Method for constructing and culturing hair follicle organoid, hair drug screening method using same, and hair transplantation material
By isolating and culturing hair follicle organoids from skin organoids at the gas-liquid interface, and treating them with hair loss inducing factors, hair follicle organoids for hair transplantation and hair loss simulation models were prepared. This solved the problems of long time consumption, large side effects, and insufficient effectiveness of hair growth products in existing hair transplantation technologies, and achieved highly efficient hair transplantation and hair loss treatment.
Patent Information
- Application Number
- CN202480019078.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-03-16
- Filing Date
- 2024-03-18
- Publication Date
- 2025-11-11
AI Technical Summary
Existing hair transplant techniques suffer from problems such as long treatment time, significant side effects, insufficient autologous hair, and difficulty in storage. Furthermore, the effectiveness and safety of hair growth products on the market are insufficient, making hair loss treatment difficult.
Hair follicles were isolated from skin organoids and cultured at the gas-liquid interface. Combined with treatment with hair loss inducing factors, hair follicle organoids were prepared for hair transplantation and hair loss simulation models. Hair growth promoters, anti-hair loss agents or hair loss treatment agents were then screened.
It provides efficient hair transplant materials and hair loss simulation models, enabling the screening of effective hair growth promoters and hair loss treatments, solving the problems in existing technologies, and improving the efficiency and safety of hair transplantation.
Smart Images
Figure CN120936706A_ABST
Abstract
Description
Technical Field
[0001] This disclosure provides methods for producing and culturing hair follicle organoids, methods for screening hair-related drugs using these methods, and hair transplant materials. Background Technology
[0002] Human hair is considered one of the most important aspects of a person's appearance. It is important not only because it primarily protects the skin and scalp, but also because it plays a unique role in social and sexual interactions. Hair loss refers to the absence of hair in areas where hair should be present, or the natural shedding of hair that has stopped growing, and typically refers to the loss of terminal hairs (thick, black hairs) on the scalp. Usually, about 50 to 100 hairs fall out per day, and the more hairs lost, the greater the likelihood of having hair loss.
[0003] South Korea is conducting numerous studies on hair loss, and recently, research on many regulatory factors involved in the mechanisms of hair growth and loss is actively underway. Although a wide variety of hair growth products are currently available on the market, most have little or no effect on hair loss prevention or hair growth, failing to meet consumer expectations. Furthermore, there is insufficient evidence regarding the effectiveness or safety of these products, and serious side effects (such as relapse of hair loss after discontinuation of product use, or sexual dysfunction) have been reported, making application difficult.
[0004] In addition to developing treatments to overcome hair loss, hair transplantation can be considered for people with very little hair (skin hair). By artificially implanting hair, it can prevent stress and reduced social adaptability caused by hair loss. Most importantly, it aims to help the mental health of hair transplant recipients.
[0005] These hair transplants include methods of transplanting natural hair and methods of transplanting artificial hair. In the case of natural hair transplantation, some of the patient's own hair is collected, and the hair roots (hair follicles) are transplanted to areas without hair to induce future hair growth.
[0006] Although this type of hair transplant has the advantages of less bleeding and less scarring because the hair roots are separated one by one and transplanted to the desired location, and the direction and location of hair growth are highly determined, it also has the disadvantage of requiring a lot of experience because the process involves separating the hair roots one by one, which takes a considerable amount of time and may cause side effects (such as pain and inflammation) due to the separation.
[0007] In particular, in the case of hair transplantation, due to the use of autologous hair, the difficulty in collecting a large number of hair roots, considering the characteristics of hair transplant recipients with insufficient hair (skin), makes it difficult to cover a large area for transplantation. Furthermore, there are also situations where there is a shortage of hair to be transplanted, which limits the use of autologous hair for transplantation. In addition, due to the difficulty in storage, the plucked hair must be transplanted immediately, which also has time limitations.
[0008] Therefore, there is a need to develop a screening method for discovering candidate substances that have a structure similar to actual hair follicles and at the same time mimic the function of hair follicle transplantation materials, or for developing a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss. Summary of the Invention
[0009] Technical goals
[0010] The purpose of this disclosure is to provide a method for preparing hair follicle organoids, comprising the following steps:
[0011] (1) Isolating hair follicles from skin organoids; and (2) culturing isolated hair follicles at the air-liquid interface.
[0012] Another object of this disclosure is to provide hair follicle organoids prepared by this preparation method.
[0013] Another objective of this disclosure is to provide hair transplant material containing the hair follicle organoid.
[0014] Another object of this disclosure is to provide a method for preparing strip-shaped hair follicle organoids, comprising the following steps:
[0015] (1) Planar sections of skin organoids were prepared and unfolded, and then vertically cut; and (2) the cut skin organoids were cultured at the air-liquid interface.
[0016] Another object of this disclosure is to provide strip-shaped hair follicle organoids prepared by this preparation method.
[0017] Another objective of this disclosure is to provide hair transplant material comprising the strip-shaped hair follicle organoid.
[0018] Another object of this disclosure is to provide a method for preparing hair follicle organoids for a hair loss simulation model, comprising the following steps:
[0019] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; and (3) Treat the hair follicle organoids with a hair loss inducing factor.
[0020] Another object of this disclosure is to provide hair follicle organoids for hair loss simulation models prepared by the method described.
[0021] Another object of this disclosure is to provide a method for preparing strip-shaped hair follicle organoids for a hair loss simulation model, comprising the following steps:
[0022] (1) The skin organoids were sliced in a plane and unfolded, and then cut vertically; (2) the cut skin organoids were cultured at the gas-liquid interface to produce strip hair follicle organoids; and (3) the strip hair follicle organoids were treated with hair loss inducing factors.
[0023] Another object of this disclosure is to provide strip-shaped hair follicle organoids for hair loss simulation models produced by the preparation method.
[0024] Another object of this disclosure is to provide a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0025] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factor to produce hair follicle organoids for a hair loss simulation model; and (4) Treat the hair follicle organoids for a hair loss simulation model with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relieving agent or hair loss treatment agent.
[0026] Another object of this disclosure is to provide a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0027] (1) Planar sections of skin organoids are prepared and then vertically cut; (2) Cut skin organoids are cultured at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) Strip-shaped hair follicle organoids are treated with hair loss inducing factors to produce strip-shaped hair follicle organoids for hair loss simulation models; and (4) Strip-shaped hair follicle organoids for hair loss simulation models are treated with candidates of hair growth promoters, anti-hair loss agents, hair loss alleviators or hair loss treatments.
[0028] Another object of this disclosure is to provide a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0029] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; and (3) Treat the hair follicle organoids with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relieving agent or hair loss treatment agent.
[0030] Another object of this disclosure is to provide a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0031] (1) Planar sectioning and unfolding of skin organoids, followed by vertical cutting; (2) Culturing the cut skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treating the strip-shaped hair follicle organoids with candidates of hair growth promoters, anti-hair loss agents, hair loss relieving agents or hair loss treatment agents.
[0032] Another object of this disclosure is to provide a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0033] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factor to produce hair follicle organoids for a hair loss simulation model; and (4) Treat the hair follicle organoids for a hair loss simulation model with a candidate substance of a cosmetic composition to promote hair growth, prevent hair loss, or alleviate hair loss.
[0034] Another object of this disclosure is to provide a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0035] (1) Planar sections of skin organoids are prepared and then vertically cut; (2) Cut skin organoids are cultured at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) Strip-shaped hair follicle organoids are treated with a hair loss inducing factors to produce strip-shaped hair follicle organoids for hair loss simulation models; and (4) Strip-shaped hair follicle organoids for hair loss simulation models are treated with a candidate substance of a cosmetic composition to promote hair growth, prevent hair loss, or alleviate hair loss.
[0036] Another object of this disclosure is to provide a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0037] (1) Isolating hair follicles from skin organoids; (2) Culturing the isolated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) Treating the hair follicle organoids with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.
[0038] Another object of this disclosure is to provide a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0039] (1) Planar sectioning and unfolding of skin organoids, followed by vertical cutting; (2) Culturing the cut skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treating the strip-shaped hair follicle organoids with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.
[0040] Technical solution
[0041] To achieve the above objectives, this disclosure provides a method for preparing hair follicle organoids, comprising the following steps:
[0042] (1) Isolation of hair follicles from skin organoids; and (2) culture of isolated hair follicles at the air-liquid interface.
[0043] In addition, this disclosure provides hair follicle organoids prepared by this preparation method.
[0044] In addition, this disclosure provides hair transplant materials comprising hair follicle organoids.
[0045] Furthermore, this disclosure provides a method for preparing strip-shaped hair follicle organoids, comprising the following steps:
[0046] (1) Planar sections of skin organoids were prepared and unfolded, and then vertically cut; and (2) the cut skin organoids were cultured at the air-liquid interface.
[0047] In addition, this disclosure provides strip-shaped hair follicle organoids prepared by this preparation method.
[0048] In addition, this disclosure provides hair transplant materials comprising strip-shaped hair follicle organoids.
[0049] Furthermore, this disclosure provides a method for preparing hair follicle organoids for a hair loss simulation model, comprising the following steps:
[0050] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; and (3) Treat the hair follicle organoids with a hair loss inducing factor.
[0051] In addition, this disclosure provides hair follicle organoids for hair loss simulation models produced by this preparation method.
[0052] Furthermore, this disclosure provides a method for preparing strip-shaped hair follicle organoids for a hair loss simulation model, comprising the following steps:
[0053] (1) The skin organoids were sliced in a plane and unfolded, and then cut vertically; (2) the cut skin organoids were cultured at the gas-liquid interface to produce strip hair follicle organoids; and (3) the strip hair follicle organoids were treated with hair loss inducing factors.
[0054] In addition, this disclosure provides strip-shaped hair follicle organoids for hair loss simulation models produced by this preparation method.
[0055] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0056] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factor to produce hair follicle organoids for a hair loss simulation model; and (4) Treat the hair follicle organoids for a hair loss simulation model with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relieving agent or hair loss treatment agent.
[0057] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0058] (1) Planar sections of skin organoids are prepared and then vertically cut; (2) Cut skin organoids are cultured at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) Strip-shaped hair follicle organoids are treated with hair loss inducing factors to produce strip-shaped hair follicle organoids for hair loss simulation models; and (4) Strip-shaped hair follicle organoids for hair loss simulation models are treated with candidates of hair growth promoters, anti-hair loss agents, hair loss alleviators or hair loss treatments.
[0059] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0060] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; and (3) Treat the hair follicle organoids with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relieving agent or hair loss treatment agent.
[0061] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0062] (1) Planar sectioning and unfolding of skin organoids, followed by vertical cutting; (2) Culturing the cut skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treating the strip-shaped hair follicle organoids with candidates of hair growth promoters, anti-hair loss agents, hair loss relieving agents or hair loss treatment agents.
[0063] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0064] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factor to produce hair follicle organoids for a hair loss simulation model; and (4) Treat the hair follicle organoids for a hair loss simulation model with a candidate substance of a cosmetic composition to promote hair growth, prevent hair loss, or alleviate hair loss.
[0065] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0066] (1) Planar sections of skin organoids are prepared and then vertically cut; (2) Cut skin organoids are cultured at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) Strip-shaped hair follicle organoids are treated with a hair loss inducing factors to produce strip-shaped hair follicle organoids for hair loss simulation models; and (4) Strip-shaped hair follicle organoids for hair loss simulation models are treated with a candidate substance of a cosmetic composition to promote hair growth, prevent hair loss, or alleviate hair loss.
[0067] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0068] (1) Isolating hair follicles from skin organoids; (2) Culturing the isolated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) Treating the hair follicle organoids with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.
[0069] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0070] (1) Planar sectioning and unfolding of skin organoids, followed by vertical cutting; (2) Culturing the cut skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treating the strip-shaped hair follicle organoids with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.
[0071] Beneficial effects
[0072] This disclosure relates to a method for screening drugs related to hair and a technique for producing and culturing hair follicle organoids for the production of hair transplant materials. More specifically, it involves isolating hair follicles in the plug stage from existing skin organoids using human induced pluripotent stem cell lines, culturing them on culture inserts coated with collagen and Matrigel, identifying the growth and degeneration of hair follicles, and observing the growth of new hair after transplanting the cultured hair follicles onto the skin, thereby potentially using them as hair transplant materials.
[0073] Furthermore, after treating the hair follicle organoids of this disclosure with hair loss inducing factors, a shortening of 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 were observed, thereby generating a hair loss simulation model. Therefore, this disclosure is expected to be used as a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatment agents, or as a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss. Attached Figure Description
[0074] Figure 1 The study demonstrates the identification of skin organoid development and hair follicle generation using human induced pluripotent stem cell lines.
[0075] Figure 2 The identification of the structural features of hair follicles observed in the resulting skin organoids is shown.
[0076] Figure 3 The identification of hair follicles at the hair germ stage or hair peg stage is shown.
[0077] Figure 4 The identification of culture results of hair follicles isolated from the resulting skin organoids at the hair germ stage or hair plug stage is shown.
[0078] Figure 5 The results of culturing hair follicles at the gas-liquid interface after generating artificial extracellular matrix (as a coating carrier) on culture inserts under various conditions (collagen only, Matrigel only, and combinations of collagen and Matrigel) are shown.
[0079] Figure 6 This study demonstrates how changes in the expression of DKK-1, β-catenin, and CD34 genes can be used to identify hair follicle growth and degeneration.
[0080] Figure 7 The results of in vivo transplantation of hair follicle organoids are shown.
[0081] Figure 8This study demonstrates the identification of changes in the boundaries of dermal papilla cell aggregates, hair follicle length, and expression of WNT3A and β-catenin genes after treatment of hair follicle organoids with dihydrotestosterone (DHT) and minoxidil (MXD).
[0082] Figure 9 illustrates the identification of changes in hair follicle and hair length depending on cell death in strip-shaped follicle organoids, the presence of cell proliferation, and treatment with hair loss-inducing hormones. Detailed Implementation
[0083] This disclosure will be described in more detail below.
[0084] This disclosure provides a method for preparing hair follicle organoids, comprising the steps of: (1) isolating hair follicles from skin organoids; and (2) culturing the isolated hair follicles at an air-liquid interface.
[0085] The skin organoids in step (1) can be prepared by including the following steps:
[0086] (a) Culture pluripotent stem cell-derived organoids in the presence of Wnt agonists; (b) Culture the culture of step (a) for at least 40 days in a medium for maturation of skin organoids; and (c) Dissect the culture of step (b) and then culture it at the gas-liquid interface.
[0087] Wnt agonists can be added between day 5 and day 7 of culturing pluripotent stem cells.
[0088] The Wnt agonist could be CHIR-99021.
[0089] Skin organoids may express one or more of the following groups, but are not limited to: keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
[0090] The hair follicle in step (1) may include, but is not limited to, any one or more of the group consisting of dermal sheath, outer sheath, inner sheath, dermal papilla cells, hair follicle cells, ridge area and sebaceous gland.
[0091] The hair follicles in step (1) can be separated at the hair germ stage or the hair plug stage.
[0092] The embryonic stage can form within 60 to 80 days after the formation of skin organoids, preferably within 70 to 80 days.
[0093] The hair plug stage can form within 80 to 100 days after the formation of skin organoids, preferably within 90 to 100 days.
[0094] The separation in step (1) can be performed by microdissection.
[0095] The cultivation in step (2) can be carried out on a culture insert.
[0096] The culture insert can be coated with collagen and Matrigel; or coated with collagen.
[0097] The ratio of collagen to Matrigel can be 1:10 to 10:1, preferably 1:1 to 3:1.
[0098] In addition, this disclosure provides hair follicle organoids prepared by this preparation method.
[0099] Hair follicle organoids may express one or more of the following groups, but are not limited to: keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
[0100] In addition, this disclosure provides hair transplant materials comprising hair follicle organoids.
[0101] Furthermore, this disclosure provides a method for preparing strip-shaped hair follicle organoids, comprising the following steps:
[0102] (1) Planar sections of skin organoids were prepared and unfolded, and then vertically cut; and (2) the cut skin organoids were cultured at the air-liquid interface.
[0103] The skin organoids of step (1) can be prepared by including the following steps:
[0104] (a) Culture pluripotent stem cell-derived organoids in the presence of Wnt agonists; (b) Culture the culture of step (a) for at least 40 days in a medium for maturation of skin organoids; and (c) Dissect the culture of step (b) and then culture it at the gas-liquid interface.
[0105] Wnt agonists can be added between day 5 and day 7 of culturing pluripotent stem cells.
[0106] The Wnt agonist could be CHIR-99021.
[0107] Skin organoids may express one or more of the following groups, but are not limited to: keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
[0108] The cutting in step (1) can be performed when the hair follicle in the skin organoid is in the hair germ stage or in the hair plug stage.
[0109] The embryonic stage can form within 60 to 80 days after the formation of skin organoids, preferably within 70 to 80 days.
[0110] The hair plug stage can form within 80 to 100 days after the formation of skin organoids, preferably within 90 to 100 days.
[0111] The cutting in step (1) can be performed by cutting the unfolded skin organoid at intervals of 1 mm to 3 mm.
[0112] In addition, this disclosure provides strip-shaped hair follicle organoids prepared by this preparation method.
[0113] Strip-shaped hair follicle organoids may express one or more of the following groups, but are not limited to: keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
[0114] In addition, this disclosure provides hair transplant materials comprising strip-shaped hair follicle organoids.
[0115] Furthermore, this disclosure provides a method for preparing hair follicle organoids for a hair loss simulation model, comprising the following steps:
[0116] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; and (3) Treat the hair follicle organoids with a hair loss inducing factor.
[0117] The skin organoids of step (1) can be prepared by including the following steps:
[0118] (a) Culture pluripotent stem cell-derived organoids in the presence of Wnt agonists; (b) Culture the culture of step (a) for at least 40 days in a medium for maturation of skin organoids; and (c) Dissect the culture of step (b) and then culture it at the gas-liquid interface.
[0119] Wnt agonists can be added between day 5 and day 7 of culturing pluripotent stem cells.
[0120] The Wnt agonist could be CHIR-99021.
[0121] Skin organoids may express one or more of the following groups, but are not limited to: keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
[0122] Hair loss can be caused by hormonal imbalances or ultraviolet radiation.
[0123] Hormones can be dihydrotestosterone (DHT), testosterone, thyroid hormones, or cortisol.
[0124] Thyroid hormones can be tyrosine.
[0125] The hair loss inducing factor in step (3) can be androgens, stress hormones, or ultraviolet radiation.
[0126] Hormones can be, but are not limited to, dihydrotestosterone, testosterone, or thyroid hormones.
[0127] Stress hormones can include cortisol.
[0128] Thyroid hormones can be tyrosine.
[0129] In addition, this disclosure provides hair follicle organoids for hair loss simulation models produced by this preparation method.
[0130] Hair follicle organoids used in hair loss simulation models may have one or more features selected from, but not limited to, the group consisting of:
[0131] 1) Shortening of 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.
[0132] Furthermore, this disclosure provides a method for preparing strip-shaped hair follicle organoids for a hair loss simulation model, comprising the following steps:
[0133] (1) The skin organoids were sliced in a plane and unfolded, and then cut vertically; (2) the cut skin organoids were cultured at the gas-liquid interface to produce strip hair follicle organoids; and (3) the strip hair follicle organoids were treated with hair loss inducing factors.
[0134] The skin organoids of step (1) can be prepared by including the following steps:
[0135] (a) Culture pluripotent stem cell-derived organoids in the presence of Wnt agonists; (b) Culture the culture of step (a) for at least 40 days in a medium for maturation of skin organoids; and (c) Dissect the culture of step (b) and then culture it at the gas-liquid interface.
[0136] Skin organoids may express one or more of the following groups, but are not limited to: keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
[0137] The cutting in step (1) can be performed when the hair follicle in the skin organoid is in the hair germ stage or in the hair plug stage.
[0138] The hair loss inducing factor in step (3) can be androgens, stress hormones, or ultraviolet radiation.
[0139] In addition, this disclosure provides strip-shaped hair follicle organoids for hair loss simulation models produced by this preparation method.
[0140] Strip-shaped hair follicle organoids used in hair loss simulation models may have one or more features selected from, but not limited to, the group consisting of:
[0141] 1) Shortening of 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.
[0142] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0143] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factor to produce hair follicle organoids for a hair loss simulation model; and (4) Treat the hair follicle organoids for a hair loss simulation model with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relieving agent or hair loss treatment agent.
[0144] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (4), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
[0145] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0146] (1) Planar sections of skin organoids are prepared and then vertically cut; (2) Cut skin organoids are cultured at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) Strip-shaped hair follicle organoids are treated with hair loss inducing factors to produce strip-shaped hair follicle organoids for hair loss simulation models; and (4) Strip-shaped hair follicle organoids for hair loss simulation models are treated with candidates of hair growth promoters, anti-hair loss agents, hair loss alleviators or hair loss treatments.
[0147] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (4), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
[0148] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0149] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; and (3) Treat the hair follicle organoids with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relieving agent or hair loss treatment agent.
[0150] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (3), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
[0151] In addition, this disclosure provides a method for screening hair growth promoters, anti-hair loss agents, hair loss alleviators, or hair loss treatments, comprising the following steps:
[0152] (1) Planar sectioning and unfolding of skin organoids, followed by vertical cutting; (2) Culturing the cut skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treating the strip-shaped hair follicle organoids with candidates of hair growth promoters, anti-hair loss agents, hair loss relieving agents or hair loss treatment agents.
[0153] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (3), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
[0154] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0155] (1) Isolate hair follicles from skin organoids; (2) Culture the isolated hair follicles at the gas-liquid interface to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factor to produce hair follicle organoids for a hair loss simulation model; and (4) Treat the hair follicle organoids for a hair loss simulation model with a candidate substance of a cosmetic composition to promote hair growth, prevent hair loss, or alleviate hair loss.
[0156] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (4), then the candidate substance is identified as a cosmetic composition that promotes hair growth, prevents hair loss or alleviates hair loss.
[0157] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0158] (1) Planar sections of skin organoids are prepared and then vertically cut; (2) Cut skin organoids are cultured at an air-liquid interface to produce strip-shaped hair follicle organoids; (3) Strip-shaped hair follicle organoids are treated with a hair loss inducing factors to produce strip-shaped hair follicle organoids for hair loss simulation models; and (4) Strip-shaped hair follicle organoids for hair loss simulation models are treated with a candidate substance of a cosmetic composition to promote hair growth, prevent hair loss, or alleviate hair loss.
[0159] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (4), then the candidate substance is identified as a cosmetic composition that promotes hair growth, prevents hair loss or alleviates hair loss.
[0160] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0161] (1) Isolating hair follicles from skin organoids; (2) Culturing the isolated hair follicles at an air-liquid interface to produce hair follicle organoids; and (3) Treating the hair follicle organoids with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.
[0162] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (3), then the candidate substance is identified as a cosmetic composition that promotes hair growth, prevents hair loss or alleviates hair loss.
[0163] In addition, this disclosure provides a method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, comprising the following steps:
[0164] (1) Planar sectioning and unfolding of skin organoids, followed by vertical cutting; (2) Culturing the cut skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treating the strip-shaped hair follicle organoids with a candidate substance of a cosmetic composition for promoting hair growth, preventing hair loss, or alleviating hair loss.
[0165] The method may include: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (3), then the candidate substance is identified as a cosmetic composition that promotes hair growth, prevents hair loss or alleviates hair loss.
[0166] The present disclosure will be described in more detail below by way of examples to aid in understanding the present disclosure. However, the following examples are intended to illustrate the present disclosure only, and the scope of the present disclosure is not limited to the following examples. Examples of the present disclosure are provided to explain the present disclosure more fully to those skilled in the art.
[0167] Example
[0168] <Example 1> Production of skin organoids derived from human induced pluripotent stem cells
[0169] 1. Culture of human induced pluripotent stem cells
[0170] Human induced pluripotent stem cell lines (iPSCs; CMC3, CMC11) were cultured in Essential 8 (Gibo) medium supplemented with Y-27632 (10 μM) on plates coated with Vitronectin (ThermoFisher). The medium was changed daily, and passages were performed using ReLeSR (Stem Cell Technology) at approximately 4-day intervals.
[0171] 2. Methods for differentiating skin organoids using human induced pluripotent stem cell lines
[0172] Referring to existing skin organoid differentiation protocols, skin organoids were cultured using human induced pluripotent stem cell lines disclosed in the 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 from human induced pluripotent stem cells through the following steps: 1) culturing embryoids, 2) inducing differentiation into non-neuroectoderm, and 3) inducing differentiation into cranial neural crest-like cells and activating Wnt signaling (…). Figure 1 A).
[0173] In skin organoids, it was found that every 1 mm 2 Each area develops approximately 10 hair follicles (typically, about 45,000 identical hair follicles develop from a single spherical skin organoid). Figure 1 B).
[0174] 3. Characteristics of hair follicles derived from skin organoids
[0175] To verify the structural features of hair follicles displayed in the resulting skin organoids, H&E and immunofluorescence staining were performed, and the microstructure of the hair follicles, including the dermal sheath, outer sheath, inner sheath, dermal papilla cells, hair follicle cells, ridge area, and sebaceous gland, was identified. Figure 2 ).
[0176] <Example 2> Isolation and culture of hair follicles from skin organoids derived from human induced pluripotent stem cells
[0177] 1. Separation and culture via microdissection
[0178] In skin organoids derived from human induced pluripotent stem cells, hair follicles are separated from the epidermis and dermis. First, because hair follicles exist in skin organoids at a very small size, a microdissection technique was developed to obtain the hair follicles without causing damage.
[0179] Skin organoids were transferred to tissue culture plates and then cut using spring micro-scissors. The target tissue was then separated from the epidermis and dermis using forceps. Due to the tendency of the target tissue to highly embed and tightly adhere to collagen fibers in the skin layers (epidermis and dermis) and hair follicles, it was carefully transferred using microneedles. The separated hair follicles were washed with culture medium to avoid contamination by other cells and then cultured on air-exposed collagen-coated 3D Transwell culture inserts.
[0180] To optimize the appropriate separation period, hair follicles are separated at different stages: the hair germ stage (the stage where the epidermis thickens and protrudes downwards, and dermal fibroblasts gather under the hair follicle substrate). Figure 3 A); and the plug stage (the end elongates into a round column, dermal fibroblasts form spherical hair papilla cells, and the concave proximal end begins to surround the aggregated cells in the stage). Figure 3 B). When separating at the hair germ stage, it is difficult to completely separate the epidermis and dermis, leading to regeneration of both layers during culture. Furthermore, it was found that when hair follicles are separated and cultured before the plug stage, new follicles form on top of the follicle. On the other hand, when hair follicles are separated after the plug stage, hair is found to grow from structurally perfect follicles without intervention from other tissues. In the case of the hair germ stage, the separation period occurs within 60 to 80 days after the formation of the skin organoid, while in the case of the plug stage, separation occurs between 80 and 100 days after the formation of the plug. Figure 4 A and Figure 4 B).
[0181] When hair follicles after the follicle plug stage are isolated and cultured in vitro, the fine structural differentiation of the hair follicles is verified by H&E and immunofluorescence staining. Figure 4 C).
[0182] 2. Optimization of conditions for hair follicle culture carriers
[0183] After generating an artificial extracellular matrix (a coated carrier on a Transwell culture insert for culturing hair follicles), hair follicles were cultured at the gas-liquid interface under various conditions. Hair follicles after the embryonic stage were isolated and cultured on collagen-only, Matrigel-only, and mixtures of extracellular matrix containing both collagen and Matrigel.
[0184] The results showed that hair follicles grew when cultured under collagen-only conditions, but the differentiation into hair and the growth rate were slower compared to conditions with an extracellular matrix containing a mixture of collagen and Matrigel. Furthermore, hair follicles clumped together and could not be cultured under Matrigel-only conditions. Finally, the growth rate of hair follicles and hair was found to be most significantly enhanced in extracellular matrices containing either collagen or a mixture of collagen and Matrigel (in the collagen and Matrigel mixed culture, the collagen:Matrigel ratio was 3:1 to 1:1). Figure 5 A). Furthermore, it was determined 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 ceased after 80 days. Figure 5 B).
[0185] Hair follicles were sorted at 10, 30-50, and over 60 days after culture, and the expression of genes related to hair growth induction and inhibition was observed by quantitative PCR. In follicles older than 60 days, the expression of the DKK-1 gene (a hair loss-inducing cytokine) was significantly increased, while the expression of β-catenin (a gene involved in hair growth) and CD34 (a major marker of hair follicle stem cells) was decreased. This indicates that hair follicle growth and regression can be observed even under in vitro culture conditions. Figure 6 ).
[0186] <Example 3> Observation of hair follicle growth in vivo during transplantation
[0187] Hair follicles used for transplantation refer to hair follicles that, after in vitro culture, have a morphological structure that can be distinguished into a hair bulb and a hair root, with obvious differentiation towards the ridge, and have begun to produce hair. Figure 7 A).
[0188] A shallow puncture wound, approximately 0.5 mm vertically and 2.5 mm horizontally, almost parallel to the skin surface, was created on the skin of 6-week-old Balb / c nu / nu mice using a 20G injection needle. Hair follicle organoids were then intradermally transplanted to the wound site. This was an attempt to assess their potential as a transplant material.
[0189] As a result, existing hair first disappeared within a week, and then new hair was observed after about a month. The growth of white or black hair was observed based on the presence of melanocytes in the hair follicle organoids. Figure 7 B Figure 7 C).
[0190] H&E staining and immunofluorescence staining using human nuclear antigen antibodies revealed that the hair originated from humans, not mice. Figure 7D). This is significant because the resulting hair follicle organoids enabled hair to grow through the skin layer of hairy mice even after transplantation, indicating that hair follicle organoids can function as potential hair transplant materials.
[0191] Example 4: Identification of its potential for drug screening
[0192] The efficacy of alleviating hair loss symptoms and promoting hair loss is mainly evaluated through animal experiments. In vitro testing methods have not yet been established, and existing known in vitro testing methods have limitations in assessing hair follicle growth and morphology as well as studying signal transduction mechanisms (e.g., treatment of hair follicle papilla cells).
[0193] In this disclosure, various effective substances can be evaluated by observing changes in hair follicle morphology and gene expression during growth.
[0194] In order to determine the likelihood of evaluating an effective substance, in addition to using 10 -5 In addition to treatment with dihydrotestosterone (DHT) (DHT is a well-known hormone that acts on hair follicles and causes hair loss), patients were also treated with 10 µM minoxidil (MXD), which is known to have hair growth effects, for 10 days simultaneously with DHT.
[0195] As a result, morphologically, in the DHT-only treatment group, hair follicles were shorter and the boundaries of dermal papilla cell aggregates were blurred and damaged, while in the MXD combined treatment group, hair follicles were longer and the boundaries of dermal papilla cell aggregates were clearer, with relatively lower damage compared to the DHT-only group. Figure 8 A).
[0196] The expression of genes associated with promoting or inhibiting hair follicle growth was detected by quantitative PCR. Results showed that in the DHT-only group, compared with the control group, the expression of WNT3A and β-catenin, which are involved in promoting hair follicle growth, was decreased, while in the MXD combined treatment group, their expression was significantly increased. Furthermore, in the DHT-only group, compared with the control group, the expression of DKK-1 and TGF-β2, which are involved in inhibiting hair follicle growth, was significantly increased, while in the MXD combined treatment group, their expression was significantly decreased. This confirms that DHT can induce alopecia-like responses in hair follicle organoids, while MXD alleviates hair loss symptoms. Figure 8 B).
[0197] In addition to androgens, factors that can cause hair loss (ultraviolet radiation or stress hormones) can also be used to evaluate the effectiveness of hair growth promoters, anti-hair loss agents, or hair loss treatments by generating hair loss simulation models.
[0198] Depending on the order of treatment of the active substance and the hair loss inducing substance, the targets for drug screening can be diverse. Among them, the effects of preventing and protecting hair loss when treated first with the active substance, and the effects of promoting and treating hair growth when treated later, can be determined. Moreover, it is not limited to this. Even when various substances treat normal hair follicle organs alone, the substance can be used as a platform to allow the identification of the effects of inhibiting or promoting hair follicle growth.
[0199] Therefore, the efficacy of the identified substance can be assessed by examining the hair follicle. It is noted that the assessment of other effective substances is also feasible, and the effects of drugs on multiple hair follicles can be assessed not only by using a single hair follicle but also by using strip-shaped hair follicle organoids.
[0200] During the 60 to 80 day period of the hair embryo stage or the 80 to 100 day period of the hair plug stage, the spherical skin organoids are sliced flat and unfolded during formation, and then cut vertically at 1 mm intervals.
[0201] The cut portions were placed face down and face up on an artificial extracellular matrix (as a coating carrier on the Transwell culture insert), which consisted of collagen alone or a mixture of collagen and Matrigel in a ratio of 1:10 to 10:1, and then cultured at the gas-liquid interface.
[0202] When the hair follicle strips cultured in this way were subjected to immunofluorescence staining to identify apoptosis (lysed caspase-3) and cell proliferation (Ki-67), it was determined that cell proliferation could occur without cell death (Figure 9A).
[0203] After 3 days of culture using strip-shaped hair follicle organoids, shortening of hair follicle and hair length was observed, and growth was inhibited upon treatment with hair loss inducing hormone (DHT). Furthermore, hair follicle atrophy was confirmed, and separation of dermal papilla cells and hair follicles began (Figs. 9B and 9C).
[0204] Thus, it was discovered that strip-shaped hair follicle organoids can also be used to evaluate effective substances for hair growth promoters, anti-hair loss agents, or hair loss treatments.
[0205] The above description is for illustrative purposes only, and those skilled in the art will understand that this disclosure can be readily modified into other specific forms without altering its technical concept or essential characteristics. Therefore, it should be understood that the above embodiments are exemplary in all respects and not restrictive.
[0206] The scope of this disclosure is indicated by the appended claims, and all variations or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included within the scope of this disclosure.
Claims
1. A method for preparing hair follicle organoids, the method comprising: (1) Isolating hair follicles from skin organoids; as well as (2) Hair follicles isolated by culture at the gas-liquid interface.
2. The method according to claim 1, wherein, The skin organoids in step (1) are prepared by the following: (a) Culture of organoids derived from pluripotent stem cells in the presence of Wnt agonists; (b) Culture the culture from step (a) in a culture medium for skin organoid maturation for at least 40 days; and (c) Cut the culture from step (b) and then culture it at the gas-liquid interface.
3. The method according to claim 2, wherein, The Wnt agonist was added between day 5 and day 7 of culturing the pluripotent stem cells.
4. The method according to claim 1, wherein, The skin organoid expression is selected from one or more of the group consisting of keratin 5 (KRT5), KRT10, KRT15, KRT17, stigmacin, filaggrin, SRY box transcription factor 2 (SOX2), and Melan-A.
5. The method according to claim 1, wherein, The hair follicle in step (1) includes any one or more selected from the group consisting of dermal sheath, outer sheath, inner sheath, dermal papilla cells, hair follicle cells, ridge area and sebaceous gland.
6. The method according to claim 1, wherein, The hair follicles in step (1) are separated at the hair germ stage or hair plug stage.
7. The method according to claim 6, wherein, The embryonic stage is formed within 60 to 80 days after the formation of skin organoids.
8. The method according to claim 6, wherein, The hair plug stage forms within 80 to 100 days after the formation of skin organoids.
9. The method according to claim 1, wherein, The separation in step (1) is performed by microdissection.
10. The method according to claim 1, wherein, The culture in step (2) is performed on a culture insert.
11. The method according to claim 10, wherein, The culture insert is coated with collagen and Matrigel; or is coated with collagen.
12. The method according to claim 11, wherein, The ratio of collagen to Matrigel is 1:10 to 10:
1.
13. A hair follicle organoid, said hair follicle organoid being prepared by the preparation method according to claim 1.
14. A hair transplant material comprising the hair follicle organoids according to claim 13.
15. A method for preparing strip-shaped hair follicle organoids, the method comprising: (1) The skin organoids were sliced in plane and unfolded, and then cut vertically; as well as (2) Culture dissected skin organoids at the gas-liquid interface.
16. The method according to claim 15, wherein, The skin organoids of step (1) are prepared by the following: (a) Culture of organoids derived from pluripotent stem cells in the presence of Wnt agonists; (b) Culture the culture from step (a) in a culture medium for skin organoid maturation for at least 40 days; and (c) Cut the culture from step (b) and then culture it at the gas-liquid interface.
17. The method according to claim 15, wherein, The cutting in step (1) is performed when the hair follicle in the skin organoid is in the hair germ stage or hair plug stage.
18. The method according to claim 15, wherein, The cutting in step (1) is performed by cutting the unfolded skin organoid at intervals of 1 mm to 3 mm.
19. A strip-shaped hair follicle organoid, said strip-shaped hair follicle organoid being prepared by the preparation method according to claim 15.
20. A hair transplant material comprising the strip-shaped hair follicle organoids according to claim 19.
21. A method for preparing hair follicle organoids for a hair loss simulation model, the method comprising: (1) Isolating hair follicles from skin organoids; (2) Hair follicles isolated at the gas-liquid interface are cultured to produce hair follicle organoids; as well as (3) Treat the hair follicle organoids with a hair loss inducing factor.
22. The method according to claim 21, wherein, The hair loss inducing factor mentioned in step (3) is androgen, stress hormone or ultraviolet radiation.
23. The method according to claim 22, wherein, The hormone in question is dihydrotestosterone, testosterone, or thyroid hormone.
24. The method according to claim 22, wherein, The stress hormone in question is cortisol.
25. A hair follicle organoid for a hair loss simulation model, said hair follicle organoid being produced by the preparation method according to claim 21.
26. The hair follicle organoid according to claim 25, wherein, The hair follicle organoids used in the hair loss simulation model have one or more characteristics selected from the group consisting of: 1) Shortening of 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 preparing strip-shaped hair follicle organoids for a hair loss simulation model, the method comprising: (1) The skin organoids were sliced in plane and unfolded, and then cut vertically; (2) Culturing dissected skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; and (3) Treat the strip-shaped hair follicle organoids with a hair loss inducing factor.
28. A strip-shaped hair follicle organoid for a hair loss simulation model, said strip-shaped hair follicle organoid being produced by the preparation method according to claim 27.
29. A method for screening hair growth promoters, anti-hair loss agents, hair loss alleviating agents, or hair loss treatment agents, the method comprising: (1) Isolating hair follicles from skin organoids; (2) Hair follicles isolated at the gas-liquid interface are cultured to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factors to generate hair follicle organoids for use in hair loss simulation models; as well as (4) Treat the hair follicle organoids used in the hair loss simulation model with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
30. The method according to claim 29, wherein, The method includes: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (4), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
31. A method for screening hair growth promoters, anti-hair loss agents, hair loss alleviating agents, or hair loss treatment agents, the method comprising: (1) The skin organoids were sliced in plane and unfolded, and then cut vertically; (2) Culture dissected skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; (3) Treating the strip-shaped hair follicle organoids with a hair loss inducing factor to produce strip-shaped hair follicle organoids for use in a hair loss simulation model; and (4) Treat the strip-shaped hair follicle organoids used in the hair loss simulation model with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
32. The method according to claim 31, wherein, The method includes: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (4), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
33. A method for screening hair growth promoters, anti-hair loss agents, hair loss alleviating agents, or hair loss treatment agents, the method comprising: (1) Isolating hair follicles from skin organoids; (2) Hair follicles isolated at the gas-liquid interface are cultured to produce hair follicle organoids; as well as (3) Treat the hair follicle organoids with a candidate substance of a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
34. The method according to claim 33, wherein, The method includes: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (3), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
35. A method for screening hair growth promoters, anti-hair loss agents, hair loss alleviating agents, or hair loss treatment agents, the method comprising: (1) The skin organoids were sliced in plane and unfolded, and then cut vertically; (2) Culture dissected skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; as well as (3) Treat the strip-shaped hair follicle organoids with a candidate substance of hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
36. The method according to claim 35, wherein, The method includes: if the expression of WNT3A or β-catenin increases or the expression of DKK-1 or TGF-β2 decreases after treatment with the candidate substance in step (3), then the candidate substance is identified as a hair growth promoter, anti-hair loss agent, hair loss relief agent or hair loss treatment agent.
37. A method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, the method comprising: (1) Isolating hair follicles from skin organoids; (2) Hair follicles isolated at the gas-liquid interface are cultured to produce hair follicle organoids; (3) Treat the hair follicle organoids with a hair loss inducing factors to generate hair follicle organoids for use in hair loss simulation models; as well as (4) Treat the hair follicle organoids used in the hair loss simulation model with a candidate substance of the cosmetic composition to promote hair growth, prevent hair loss or alleviate hair loss.
38. A method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, the method comprising: (1) The skin organoids were sliced in plane and unfolded, and then cut vertically; (2) Culture dissected skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; (3) Treat the strip-shaped hair follicle organoids with a hair loss inducing factor to produce strip-shaped hair follicle organoids for use in hair loss simulation models; as well as (4) Treat the strip-shaped hair follicle organoids used in the hair loss simulation model with a candidate substance of the cosmetic composition to promote hair growth, prevent hair loss or alleviate hair loss.
39. A method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, the method comprising: (1) Isolating hair follicles from skin organoids; (2) Hair follicles isolated at the gas-liquid interface are cultured to produce hair follicle organoids; as well as (3) Treat the hair follicle organoids with a candidate substance of a cosmetic composition for the purpose of promoting hair growth, preventing hair loss or alleviating hair loss.
40. A method for screening cosmetic compositions for promoting hair growth, preventing hair loss, or alleviating hair loss, the method comprising: (1) The skin organoids were sliced in plane and unfolded, and then cut vertically; (2) Culture dissected skin organoids at the gas-liquid interface to produce strip-shaped hair follicle organoids; as well as (3) Treat the strip-shaped hair follicle organoids with a candidate substance of a cosmetic composition for the purpose of promoting hair growth, preventing hair loss or alleviating hair loss.