Method for preparing and culturing hair follicle organoids, a screening method for hair-related drugs using them, and hair transplant materials.

Abstract

This invention relates to a screening method for hair-related drugs and a technology for producing and culturing hair follicle organoids for the creation of hair transplant materials. More specifically, it involves isolating hair follicles from existing skin organoids using human induced pluripotent stem cell lines during the hair follicle stage, culturing them on culture inserts coated with collagen and Matrigel to confirm hair follicle growth and degeneration, and confirming that new hair grows when the cultured hair follicles are transplanted into the skin, thus enabling their use as hair transplant materials. Furthermore, by treating hair follicle organoids with hair loss-inducing factors, a hair loss simulation model was generated by confirming a decrease in hair follicle length, damage to dermal papilla cell aggregates, decreased expression of WNT3A or β-catenin, and increased expression of DKK-1 or TGF-β2. This is expected to be widely applicable as a screening method for hair growth promoters, hair loss preventives, hair loss mitigating agents, or hair loss treatments, or as a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or mitigating hair loss.

Description

Technical Field

[0001] The present invention provides a method for producing and culturing hair follicle organoids, a method for screening hair drugs using the same, and a hair transplantation material.

Background Art

[0002] Human hair is regarded as one of the most important aspects of human appearance. Human hair is not only of primary importance in protecting the skin and scalp, but also plays a unique role in social and sexual communication, and thus is very important. Alopecia refers to the state where there is no hair in a part where hair should normally exist, or the phenomenon where hair that has stopped growing falls out naturally. Generally, it means the loss of terminal hair (thick and black hair) on the scalp. Hair usually falls out about 50 to 100 or more per day, and the more hair falls out, the more it is defined as alopecia.

[0003] In South Korea as well, many studies related to alopecia are being conducted. Recently, research on many regulatory factors involved in hair growth and alopecia mechanisms has been actively carried out. Currently, the hair growth-related products distributed in the market are diverse, but most of them have insufficient hair loss prevention and hair growth effects, or are temporary and do not meet the requirements of users. In addition, the supporting materials regarding the efficacy and safety of the products are not sufficient, and when the use of the products is interrupted, serious side effects such as recurrence of alopecia or occurrence of sexual dysfunction are reported, and the actual situation is that there are difficulties in using them.

[0004] In order to overcome alopecia, in addition to the development of therapeutic agents, hair transplantation can be considered. Hair transplantation aims to prevent the decline of stress and social adaptability due to hair, etc., and above all, to contribute to the mental health of hair transplant recipients by artificially implanting hair into humans with little hair.

[0005] Hair transplantation can be performed using either live hair or artificial hair. In the case of live hair transplantation, a portion of the patient's own hair is taken and the hair follicles are transplanted to areas without hair, allowing hair to grow in the future.

[0006] This type of hair transplantation, which involves separating hair follicles one by one and then transplanting them to the desired location, has the advantages of less bleeding, minimal scarring, and the ability to freely control the direction and position of growing hair. However, because the procedure involves separating each hair follicle, it is time-consuming, and there is a risk of side effects such as pain and inflammation from the separation, as well as the procedure requiring considerable experience.

[0007] In particular, in the case of hair transplantation, since the transplanted hair is the patient's own, considering the characteristics of hair transplant recipients who have insufficient hair, it is difficult to harvest a large number of hair follicles, making extensive transplantation challenging. This can lead to a shortage of hair to transplant, thus limiting the capabilities of autologous hair transplantation. Furthermore, the difficulty in storing the plucked hair and the need to transplant it immediately in real time also presented time constraints.

[0008] Therefore, there is a need to develop screening methods for finding candidate substances that have a structure more similar to actual hair follicles, can mimic their function and be used as hair follicle transplant materials, and can be used as hair growth agents or hair loss preventatives, hair loss alleviants, and hair loss treatments, or for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss. [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a method for producing hair follicle organoids, comprising the following steps:

[0010] (1) the step of separating hair follicles from skin organoids; and (2) the step of culturing the separated hair follicles at an air-liquid interface.

[0011] Another object of the present invention is to provide hair follicle organoids manufactured by the above manufacturing method.

[0012] Another object of the present invention is to provide a hair transplant material comprising the hair follicle organoid.

[0013] Another object of the present invention is to provide a method for producing strip-shaped hair follicle organoids, comprising the following steps:

[0014] (1) Cutting the skin organoid into a flat surface and then cutting it vertically; and (2) Culturing the cut skin organoid at an air-liquid interface.

[0015] Another object of the present invention is to provide strip-shaped hair follicle organoids manufactured by the above manufacturing method.

[0016] Another object of the present invention is to provide a hair transplant material comprising the strip-shaped hair follicle organoid.

[0017] Another object of the present invention is to provide a method for manufacturing hair follicle organoids of a hair loss simulating model, comprising the following steps:

[0018] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with a hair loss inducing factor.

[0019] Another object of the present invention is to provide a hair follicle organoid, a hair loss simulating model, manufactured by the above-described method.

[0020] Another object of the present invention is to provide a method for manufacturing strip-shaped hair follicle organoids of a hair loss simulating model, comprising the following steps:

[0021] (1) After cutting and spreading the skin organoid on a flat surface, the step of cutting it vertically; (2) The step of culturing the cut skin organoid at an air-liquid interface to produce strip-shaped hair follicle organoids; and (3) The step of treating the strip-shaped hair follicle organoids with a hair loss induction factor.

[0022] Still another object of the present invention is to provide strip-shaped hair follicle organoids of a hair loss imitation model produced by the above manufacturing method.

[0023] Still another object of the present invention is to provide a screening method for a hair growth promoter, a hair loss preventive agent, a hair loss relieving agent or a hair loss treatment agent including the following steps:

[0024] (1) The step of separating hair follicles from skin organoids; (2) The step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; (3) The step of treating the hair follicle organoids with a hair loss induction factor to produce hair follicle organoids of a hair loss imitation model; and (4) The step of treating the hair follicle organoids of the hair loss imitation model with a candidate substance of a hair growth promoter, a hair loss preventive agent, a hair loss relieving agent or a hair loss treatment agent.

[0025] Still another object of the present invention is to provide a screening method for a hair growth promoter, a hair loss preventive agent, a hair loss relieving agent or a hair loss treatment agent including the following steps:

[0026] (1) After cutting and spreading the skin organoid on a flat surface, the step of cutting it vertically; (2) The step of culturing the cut skin organoid at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) The step of treating the strip-shaped hair follicle organoids with a hair loss induction factor to produce strip-shaped hair follicle organoids of a hair loss imitation model; and (4) The step of treating the strip-shaped hair follicle organoids of the hair loss imitation model with a candidate substance of a hair growth promoter, a hair loss preventive agent, a hair loss relieving agent or a hair loss treatment agent.

[0027] Still another object of the present invention is to provide a method for screening a hair growth promoter, a hair loss preventive agent, a hair loss alleviating agent or a hair loss therapeutic agent, comprising the following steps:

[0028] (1) separating hair follicles from a skin organoid; (2) culturing the separated hair follicles at an air-liquid interface to produce a hair follicle organoid; and (3) treating the hair follicle organoid with a candidate substance for a hair growth promoter, a hair loss preventive agent, a hair loss alleviating agent or a hair loss therapeutic agent.

[0029] Still another object of the present invention is to provide a method for screening a hair growth promoter, a hair loss preventive agent, a hair loss alleviating agent or a hair loss therapeutic agent, comprising the following steps:

[0030] (1) cutting a skin organoid flatwise and spreading it, then cutting it vertically; (2) culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) treating the strip-shaped hair follicle organoid with a candidate substance for a hair growth promoter, a hair loss preventive agent, a hair loss alleviating agent or a hair loss therapeutic agent.

[0031] Still another object of the present invention is to provide a method for screening a cosmetic composition for hair growth promotion, hair loss prevention or hair loss alleviation, comprising the following steps:

[0032] (1) separating hair follicles from a skin organoid; (2) culturing the separated hair follicles at an air-liquid interface to produce a hair follicle organoid; (3) treating the hair follicle organoid with a hair loss-inducing factor to produce a hair follicle organoid of a hair loss mimic model; and (4) treating the hair follicle organoid of the hair loss mimic model with a candidate substance for a cosmetic composition for hair growth promotion, hair loss prevention or hair loss alleviation.

[0033] Still another object of the present invention is to provide a method for screening a cosmetic composition for hair growth promotion, hair loss prevention or hair loss alleviation, comprising the following steps:

[0034] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor to produce a strip-shaped hair follicle organoid for a hair loss simulation model; and (4) Treating the strip-shaped hair follicle organoid for the hair loss simulation model with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0035] Another object of the present invention is to provide a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0036] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with candidate substances for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0037] Another object of the present invention is to provide a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0038] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss. [Means for solving the problem]

[0039] To achieve the above objective, the present invention provides a method for producing hair follicle organoids, comprising the following steps:

[0040] (1) the step of separating hair follicles from skin organoids; and (2) the step of culturing the separated hair follicles at an air-liquid interface.

[0041] Furthermore, the present invention provides hair follicle organoids manufactured by the above manufacturing method.

[0042] Furthermore, the present invention provides a hair transplant material containing the hair follicle organoid.

[0043] Furthermore, the present invention provides a method for producing strip-shaped hair follicle organoids, comprising the following steps:

[0044] (1) Cutting the skin organoid into a flat surface and then cutting it vertically; and (2) Culturing the cut skin organoid at an air-liquid interface.

[0045] Furthermore, the present invention provides strip-shaped hair follicle organoids manufactured by the above manufacturing method.

[0046] Furthermore, the present invention provides a hair transplant material that includes the strip-shaped hair follicle organoid.

[0047] Furthermore, the present invention provides a method for manufacturing hair follicle organoids for hair loss simulation models, which includes the following steps:

[0048] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with a hair loss inducing factor.

[0049] Furthermore, the present invention provides a hair follicle organoid, a hair loss simulation model, manufactured by the above-described method.

[0050] Furthermore, the present invention provides a method for manufacturing strip-shaped hair follicle organoids of a hair loss simulating model, which includes the following steps:

[0051] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor.

[0052] Furthermore, the present invention provides strip-shaped hair follicle organoids of hair loss simulation models produced by the above manufacturing method.

[0053] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0054] (1) the step of separating hair follicles from skin organoids; (2) the step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; (3) the step of treating the hair follicle organoids with hair loss inducing factors to produce hair follicle organoids for hair loss simulation models; and (4) the step of treating the hair follicle organoids for hair loss simulation models with candidate substances for hair growth promoters, hair loss preventives, hair loss mitigants, or hair loss treatments.

[0055] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0056] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor to produce a strip-shaped hair follicle organoid for a hair loss simulation model; and (4) Treating the strip-shaped hair follicle organoid for the hair loss simulation model with a candidate substance for a hair growth promoter, hair loss preventive agent, hair loss mitigating agent, or hair loss treatment agent.

[0057] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0058] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with candidate substances for hair growth promoters, hair loss preventives, hair loss mitigants, or hair loss treatments.

[0059] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0060] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a candidate substance for a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

[0061] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0062] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; (3) a step of treating the hair follicle organoids with hair loss inducing factors to produce hair follicle organoids for hair loss simulation models; and (4) a step of treating the hair follicle organoids for hair loss simulation models with candidate substances for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0063] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0064] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor to produce a strip-shaped hair follicle organoid for a hair loss simulation model; and (4) Treating the strip-shaped hair follicle organoid for the hair loss simulation model with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0065] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0066] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with candidate substances for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0067] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0068] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss. [Effects of the Invention]

[0069] This invention relates to a screening method for hair drugs and a technique for producing and culturing hair follicle organoids for hair transplantation materials. More specifically, it involves isolating hair follicles from existing skin organoids using human induced pluripotent stem cell lines during the hair follicle stage, culturing them on culture inserts coated with collagen and Matrigel to confirm hair follicle growth and degeneration, and confirming that new hair grows when the cultured hair follicles are transplanted into the skin. As a result, it is expected that these follicles can be used as hair transplantation materials.

[0070] Furthermore, by treating the hair follicle organoids of the present invention with hair loss-inducing factors, we confirmed a decrease in hair follicle length, damage to dermal papilla cell aggregates, decreased expression of WNT3A or β-catenin, and increased expression of DKK-1 or TGF-β2, thereby generating a hair loss simulation model. It is expected that this can be utilized as a screening method for hair growth promoters, hair loss preventives, hair loss mitigating agents, or hair loss treatments, or as a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or mitigating hair loss. [Brief explanation of the drawing]

[0071] [Figure 1] This study confirmed the generation of skin organoids and hair follicles using human-induced pluripotent stem cell lines. [Figure 2] This study confirmed the structural characteristics of hair follicles as expressed in the fabricated skin organoids. [Figure 3] This image shows a hair follicle in the hair germ or hair peg stage. [Figure 4] Hair follicles in the hair bud or hair stalk stage were isolated from the prepared skin organoids, and the culture results were confirmed. [Figure 5] This study involved preparing artificial extracellular matrix coatings for culture inserts under various conditions (collagen alone, Matrigel alone, and a mixture of collagen and Matrigel), and then culturing hair follicles at an air-liquid interface. [Figure 6]This study observed changes in the expression of DKK-1, β-catenin, and CD34 genes to confirm hair follicle growth and degeneration. [Figure 7] This study confirmed the results of in vivo transplantation of hair follicle organoids. [Figure 8] Hair follicle organoids were treated with dihydrotestosterone (DHT) and minoxidil (MXD), and changes in the boundaries of the dermal papilla cell aggregates, hair follicle length, and the expression of the genes WNT3A and β-catenin were examined. [Figure 9] This study examined the presence or absence of cell death and cell proliferation in strip-shaped hair follicle organoids, as well as changes in hair follicle and hair length induced by hair loss-inducing hormones. [Modes for carrying out the invention]

[0072] The present invention will be described in more detail below.

[0073] The present invention provides a method for producing hair follicle organoids, comprising the following steps: (1) separating hair follicles from skin organoids; and (2) culturing the separated hair follicles at an air-liquid interface.

[0074] The skin organoid of step (1) above can be manufactured by the following steps:

[0075] (a) A step of culturing organoids derived from pluripotent stem cells in the presence of a Wnt agonist; (b) A step of culturing the culture from step (a) in a medium for maturing skin organoids for 40 days or more; and (c) A step of cutting the culture from step (b) and culturing it at an air-liquid interface.

[0076] The Wnt agonist is added to pluripotent stem cells on days 5-7 of culture.

[0077] The aforementioned Wnt agonist is also CHIR-99021.

[0078] The aforementioned skin organoids may express one or more selected from the group consisting of KRT5 (Keratin 5), KRT10, KRT15, ​​KRT17, Loricrin, Filaggrin, SOX2 (SRY-Box Transcription Factor 2), and Melan-A, but are not limited to these.

[0079] The hair follicle in stage (1) above may include, but is not limited to, one or more selected from the group consisting of the dermal sheath, outer root sheath, inner root sheath, dermal papilla cells, matrix cells, bulge region, and sebaceous gland.

[0080] The hair follicles in stage (1) above can be separated during the hair bud stage or hair stalk stage.

[0081] The aforementioned hair bud stage is formed within 60 to 80 days after the formation of skin organoids, and preferably within 70 to 80 days.

[0082] The aforementioned hair-scratching stage is formed within 80 to 100 days after the formation of skin organoids, and preferably within 90 to 100 days.

[0083] The separation in step (1) above is performed using microexcision.

[0084] The culture in step (2) above can be carried out on a culture insert.

[0085] The culture inserts are coated with collagen and Matrigel; or with collagen.

[0086] The collagen and Matrigel are in a ratio of 1:10 to 10:1, and preferably 1:1 to 3:1.

[0087] Furthermore, the present invention provides hair follicle organoids manufactured by the above manufacturing method.

[0088] The hair follicle organoid may express one or more selected from the group consisting of KRT5, KRT10, KRT15, ​​KRT17, loricrin, filaggrin, SOX2, and melan-A, but is not limited to these.

[0089] Furthermore, the present invention provides a hair transplant material containing the hair follicle organoid.

[0090] Furthermore, the present invention provides a method for producing strip-shaped hair follicle organoids, comprising the following steps:

[0091] (1) Cutting the skin organoid into a flat surface and then cutting it vertically; and (2) Culturing the cut skin organoid at an air-liquid interface.

[0092] The skin organoid of step (1) above can be manufactured by the following steps:

[0093] (a) A step of culturing organoids derived from pluripotent stem cells in the presence of a Wnt agonist; (b) A step of culturing the culture from step (a) in a medium for maturing skin organoids for 40 days or more; and (c) A step of cutting the culture from step (b) and culturing it at an air-liquid interface.

[0094] The Wnt agonist is added to pluripotent stem cells on days 5-7 of culture.

[0095] The aforementioned Wnt agonist is also CHIR-99021.

[0096] The aforementioned skin organoids may express one or more selected from the group consisting of KRT5, KRT10, KRT15, ​​KRT17, loricrin, filaggrin, SOX2, and melan-A, but are not limited to these.

[0097] The cutting described in (1) above may be performed when the hair follicle within the skin organoid is in the dermal bud stage or the hair stalk stage.

[0098] The aforementioned hair bud stage is formed within 60 to 80 days after the formation of skin organoids, and preferably within 70 to 80 days.

[0099] The aforementioned hair-scratching stage is formed within 80 to 100 days after the formation of skin organoids, and preferably within 90 to 100 days.

[0100] The cutting in step (1) above allows the opened skin organoid to be cut at intervals of 1 to 3 mm.

[0101] Furthermore, the present invention provides strip-shaped hair follicle organoids manufactured by the above manufacturing method.

[0102] The aforementioned strip-shaped hair follicle organoids can express one or more selected from the group consisting of KRT5, KRT10, KRT15, ​​KRT17, loricrin, filaggrin, SOX2, and melan-A, but are not limited to these.

[0103] Furthermore, the present invention provides a hair transplant material that includes the strip-shaped hair follicle organoid.

[0104] Furthermore, the present invention provides a method for manufacturing hair follicle organoids for hair loss simulation models, which includes the following steps:

[0105] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with a hair loss inducing factor.

[0106] The skin organoid of step (1) above can be manufactured by the following steps:

[0107] (a) A step of culturing organoids derived from pluripotent stem cells in the presence of a Wnt agonist; (b) A step of culturing the culture from step (a) in a medium for maturing skin organoids for 40 days or more; and (c) A step of cutting the culture from step (b) and culturing it at an air-liquid interface.

[0108] The Wnt agonist is added to pluripotent stem cells on days 5-7 of culture.

[0109] The aforementioned Wnt agonist is also CHIR-99021.

[0110] The aforementioned skin organoids may express one or more selected from the group consisting of KRT5, KRT10, KRT15, ​​KRT17, loricrin, filaggrin, SOX2, and melan-A, but are not limited to these.

[0111] The aforementioned hair loss is caused by hormonal imbalance or ultraviolet radiation.

[0112] The aforementioned hormones are also dihydrotestosterone (DHT), testosterone, thyroid hormone, or cortisol.

[0113] The aforementioned thyroid hormone is also tyrosine.

[0114] The hair loss inducing factors in stage (3) above include male hormones, stress hormones, or ultraviolet light.

[0115] The aforementioned hormones are, but are not limited to, dihydrotestosterone, testosterone, or thyroid hormones.

[0116] The aforementioned stress hormone is also cortisol.

[0117] The aforementioned thyroid hormone is also tyrosine.

[0118] Furthermore, the present invention provides a hair follicle organoid, a hair loss simulation model, manufactured by the above-described method.

[0119] The hair follicle organoids in the hair loss simulation model may have, but are not limited to, one or more features selected from the group consisting of the following features:

[0120] 1) Decreased hair follicle length; 2) Damage to dermal papilla cell aggregates; 3) Decreased expression of WNT3A or β-catenin; or 4) Increased expression of DKK-1 or TGF-β2.

[0121] Furthermore, the present invention provides a method for manufacturing strip-shaped hair follicle organoids of a hair loss simulating model, which includes the following steps:

[0122] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor.

[0123] The skin organoid of step (1) above can be manufactured by the following steps:

[0124] (a) A step of culturing organoids derived from pluripotent stem cells in the presence of a Wnt agonist; (b) A step of culturing the culture from step (a) in a medium for maturing skin organoids for 40 days or more; and (c) A step of cutting the culture from step (b) and culturing it at an air-liquid interface.

[0125] The aforementioned skin organoids may express one or more selected from the group consisting of KRT5, KRT10, KRT15, ​​KRT17, loricrin, filaggrin, SOX2, and melan-A, but are not limited to these.

[0126] The cutting described in (1) above may be performed when the hair follicle within the skin organoid is in the dermal bud stage or the hair stalk stage.

[0127] The hair loss inducing factors in stage (3) above include male hormones, stress hormones, or ultraviolet light.

[0128] Furthermore, the present invention provides strip-shaped hair follicle organoids of hair loss simulation models produced by the above manufacturing method.

[0129] The strip-shaped hair follicle organoids of the hair loss simulation model may have, but are not limited to, one or more features selected from the group consisting of the following features:

[0130] 1) Decreased hair follicle length; 2) Damage to dermal papilla cell aggregates; 3) Decreased expression of WNT3A or β-catenin; or 4) Increased expression of DKK-1 or TGF-β2.

[0131] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0132] (1) the step of separating hair follicles from skin organoids; (2) the step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; (3) the step of treating the hair follicle organoids with hair loss inducing factors to produce hair follicle organoids for hair loss simulation models; and (4) the step of treating the hair follicle organoids for hair loss simulation models with candidate substances for hair growth promoters, hair loss preventives, hair loss mitigants, or hair loss treatments.

[0133] The process may include a step in which, after treatment with the candidate substance in step (4), if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined that the substance is a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

[0134] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0135] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor to produce a strip-shaped hair follicle organoid for a hair loss simulation model; and (4) Treating the strip-shaped hair follicle organoid for the hair loss simulation model with a candidate substance for a hair growth promoter, hair loss preventive agent, hair loss mitigating agent, or hair loss treatment agent.

[0136] The process may include a step in which, after treatment with the candidate substance in step (4), if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined that the substance is a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

[0137] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0138] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with candidate substances for hair growth promoters, hair loss preventives, hair loss mitigants, or hair loss treatments.

[0139] The procedure may include a step in which, after treatment with the candidate substance in step (3) above, if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined to be a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

[0140] Furthermore, the present invention provides a screening method for hair growth stimulants, hair loss preventatives, hair loss mitigating agents, or hair loss treatment agents, comprising the following steps:

[0141] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a candidate substance for a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

[0142] The procedure may include a step in which, after treatment with the candidate substance in step (3) above, if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined to be a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

[0143] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0144] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; (3) a step of treating the hair follicle organoids with hair loss inducing factors to produce hair follicle organoids for hair loss simulation models; and (4) a step of treating the hair follicle organoids for hair loss simulation models with candidate substances for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0145] The process may include a step in which, after treatment with the candidate substance in step (4), if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined to be a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0146] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0147] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; (3) Treating the strip-shaped hair follicle organoid with a hair loss inducing factor to produce a strip-shaped hair follicle organoid for a hair loss simulation model; and (4) Treating the strip-shaped hair follicle organoid for the hair loss simulation model with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0148] The process may include a step in which, after treatment with the candidate substance in step (4), if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined to be a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0149] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0150] (1) a step of separating hair follicles from skin organoids; (2) a step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) a step of treating the hair follicle organoids with candidate substances for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0151] The process may include a step in which, after treatment with the candidate substance in step (3), if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined to be a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0152] Furthermore, the present invention provides a screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:

[0153] (1) Cutting a skin organoid into a flat surface and then cutting it vertically; (2) Culturing the cut skin organoid at an air-liquid interface to produce a strip-shaped hair follicle organoid; and (3) Treating the strip-shaped hair follicle organoid with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0154] The process may include a step in which, after treatment with the candidate substance in step (3), if the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases, it is determined to be a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

[0155] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to those skilled in the art to explain the present invention more completely.

[0156] <Example 1> Preparation of skin organoids derived from human induced pluripotent stem cells

[0157] 1. Culture of human induced pluripotent stem cells

[0158] Human induced pluripotent stem cell lines (iPSCs; CMC3, CMC11) were cultured in Essential 8 (Gibo) culture medium supplemented with Y-27632 (10 μM) on culture dishes coated with Vitronectin (ThermoFisher). The culture medium was changed daily, and subculturing was performed using ReLeSR (Stem Cell Technology) in cycles of approximately 4 days.

[0159] 2. Differentiation method for skin organoids using human induced pluripotent stem cell lines

[0160] Skin organoids were cultured using existing differentiation protocols for skin organoids utilizing human induced pluripotent stem cell lines, based on literature and patents (Jung, S. et al. Wnt-activating human skin organoid model of atopic dermatitis induced by Staphylococcus aureus and its protective effects by Cutibacterium acnes. iScience, 25(10), 105150 (2022), METHOD FOR CONSTRUCTION OF ATOPIC DERMATITS MODEL BY USING PLURIPOTENT STEM CELL-DERIVED SKIN ORGANOID, PCT / KR2021 / 014810 (2021.10.21.)). Skin organoids were cultured using human induced pluripotent stem cells through the following steps: 1) culturing germinal blasts, 2) inducing differentiation into non-neuroectoderm, and 3) inducing differentiation into cranial nerve ridge-like cells and activating Wnt signals (Figure 1A).

[0161] In skin organoids, hair follicles are 1 mm 2 We confirmed that approximately 10 hair follicles were generated per unit area (normally, nearly 45,000 identical hair follicles are generated in a single spherical skin organoid) (Figure 1B).

[0162] 3. Verification of the characteristics of skin organoid hair follicles

[0163] To verify the structural characteristics of hair follicles expressed in the fabricated skin organoids, H&E and immunofluorescence staining were performed. As a result, all of the hair follicle's fine structures, including the dermal sheath, outer root sheath, inner root sheath, dermal papilla cells, matrix cells, bulge region, and sebaceous glands, were identified (Figure 2).

[0164] <Example 2> Hair follicle isolation and culture in skin organoids derived from human induced pluripotent stem cells

[0165] 1. Isolation and culture via microdissection

[0166] Hair follicles were isolated from the epidermis and dermis layers using skin organoids derived from human-induced pluripotent stem cells. First, because hair follicles exist at a very small size in skin organoids, micro-excision techniques were developed to harvest hair follicles without damage.

[0167] Skin organoids were transferred to tissue culture plates, dissected using a Spring Micro-Scissor, and the target tissue was separated from the epidermis and dermis using forceps. Because hair follicles and skin layers (epidermis and dermis) are highly embedded and tend to adhere firmly to collagen fibers, they were carefully removed with microneedles. After washing the separated hair follicles with culture medium to avoid contamination from other cells, they were cultured on Transwell culture inserts coated with collagen in a 3D manner, exposed to air.

[0168] To optimize the optimal isolation period, hair follicles were isolated at different stages: the trichome stage (where the epidermis thickens and protrudes downward, and dermal fibroblasts are condensed beneath the hair placode (Figure 3A)) and the pilosa stage (where the end elongates into a rounded columnar shape, dermal fibroblasts form bulb-shaped dermal papilla cells, and the concave proximal end begins to surround the condensed cells (Figure 3B)). When separating during the trichome stage, complete separation of the epidermal and dermal layers was difficult, resulting in regeneration of both layers during culture. Furthermore, when hair follicles were isolated and cultured before the pilosa stage, it was confirmed that new hair follicles formed at the upper end of the existing follicles. On the other hand, when hair follicles at stages beyond the pilosa stage were isolated, hair growth was observed in perfectly structured follicles without intervention from other tissues. The separation timing was determined to be within 60-80 days after the formation of skin organoids in the hair bud stage, and within 80-100 days in the hair stalk stage (Figure 4A, Figure 4B).

[0169] When hair follicles at the pilaris stage or later were isolated and cultured in vitro, their ultrastructural differentiation was examined through H&E and immunofluorescence staining (Figure 4C).

[0170] 2. Optimization of conditions for hair follicle culture support

[0171] Artificial extracellular matrix, which serves as a coating support on Transwell culture inserts used for culturing hair follicles, was prepared under various conditions, and then the hair follicles were cultured at an air-liquid interface. Hair follicles at the blast stage or higher were isolated and cultured on collagen alone, Matrigel alone, and an extracellular matrix mixture containing collagen and Matrigel.

[0172] As a result, when hair follicles were cultured under collagen-only conditions, hair follicle growth was observed, but the differentiation and growth rate to hair were slower than under conditions where an extracellular matrix containing collagen and Matrigel was mixed. When hair follicles were cultured with Matrigel alone, they aggregated with each other, resulting in culture failure. Ultimately, it was confirmed that the growth rate of hair follicles and hair was most enhanced with an extracellular matrix containing collagen or a mixture of collagen and Matrigel (collagen:Matrigel ratio of 3:1 to 1:1 in mixed culture of collagen and Matrigel) (Figure 5A). Furthermore, it was confirmed that hair follicles could be cultured in vitro for more than 60 days on an extracellular matrix mixture containing collagen and Matrigel, and that hair follicle growth stopped after 80 days (Figure 5B).

[0173] After culturing hair follicles, we classified them into those cultured within 10 days, 30-50 days, and 60 days or more, and confirmed the expression of genes associated with hair growth induction and inhibition using quantitative PCR. The expression of the hair loss-inducing cytokine DKK-1 gene was significantly increased in hair follicles cultured for 60 days or more, while the expression of β-catenin, a gene involved in hair growth, and CD34, a major marker of hair follicle stem cells, decreased. This demonstrated that hair follicle growth and degeneration can be confirmed even under in vitro culture conditions (Figure 6).

[0174] <Example 3> Confirmation of hair follicle growth in vivo during transplantation

[0175] For transplantation, we used hair follicles that, after in vitro culture, had a morphological structure that allowed for differentiation into the hair bulb and hair root, and that had reliably progressed to the bulge region and were beginning to produce hair (Figure 7A).

[0176] In 6-week-old Balb / c nu / nu mice, shallow puncture wounds were made in the skin using a 20G needle, approximately parallel to the surface, about 0.5 mm vertically, and 2.5 mm horizontally. Hair follicle organoids were then transplanted into the wound sites. This was done to confirm their potential as a transplant material.

[0177] As a result, existing hair disappeared within one week, and new hair was observed approximately one month later. The presence or absence of melanin cells in the hair follicle organoids determined the growth of either white or black hair (Figure 7B, Figure 7C).

[0178] We confirmed that the hair in question was of human origin, not mouse origin, through H&E staining and immunofluorescence staining using human nuclear antigen antibodies (Figure 7D). This is significant because the hair follicle organoids created opened up the skin layer of the hair mice and allowed hair to grow even after transplantation, demonstrating that hair follicle organoids can function as a potential hair transplant material.

[0179] <Example 4> Confirmation of the possibility of drug screening

[0180] Methods for evaluating the efficacy of promoting hair loss and alleviating hair loss symptoms have mainly been conducted through animal experiments, and in vitro testing methods have not yet been established. Existing in vitro testing methods evaluate hair follicle growth and morphology by treating hair follicle papilla cells, and there are limitations in studying signal transmission mechanisms.

[0181] In this invention, changes in morphology and gene expression of hair follicles were observed during growth, making it possible to evaluate a variety of active substances.

[0182] To confirm the possibility of evaluating the effective substance, dihydrotestosterone (DHT), a substance well known as a hormone that acts on hair follicles and causes hair loss, was used in a 10-minute test. -5 One group was treated with M, while the other was treated with minoxidil (MXD), known to promote hair growth, at a concentration of 10 μM for 10 days, along with DHT.

[0183] As a result, morphologically, the DHT-only treatment group showed shorter hair follicle lengths, blurred boundaries in the dermal papilla cell aggregates, and damage, while the MXD concurrent treatment group showed longer hair follicle lengths, sharper boundaries in the dermal papilla cell aggregates, and a relatively reduced degree of damage compared to the DHT-only group (Figure 8A).

[0184] The expression of genes related to hair follicle growth promotion or suppression was confirmed via quantitative PCR. The results showed that in the DHT-only group, the expression of WNT3A and β-catenin, which are involved in promoting hair follicle growth, decreased compared to the control group, while it significantly increased in the MXD concurrent treatment group. Furthermore, in the DHT-only group, the expression of DKK-1 and TGF-β2, which are involved in suppressing hair follicle growth, significantly increased compared to the control group, while it significantly decreased in the MXD concurrent treatment group. This confirmed that DHT induced a reaction similar to hair loss in hair follicle organoids, and that MXD alleviated the hair loss symptoms (Figure 8B).

[0185] In addition to male hormones, other factors that can induce hair loss (such as ultraviolet light or stress hormones) can also be used to create hair loss-like models and evaluate effective substances for hair growth stimulants, hair loss preventatives, or hair loss treatments.

[0186] The target of drug screening varies depending on the treatment order of the active substance and the hair loss inducing substance. However, it is possible to confirm the hair loss prevention and protective effects when the active substance is applied first, and the hair growth and therapeutic effects when applied second. Furthermore, even when various substances are applied individually to normal hair follicle organoids, this can be used as a platform to confirm the inhibitory or promoting effect of those substances on hair follicle growth.

[0187] Therefore, by confirming that the efficacy of the substance identified through this method can be evaluated through the hair follicle, it was found that it is also possible to evaluate other active substances, and that in order to evaluate the effects of drugs on a large number of hair follicles, it is possible to use not only single hair follicles but also strip-shaped hair follicle organoids.

[0188] Spherical skin organoids were cut flat and spread out during the hair bud stage (60-80 days) or the hair stalk stage (80-100 days), and then cut vertically at 1 mm intervals.

[0189] The Transwell culture inserts were cultured at an air-liquid interface on an artificial extracellular matrix consisting of collagen alone or a mixture of collagen and Matrigel in a ratio of 1:10 to 10:1, with the cut surfaces facing downwards and upwards.

[0190] When cell death (Cleaved Caspase-3) and cell proliferation (Ki-67) were examined in the hair follicle strips cultured in this manner using immunofluorescence staining, it was confirmed that proliferation was possible without cell death (Figure 9A).

[0191] In strip-shaped hair follicle organoids, we observed a decrease in hair follicle and hair length and suppression of growth when treated with a hair loss-inducing hormone (DHT) from 3 days after culture. We also confirmed that the hair follicles atrophied and that the dermal papilla cells and hair spindles began to separate (Figure 9B, Figure 9C).

[0192] Through this process, it was found that strip-shaped hair follicle organoids can also be used to evaluate active substances for hair growth stimulants, hair loss preventatives, or hair loss treatments.

[0193] The above description of the present invention is illustrative, and those skilled in the art will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not limiting.

[0194] The scope of this invention is defined by the claims described below, and all modified or altered forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be interpreted as being included within the scope of this invention.

Claims

1. A method for producing hair follicle organoids, including the following steps: (1) The step of separating hair follicles from skin organoids, (2) The step of culturing the separated hair follicles at the air-liquid interface.

2. The method for producing a hair follicle organoid according to claim 1 is characterized in that the skin organoid of step (1) is produced by comprising the following steps: (a) A step of culturing organoids derived from pluripotent stem cells in the presence of a Wnt agonist, (b) A step of culturing the culture from step (a) in a medium for skin organoid maturation for 40 days or more, (c) A step in which the culture from step (b) is cut and cultured at the air-liquid interface.

3. The method for producing hair follicle organoids according to claim 2, characterized in that the Wnt activator is added to pluripotent stem cells on the 5th to 7th day of culture.

4. The method for producing a hair follicle organoid according to claim 1, characterized in that the skin organoid expresses one or more selected from the group consisting of KRT5, KRT10, KRT15, ​​KRT17, loricrin, filaggrin, SOX2, and melan-A.

5. The method for producing a hair follicle organoid according to claim 1, characterized in that the hair follicle in step (1) above includes one or more selected from the group consisting of the dermal sheath, outer root sheath, inner root sheath, dermal papilla cells, hair matrix cells, bulge region, and sebaceous gland.

6. The method for producing hair follicle organoids according to claim 1, characterized in that the hair follicles in step (1) are separated during the hair bud stage or hair stalk stage.

7. The method for producing hair follicle organoids according to claim 6, characterized in that the hair bud stage is formed within 60 to 80 days after the formation of skin organoids.

8. The method for producing a hair follicle organoid according to claim 6, characterized in that the hair stalking stage is formed within 80 to 100 days after the formation of the skin organoid.

9. The method for producing hair follicle organoids according to claim 1, characterized in that the separation in step (1) is performed using microexcision.

10. The method for producing a hair follicle organoid according to claim 1, characterized in that the culture in step (2) is performed on a culture insert.

11. The method for producing a hair follicle organoid according to claim 10, characterized in that the culture insert is coated with collagen and Matrigel; or collagen.

12. The method for producing a hair follicle organoid according to claim 11, characterized in that the collagen and Matrigel are in a ratio of 1:10 to 10:

1.

13. A hair follicle organoid manufactured by the manufacturing method described in claim 1.

14. A hair transplant material comprising the hair follicle organoid described in claim 13.

15. A method for producing strip-shaped hair follicle organoids, including the following steps: (1) After cutting the skin organoid into a flat surface and spreading it out, the next step is to cut it vertically, (2) The step of culturing the severed skin organoids at an air-liquid interface.

16. The method for producing a strip-shaped hair follicle organoid according to claim 15 is characterized in that the skin organoid of step (1) is produced by comprising the following steps: (a) A step of culturing organoids derived from pluripotent stem cells in the presence of a Wnt agonist, (b) A step of culturing the culture from step (a) in a medium for skin organoid maturation for 40 days or more, (c) A step in which the culture from step (b) is cut and cultured at the air-liquid interface.

17. The method for producing a strip-shaped hair follicle organoid according to claim 15, characterized in that the cutting in step (1) is performed when the hair follicle within the skin organoid is in the hair bud stage or hair stalk stage.

18. The method for producing a strip-shaped hair follicle organoid according to claim 15, characterized in that the cutting in step (1) above involves cutting the opened skin organoid at intervals of 1 to 3 mm.

19. A strip-shaped hair follicle organoid manufactured by the manufacturing method described in claim 15.

20. A hair transplant material comprising the strip-shaped hair follicle organoid described in claim 19.

21. A method for manufacturing hair follicle organoids in hair loss simulation models, including the following steps: (1) The step of separating hair follicles from skin organoids, (2) A step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids, (3) The step of treating the hair follicle organoid with a hair loss inducing factor.

22. The method for producing a hair follicle organoid of a hair loss simulation model according to claim 21, characterized in that the hair loss inducing factor in step (3) is male hormones, stress hormones, or ultraviolet light.

23. The method for producing hair follicle organoids of a hair loss simulation model according to claim 22, characterized in that the hormone is dihydrotestosterone, testosterone, or thyroid hormone.

24. The method for producing a hair follicle organoid of a hair loss simulation model according to claim 22, characterized in that the stress hormone is cortisol.

25. A hair follicle organoid of a hair loss simulation model, manufactured by the manufacturing method described in claim 21.

26. The hair follicle organoid of the hair loss simulation model is characterized by having one or more features selected from the group consisting of the following features, as described in claim 25: 1) Decreased hair follicle length; 2) Damage to dermal papilla cell aggregates; 3) Decreased expression of WNT3A or β-catenin; or 4) Increased expression of DKK-1 or TGF-β2.

27. A method for manufacturing strip-shaped hair follicle organoids of a hair loss simulation model, including the following steps: (1) After cutting the skin organoid into a flat surface and spreading it out, the next step is to cut it vertically. (2) A step of culturing the severed skin organoids at an air-liquid interface to produce strip-shaped hair follicle organoids, (3) The step of treating the strip-shaped hair follicle organoid with a hair loss inducing factor.

28. A strip-shaped hair follicle organoid, a hair loss imitation model, manufactured by the manufacturing method described in claim 27.

29. Screening methods for hair growth stimulants, hair loss preventatives, hair loss relief agents, or hair loss treatment agents, including the following stages: (1) The step of separating hair follicles from skin organoids, (2) A step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids, (3) The step of preparing a hair follicle organoid model for hair loss simulation by treating the hair follicle organoid with a hair loss inducing factor, (4) A step of treating the hair follicle organoid of the hair loss simulation model with a candidate substance for a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

30. A method for screening a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent according to claim 29, further comprising the step of determining that the agent is a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent if, after processing the candidate substance in step (4) above, the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases.

31. Screening methods for hair growth stimulants, hair loss preventatives, hair loss relief agents, or hair loss treatment agents, including the following stages: (1) After cutting the skin organoid into a flat surface and spreading it out, the next step is to cut it vertically, (2) A step of culturing the severed skin organoids at an air-liquid interface to produce strip-shaped hair follicle organoids, (3) The step of preparing a strip-shaped hair follicle organoid for a hair loss simulation model by treating the strip-shaped hair follicle organoid with a hair loss inducing factor, (4) A step of treating the strip-shaped hair follicle organoids of the hair loss simulation model with candidate substances for hair growth stimulants, hair loss preventants, hair loss mitigants, or hair loss treatments.

32. A method for screening a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent according to claim 31, further comprising the step of determining that the agent is a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent if, after processing the candidate substance in step (4) above, the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases.

33. Screening methods for hair growth stimulants, hair loss preventatives, hair loss relief agents, or hair loss treatment agents, including the following stages: (1) The step of separating hair follicles from skin organoids, (2) A step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids, (3) A step of treating the hair follicle organoid with a candidate substance for a hair growth stimulant, a hair loss preventative, a hair loss mitigating agent, or a hair loss treatment agent.

34. A method for screening a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent according to claim 33, further comprising the step of determining that the agent is a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent if, after processing the candidate substance in step (3) above, the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases.

35. Screening methods for hair growth stimulants, hair loss preventatives, hair loss relief agents, or hair loss treatment agents, including the following stages: (1) After cutting the skin organoid into a flat surface and spreading it out, the next step is to cut it vertically, (2) A step of culturing the severed skin organoids at an air-liquid interface to produce strip-shaped hair follicle organoids, (3) A step of treating the strip-shaped hair follicle organoid with a candidate substance for a hair growth stimulant, hair loss preventative, hair loss mitigating agent, or hair loss treatment agent.

36. A method for screening a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent according to claim 35, further comprising the step of determining that the agent is a hair growth stimulant, hair loss preventive, hair loss reliever, or hair loss treatment agent if, after processing the candidate substance in step (3) above, the expression of WNT3A or β-catenin increases, or the expression of DKK-1 or TGF-β2 decreases.

37. A screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, including the following steps: (1) The step of separating hair follicles from skin organoids, (2) A step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids, (3) The step of preparing a hair follicle organoid model for hair loss simulation by treating the hair follicle organoid with a hair loss inducing factor, (4) A step of treating the hair follicle organoid of the hair loss simulation model with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

38. A screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, including the following steps: (1) After cutting the skin organoid into a flat surface and spreading it out, the next step is to cut it vertically, (2) A step of culturing the severed skin organoids at an air-liquid interface to produce strip-shaped hair follicle organoids, (3) The step of preparing a strip-shaped hair follicle organoid for a hair loss simulation model by treating the strip-shaped hair follicle organoid with a hair loss inducing factor, (4) A step of treating the strip-shaped hair follicle organoids of the hair loss simulation model with candidate substances for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

39. A screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, including the following steps: (1) The step of separating hair follicles from skin organoids, (2) A step of culturing the separated hair follicles at an air-liquid interface to produce hair follicle organoids, (3) A step of treating the hair follicle organoid with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

40. A screening method for cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, including the following steps: (1) After cutting the skin organoid into a flat surface and spreading it out, the next step is to cut it vertically, (2) A step of culturing the severed skin organoids at an air-liquid interface to produce strip-shaped hair follicle organoids, (3) A step of treating the strip-shaped hair follicle organoid with a candidate substance for a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.

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