial cells and mesenchymal cells. to form a cell aggregate having hair regeneration ability, wherein the co-culturing retains the epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed It includes a method for producing cell aggregates, including a matrix treatment to perform the treatment.
  • the inventors of the present invention have extensively studied technical means for producing in vitro cell aggregates having hair regeneration ability, and unexpectedly found that co-culture of epithelial cells and mesenchymal cells
  • co-culture of epithelial cells and mesenchymal cells by performing a matrix treatment to retain the epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, it is possible to effectively improve the hair regeneration ability of the cell aggregates.
  • the present inventors have independently discovered what they can do, and have completed the present invention.
  • the present method includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain a cell aggregate having hair regeneration ability.
  • a method for enhancing the hair regrowth ability of clumps is included.
  • the present method includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain a cell aggregate having hair regeneration ability.
  • a cell aggregate having hair regeneration ability In the production of cell aggregates, which comprises forming The use of collagen or fibronectin for improving the hair regeneration ability of the cell aggregates is included.
  • epithelial cells and mesenchymal cells are seeded, and the seeded epithelial cells and mesenchymal cells are co-cultured to form cell aggregates having hair regeneration ability. .
  • the epithelial cells used to form the cell aggregates are not particularly limited as long as the effects of the present invention are obtained.
  • one or more selected from the group consisting of hair follicle epithelial cells and their precursor cells. is preferred.
  • Hair follicle epithelial cells are epithelial cells that contribute to hair growth (more specifically, for example, epithelial cells that contribute to hair growth in cooperation with hair follicle mesenchymal cells).
  • the hair follicle epithelial cells may be those collected from hair follicles in vivo, or may be those induced to differentiate from undifferentiated cells in vitro.
  • Undifferentiated cells used for inducing differentiation of hair follicle epithelial cells in vitro are not particularly limited as long as they are cells capable of differentiating into the hair follicle epithelial cells in vitro. (e.g., iPS (induced pluripotent stem) cells, ES (embryonic stem) cells, Muse (multilineage-differentiating stress-ending) cells or EG (embryonic germ) cells), and stem cells other than the pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells).
  • the hair follicle epithelial lineage cells are selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, outer root sheath cells, and inner root sheath cells. is preferably one or more, and particularly preferably one or more selected from the group consisting of hair follicle epithelial stem cells, hair matrix cells, and outer root sheath cells.
  • the progenitor cells of hair follicle epithelial cells are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle epithelial cells in vitro.
  • epithelial cells derived from the epidermal layer of neonatal skin), and stem cells other than pluripotent stem cells that have the ability to differentiate into the hair follicle epithelial cells in vitro. is preferred.
  • the mesenchymal cells used for forming the cell aggregates are not particularly limited as long as the effects of the present invention can be obtained.
  • Hair follicle mesenchymal cells are mesenchymal cells that contribute to hair growth (more specifically, for example, mesenchymal cells that contribute to hair growth in cooperation with hair follicle epithelial cells).
  • the hair follicle mesenchymal cells may be those collected from hair follicles in vivo, or may be those induced to differentiate from undifferentiated cells in vitro.
  • the undifferentiated cells used for inducing differentiation of hair follicle mesenchymal cells in vitro are not particularly limited as long as they are cells capable of differentiating into the hair follicle mesenchymal cells in vitro.
  • stem cells e.g., iPS cells, ES cells, Muse cells or EG cells
  • stem cells other than the pluripotent stem cells e.g., stem cells obtained by reprogramming differentiated cells, and mesenchymal stem cells (e.g., , adipose tissue-derived mesenchymal stem cells)).
  • the hair follicle mesenchymal cells are preferably one or more selected from the group consisting of dermal papilla cells and dermal sheath cup cells, and are dermal papilla cells. is particularly preferred.
  • Progenitor cells of hair follicle mesenchymal cells are not particularly limited as long as they are cells that have the ability to differentiate into the hair follicle mesenchymal cells in vitro. , mesenchymal cells derived from the dermal layer of fetal or neonatal skin), and stem cells other than pluripotent stem cells that have the ability to differentiate into the hair follicle mesenchymal cells in vitro (e.g., derived from adipose tissue mesenchymal stem cells).
  • adipose tissue-derived mesenchymal stem cells are not particularly limited as long as the effects of the present invention can be obtained, but are, for example, collected from adipose tissue (subcutaneous adipose tissue and/or other adipose tissue) in a living body.
  • epithelial cells and mesenchymal cells may be co-cultured to form cell aggregates, but other cells may be added to co-culture to form cell aggregates. good.
  • Other cells are not particularly limited as long as the effects of the present invention can be obtained, but are preferably one or more selected from the group consisting of melanocyte, melanocyte precursor cell, and melanocyte stem cell, for example.
  • Other cells may be those collected from hair follicles in vivo, or those induced to differentiate from undifferentiated cells in vitro.
  • Undifferentiated cells used for inducing differentiation of other cells in vitro are not particularly limited as long as they are cells that have the ability to differentiate into the other cells in vitro. cells, ES cells, Muse cells, or EG cells), and stem cells other than the pluripotent stem cells (e.g., stem cells obtained by reprogramming differentiated cells). .
  • the cells used for co-culture are not particularly limited as long as they are derived from animals having hair follicles, and may be cells derived from humans or non-human animals (non-human animals, e.g., primates (e.g., , monkeys), rodents (e.g. mice, rats, hamsters, guinea pigs, rabbits), carnivorous animals (e.g. dogs, cats), and ungulates (e.g. pigs, cows, horses, goats, sheep), etc. non-human mammals))).
  • non-human animals e.g., primates (e.g., , monkeys), rodents (e.g. mice, rats, hamsters, guinea pigs, rabbits), carnivorous animals (e.g. dogs, cats), and ungulates (e.g. pigs, cows, horses, goats, sheep), etc. non-human mammals)
  • human cells are used for transplantation into humans.
  • the cells used for co-culture are preferably derived from the individual to whom the cells are to be transplanted, but may be derived from an individual other than the individual to which the cells are to be transplanted.
  • the human cells used for co-culture are preferably derived from a human patient to whom the human cells are to be transplanted, but are derived from a human other than the patient (e.g., multiple cells derived from a human other than the patient).
  • Potential stem cells for example, cells induced to differentiate in vitro from iPS cells, ES cells, Muse cells or EG cells stored in cell banks may be used.
  • the epithelial cells and mesenchymal cells are seeded first.
  • induced pluripotent stem cells are seeded. Therefore, a long culture time and complicated operations for differentiating induced pluripotent stem cells are essential in culture after seeding.
  • special components such as Matrigel (registered trademark) to the culture solution. Tissues other than skin tissue may also be formed in methods involving seeding of pluripotent stem cells.
  • the method according to this embodiment may not include seeding pluripotent stem cells. Also, the method according to this embodiment may not include differentiating pluripotent stem cells. Also, the method according to this embodiment may not include culturing pluripotent stem cells.
  • Seeding of epithelial cells and mesenchymal cells is performed by placing the epithelial cells and mesenchymal cells in a culture vessel (eg, well for cell culture).
  • the culture vessel for co-cultivating epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained.
  • a relatively small volume culture vessel suitable for forming a single cell aggregate from cells including mesenchymal cells is preferably used.
  • the area of the bottom surface of the culture vessel (for example, the bottom surface of one well) may be, for example, 1000 mm 2 or less, preferably 500 mm 2 or less, and more preferably 100 mm 2 or less. , 50 mm 2 or less, and particularly preferably 20 mm 2 or less.
  • the area of the bottom surface of the culture vessel may be, for example, 0.01 mm 2 or more, preferably 0.10 mm 2 or more, more preferably 0.30 mm 2 or more, and 0.50 mm 2 or more . It is more preferably 0.70 mm 2 or more, and particularly preferably 0.70 mm 2 or more.
  • the area of the bottom surface of the culture vessel may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.
  • epithelial cells and mesenchymal cells it is preferable to seed the epithelial cells and the mesenchymal cells at the same time. , the other may be sown further.
  • the time interval between the seeding of one of epithelial cells and mesenchymal cells and the seeding of the other is the effect of the present invention. However, for example, it may be 48 hours or less (0 hours or more and 48 hours or less). That is, in this case, within 48 hours after seeding one of epithelial cells and mesenchymal cells, the other is also seeded to complete seeding of both the epithelial cells and mesenchymal cells.
  • the time interval between the seeding of one of epithelial cells and mesenchymal cells and the other is, for example, preferably 45 hours or less, more preferably 36 hours or less, and 30 hours or less. More preferably, it is particularly preferably 24 hours or less.
  • the time interval between the seeding of one of the epithelial cells and the mesenchymal cells and the seeding of the other is, for example, preferably 18 hours or less, more preferably 15 hours or less, and 12 hours or less. is even more preferable, and 9 hours or less is particularly preferable.
  • the time interval between seeding one of epithelial cells and mesenchymal cells and seeding the other is, for example, preferably 6 hours or less, more preferably 3 hours or less, and 1 hour or less. is even more preferable, and 0 hours (that is, seeding epithelial cells and mesenchymal cells at the same time) is particularly preferable.
  • the dispersed epithelial cells and the dispersed mesenchymal cells it is preferable to seed the dispersed epithelial cells and the dispersed mesenchymal cells. That is, when epithelial cells and mesenchymal cells are seeded at the same time, a cell suspension in which the epithelial cells and mesenchymal cells are dispersed is placed in a culture vessel. When one of the epithelial cells and mesenchymal cells is first seeded and then the other is further seeded, the cell suspension in which the one of the cells is dispersed is first placed in a culture vessel, and then the cell suspension is placed in a culture vessel. A cell suspension in which the other cells are dispersed is additionally put into the culture vessel.
  • the epithelial cells and mesenchymal cells seeded using the cell suspension as described above are dispersed and mixed in the culture medium in the culture vessel.
  • Individual cells dispersed in the culture medium are not substantially bound to other cells, or adhere to other cells, but the culture medium can be fluidized by an operation such as pipetting. It is easily separated from the other cells.
  • the seeding density of epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained. Density is preferred.
  • the seeding density of epithelial cells and mesenchymal cells is, for example, 0.1 ⁇ 10 4 cells/cm.
  • the number may be 2 or more, preferably 0.5 ⁇ 10 4 pieces/cm 2 or more, more preferably 1.0 ⁇ 10 4 pieces/cm 2 or more, and 2.5 ⁇ 10 4 pieces/cm 2 or more.
  • /cm 2 or more and particularly preferably 5.0 ⁇ 10 4 /cm 2 or more.
  • the seeding density of epithelial cells and mesenchymal cells may be, for example, 1000 ⁇ 10 4 cells/cm 2 or less, preferably 700 ⁇ 10 4 cells/cm 2 or less, and 500 ⁇ 10 cells/cm 2 or less. It is more preferably 4 pieces/cm 2 or less, even more preferably 400 ⁇ 10 4 pieces/cm 2 or less, and particularly preferably 300 ⁇ 10 4 pieces/cm 2 or less.
  • the seeding density of epithelial cells and mesenchymal cells may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.
  • the ratio of the total number of epithelial cells and mesenchymal cells to the total number of cells seeded in coculture is not particularly limited as long as the effect of the present invention can be obtained, but for example, it is 50% or more. 60% or more is preferable, 70% or more is more preferable, 80% or more is even more preferable, and 90% or more is particularly preferable.
  • the ratio of the total number of epithelial cells and mesenchymal cells to the total number of cells composing the cell aggregates formed by co-culturing is particularly limited as long as the effects of the present invention are obtained. However, for example, it may be 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and 90% or more. is particularly preferred.
  • the ratio of the number of seeded epithelial cells and the number of seeded mesenchymal cells in co-culture is not particularly limited as long as the effects of the present invention can be obtained.
  • it may be in the range of 1:10 to 10:1, preferably in the range of 1:9 to 9:1, and preferably in the range of 1:8 to 8:1. It is more preferably in the range of 1:7 to 7:1, even more preferably in the range of 1:6 to 6:1.
  • the mesenchymal: epithelial seeding ratio is, for example, preferably in the range of 1:5 to 5:1, more preferably in the range of 1:4 to 4:1, 1:3 to 3 :1 is more preferred, and 1:2 to 2:1 is particularly preferred.
  • epithelial cells and mesenchymal cells are mixed and cultured in a culture solution to aggregate the epithelial cells and mesenchymal cells to form cell aggregates. More specifically, in co-cultivation, epithelial cells and mesenchymal cells are seeded in a culture solution in a dispersed and mixed state, and the epithelial cells aggregate with the passage of culture time. causing the mesenchymal cells to aggregate and form intercellular connections between some epithelial cells and some mesenchymal cells.
  • epithelial cell aggregates formed by aggregation of epithelial cells mesenchymal cell aggregates formed by aggregation of mesenchymal cells, and between some epithelial cells and some Cell clumps containing cell-to-cell junctions with leaf lineage cells are obtained.
  • epithelial cells and mesenchymal cells it is preferable to perform suspension culture of epithelial cells and mesenchymal cells to form cell aggregates.
  • suspension culture epithelial cells and mesenchymal cells are cultured in a non-adhesive state to form non-adherent cell aggregates.
  • a culture vessel with a non-cell-adhesive bottom surface is preferably used for suspension culture.
  • the epithelial cells and mesenchymal cells are cultured on the non-cell-adhesive bottom surface without substantially adhering to the bottom surface (that is, in a non-adherent state). That is, for example, epithelial cells and mesenchymal cells sedimented on a non-cell-adhesive bottom surface do not adhere to the bottom surface in the culture solution, or the culture solution is made to flow by an operation such as pipetting. It adheres to the bottom surface weakly enough to be easily detached from the bottom surface.
  • the shape of epithelial cells and mesenchymal cells cultured on the non-cell-adhesive bottom surface is maintained approximately spherical.
  • cell aggregates are formed on a non-cell-adhesive bottom surface without substantially adhering to the bottom surface (that is, in a non-adhesive state). That is, for example, cell aggregates formed on a non-adhesive bottom surface do not adhere to the bottom surface in the culture medium, or are easily removed from the bottom surface by flowing the culture medium by pipetting or the like. It adheres to the bottom surface so weakly that it detaches from the bottom surface.
  • one cell aggregate in co-culture, in one culture vessel (e.g., one well), one cell aggregate can be formed from cells including epithelial cells and mesenchymal cells seeded in the culture vessel. preferable.
  • the hair regeneration ability of a cell aggregate is the ability to induce hair growth at the site where the cell aggregate is transplanted when the cell aggregate is transplanted into a living body.
  • co-culturing is performed by co-cultivating epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin (hereinafter collectively referred to as "matrix”) is dispersed.
  • matrix type I collagen or fibronectin
  • a matrix treatment is, for example, a treatment for retaining epithelial cells and mesenchymal cells in a culture solution in which type I collagen is dispersed as a matrix.
  • Matrix treatment is, for example, a treatment for retaining epithelial cells and mesenchymal cells in a culture solution in which fibronectin is dispersed as a matrix.
  • the matrix-dispersed culture medium used for matrix treatment (hereinafter sometimes referred to as "matrix treatment culture medium”) is a basal culture medium (e.g., used for co-cultivating epithelial cells and mesenchymal cells). possible culture medium) by adding the matrix. That is, the matrix treatment medium contains an exogenously added matrix.
  • the matrix treatment culture medium contains the added matrix in a solubilized state (not insolubilized state). That is, the matrix treatment culture medium contains solubilized type I collagen (non-insolubilized type I collagen) or solubilized fibronectin (non-insolubilized fibronectin) as a dispersed matrix.
  • the basal culture medium is not particularly limited as long as the effects of the present invention can be obtained, but for example, 1% A culture solution prepared by adding GultaMax Supplement (GIBCO (registered trademark)) and 0.2% Normocin (InvivoGen) is preferably used.
  • GultaMax Supplement GultaMax Supplement
  • Normocin InvivoGen
  • the matrix added in the preparation of the matrix treatment culture medium is not particularly limited as long as the effects of the present invention can be obtained.
  • commercially available matrix products such as those used in the examples below is preferably used.
  • the matrix to be added may be derived from animals (human or non-human animals), may be derived from cultured cells, or may be synthesized using genetic recombination technology. may be
  • the matrix to be added may be treated to reduce antigenicity.
  • the collagen may be atelocollagen with the telopeptide portion removed.
  • the matrix-processing culture medium in which a specific matrix is dispersed may be added with another matrix, or may be added with no other matrix.
  • the matrix treatment culture medium in which type I collagen is dispersed may be further added with fibronectin, laminin, entactin or type IV collagen, or fibronectin, laminin, entactin or type IV collagen may be added. It may be assumed that it is not.
  • the fibronectin-dispersed matrix treatment culture medium may further contain type I collagen, laminin, entactin, or type IV collagen, or type I collagen, laminin, entactin, or type IV collagen. may not be added.
  • the matrix treatment culture medium a matrix treatment culture medium in which type I collagen is dispersed is particularly preferable.
  • the matrix treatment culture medium mainly contains type I collagen as a matrix. That is, with respect to the sum of the content of type I collagen, the content of fibronectin, the content of laminin, the content of entactin, and the content of type IV collagen in the matrix treatment medium in which type I collagen is dispersed,
  • the weight percentage of the type I collagen content may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and 80% by weight or more. is even more preferable, and 90% by weight or more is particularly preferable.
  • the matrix-processing culture medium in which fibronectin is dispersed may mainly contain fibronectin as a matrix. That is, the fibronectin content relative to the sum of the type I collagen content, the fibronectin content, the laminin content, the entactin content, and the type IV collagen content in the matrix treatment medium in which the fibronectin is dispersed.
  • the weight percentage of the content may be, for example, 50% by weight or more, preferably 60% by weight or more, more preferably 70% by weight or more, and even more preferably 80% by weight or more. It is preferably 90% by weight or more, and particularly preferably 90% by weight or more.
  • the matrix treatment culture medium contains matrix at a concentration within the range in which the culture medium as a whole maintains fluidity in the co-culture of epithelial cells and mesenchymal cells.
  • the cells and gelation are first allowed to occur under conditions that do not cause gelation of the matrix (for example, a temperature that does not cause gelation).
  • a suspension containing the matrix at a suitable concentration is prepared, and then a suspension containing the cells and the matrix in a culture vessel under conditions suitable for gelation of the matrix (e.g., temperature at which gelation occurs). to gel the whole.
  • an immobile hydrogel filling the culture vessel is formed.
  • the matrix treatment culture medium does not allow co-cultivation of epithelial cells and mesenchymal cells in the culture vessel (for example, well).
  • the matrix is contained at a concentration within a range in which the treatment medium as a whole maintains fluidity (that is, in a concentration within which a non-fluid hydrogel filling the culture vessel is not formed).
  • the matrix-processing culture medium that maintains fluidity as a whole is, for example, a gel-like liquid floating in the culture medium that is formed due to local unevenness in the concentration of the dispersed matrix in the culture medium. may contain objects.
  • the gel-like material in this case is not, for example, intentionally formed hydrogel beads (eg, hydrogel beads in which cells are embedded), and does not have a specific shape such as a spherical shape.
  • the concentration of the matrix in the culture medium is generally Low compared to the concentrations used.
  • the concentration of the type I collagen in the matrix treatment medium in which the type I collagen is dispersed is not particularly limited as long as the effects of the present invention can be obtained. It is more preferably 460 ⁇ g/mL or less, even more preferably 420 ⁇ g/mL or less, and particularly preferably 400 ⁇ g/mL or less.
  • the concentration of type I collagen in the matrix treatment culture medium is, for example, preferably 380 ⁇ g/mL or less, more preferably 350 ⁇ g/mL or less, and even more preferably 300 ⁇ g/mL or less. Preferably, it is particularly preferably 260 ⁇ g/mL or less.
  • the concentration of type I collagen in the matrix treatment culture medium is, for example, preferably 3 ⁇ g/mL or higher, more preferably 5 ⁇ g/mL or higher, and even more preferably 10 ⁇ g/mL or higher. 12 ⁇ g/mL or more is particularly preferred.
  • the concentration of type I collagen in the matrix treatment culture medium is, for example, preferably 14 ⁇ g/mL or more, more preferably 16 ⁇ g/mL or more, and even more preferably 18 ⁇ g/mL or more. Preferably, it is particularly preferably 20 ⁇ g/mL or more.
  • the concentration of type I collagen in the matrix-treatment culture medium may be specified by arbitrarily combining one of the above upper limits and one of the above lower limits.
  • the concentration of fibronectin in the matrix treatment culture medium in which fibronectin is dispersed is not particularly limited as long as the effects of the present invention can be obtained, but for example, it is preferably 450 ⁇ g/mL or less, and 400 ⁇ g/mL or less. is more preferably 350 ⁇ g/mL or less, and particularly preferably 300 ⁇ g/mL or less.
  • the concentration of fibronectin in the matrix treatment culture medium is preferably, for example, 250 ⁇ g/mL or less, more preferably 200 ⁇ g/mL or less, and even more preferably 150 ⁇ g/mL or less, 120 ⁇ g/mL or less is particularly preferred.
  • the concentration of fibronectin in the matrix treatment culture medium is, for example, preferably 1 ⁇ g/mL or more, more preferably 2 ⁇ g/mL or more, even more preferably 3 ⁇ g/mL or more, and 4 ⁇ g/mL. mL or more is particularly preferred.
  • the concentration of fibronectin in the matrix treatment culture medium may be specified by any combination of one of the above upper limits and one of the above lower limits.
  • the matrix that is brought into contact with epithelial cells and mesenchymal cells in matrix treatment is a solubilized matrix that is dispersed in a matrix treatment culture medium that has fluidity as a whole. That is, the dispersed matrix that is brought into contact with epithelial cells and mesenchymal cells in matrix treatment is, for example, a hydrogel that does not have fluidity as a whole (for example, a solution containing a hydrogel polymer is poured into a culture vessel, and the culture is When epithelial cells and mesenchymal cells are embedded in a hydrogel formed by gelling the entire solution in a container, it is not a matrix that constitutes the hydrogel.
  • a hydrogel that does not have fluidity as a whole for example, a solution containing a hydrogel polymer is poured into a culture vessel, and the culture is When epithelial cells and mesenchymal cells are embedded in a hydrogel formed by gelling the entire solution in a container, it is not a matrix
  • the dispersed matrix that is brought into contact with the epithelial cells and mesenchymal cells in the matrix treatment is, for example, the case where the epithelial cells and mesenchymal cells are held on the surface of a hydrogel that does not have fluidity as a whole. is not the matrix that constitutes the hydrogel.
  • the dispersed matrix that is brought into contact with the epithelial cells and mesenchymal cells in the matrix treatment is, for example, a matrix that is preliminarily immobilized on the culture surface on which the epithelial cells and mesenchymal cells are held (e.g., pre-coated matrix on the bottom of culture vessels such as wells).
  • hydrogel In matrix treatment, epithelial cells and mesenchymal cells retained on the surface of a hydrogel that does not have fluidity as a whole (hydrogel containing a matrix or hydrogel that does not contain a matrix) are treated as a whole. It may be held in a fluid matrix treatment culture medium and brought into contact with the matrix dispersed in the culture medium.
  • the epithelial cells and mesenchymal cells retained on the surface of the hydrogel containing the matrix and having no fluidity as a whole are retained in the matrix treatment culture medium, may not include contacting with the matrix dispersed in the
  • the epithelial cells and mesenchymal cells held on the surface of the hydrogel that does not contain a matrix and have no fluidity as a whole are held in a culture medium for matrix treatment, and the culture is performed. It may not include contacting with a matrix dispersed in a liquid.
  • epithelial cells and mesenchymal cells retained on the surface of a hydrogel that does not have fluidity as a whole are used for matrix treatment. It may not include being held in a culture medium and brought into contact with a matrix dispersed in the culture medium.
  • the epithelial cells and mesenchymal cells retained on the culture surface on which the matrix has been immobilized in advance are retained in a culture medium for matrix treatment, and the cells dispersed in the culture medium are treated. Contacting with a matrix may be included.
  • the epithelial cells and mesenchymal cells held on the culture surface on which the matrix is previously immobilized are held in the matrix treatment culture medium and dispersed in the culture medium. It is good also as not including contacting with the said matrix.
  • Matrix treatment may not include embedding and culturing epithelial cells and mesenchymal cells in a hydrogel containing a matrix before cell aggregates are formed.
  • the matrix treatment may not include embedding and culturing the epithelial cells and mesenchymal cells in a matrix-free hydrogel before the cell aggregates are formed.
  • matrix treatment involves embedding and culturing epithelial cells and mesenchymal cells in a hydrogel (regardless of whether the hydrogel contains a matrix) before cell aggregates are formed. may not be included.
  • the temperature at which the epithelial cells and mesenchymal cells are maintained in the matrix treatment medium is not particularly limited as long as the effects of the present invention can be obtained. (e.g., 30 ° C. or higher and 45 ° C. or lower, preferably 33 ° C. or higher and 41 ° C. or lower, more preferably 34 ° C. or higher and 40 ° C. or lower, still more preferably 35 ° C. Above, the temperature is preferably 39° C. or less, particularly preferably 36° C. or more and 38° C. or less.
  • the time for which the epithelial cells and mesenchymal cells are retained in the matrix treatment culture medium in the matrix treatment is not particularly limited as long as the effects of the present invention can be obtained. It is preferably 6 hours or longer, more preferably 9 hours or longer, and particularly preferably 12 hours or longer.
  • the epithelial cells and mesenchymal cells are preferably retained in the matrix treatment medium for 15 hours or longer, more preferably 18 hours or longer, and more preferably 21 hours or longer. More preferably, holding for 24 hours or more is particularly preferable.
  • the timing of starting the matrix treatment is not particularly limited as long as the effects of the present invention can be obtained. is preferred.
  • time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, more preferably 66 hours or less, even more preferably 60 hours or less, and 54 hours. The following are particularly preferred.
  • the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 48 hours or less, more preferably 45 hours or less, and 42 hours or less. is even more preferable, and 39 hours or less is particularly preferable.
  • the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 36 hours or less, more preferably 33 hours or less, and 30 hours or less. is even more preferable, and 27 hours or less is particularly preferable.
  • the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 24 hours or less, more preferably 21 hours or less, and 18 hours or less. is even more preferable, and 15 hours or less is particularly preferable.
  • the time interval from the seeding of epithelial cells and mesenchymal cells to the start of matrix treatment is, for example, preferably 12 hours or less, more preferably 5 hours or less, and 3 hours or less. is even more preferable, and one hour or less is particularly preferable.
  • the time interval from seeding epithelial cells and mesenchymal cells to starting matrix treatment is 0 hours. That is, in this case, for example, by placing a cell suspension prepared by dispersing epithelial cells and mesenchymal cells in a matrix treatment culture medium into a culture vessel, the epithelial cells and mesenchymal cells Start matrix treatment at the same time as seeding.
  • the amount of matrix required for matrix treatment (for example, the concentration in the culture medium after addition is one of the above-mentioned upper limits and / or the above-mentioned A quantity of matrix that provides a concentration within the range specified by one of the lower limits specified above) is added to the culture solution containing the epithelial cells and mesenchymal cells to initiate matrix treatment.
  • the sedimented epithelial cells and mesenchymal cells in the matrix treatment culture medium. That is, in this case, co-culturing involves sedimenting the seeded epithelial cells and mesenchymal cells in a culture medium, and placing the sedimented epithelial cells and mesenchymal cells in a culture medium for matrix treatment. Including matrix processing that holds in .
  • the dispersed epithelial cells and mesenchymal cells are allowed to settle in the culture solution and deposited on the bottom surface of the culture vessel. Then, the epithelial cells and mesenchymal cells deposited on the bottom surface are held in a matrix treatment culture medium having fluidity as a whole, and the epithelial cells and mesenchymal cells are dispersed in the culture medium. contact with the matrix.
  • the method for sedimenting epithelial cells and mesenchymal cells is not particularly limited as long as the effects of the present invention can be obtained. and/or a method of centrifuging the culture vessel is preferably used.
  • the sedimentation of epithelial cells and mesenchymal cells is preferably performed before starting matrix treatment at a temperature at which the epithelial cells and mesenchymal cells are co-cultured. That is, for example, the culture for matrix treatment is first performed at a temperature lower than the co-culturing temperature (e.g., preferably 10°C or lower (more than 0°C, 10°C or lower), more preferably 7°C or lower, particularly preferably 5°C or lower).
  • a temperature at which epithelial cells and mesenchymal cells are precipitated in a liquid and then co-cultured for example, a temperature of 30 ° C. or higher and 45 ° C. or lower, preferably a temperature of 33 ° C.
  • the matrix treatment may be performed at a temperature of 34° C. or higher and 40° C. or lower, more preferably 35° C. or higher and 39° C. or lower, particularly preferably 36° C. or higher and 38° C. or lower.
  • epithelial cells and mesenchymal cells are first precipitated in a culture medium to which no matrix is added, and then the amount of matrix required for matrix treatment is added to the culture medium (for example, The concentration of matrix is added in an amount specified by one of the upper limit values and/or one of the lower limit values described above), and matrix treatment is performed at a temperature suitable for co-cultivation.
  • the addition of the matrix to the sedimented epithelial cells and mesenchymal cells is carried out at a temperature lower than the co-culturing temperature (e.g., preferably 10°C or lower (greater than 0°C, 10°C or lower), more preferably 7 C. or lower, particularly preferably 5.degree.
  • Matrix treatment is performed as part or all of the co-culture of epithelial cells and mesenchymal cells. That is, the co-cultivation may be performed entirely in the matrix treatment medium. Alternatively, co-cultivation may be performed in a matrix treatment medium until cell aggregates are formed. Also, the co-cultivation may be carried out partly in a matrix treatment culture medium and the other part in a culture medium having a lower matrix concentration than the matrix treatment culture medium.
  • co-culturing includes performing matrix treatment of epithelial cells and mesenchymal cells in a matrix treatment culture medium containing a matrix at a first concentration, Continuing the co-culturing of the epithelial and mesenchymal cells in a medium containing the matrix at a second concentration that is less than the one concentration.
  • the second concentration is not particularly limited as long as the effect of the present invention is obtained, but the ratio of the second concentration to the first concentration may be, for example, 90% or less, or 70%. % or less, 50% or less, 30% or less, or 10% or less. Also, two or more different densities may be employed as the second density. That is, the second concentration may change (for example, the second concentration may decrease) as the culture time elapses.
  • the method for reducing the matrix concentration in the culture medium during co-cultivation to be lower than that in the matrix-treatment culture medium is not particularly limited as long as the effects of the present invention can be obtained.
  • part of the matrix-treatment culture medium in the culture vessel is removed, and instead a culture medium with a matrix concentration lower than that of the matrix-treatment culture medium (for example, a culture medium to which the matrix is not added) is added. liquid) may be added.
  • the same culture vessel for example, the same well. That is, once the epithelial cells and mesenchymal cells are seeded in the culture vessel, matrix treatment and cell aggregate formation are performed in the culture vessel without removing the epithelial cells and mesenchymal cells from the culture vessel. Forming is preferred.
  • cell aggregates with excellent hair regeneration ability can be produced. That is, in the present method, co-cultivation including the above-described matrix treatment is performed, and a culture medium in which the matrix is not dispersed (that is, a culture medium to which the matrix is not added) is used instead of the culture medium for matrix treatment.
  • a cell aggregate with improved hair regrowth ability is produced as compared with a cell aggregate formed under the same conditions (hereinafter sometimes referred to as "control cell aggregate") except for the addition.
  • control cell aggregate a cell aggregate is formed in which the expression level of one or more hair growth-related genes is at least twice as high as that of the control cell aggregate.
  • the hair growth-related gene expressed by the cell aggregates is not particularly limited as long as it is a gene related to hair growth. , Edaradd, Pdgfa, and Lgr4).
  • the cell aggregates obtained by co-culturing including matrix treatment may have twice or more the expression level of the Tgfb2 gene and two times or more the expression level of the Sox21 gene as compared with the control cell aggregates.
  • the expression level of the Lgr5 gene may be 2 times or more
  • the expression level of the Lhx2 gene may be 2 times or more
  • the Edaradd gene expression level may be 2 times or more
  • the expression level of the Pdgfa gene may be doubled or more
  • the expression level of the Lgr4 gene may be doubled or more.
  • co-culturing including the matrix treatment described above, for example, it is possible to form cell aggregates that have the ability to form hair-like tissue on their surface in vitro.
  • co-cultivation involving matrix treatment can be performed to form cell aggregates having hair-like tissue on their surface.
  • cell aggregates that have the ability to form hairy tissue on their surface in vitro but do not have hairy tissue formed on their surface (i.e., By continuing the co-cultivation, it is also possible to produce a cell aggregate that has the ability to form a hairy tissue on its surface but has no hairy tissue formed on its surface yet.
  • the present embodiment includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain cells having hair regeneration ability.
  • cell aggregates including forming aggregates, by performing a matrix treatment that retains the epithelial cells and mesenchymal cells in a culture medium in which type I collagen or fibronectin is dispersed, A method of promoting the formation of ciliary tissue in cell clumps is included.
  • this embodiment includes seeding epithelial cells and mesenchymal cells, and co-culturing the epithelial cells and mesenchymal cells to obtain cell aggregates having hair regeneration ability.
  • cell aggregates including forming clumps, I
  • type collagen or fibronectin to promote the formation of hair-like tissue in the cell aggregates is included.
  • the hair-like tissue of cell aggregates contains epithelial cells and mesenchymal cells, and is formed as a structure protruding from the surface of the cell aggregates.
  • the hair-like tissue formed on the surface of the cell aggregate has, for example, a dermal papilla-like structure at its free end, the tip portion.
  • the hair-like tissue formed on the surface of the cell aggregate does not have a dermal papilla-like structure at its root portion (the end opposite to the tip portion, which is a free end).
  • the dermal papilla-like structure of the hair-like tissue has a structure similar to the dermal papilla in the hair follicle of the living body. That is, the dermal papilla-like structure has a spherical shape.
  • Dermal papilla-like structures also contain mesenchymal cells (eg, dermal papilla cells). That is, dermal papilla-like structures are identified, for example, as aggregates of Versican-positive cells.
  • the ratio of the number of mesenchymal cells (for example, dermal papilla cells) contained in the dermal papilla-like structure to the total number of cells constituting the dermal papilla-like structure is, for example, 50% or more.
  • it is preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and particularly preferably 90% or more.
  • the hair-like tissue has, for example, a hair shaft-like structure.
  • the hair shaft-like structure of the hair-like tissue has a structure similar to the hair shaft in the hair follicle of the body. That is, the hair shaft-like structure contains keratin.
  • the hair shaft-like structure also has a cuticle structure.
  • the hair shaft-like structure of the hair-like tissue extends from the vicinity of the dermal papilla-like structure included in the tip portion of the hair-like tissue toward the root portion of the hair-like tissue.
  • the shaft-like structure preferably contains melanin.
  • ciliary tissue does not contain capillaries. That is, for example, a dermal papilla-like structure contained in a hair-like tissue does not contain capillaries, unlike a dermal papilla contained in a hair follicle collected from a living body.
  • the length of the hairy tissue protruding from the surface of the cell aggregate changes with the passage of culture time, but may be, for example, 30 ⁇ m or more, preferably 50 ⁇ m or more, and 100 ⁇ m or more. is more preferable, 500 ⁇ m or more is even more preferable, and 1000 ⁇ m or more is particularly preferable.
  • the length of the hairy tissue protruding from the surface of the cell aggregate is, for example, preferably 2 mm (2000 ⁇ m) or more, more preferably 4 mm or more, and even more preferably 6 mm or more, 8 mm or more is particularly preferred.
  • the length of the hair-like tissue protruding from the surface of the cell aggregate may be, for example, 100 mm or less.
  • a cell aggregate having a hairy tissue may have a cyst-like structure inside.
  • This cyst-like structure is identified as a non-nucleated structure surrounded by cells with a nucleus.
  • the cyst-like structure is, for example, in the central part of the HE (hematoxylin-eosin)-stained cross-section of the cell aggregate, the periphery is covered with cells having cell nuclei, and is colored pink. Observed as a stained structure.
  • the cell aggregates formed by co-culture including matrix treatment do not contain piloerection muscle structures and/or sebaceous gland structures.
  • Co-cultivation is performed by performing suspension culture of epithelial cells and mesenchymal cells to form cell aggregates, and embedding the cell aggregates formed by the suspension culture in hydrogel and further culturing may include doing.
  • cell aggregates that do not have hairy tissue on their surface may be formed, or cell aggregates having hair-like tissue on their surface may be formed.
  • hydrogel-forming solution a solution for forming hydrogel
  • the hydrogel-forming solution is not particularly limited as long as the effects of the present invention can be obtained, but preferably contains a matrix, for example. That is, the hydrogel-forming solution preferably contains type I collagen or fibronectin. Moreover, the hydrogel in which the cell aggregates are embedded preferably contains type I collagen or fibronectin.
  • the matrix-containing hydrogel-forming solution and the matrix-containing hydrogel may or may not have other matrices added.
  • the hydrogel-forming solution containing type I collagen and the hydrogel containing type I collagen may further contain fibronectin, laminin, entactin, or type IV collagen. , entactin or type IV collagen may not be added.
  • the hydrogel-forming solution containing the matrix may be a basic solution (e.g., a culture medium that can be used for co-culturing epithelial cells and mesenchymal cells), similar to the matrix-processing culture medium. and an aqueous solution capable of sustaining the survival of mesenchymal cells), by adding the matrix.
  • a basic solution e.g., a culture medium that can be used for co-culturing epithelial cells and mesenchymal cells
  • an aqueous solution capable of sustaining the survival of mesenchymal cells
  • the concentration of the matrix contained in the hydrogel-forming solution is not particularly limited as long as the effects of the present invention can be obtained. is preferred.
  • the concentration of type I collagen in the hydrogel-forming solution and the concentration of type I collagen in the hydrogel obtained by gelling the entire solution are, for example, It is preferably 500 ⁇ g/mL or more, more preferably 1000 ⁇ g/mL or more, even more preferably 1500 ⁇ g/mL or more, and particularly preferably 2000 ⁇ g/mL or more.
  • the type I collagen concentration in the hydrogel-forming solution and the type I collagen concentration in the hydrogel obtained by gelling the entire solution may be, for example, 3500 ⁇ g/mL or less.
  • the fibronectin concentration in the hydrogel-forming solution and the fibronectin concentration in the hydrogel obtained by gelling the entire solution are, for example, 500 ⁇ g/mL or more. It is preferably 1000 ⁇ g/mL or more, even more preferably 1500 ⁇ g/mL or more, and particularly preferably 2000 ⁇ g/mL or more.
  • the fibronectin concentration in the hydrogel-forming solution and the fibronectin concentration in the hydrogel obtained by gelling the entire solution may be, for example, 3500 ⁇ g/mL or less.
  • the type of matrix contained in the hydrogel that embeds the cell aggregates may be the same as or different from the type of matrix used in the matrix treatment in the co-culture for forming the cell aggregates. good too.
  • cell aggregates may be formed by co-culturing including matrix treatment using type I collagen or fibronectin, and then the cell aggregates may be embedded in a hydrogel containing type I collagen or fibronectin and cultured. .
  • cell aggregates are formed by co-culturing including matrix treatment using type I collagen or fibronectin, and then one or more matrices used in the matrix treatment selected from the group consisting of type I collagen and fibronectin.
  • the cell aggregate may be embedded in a hydrogel containing and cultured.
  • cell aggregates are formed by co-cultivation including matrix treatment using type I collagen, and then the cell aggregates are embedded in a hydrogel containing type I collagen and cultured. is particularly preferred.
  • Hydrogel-embedded culture of cell aggregates is preferably carried out, for example, by adding a culture medium onto the hydrogel in a culture vessel and culturing the cell aggregates inside the hydrogel.
  • the use of the cell aggregates obtained in this embodiment is not particularly limited, but the cell aggregates have excellent hair regeneration ability and are useful as grafts to living organisms. Therefore, the method for producing cell aggregates may further include collecting the cell aggregates formed by co-culturing including matrix treatment for transplantation into a living body.
  • a cell aggregate having a hairy tissue on its surface may be used as a graft to a living body, but it is preferable to use a cell aggregate on which no hairy tissue has yet been formed.
  • a cell aggregate having the ability to form a hairy tissue on its surface and having no hairy tissue formed on its surface formed by co-culture including matrix treatment is used for transplantation into a living body. It is preferable to collect at
  • the cell aggregates are collected before the hair-like tissue is formed on the surface.
  • collection of cell aggregates for transplantation into a living body is preferably performed within 168 hours, more preferably within 144 hours, from the seeding of epithelial cells and mesenchymal cells, for example. It is even more preferable to carry out within 120 hours, and particularly preferably within 96 hours.
  • collection of cell aggregates for transplantation into a living body is preferably performed, for example, within 84 hours, more preferably within 72 hours, and 60 hours after seeding epithelial cells and mesenchymal cells. It is more preferable to carry out within 48 hours, and particularly preferably within 48 hours.
  • collection of cell aggregates for transplantation into a living body may be performed, for example, after 12 hours, preferably after 15 hours, and preferably after 18 hours from the seeding of epithelial cells and mesenchymal cells. It is more preferable to carry out after 21 hours, even more preferably after 24 hours, and particularly preferably after 24 hours.
  • the timing of collecting cell aggregates for transplantation into a living body may be specified by arbitrarily combining one of the above lower limits and one of the above upper limits.
  • a cell aggregate that does not have a hair-like tissue on its surface formed by co-culturing may not have a cyst-like structure inside.
  • the collection of cell aggregates is performed by taking out the cell aggregates from the culture vessel in which the cell aggregates were cultured.
  • the collected cell aggregates may be transferred to a container other than the culture container.
  • the method may further comprise cold storage of the cell aggregate formed by co-culturing including matrix treatment for transplantation into a living body.
  • cell aggregates formed by co-cultivation are collected, and the collected cell aggregates are stored in a cold state until they are transplanted into a living body.
  • the cell aggregate collected from the culture vessel is transferred to a container separate from the culture vessel and stored in a cold state.
  • cell aggregates formed by co-cultivation may be stored in a cold state without being collected. That is, in this case, for example, the cell aggregates in the culture vessel are stored by cooling as they are.
  • the temperature for cold storage of cell aggregates is not particularly limited as long as the effects of the present invention can be obtained, but for example, it is preferably 10 ° C. or lower (more than 0 ° C. and 10 ° C. or lower), and 7 ° C. or lower. is more preferable, and 5° C. or less is particularly preferable.
  • this method includes a hair regeneration method including transplanting the cell aggregate collected as described above into a living body.
  • a living body into which the cell aggregates are transplanted may be a human or a non-human animal, but is preferably a human. Transplantation of the cell aggregates into the living body is preferably to the skin of the living body, and particularly preferably to the scalp of the living body.
  • the transplantation of cell aggregates into a living body may be for medical or research purposes.
  • Transplantation of cell aggregates into a living body is preferably for the treatment or prevention of diseases associated with hair loss. That is, transplantation of cell aggregates into a living body is preferably transplantation into a human patient suffering from or likely to suffer from a disease associated with hair loss. Therefore, the hair regeneration method according to this embodiment is preferably a method for treating or preventing a disease associated with hair loss.
  • AGA androgenetic alopecia
  • FGA female androgenetic alopecia
  • postpartum alopecia diffuse alopecia
  • seborrhea Composed of alopecia areata, alopecia pityriasis, traction alopecia, metabolic alopecia, alopecia areata compressive, alopecia areata, alopecia areata, alopecia nervosa, trichotillomania, alopecia generalis, and symptomatic alopecia It may be one or more selected from the group.
  • the cell aggregates obtained in this embodiment can be used, for example, to search for substances that can be used for the treatment or prevention of diseases associated with hair loss, to search for substances involved in the diseases, and to study the mechanisms of the diseases. It may be possible to
  • type I collagen undiluted solution Cellmatrix (registered trademark) Type IA, type I collagen concentration 2.4 mg/mL, Nitta Gelatin Co., Ltd.
  • Basal medium cooled to 4°C. It was added to concentrations of 2.4 ⁇ g/mL, 24 ⁇ g/mL, 120 ⁇ g/mL, 240 ⁇ g/mL, 360 ⁇ g/mL, or 480 ⁇ g/mL to prepare six culture solutions with different concentrations of type I collagen.
  • each culture solution at 4°C the amount of epithelial cells and mesenchymal cells that each cell density becomes 5 ⁇ 10 4 cells / mL (the total cell density becomes 1 ⁇ 10 5 cells / mL) was suspended and a cell suspension (i.e., epithelial and mesenchymal cells dispersed and 2.4 ⁇ g/mL, 24 ⁇ g/mL, 120 ⁇ g/mL, 240 ⁇ g/mL, 360 ⁇ g/mL, or 480 ⁇ g/mL A culture medium for matrix treatment in which type I collagen was dispersed at a concentration of mL was prepared. Then, 200 ⁇ L of the cell suspension at 4° C. was poured into each well of a 96-well plate to seed 1 ⁇ 10 4 cells/well of epithelial cells and 1 ⁇ 10 4 cells/well of mesenchymal cells. .
  • a cell suspension i.e., epithelial and mesenchymal cells dispersed and 2.4
  • the 96-well plate was left in a refrigerator at 4°C for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).
  • the culture solution was exchanged once every 2 days.
  • the culture medium was exchanged by removing half of the culture medium (100 ⁇ L) from each well and then adding 100 ⁇ L of the culture medium to which type I collagen was not added (basic medium). Under all conditions, the culture medium in the wells maintained fluidity as a whole throughout the culture period.
  • FIG. 1 shows micrographs on day 8 of culture.
  • epithelial cells and mesenchymal cells aggregated in all six culture systems with different concentrations of type I collagen in the culture medium at the time of seeding (that is, the concentration of type I collagen in the matrix treatment).
  • cell clumps were formed, one in each well.
  • hairy tissue in four culture systems in which the concentration of type I collagen in the culture medium at the time of seeding was 24 ⁇ g/mL, 120 ⁇ g/mL, 240 ⁇ g/mL or 360 ⁇ g/mL, hairy tissue (four photographs included in FIG. In each case, the formation of cell aggregates having a portion indicated by an arrow) was confirmed.
  • epithelial cells and mesenchymal cells began to aggregate after the start of suspension culture, and cell aggregates were formed on the first day of culture. Thereafter, on the 4th to 6th days of culture, hairy tissue began to form on the surface of the cell aggregates. The hair-like tissue of cell aggregates elongated with the passage of culture time.
  • Fig. 2 shows the relationship between the concentration of type I collagen in the culture solution at the time of seeding (at the time of matrix treatment) and the formation efficiency of hairy tissue in cell aggregates on day 8 of culture.
  • the type I collagen concentration was 24 ⁇ g/mL, 120 ⁇ g/mL or 240 ⁇ g/mL
  • hairy tissue was formed in all cell aggregates (hairy tissue formation efficiency 100%).
  • hairy tissue was formed in 3 out of 6 cell aggregates (hairy tissue formation efficiency: 50%).
  • no hairy tissue was formed in any of the cell aggregates (hairy tissue formation efficiency 0%).
  • Fig. 3 shows the relationship between the type I collagen concentration in the culture solution at the time of seeding and the number of hair-like tissues formed per cell aggregate on day 8 of culture. That is, in FIG. 3, the horizontal axis indicates the type I collagen concentration in the culture medium at the time of seeding, and the vertical axis indicates the total number of hairy tissue formed in the cell aggregates divided by the number of the cell aggregates. The arithmetic mean value is shown.
  • FIG. 4 shows a micrograph of cell aggregates on day 8 of culture in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 ⁇ g/mL.
  • Photograph (ii) in FIG. 4 shows an enlarged portion of the square frame shown in photograph (i).
  • FIG. 5 shows the results of HE staining of sections of cell aggregates on day 8 of culture in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 ⁇ g/mL.
  • Photograph (ii) in FIG. 5 shows an enlarged portion of the square frame shown in photograph (i).
  • cyst-like structure was confirmed in the central portion of the cell aggregate.
  • This cyst-like structure was a structure without a cell nucleus.
  • the periphery of the cyst-like structure was covered by cells with cell nuclei.
  • FIG. 6 shows the results of fluorescent staining of Versican on sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 ⁇ g/mL.
  • Photograph (ii) in FIG. 6 shows an enlarged portion of the square frame shown in photograph (i). The part enclosed by the white dotted line in photograph (ii) shows the hair shaft-like structure.
  • the hair-like tissue formed in the cell aggregates has Versican-positive cell aggregates, ie, dermal papilla cell aggregates (dermal papilla-like structure) at its tip. confirmed.
  • FIG. 7 shows the results of fluorescent staining of CD34 on sections of cell aggregates on day 8 of culture formed in a culture system in which the concentration of type I collagen in the culture solution at the time of seeding was 120 ⁇ g/mL.
  • the portion surrounded by the white dotted line indicates the tip portion of the ciliary tissue.
  • CD34-positive cells i.e., hair follicle epithelial stem cells
  • hair follicle epithelial stem cells were contained in the periphery of the hair shaft-like structure close to the dermal papilla-like structure at the tip of the hair-like tissue.
  • FIG. 8 schematically shows a cell aggregate having a hairy tissue obtained in this example based on the observation results described above. Note that FIG. 8 is only a schematic diagram, and the size and arrangement of the cell aggregates, the hairy tissue, and the cells and structures contained therein do not limit the cell aggregates according to the present invention.
  • the cell aggregate formed by co-culturing epithelial cells and mesenchymal cells in this example had a cyst-like structure in its central part.
  • the cell clumps had one or more hair-like tissue on their surface.
  • This hair-like tissue consists of a dermal papilla-like structure (agglomerate of Versican-positive cells) formed at the tip, which is the free end, and hair extending from the vicinity of the dermal papilla-like structure to the root of the hair-like tissue. It had a stem-like structure.
  • the hair-like tissue contained hair follicle epithelial stem cells (CD34-positive cells) around the periphery of the portion of the hair shaft-like structure close to the dermal papilla-like structure.
  • the 96-well plate was left in a refrigerator at 4°C for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).
  • the culture solution was exchanged in the same manner as in Example 1 above. On day 4 of culture, one cell aggregate was formed in each well. However, formation of hairy tissue was not confirmed in the cell aggregates on day 4 of culture.
  • a hydrogel-forming solution containing cell aggregates and type I collagen was dropped into one well of a 6-well plate and held in an incubator at 37°C for 20 minutes to gel type I collagen. .
  • the entire solution in the well gelled to form a hydrogel having no fluidity.
  • the seven cell aggregates were three-dimensionally dispersed and embedded separately from each other.
  • FIG. 9 shows one cell aggregate embedded in the hydrogel on day 8 of culture (day 4 after starting hydrogel embedding culture), day 12, day 18, and day 22. Photomicrographs taken on day 27 and day 27 are shown.
  • FIG. 10 shows an enlarged portion (ciliary tissue) surrounded by a dotted box in each of the five photographs included in FIG.
  • FIG. 11 shows changes over time in the length of the hair-like tissue shown in FIGS.
  • the horizontal axis indicates the number of culture days (days), and the vertical axis indicates the length of the hairy tissue ( ⁇ m).
  • the hair-like tissue on the surface of the cell aggregates elongated with the passage of the culture time. increased.
  • the length of the ciliary tissue reached approximately 2 mm (2000 ⁇ m) on day 22 of culture.
  • fibronectin undiluted solution (fibronectin solution (derived from human plasma), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with a final concentration of 6 ⁇ g / mL, 12 ⁇ g / mL, 25 ⁇ g / mL, 50 ⁇ g / mL or 100 ⁇ g /
  • fibronectin solution derived from human plasma
  • fibronectin solution derived from human plasma
  • 50 ⁇ g / mL 100 ⁇ g /
  • 1 ⁇ 10 4 cells/well of epithelial cells were added in the same manner as in Example 1 above, except that the culture solution prepared by adding to the basal medium was used so as to make the volume of mL. and 1 ⁇ 10 4 cells/well of mesenchymal cells were seeded.
  • the 96-well plate was left in a refrigerator at 4°C for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).
  • the culture solution was exchanged in the same manner as in Example 1 above. Under all conditions, the culture medium in the wells maintained fluidity as a whole throughout the culture period.
  • FIG. 12 shows micrographs on day 8 of culture. As shown in FIG. 12, epithelial cells and mesenchymal cells were aggregated in all five culture systems with different fibronectin concentrations in the culture medium at the time of seeding, and one cell aggregate was formed in each well. rice field.
  • FIG. 13 shows the correspondence between the fibronectin concentration in the culture solution at the time of seeding and the formation efficiency of hairy tissue in the cell aggregates on day 8 of culture. As shown in FIG. 13, the formation efficiency of hair-like tissue was 40% to 70%.
  • Fig. 14 shows the relationship between the fibronectin concentration in the culture medium at the time of seeding and the number of hairy tissues formed per cell aggregate on day 8 of culture. As shown in FIG. 14, 1 to 3 hair-like tissues were formed on average per cell aggregate.
  • Fig. 15 shows the results of HE staining of sections of cell aggregates on day 8 of culture in a culture system in which fibronectin in the culture medium at the time of seeding was 100 ⁇ g/mL. As indicated by arrows in FIG. 15, two strands of hairy tissue were identified in this section. In addition, a dermal papilla-like structure was confirmed at the tip of the hair-like tissue. On the other hand, a cyst-like structure was confirmed in the central portion of the cell aggregate.
  • a basal medium to which type I collagen was not added was prepared in the same manner as in Example 1 described above. Then, in a basal medium cooled to 4°C, epithelial cells and mesenchymal cells were added in an amount that each gave a cell density of 1 x 10 5 cells/mL (an amount that gave a total cell density of 2 x 10 5 cells/mL). Cells were suspended to prepare a cell suspension. On the other hand, a culture solution containing type I collagen at a concentration of 240 ⁇ g/mL was prepared by adding type I collagen undiluted solution cooled to 4° C. to a basal medium cooled to 4° C.
  • Example 4-1 Immediately after seeding (0 hours after seeding), 100 ⁇ L of a culture solution containing type I collagen at a concentration of 240 ⁇ g/mL and cooled to 4° C. was added to the wells of Example 4-1. As a result, in the wells of Example 4-1, epithelial cells and mesenchymal cells were retained in the matrix treatment culture medium in which type I collagen was dispersed at a concentration of 120 ⁇ g/mL.
  • the 96-well plate was then left in a refrigerator at 4°C for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).
  • the 96-well plate When 5 hours and 40 minutes had passed since the cells were seeded, the 96-well plate was transferred to a refrigerator at 4°C and allowed to stand for 20 minutes to cool the culture medium containing the cells in the wells. After cooling for 20 minutes, that is, when 6 hours had passed since seeding the cells, 100 ⁇ L of the culture medium containing type I collagen at a concentration of 240 ⁇ g/mL and cooled to 4° C. was added to the wells of Example 4-2. As a result, in the wells of Example 4-2, epithelial cells and mesenchymal cells were retained in the matrix treatment medium in which type I collagen was dispersed at a concentration of 120 ⁇ g/mL.
  • the 96-well plate was shaken in a refrigerator at 4°C for 20 minutes. After that, the 96-well plate was transferred to a 37° C. incubator to continue co-cultivation (suspension culture).
  • Example 4-3, Example 4-4, and Example Matrix treatment was initiated by adding type I collagen to the wells of Examples 4-5 and 4-6.
  • the culture solution was changed for the first time on the third day of culture, and thereafter, the culture solution was changed once every two days. Exchange of the culture solution was performed in the same manner as in Example 1 above.
  • FIG. 16 shows micrographs on day 8 of culture. As shown in FIG. 16, epithelial cells and mesenchymal cells aggregated to form one cell aggregate in each well in all of the six culture systems with different addition timings of type I collagen.
  • Fig. 17 shows the timing of addition of type I collagen (time from seeding of cells to addition of type I collagen) in correspondence with the formation efficiency of hairy tissue in cell aggregates on day 8 of culture.
  • type I collagen time from seeding of cells to addition of type I collagen
  • hairy tissue was formed in 9 out of 12 cell aggregates (hair formation efficiency of like tissue 75%).
  • the formation efficiencies of hairy tissue in the culture system to which type I collagen was added were 58%, 58%, and 58%, respectively. 50%, 33% and 25%. That is, it was confirmed that the efficiency of hair-like tissue formation tends to decrease as the timing of adding type I collagen is delayed.
  • Fig. 18 shows the relationship between the timing of addition of type I collagen and the number of hair-like tissues formed per cell aggregate on day 8 of culture. As shown in FIG. 18, it was confirmed that as the timing of adding type I collagen was delayed, the number of hair-like tissues formed in one cell aggregate tended to decrease.
  • FIG. 19 shows a micrograph of cell aggregates on day 8 of culture in a culture system to which type I collagen was added immediately after cell seeding.
  • Photograph (ii) in FIG. 19 shows an enlarged portion of the square frame shown in photograph (i).
  • a swollen structure was observed at the tip, which is the free end of the hair-like tissue protruding from the surface of the cell aggregate.
  • Fig. 20 shows an enlarged photograph of hairy tissue formed on the surface of cell aggregates on day 8 of culture in a culture system in which type I collagen was added immediately after cell seeding.
  • the portion surrounded by the white dotted line in FIG. 20 is the ciliary tissue.
  • a bulging structure is observed at the tip portion, which is the free end of the hairy tissue, and a black hair shaft extending from the bulging structure toward the root of the hairy tissue. A similar structure was observed.
  • Example 5-1 a culture medium to which type I collagen was added to a final concentration of 120 ⁇ g/mL was used, and in the same manner as in Example 1 above, epithelial cells and mesenchymal cells were co-cultured (suspended culture) was started.
  • Example 5-2 co-culture of epithelial cells and mesenchymal cells ( Floating culture) was started.
  • FIG. 21 shows cell aggregates formed using a culture solution in which type I collagen is dispersed in Example 5-1, and cell aggregates formed using a culture solution in which type I collagen is not dispersed in Example 5-2.
  • the results of GO (Gene Ontology) analysis using a microarray are shown for the cell aggregates obtained. That is, FIG. 21 shows a group of genes significantly (Fold change>2) increased in the cell aggregates of Example 5-1 compared to the cell aggregates of Example 5-2 (i.e., the cell aggregates of Example 5-1 Gene groups whose expression levels in clumps were at least twice that of cell clumps in Example 5-2).
  • Example 5-1 formed by co-culture including matrix treatment using type I collagen
  • Example 5-2 formed by co-culture without the matrix treatment
  • the expression levels of hair growth-related genes were significantly increased.
  • the expression levels of Tgfb2, Sox21, Lgr5, Lhx2, Edaradd, Pdgfa, and Lgr4, which are marker genes related to hair follicle development, are all It was more than double that of 2 cell clumps.
  • Example 5-1 formed by co-culture including the matrix treatment using type I collagen were compared to the cell aggregates of Example 5-2 formed by co-culture without the matrix treatment. Therefore, it was considered that the hair regrowth ability was remarkably improved.
  • Example 6-1 a culture medium to which type I collagen was added to a final concentration of 120 ⁇ g/mL was used, and 1 ⁇ 10 4 cells were added to each well of a 96-well plate in the same manner as in Example 1 above. Epithelial cells/well and 1 ⁇ 10 4 cells/well of mesenchymal cells were seeded.
  • the 96-well plate was left in a refrigerator at 4°C for 30 minutes to allow the cells to settle and deposit on the bottom of the wells. After that, the 96-well plate was transferred to a 37° C. incubator to initiate co-culture (suspension culture).
  • Example 6-2 instead of type I collagen, a culture solution was used in which Matrigel undiluted solution (Matrigel (registered trademark) Basement Membrane Matrix, CORNING (registered trademark)) was added at a final concentration of 2 v/v%.
  • Co-culture (suspension culture) of epithelial cells and mesenchymal cells was started in the same manner as in Example 6-1 except for the above.
  • the Matrigel undiluted solution contains 10.6 mg/mL (protein amount measured by the Lowry method) of soluble basement membrane matrix extracted from EHS (Engelbreth-Holm-Swarm) mouse tumor, and the composition ratio in the basement membrane matrix is , 56% laminin, 8% entactin, and 31% type IV collagen.
  • the culture solution to which the Matrigel stock solution was added in an amount to give a final concentration of 2 v/v% contained 118 ⁇ g/mL laminin, 16 ⁇ g/mL entactin, and 66 ⁇ g/mL type IV collagen. be.
  • Example 6-3 epithelial cells and mesenchymal cells were obtained in the same manner as in Example 6-1 above, except that a culture medium (basic medium) to which neither type I collagen nor Matrigel was added was used. Co-culture (suspension culture) was started.
  • FIG. 22 shows the results of visually counting the number of regenerated hairs at each transplantation site of the mouse 4 weeks after transplantation.
  • FIG. 22 shows the results of visually counting the number of regenerated hairs at each transplantation site of the mouse 4 weeks after transplantation.
  • a culture solution basic medium
  • no matrix in which neither type I collagen nor Matrigel was dispersed
  • the number of regenerated hairs per cell aggregate formed using type I collagen was 8 times that of cell aggregates formed without using type I collagen and Matrigel
  • the number of regenerated hairs using Matrigel was 8 times that of cell aggregates formed without using type I collagen and Matrigel. 3.6 times that of the formed cell clumps.

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Abstract

Ce procédé de production d'un agrégat cellulaire comprend : le placage de cellules épithéliales et de cellules mésenchymateuses ; et la co-culture des cellules épithéliales et des cellules mésenchymateuses pour former un agrégat cellulaire ayant une capacité de régénération capillaire. La co-culture comprend un processus matriciel pour maintenir les cellules épithéliales et les cellules mésenchymateuses dans une solution de culture dans laquelle est dispersé du collagène de type I ou de la fibronectine.
PCT/JP2022/034628 2021-10-08 2022-09-15 Procédé de production d'agrégat cellulaire ayant une capacité de régénération capillaire, et procédé associé WO2023058429A1 (fr)

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