Skin injury organ model and method for screening drugs by using same

Abstract

The invention relates to a skin injury organ model and a method for screening drugs by using the skin injury organ model, and the skin injury organ model is characterized in that skin cells, accessory structures, nerve cells, fat cells and the like form structures similar to actual skin by using an air-liquid interface skin organ preparation technology; and the damaged skin is prepared by irradiating the skin-like organ with ultraviolet rays which actually reach the ground surface. In the present invention, the skin barrier is damaged and the activity of a matrix proteolytic enzyme is improved to promote the decomposition of collagen while increasing the secretion of pro-inflammatory cytokines by irradiating with solar-simulated ultraviolet light comprising 94.5% ultraviolet light (UV-A) and 5.5% ultraviolet light (UV-B), thereby well simulating the characteristics of skin injury caused by ultraviolet light. Therefore, the invention can be used as a model of skin injury and photoaging caused by ultraviolet rays, and can be useful in potential therapeutic drug screening.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2023-0134662, filed on October 10, 2023, the entire description of which is incorporated herein by reference.

[0002] This invention relates to a skin damage organoid model and a method for drug screening using the same, specifically a method for preparing a photoaging-like skin model caused by ultraviolet damage by irradiating differentiated human skin organoids with ultraviolet light that simulates actual ultraviolet radiation reaching the Earth's surface, and a method for evaluating the efficacy of potential therapeutic drugs.

[0003] This invention was completed with the support of the Ministry of Science and ICT of Korea, under project number 00215812 (1711195569). Background Technology

[0004] With the improvement of living standards, modern people are paying more attention to maintaining healthy skin while preserving their overall health. Therefore, there is increasing focus not only on skin aesthetics but also on improving skin damage and aging. Skin is the largest organ in the human body, accounting for about 15% of body weight. Covering the body surface and directly exposed to the external environment, it is a vital organ. Skin cells can be damaged by various external factors attempting to invade the body. Among these, ultraviolet (UV) radiation exposure is widely recognized as an exogenous factor in skin aging; exposure alone can cause various types of skin damage. In recent years, the reduction of the ozone layer, which provides protection at the top of the atmosphere, has led to an increase in the amount of UV radiation reaching the Earth's surface, potentially causing skin damage.

[0005] Currently, methods to improve damage and aging-related phenomena caused by ultraviolet radiation are being actively researched, especially to uncover the main mechanisms that cause skin damage or aging and to identify substances that can alleviate them.

[0006] Existing technical documents Patent documents Korean Patent No. 10-2023-0115654 (published on August 3, 2023). Summary of the Invention

[0007] The technical problem that the invention aims to solve The purpose of this invention is to provide a method for preparing skin-damaged organoids or hair follicle-damaged organoids by irradiating ultraviolet light that simulates sunlight.

[0008] Furthermore, another object of the present invention is to provide a skin damage organoid or hair follicle damage organoid prepared by the preparation method described above.

[0009] Furthermore, another object of the present invention is to provide a method for screening skin injury treatment agents, preventive agents or ameliorators utilizing the aforementioned skin injury organoids.

[0010] Furthermore, another object of the present invention is to provide a method for screening hair growth promoters, hair loss preventive agents, hair loss alleviators, or hair loss treatment agents utilizing the aforementioned hair follicle damaged organoids.

[0011] Furthermore, another object of the present invention is to provide a biomarker composition for detecting hair follicle damage, comprising NF-κB as an active ingredient.

[0012] Furthermore, another object of the present invention is to provide a kit for detecting hair follicle damage, comprising an agent capable of detecting NF-κB.

[0013] Furthermore, another object of the present invention is to provide a method for providing information needed for the detection of hair follicle damage by measuring the expression level of NF-kB.

[0014] Furthermore, another object of the present invention is to provide a pharmaceutical composition for use as a hair growth promoter, hair loss prevention agent, hair loss relief agent or hair loss treatment agent, comprising an NF-κB activity or expression inhibitor as an active ingredient.

[0015] Furthermore, another object of the present invention is to provide a pharmaceutical composition or cosmetic composition for use as a hair removal agent, comprising an NF-κB activity or expression promoter as an active ingredient.

[0016] Furthermore, another object of the present invention is to provide a hair growth promotion, hair loss prevention, hair loss relief, or hair loss treatment method, comprising the step of administering an NF-κB activity or expression inhibitor in an effective amount to an individual in need of the treatment.

[0017] Furthermore, another object of the present invention is to provide a hair removal method comprising the step of applying an NF-κB activity or expression promoter in an effective amount to an individual in need of the treatment.

[0018] means for solving problems To achieve the above objectives, the present invention provides a method for preparing organoids of skin damage, comprising the following steps: (1) culturing organoids derived from pluripotent stem cells in the presence of Wnt agonists; (2) Culture the culture of step (1) in skin organoid maturation medium; (3) The culture from step (2) is cut and cultured at the gas-liquid interface to prepare skin organoids; and (4) Irradiate the skin organoids with ultraviolet light that simulates sunlight. The Wnt agonist is added when non-neuroectoderm is induced to differentiate into cranial neural crest cells (CNCC).

[0019] Furthermore, the present invention provides a skin damage organoid or hair follicle damage organoid prepared by the preparation method described above.

[0020] In addition, the present invention provides a method for screening skin injury treatment agents, preventive agents or improvers, including the step of treating the skin injury treatment agent, preventive agent or improver candidate substance on the skin injury organoid.

[0021] In addition, the present invention provides a method for screening hair growth promoters, hair loss preventive agents, hair loss alleviating agents or hair loss treatment agents, including the step of treating the hair follicle damaged organoid with the candidate substances of hair growth promoters, hair loss preventive agents, hair loss alleviating agents or hair loss treatment agents.

[0022] In addition, the present invention provides a biomarker composition for detecting hair follicle damage, which includes NF-κB as an active ingredient.

[0023] In addition, the present invention provides a kit for detecting hair follicle damage, which includes a formulation capable of detecting NF-κB.

[0024] Furthermore, the present invention provides a method for obtaining information required for hair follicle damage detection, comprising the following steps: (1) Measure the expression level of NF-κB in the isolated samples; (2) The expression level of NF-kB was compared with that of the control group sample; (3) If the expression level of NF-kB is higher than that of the control group sample, the hair follicle is considered to be damaged.

[0025] Furthermore, the present invention provides a pharmaceutical composition for use as a hair growth promoter, hair loss prevention agent, hair loss relief agent, or hair loss treatment agent, comprising an NF-κB activity or expression inhibitor as an active ingredient.

[0026] Furthermore, the present invention provides a pharmaceutical composition for use as a hair removal agent, comprising an NF-κB activity or expression promoter as an active ingredient.

[0027] Furthermore, the present invention provides a cosmetic composition for use in hair removal agents, comprising an NF-κB activity or expression promoter as an active ingredient.

[0028] In addition, the present invention provides a hair growth promotion, hair loss prevention, hair loss relief, or hair loss treatment method, including the step of administering an NF-κB activity or expression inhibitor in an effective amount to an individual in need of the treatment.

[0029] In addition, the present invention provides a hair removal method, comprising the step of applying an NF-κB activity or expression promoter in an effective amount to an individual requiring the treatment.

[0030] The present invention provides a method for preparing organoids of skin damage, characterized by comprising the following steps: (1) culturing organoids derived from pluripotent stem cells in the presence of Wnt agonist; (2) Culture the culture of step (1) in skin organoid maturation medium; (3) The culture from step (2) is cut and cultured at the gas-liquid interface to prepare skin organoids; and (4) Irradiate the skin organoids with ultraviolet light that simulates sunlight. The Wnt agonist is added when non-neuroectoderm is induced to differentiate into cranial neural crest cells (CNCC).

[0031] Preferably, the skin injury organoid can be a hair follicle injury organoid among the skin's appendages, but it is not limited thereto.

[0032] Preferably, the simulated solar ultraviolet radiation can be treated with 50-94.5% UV-A and 5.5-50% UV-B, but is not limited thereto.

[0033] Preferably, the simulated sunlight ultraviolet light can be emitted at a rate of 25 to 75 kJ / m² after the skin organoids have been cultured under dry conditions to allow the stratum corneum to mature. 2 Treatment may be carried out using sUV doses, but is not limited to this.

[0034] Preferably, the Wnt agonist may be selected from the group consisting of CHIR-99021, Wnt3A, WNT5A and RSPO1, but is not limited thereto.

[0035] Preferably, the Wnt agonist can be added on days 5 to 7 of pluripotent stem cell culture, but is not limited thereto.

[0036] Preferably, the skin organoid maturation culture medium may include one or more components selected from the group consisting of GlutaMAX, 2-mercaptoethanol, B-27, N2 and Normocin, but is not limited thereto.

[0037] Preferably, step (3) can cut the culture from step (2) into 4 uniformly sized pieces and perform gas-liquid interface culture on a collagen-coated Transwell culture insert, so that the dermis faces the collagen side and the epidermis is exposed to air. More preferably, the gas-liquid interface culture can be performed in a skin organoid maturation culture medium, but is not limited thereto.

[0038] Preferably, the skin damage organoid or hair follicle damage organoid can be an organoid that damages the skin or hair follicle through NF-κB activation, but is not limited thereto.

[0039] Preferably, the skin injury organoid can be an organoid whose expression of proteins forming the epidermal barrier is downregulated, selected from the group consisting of Filaggrin, Loricrin, and CK10, but is not limited thereto.

[0040] Preferably, the skin injury organoid can be an organoid with decreased expression of type I collagen and increased expression of MMP-1 in the dermis, but is not limited thereto.

[0041] Preferably, the hair follicle injury organoid can be an organoid with reduced expression of one or more genes selected from the group consisting of dermal sheath marker α-SMA, outer root sheath marker KRT5 or KRT15, ​​hair follicle stem cell marker KRT15 or LHX2, and dermal papilla marker SOX2, but is not limited thereto.

[0042] Preferably, the hair follicle damage organoid can be an organoid with increased gene expression of inflammation-induced cytokines COX-2, TNF-α, or IL-1β, but is not limited thereto.

[0043] In this specification, "air-liquid interface culture (ALI culture)" refers to culture in a partially open culture vessel or a culture vessel partially filled with culture medium, but is not limited to these. Air-liquid interface culture means exposing the surface of cells or organoids to air.

[0044] In this specification, "Filaggrin" is one of the many structural proteins expressed by keratinocytes during the differentiation stage. It participates in the differentiation process from the basal layer of the epidermis to the stratum corneum. At the same time, it constitutes the main component of the natural moisturizing factor (NMF), which is essential for maintaining skin moisture, and is used as an important indicator of skin moisture maintenance and skin barrier function.

[0045] In this specification, "Loricrin" refers to one of the skin barrier proteins expressed by keratinocytes during the differentiation stage.

[0046] Furthermore, the present invention provides a skin lesion organoid prepared by the aforementioned preparation method.

[0047] In addition, the present invention provides a method for screening skin injury treatment agents, preventive agents or improvers, including the step of treating the skin injury treatment agent, preventive agent or improver candidate substance on the skin injury organoid.

[0048] Furthermore, the present invention provides a hair follicle lesion organoid prepared by the aforementioned preparation method.

[0049] Preferably, the hair follicle injury organoid can be an organoid with reduced expression of one or more genes selected from the group consisting of dermal sheath marker α-SMA, outer root sheath marker KRT5 or KRT15, ​​hair follicle stem cell marker KRT15 or LHX2, and dermal papilla marker SOX2, but is not limited thereto.

[0050] In addition, the present invention provides a method for screening hair growth promoters, hair loss preventive agents, hair loss alleviating agents or hair loss treatment agents, including the step of treating the hair follicle damaged organoid with the candidate substances of hair growth promoters, hair loss preventive agents, hair loss alleviating agents or hair loss treatment agents.

[0051] Preferably, the candidate substance can inhibit NF-κB activated in hair follicle damaged organoids, but is not limited thereto.

[0052] The term "candidate substance" used in the screening method of this invention refers to an unknown candidate substance used in the screening to detect whether it affects gene expression levels or protein expression or activity. The samples include, but are not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural extracts.

[0053] In this invention, "organoid" refers to a three-dimensional assembly composed of one or more cell types that mimics the surface appearance, actual structure, or function of a tissue or organ. Furthermore, organoids can similarly reproduce the physiological functions of the human body, and by constructing organoids from patient tissues, disease modeling based on the patient's genetic information and drug screening through repeated trials are possible.

[0054] In this invention, "organoid culture" encompasses all behaviors that enable the generation or maintenance of organoids. For example, it can differentiate cells isolated from cells or specific tissues into tissue or organ cells with specific functions, and / or enable organoids to survive, grow, or proliferate.

[0055] In addition, the present invention provides a biomarker composition for detecting hair follicle damage, which includes NF-κB as an active ingredient.

[0056] In addition, the present invention provides a kit for detecting hair follicle damage, which includes a formulation capable of detecting NF-κB.

[0057] Preferably, the formulation may be selected from one or more of the group consisting of NF-κB specific primers, probes, antisense oligonucleotides, aptamers and antibodies, but is not limited thereto.

[0058] In this specification, the term "primer" refers to a short nucleic acid sequence having a free 3'-hydroxyl group, capable of forming a base pair with a complementary template and serving as the initiation site for template strand replication. Primers can initiate DNA synthesis in the presence of reagents used for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates under appropriate buffer conditions and temperature. PCR conditions and the lengths of the forward and antisense primers can be appropriately selected according to techniques known in the art.

[0059] In this specification, the term "probe" refers to a nucleic acid fragment capable of specifically binding to mRNA, which can be from a few bases to hundreds of bases in length, and can be labeled to confirm the presence and expression level of a specific mRNA. The probe can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, or RNA probes. Appropriate probe selection and hybridization conditions can be chosen according to techniques known in the art.

[0060] In this specification, the term "antisense oligonucleotide" refers to an oligomer having a nucleotide base sequence and an inter-subunit backbone that hybridizes with a target sequence within RNA via Watson-Crick base pairing, thereby typically allowing the formation of a heteroduplex structure of mRNA and RNA:oligomer within the target sequence. The oligomer may have exact or near-complementarity with the target sequence. This antisense oligomer can block or inhibit mRNA translation and can alter the mRNA processing that produces mRNA splicing variants.

[0061] In this specification, the term "aptamer" refers to a special type of polynucleotide composed of single-stranded nucleic acids (DNA, RNA, or modified nucleic acids), possessing a stable three-dimensional structure, and capable of binding to target molecules with high affinity and specificity. As mentioned above, aptamers, similar to antibodies, can specifically bind to antigenic substances, while exhibiting higher stability than proteins, simpler structure, and ease of synthesis, thus making them a viable alternative to antibodies.

[0062] In this specification, the term "antibody" is a well-known term in the art, referring to a specific immunoglobulin targeting an antigenic site. In this invention, the antibody refers to an antibody capable of specifically binding to the biomarker of this invention, and can be prepared according to conventional methods in the art. The antibody may be in the form of a polyclonal antibody or a monoclonal antibody, and includes all immunoglobulin antibodies. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. Furthermore, the antibody also includes special antibodies such as humanized antibodies.

[0063] Furthermore, the present invention provides a method for obtaining information required for hair follicle damage detection, comprising the following steps: (1) Measure the expression level of NF-κB in the isolated samples; (2) The expression level of NF-kB was compared with that of the control group sample; (3) If the expression level of NF-kB is higher than that of the control group sample, the hair follicle is considered to be damaged.

[0064] In this specification, the term "isolated sample" refers to, but is not limited to, samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that show differences in expression levels of the biomarker NF-κB of the present invention compared to the control group.

[0065] Furthermore, the present invention provides a pharmaceutical composition for use as a hair growth promoter, hair loss prevention agent, hair loss relief agent, or hair loss treatment agent, comprising an NF-κB activity or expression inhibitor as an active ingredient.

[0066] Preferably, the NF-κB activity inhibitor can be selected from the group consisting of small molecule compounds, peptides, peptide mimics, aptamers, antibodies, and natural products that specifically bind to the NF-κB protein. The NF-κB expression inhibitor can be selected from the group consisting of antisense nucleotides that bind complementary to the mRNA of the NF-κB gene, small interfering RNA (siRNA), and short hairpin RNA (shRNA), but is not limited thereto.

[0067] Furthermore, the present invention provides a pharmaceutical composition for use as a hair removal agent, comprising an NF-κB activity or expression promoter as an active ingredient.

[0068] Preferably, the NF-kB activity promoter can be selected from the group consisting of small molecule compounds, peptides, peptide mimics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein, and the NF-kB expression promoter can be selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, and retrovirus that contain an NF-kB encoding gene that increases the expression of NF-kB gene mRNA.

[0069] The pharmaceutical compositions of the present invention may include chemical substances, nucleotides, antisense nucleotides, siRNA oligonucleotides, and natural extracts as active ingredients. The pharmaceutical compositions or compound formulations of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable excipients, which may include solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, flow aids, or flavoring agents. For administration, the pharmaceutical compositions of the present invention may further include one or more pharmaceutically acceptable carriers in addition to the active ingredient, thereby preferably being prepared as pharmaceutical compositions. In compositions prepared as liquid solutions, the pharmaceutically acceptable carrier is a sterile carrier suitable for living organisms, and may use physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of the above components, and may add other conventional additives such as antioxidants, buffers, and antibacterial agents as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be further added to prepare aqueous solutions, suspensions, emulsions, or other injectable dosage forms, or to prepare pills, capsules, granules, or tablets.

[0070] The pharmaceutical compositions of the present invention can be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, oral liquids, suspensions, emulsions, drops, or injectable solutions, as well as sustained-release formulations of the active compound. The pharmaceutical compositions of the present invention can be administered via conventional routes such as intravenous, intra-arterial, intraperitoneal, intramuscular, intrasternal, percutaneous, intranasal, inhalation, local, rectal, oral, intraocular, or intradermal. The effective amount of the active ingredient in the pharmaceutical compositions of the present invention refers to the amount required for the prevention or treatment of disease. Therefore, it can be adjusted according to various factors such as the type and severity of the disease, the type and content of the active ingredient and other components contained in the composition, the type of dosage form, and the patient's age, weight, general health status, gender, diet, administration time, route of administration, release rate of the composition, treatment period, and concurrent medications. Although not limited thereto, for example, in the case of adults, when administered once or several times daily, the compositions of the present invention, when administered once or several times daily, may be administered at a dose of 0.1 ng / kg to 10 g / kg as compounds, at a dose of 0.1 ng / kg to 10 g / kg as peptides, proteins or antibodies, and at a dose of 0.01 ng / kg to 10 g / kg as antisense nucleotides, siRNA, shRNAi or miRNA.

[0071] Furthermore, the present invention provides a cosmetic composition for use in hair removal agents, comprising an NF-κB activity or expression promoter as an active ingredient.

[0072] In addition to the active ingredients, the cosmetic composition of the present invention may also include conventional excipients such as stabilizers, solubilizers, vitamins, pigments, and fragrances, as well as a carrier.

[0073] The dosage form of the cosmetic composition can be any dosage form commonly prepared in the art, such as, but not limited to, a group selected from topical ointments, creams, lotions, nourishing lotions, masks, serums, scalp care solutions, shampoos, conditioners, hair treatments, gels, skin lotions, toners, astringents, emulsions, moisturizing lotions, nourishing lotions, massage creams, nourishing creams, eye creams, moisturizing creams, hand creams, foundations, sunscreens, soaps, facial foaming agents, facial cleansing lotions, facial cleansing creams, body lotions, and shower gels. The compositions of the above dosage forms may contain various base agents and additives suitable for their formulation, and the types and amounts of these ingredients can be readily selected by those skilled in the art.

[0074] When the dosage form is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier ingredients.

[0075] When the dosage form is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as the carrier component. In particular, in the case of spray, it may further include propellants such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0076] When the dosage form is a solution or emulsion, a solvent, solubilizer, or emulsifier can be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol fatty acid ester, polyethylene glycol, or sorbitan fatty acid ester.

[0077] When the dosage form is a suspension, liquid diluents such as water, ethanol or propylene glycol can be used as carrier components, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, as well as microcrystalline cellulose, aluminum hydroxide, bentonite, agar or tragacanth gum, etc.

[0078] In addition, the present invention provides a hair growth promotion, hair loss prevention, hair loss relief, or hair loss treatment method, including the step of administering an NF-κB activity or expression inhibitor in an effective amount to an individual in need of the treatment.

[0079] Preferably, the NF-κB activity inhibitor can be selected from the group consisting of small molecule compounds that specifically bind to NF-κB protein, peptides, peptide mimics, aptamers, antibodies, and natural products. The NF-κB expression inhibitor can be selected from the group consisting of antisense nucleotides that bind complementary to the mRNA of the NF-κB gene, small interfering RNA (siRNA), and short hairpin RNA (shRNA), but is not limited thereto.

[0080] In addition, the present invention provides a hair removal method, comprising the step of applying an NF-κB activity or expression promoter in an effective amount to an individual in need of the treatment.

[0081] Preferably, the NF-kB activity promoter can be selected from the group consisting of small molecule compounds, peptides, peptide mimics, aptamers, antibodies, and natural products that specifically bind to the NF-kB protein, and the NF-kB expression promoter can be selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, and retrovirus that contain an NF-kB encoding gene that increases the expression of NF-kB gene mRNA.

[0082] The effects of the invention This invention relates to a skin injury organoid model and a method for drug screening using it. It utilizes skin organoids derived from human induced pluripotent stem cells to model skin damage caused by external environmental factors (ultraviolet radiation). Using organoid technology that simulates the actual human development process, skin organoids containing skin-forming cells, hair follicle-forming cells, and all microstructures are prepared. By irradiating with ultraviolet light, external stimuli in the actual external environment are simulated, thus establishing an in vitro damaged skin model. Therefore, this technology allows for the confirmation of ultraviolet-induced damage and aging mechanisms even in vitro, and enables the development of a platform for screening candidate substances that can alleviate this damage. The platform established by this invention can be used to evaluate and elucidate disease mechanisms and to assess and screen the efficacy of various drug candidates. Attached Figure Description

[0083] Figure 1 To demonstrate a method for differentiating skin organoids using human induced pluripotent stem cell lines.

[0084] Figure 2 To illustrate the results of comparing the skin structure presented in the prepared skin organoids with the skin structure of adult humans.

[0085] Figure 3a To illustrate the effect of different sUV doses on SkOs.

[0086] Figure 3b To demonstrate the effects of sUV irradiation on the epidermis, dermis, or hair follicles of SkOs.

[0087] Figure 3c To demonstrate the effects of sUV extending to the deep dermal layer of SkOs.

[0088] Figure 4a To demonstrate the results of a study on the effects of sUV irradiation on skin appendages in the dermis of SkOs.

[0089] Figure 4b To demonstrate the results of an investigation into whether sUV irradiation affects hair follicles and can induce an inflammatory response in the dermis of SkOs.

[0090] Figure 5a To illustrate the results of investigating the effects of exosomes (UCB-Exos) on photodamage using a SkOs model with sUV irradiation.

[0091] Figure 5b To demonstrate that UCB-Exos treatment effectively reduces epidermal thickness and alleviates damage to skin barrier proteins (Filaggrin and Loricrin), thereby confirming the improvement of epidermal damage caused by sUV irradiation in SkOs.

[0092] Figure 6 To demonstrate the effect of UCB-Exos on hair follicles within SkOs.

[0093] Figure 7a To demonstrate the results of sUV inducing phosphorylation of IκBα in the cytoplasmic portion of SkOs and subsequently triggering IκBα degradation.

[0094] Figure 7b To demonstrate the results of sUV inducing phosphorylation of IκBα in the cytoplasmic components of SkOs and subsequently triggering IκBα degradation.

[0095] Figure 7c To demonstrate the effect of UCB-Exos in inhibiting sUV-induced IκB degradation and NF-κB activation, thereby alleviating the inflammatory response in fibroblasts and keratinocytes in hair follicles.

[0096] Figure 8a The results demonstrate that inhibition of NF-κB activation via UCB-Exos can alleviate the harmful effects of SASP and thus has the potential to create a more favorable environment for tissue regeneration.

[0097] Figure 8b The results demonstrate that inhibition of NF-κB activation via UCB-Exos can alleviate the harmful effects of SASP and thus has the potential to create a more favorable environment for tissue regeneration.

[0098] Figure 8c To demonstrate the results of UCB-Exos effectively restoring hair follicles damaged by sUV by inhibiting NF-κB.

[0099] Figure 9 To demonstrate the effect of NF-κB signaling inhibitors on promoting hair follicle growth in normal hair follicle organoids. Detailed Implementation

[0100] Hereinafter, to aid in understanding the present invention, detailed description will be provided through embodiments and the like. However, the following embodiments and the like are merely illustrative and do not limit the scope of the invention. The embodiments of the present invention are provided to enable those skilled in the art to more fully understand the present invention.

[0101] <Example 1> Preparation and Validation of Skin Organoids Derived from Human Induced Pluripotent Stem Cells Culture of human induced pluripotent stem cells: Human induced pluripotent stem cell lines (iPSCs; CMC3, CMC11) were cultured in Essential 8 (Gibo) medium supplemented with Y-27632 (10 μM) on Vitronectin (ThermoFisher) coated culture dishes. The medium was changed daily, and passaged using ReLeSR (Stem Cell Technology) at approximately 4-day intervals.

[0102] Skin organoid differentiation using human induced pluripotent stem cell lines: Induced pluripotent stem cell clones were isolated into single cells using Accutase (Gibco). To form embryoid bodies, 1500 single cells were seeded into each well of a U-bottom low-attachment 96-well plate (SPL). The embryoid bodies were cultured using StemFlex (Gibco) containing Y-27632 (20 μM). Cultured until the embryoid bodies reached a size of 250–300 μm. The formed embryoid bodies were then placed in Essential 6 (Gibco) medium containing 2% Matrigel (Corning), 10 μM SB431542 (Tocris), 4 ng / ml FGF2 (PeproTech), and 2.5–15 ng / ml BMP4 (PeproTech) to induce non-neuralectoderm differentiation. Starting from day 3 or 4, to induce cranial neural crest cells, 200 nM LDN193189 (Torcris), 50 ng / ml FGF2, and 3 μM CHIR-99021 were added to the culture medium. Furthermore, on day 12, the organoids were replaced with skin organoid maturation medium. The medium was changed every 3 days. On day 20, the organoids were placed in 6-well plates (low-attachment 6-well plate; SPL) containing 3 ml of skin organoid maturation medium and cultured in an orbital shaker (65 rpm; Thermo Fisher). The medium was changed every 3 days. The maturation medium for skin organoids was a 1:1 mixture of Advanced DMEM / F12 (Gibco) and Neurobasal (Gibco) medium, containing 1X GlutaMax (Gibco), 0.5X B-27 minus vitamin A (Gibco), 0.5X N2 (Gibco), 0.1mM 2-mercaptoethanol (Gibco), and 100μg / ml Primocin (Invivogen).After culturing skin organoids for approximately 40 to 100 days, the organoids were cut into 4 to 12 uniformly sized pieces using Dumont #3 forceps (Fine Science Tools) and spring scissors (Fine Science Tools). These pieces were then placed in collagen-coated Transwell culture inserts (0.4 μm pores, located on 12-well plates), with the dermis facing the collagen side and the epidermis exposed to air. ALI-skin organoids were further cultured for 3 to 4 weeks in an air-liquid interface at 100% humidity under skin maturation medium conditions, followed by 2 to 6 days of drying (0% humidity) to promote the maturation of the lamellar epithelium. Figure 1 A and Figure 1 B).

[0103] Validation of skin organoid characteristics: The skin structures presented in the prepared skin organoids were compared with those of adult human skin. H&E staining and immunofluorescence staining confirmed the basal layer (KRT5) and squamous epithelium (KRT10) of the epidermis. Furthermore, the similarity to the real skin epidermis was verified by confirming the expression of skin barrier markers Filaggrin (upper layer, squamous epithelium) and Loricrin (stratum corneum).

[0104] Furthermore, by confirming the expression of Collagen 3, a structural protein mainly expressed in the dermis, and Vimentin, one of the intermediate filaments in the skin, the similarity between it and real human skin was further verified. Figure 2 A and Figure 2 B).

[0105] To verify the structural characteristics of hair follicles in skin organoids, H&E staining and immunofluorescence staining were performed. The results confirmed the fine structure of the hair follicle, namely the dermal sheath (α-SMA), outer root sheath (KRT17), inner root sheath (KRT71), and follicular cortex (AE13). Simultaneously, the dermal papilla cells (SOX2), hair matrix cells (Ki67, P63), and the raised area (NFATc1) responsible for hair follicle growth and function were also identified. Figure 2 CF).

[0106] <Example 2> Establishing a skin damage model by irradiating simulated solar ultraviolet rays. There are two types of sUVs that reach the skin: UV-A and UV-B. UV-A can penetrate from the epidermis to the deepest layer of the dermis, while UV-B can only penetrate to the epidermis and the upper dermis. sUV exposure can cause sunburn and signs of aging such as pigmentation by damaging the components and major proteins of the ECM (extracellular matrix). To simulate the above process and reproduce sUV-induced skin damage in vitro, SkOs was used to irradiate the skin three times with a combination of UV-A and UV-B (94.5% UV-A and 5.5% UV-B) at intervals of two days.

[0107] First, the effect of different sUV doses on SkOs was investigated. The results showed that a high dose of 75 kJ / m²... 2 sUVs can cause the entire epidermal layer of SkOs to peel off, resulting in severe damage to the integrity of the skin structure. Figure 3a (A). Immunofluorescence staining results showed that 50 kJ / m 2 The degree of sUV-induced dermal damage was related to 75 kJ / m 2 The damage caused by sUV is similar, and 25 kJ / m 2 The sUV did not cause significant damage to SkOs. Figure 3a (B and C). Therefore, 50 kJ / m 2 The sUV is the optimal irradiation dose for inducing photodamage in SkOs ( Figure 3a (D).

[0108] Following SUV exposure, the number of sunburned cells characterized by nuclear fragmentation or aggregation, as well as the epidermal thickness, significantly increased in both the epidermis and dermis of SkOs. Figure 3b EG). Furthermore, compared to the control SkOs, the number of melanocytes in the epidermis and hair follicles increased, indicating skin pigmentation (EG). Figure 3b (H, I). In particular, sUV also affects the outer epidermal layer of SkOs, which plays a role in the skin's protective barrier. The mRNA expression of skin barrier-related genes Filaggrin and Loricrin was significantly reduced in SkOs exposed to sUV, suggesting that skin barrier function was impaired after irradiation. Figure 3b Similarly, in sUV-exposed SkOs epidermis, the epidermal barrier proteins Filaggrin, Loricrin, and CK10 were significantly downregulated compared to control SkOs. Figure 3b (K, L).

[0109] Furthermore, the effects of sUV extend to the deep dermis of SkOs. In the dermal layer of SkOs exposed to sUV, the density of collagen fibers is significantly reduced ( Figure 3cThe expression of COL1A1 was significantly decreased, while the expression of matrix metalloproteinase-1 (MMP-1), an enzyme directly involved in ECM fiber degradation, was increased. Figure 3c Specifically, immunofluorescence staining revealed a significant decrease in type I collagen expression and an increase in MMP-1 expression in the dermal layer exposed to sUV. Figure 3c (P, Q). Based on the above results, it is shown that sUV reaching the epidermis and dermis induces overall skin damage in SkOs. Therefore, the inventors have successfully established in vitro conditions for reproducing sUV-induced photodamage in SkOs.

[0110] <Example 3> Evaluation of hair follicle damage by simulating solar ultraviolet radiation The effects of sUV exposure on skin appendages in the dermis of SkOs were investigated. The results showed decreased expression of the KRT5 marker, which constitutes the outer layer of the SkOs hair follicle. Furthermore, apoptosis was significantly increased in SkOs, particularly in cells co-localized with KRT5-expressing cells within the hair follicle. Figure 4a (A, B, C). Visual observation revealed significant damage to the DS (dermal sheath) and ORS (outer root sheath) of the hair follicles, and the hair shafts were relatively thinner in the sUV-treated group, indicating a morphological transition of the hair follicle from the anagen phase to a catagen-like phase. Figure 4a (D, E). Therefore, to more systematically compare the degree of hair follicle damage, hair follicles were isolated from various skin organoids and analyzed by qRT-PCR. The results showed that in the sUV treatment group, the gene expression of DS markers (a-SMA), ORS markers (KRT5, KRT15), hair follicle stem cell markers (KRT15, ​​LHX2), and DP marker (SOX2) were all significantly reduced, indicating that sUV can penetrate into the dermis and have an adverse effect on hair follicles. Figure 4a (F).

[0111] Previous studies have shown that sUV irradiation leads to the secretion of inflammation-inducing cytokines in skin tissue, thereby triggering skin inflammation and subsequent skin damage. Based on this, further investigation was conducted to determine whether sUV exposure also affects hair follicles and induces an inflammatory response in the dermis of SkOs. Firstly, the gene expression of the inflammation-inducing cytokines cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) was significantly increased in sUV-exposed SkOs hair follicles. Figure 4b (G).

[0112] Subsequently, immunofluorescence analysis was performed to confirm the local inflammatory response in SkOs. The results showed that increased production of inflammatory-inducing cytokines was expressed throughout the skin layer, but was particularly pronounced in SkOs hair follicles exposed to sUV irradiation. Figure 4b (H, I). The above results indicate that sUV irradiation can induce structural damage and inflammatory responses in the hair follicles of SkOs.

[0113] In conclusion, this invention demonstrates that sUV exposure can not only affect skin tissue but may also damage hair follicles.

[0114] <Example 4> Screening and evaluating therapeutic candidate substances using a skin organoid model of ultraviolet damage This study explored the potential application of sUV-exposed SkOs as a model for investigating the mechanisms of photodamage in drug treatment. Currently, exosomes derived from hUCB-MSCs are considered potential therapeutic candidates for various skin problems due to their ability to promote skin regeneration and reduce skin inflammation. Against this backdrop, the effects of UCB-Exos on photodamage were investigated using a sUV-exposed SkOs model.

[0115] To determine the optimal concentration of UCB-Exos for mitigating SkOs damage caused by sUV exposure, SkOs were treated with different concentrations of UCB-Exos two hours after sUV exposure, and this was repeated three times. Figure 5a (A). The results showed that UCB-Exos treatment effectively reduced SkOs damage in a dose-dependent manner.

[0116] Especially when treating UCB-Exos particles larger than 1 × 10^8, it can effectively alleviate SkOs dermal cell apoptosis induced by sUV exposure and within the hair follicles. Figure 5a (B and C). However, at a UCB-Exos concentration of 1 × 10^9 particles, the effect on alleviating the inflammatory response was more significant ( Figure 5a Based on the above results, it was confirmed that at least 1 × 10^9 UCB-Exos particles are required to fully exert their beneficial effects of inhibiting apoptosis, restoring skin barrier function, and regulating the secretion of inflammatory cytokines.

[0117] UCB-Exos (1 × 10^9) treatment can effectively reduce epidermal thickness ( Figure 5b The study found that the proteins E and F in SkOs could alleviate the damage to skin barrier proteins (Filaggrin and Loricrin), thus indicating that SkOs could improve epidermal damage caused by sUV exposure. Figure 5b (G, H, I).

[0118] The reduced collagen fiber density after SUV exposure was restored. Figure 5b (J, K). Consistently, in SkOs treated with UCB-Exos, MMP-1 expression decreased, while COL1A1 expression increased (J, K). Figure 5b Immunofluorescence analysis showed that the dermal layer of SkOs treated with UCB-Exos maintained a higher level of collagen fibers, while MMP-1 expression was reduced, suggesting that collagen degradation was inhibited in sUV-damaged SkOs. Figure 5b (M, N).

[0119] The effects of UCB-Exos on hair follicles within skin organoids (SkOs) were investigated. Within the hair follicles, apoptosis (Cleaved Caspase-3) was observed. + The number of regenerated cells (Ki67) decreased, while the number of regenerated cells (Ki67) decreased. + The number of ) increased ( Figure 6 (A and B). Furthermore, it is known that senescent follicular keratinocytes and fibroblasts induced by sUV secretion secretion phenotype (SASP) contain MMPs and inflammatory cytokines. In the hair follicles of the UCB-Exos treatment group, the results showed a significant downregulation of SASP-related genes, indicating an improved state. Figure 6 (C). The above results indicate that UCB-Exos can effectively alleviate sUV-induced apoptosis and senescence of hair follicle cells, while promoting hair follicle regeneration.

[0120] SUV-induced hair follicle damage, such as shrinking hair bulbs and disruption of external structures like the DS and ORS, was significantly restored in the UCB-Exos treatment group. Furthermore, the phenomenon of abnormal hair shafts entering the regression phase was reversed after UCB-Exos treatment. Figure 6 D, E). Similarly, in the UCB-Exos treatment group, the expression of genes related to hair follicle structural composition was upregulated (D, E). Figure 6 Furthermore, hair growth-related genes (β-catenin and LEF1) were upregulated, while hair loss-related genes (DKK1) were downregulated, suggesting that UCB-Exos restored the hair formation capacity of skin organoids (SkOs). Figure 6 (G).

[0121] Melanocytes were also affected by UCB-Exos treatment. The number of melanocytes was reduced in both the epidermis and hair follicles. Figure 6 The expression of H and I, and genes related to melanocyte development and pigmentation (KIT and MITF) were also reduced. Figure 6 J).

[0122] In summary, this invention demonstrates the therapeutic potential of UCB-Exos in sUV-exposed SkOs to improve skin damage and promote hair follicle regeneration.

[0123] <Example 5> Using a skin organoid model of ultraviolet damage to confirm the IκB / NF-κB-mediated damage and treatment mechanism Multiple studies have shown that sUV radiation exposure can activate an inflammatory cascade in the skin, thereby inducing apoptosis. NF-κB is known to play an important role in regulating the expression of immune mediators. In its inactive state, NF-κB binds to the inhibitory protein IκBα, forming an NF-κB-IκB complex in the cytoplasm. When activated by external stimuli such as inflammatory cytokines or cellular stress, IκBα protein is phosphorylated and subsequently degraded, releasing NF-κB from the NF-κB-IκB complex. The released NF-κB translocates to the nucleus, promoting the transcription of genes involved in the inflammatory response and inducing cytokine production. Based on this understanding, the inventors further investigated whether IκB-dependent NF-κB activation mediates the inflammatory response in hair follicles.

[0124] This invention reveals that sUV induces phosphorylation of IκBα in the cytoplasmic region of SkOs, subsequently leading to IκBα degradation. This process results in the dissociation of the NF-κB-IκB complex, thereby activating and translocating NF-κB into the nucleus, as evidenced by increased NF-κB expression in the nuclear region of sUV-exposed SkOs. Treatment of UCB-Exos with sUV-exposed SkOs resulted in reduced phosphorylation and degradation of IκBα, while nuclear translocation of NF-κB was inhibited. Figure 7a (A, B). Immunofluorescence staining also confirmed the local expression of IκBα and activated NF-κB, namely phosphorylated NF-κB (p-NF-κB), in the cells that constitute the hair follicle structure. Generally, hair follicles are composed of keratinocytes and fibroblasts, both of which play important roles in skin inflammation. In particular, in the sUV treatment group, IκBα expression was significantly inhibited throughout the hair follicle, including ORS keratinocytes. Therefore, sUV exposure significantly increased the expression of p-NF-κB in ORS and the hair follicle matrix. Figure 7a (C, D). In sUV-induced SkOs, increased nuclear translocation of p-NF-κB in these structures indicates NF-κB activation following sUV exposure. Notably, the NF-κB-mediated inflammatory response primarily occurs in hair follicles, rather than dermal fibroblasts (C, D). Figure 7b E and F). On the other hand, the outermost SkOs epidermis also becomes inflamed after sUV exposure and extends into the dermis, which may indirectly damage the hair follicles (E and F). Figure 7b The expression patterns of IκBα and NF-κB were effectively reversed after UCB-Exos treatment (G, H). Figure 7b (EH).

[0125] Furthermore, in hair follicles isolated after UCB-Exos treatment, the mRNA expression of inflammation-related genes TNF-α and IL-6 was significantly reduced. Figure 7c In the vector control group, the levels of TNF-α and IL-6 proteins, which were highly expressed throughout the hair bulb, were significantly reduced in the matrix region after UCB-Exos treatment. Figure 7c J and K). This result was also consistent with the TNF-α and IL-6 secretion levels in SkOs detected by ELISA (J and K). Figure 7c (L). Overall, these results indicate that UCB-Exos alleviates the inflammatory response of fibroblasts and keratinocytes in hair follicles by inhibiting sUV exposure-induced IκB degradation and NF-κB activation.

[0126] <Example 6> Therapeutic Effect of Exosomes on Hair Follicle Photodamage Mediated by NF-κB Signal Inhibition To evaluate the direct effect of UCB-Exos in alleviating sUV-induced damage through NF-κB activation, the inventors treated sUV-damaged organoids with the NF-κB inhibitor BAY11-7082. BAY11-7082 is known to inhibit NF-κB translocation to the nucleus by suppressing the degradation of IκBα protein, thereby further preventing the sustained activation of NF-κB signaling.

[0127] Western blot analysis confirmed that BAY11-7082 treatment reduced phosphorylated IκBα levels in sUV-damaged SkOs, inhibiting IκBα degradation and thus reducing NF-κB nuclear translocation. Similarly, UCB-Exos treatment significantly reduced IκBα phosphorylation in sUV-exposed SkOs, resulting in inhibition of both IκBα degradation and NF-κB activation. In particular, the combined treatment with BAY11-7082 and UCB-Exos synergistically reduced NF-κB and phosphorylated IκBα levels. As expected, IL-6 expression was positively correlated with NF-κB expression levels. Figure 8a (A and B).

[0128] Furthermore, the increased secretion of inflammatory cytokines IL-6, TNF-α, and IL-1β after sUV exposure was assessed by ELISA. No significant difference in IL-6 and TNF-α secretion was observed between SkOs treated with BAY11-7082 and those treated with UCB-Exos. However, in the combined treatment group, TNF-α and IL-1β levels were significantly reduced, demonstrating a synergistic effect between BAY11-7082 and UCB-Exos. Figure 8a (C).

[0129] Against the backdrop of inflammatory responses, NF-κB signaling is known to be a key regulator of the aging-associated secretory phenotype (SASP) component, which delays tissue regeneration by promoting the accumulation of senescent cells, abnormal matrix remodeling, and chronic inflammation.

[0130] The study further investigated whether UCB-Exos treatment, which inhibits NF-κB activation, could regulate SASP within the hair follicle. Results showed that after treatment with UCB-Exos or BAY11-7082, the mRNA expression of inflammatory cytokines and SASP-related ECM components was significantly reduced. Figure 8b These results indicate that UCB-Exos can alleviate the adverse effects of SASP by inhibiting NF-κB activation, thereby creating an environment conducive to tissue regeneration.

[0131] Immunofluorescence staining revealed that after treatment with BAY11-7082 or UCB-Exos, the levels of NF-κB translocated to the nucleus in hair follicles and IL-6 in the cytoplasm were significantly reduced. Figure 8c The results (E, F, G) showed that after treatment with BAY11-7082 or UCB-Exos, apoptosis in these hair follicles was reduced, and the apoptosis rate was lowest after combined treatment. Figure 8c The final results showed that NF-κB inhibition could restore the structural integrity of hair follicles, including DS, ORS, and hair bulb, and prevent hair follicles from transitioning to the regression phase. Figure 8c (J). In summary, this invention demonstrates that UCB-Exos can effectively restore hair follicles damaged by sUV by inhibiting NF-κB.

[0132] <Example 7> Confirmation of the effect of NF-κB signaling inhibitors on promoting hair follicle growth in normal hair follicle organoids To evaluate the inhibitory effect of NF-κB signaling in normal hair follicles, the NF-κB inhibitor BAY11-7082 was treated on skin organoids with existing hair follicles, and hair follicle growth was observed. Simultaneous treatment with minoxidil, known to promote hair follicle growth, and dihydrotestosterone (DHT), an endogenous hormone known to induce hair loss, was used for comparison.

[0133] The results of hair follicle morphology observation on day 14 after drug treatment showed that, compared with the control group, the hair follicle length in the BAY11-7082 treatment group increased by approximately 1.4 times, while that in the Minoxidil treatment group increased by approximately 1.3 times. Conversely, in the DHT treatment group, the hair follicle length decreased to approximately 0.7 times that of the control group. Therefore, it can be concluded that the NF-κB inhibitor BAY11-7082, similar to Minoxidil, has a hair follicle growth-promoting effect. Figure 9 A and Figure 9 B).

[0134] Furthermore, the expression of genes related to hair growth promotion (IGF-1, WNT3A) was upregulated compared to the control group, while the expression of genes related to hair loss (DKK1, TGFb-2) was downregulated. These results indicate that the NF-κB inhibitor BAY11-7082 has a hair follicle growth promoting effect. Figure 9 C).

[0135] This invention demonstrates that BAY11-7082 can also promote hair follicle growth in normal hair follicles by inhibiting NF-κB signaling, thus confirming its potential as a novel therapeutic agent to promote hair growth.

[0136] In summary, based on the above results, this invention confirms that skin organoids derived from human induced pluripotent stem cells can simulate skin damage caused by actual external environmental factors in an in vitro model through the same mechanism as real skin. Furthermore, in a UV-induced skin damage model, the efficacy of potential therapeutic candidates was evaluated, verifying the alleviation of various disease phenotypes, thus confirming its applicability as a drug screening platform. This invention can be applied to research on UV-induced skin damage and aging-related issues, contributing to improved patient quality of life, and is expected to have wide applications in drug and toxicity evaluation, preclinical research, regenerative medicine, and tissue engineering research.

[0137] The above description of the present invention is merely illustrative, and those skilled in the art can make various modifications or improvements without departing from the technical concept and essential characteristics of the present invention. Therefore, it should be understood that the above embodiments are exemplary and not restrictive in all respects. The scope of protection of the present invention is defined by the following claims and should be interpreted to include the meaning and scope of the claims and all modifications or variations derived from their equivalents.

[0138] [Industrial Applicability] This invention relates to a skin damage organoid model and its application in drug screening. It utilizes skin organoids derived from human induced pluripotent stem cells to model skin damage caused by external environmental factors (ultraviolet radiation). By employing organoid technology that simulates the real human developmental process, skin organoids containing skin constituent cells, hair follicle-related cells, and all microstructures are constructed. Through ultraviolet irradiation, a modeling technique is established to simulate skin damage caused by real external stimuli in vitro. Therefore, this technology can elucidate the mechanisms of damage and aging caused by ultraviolet radiation in vitro and establish a platform for screening candidates to alleviate this damage. The platform established by this invention can be used to evaluate and screen disease mechanisms and the efficacy of various drug candidates, thus possessing industrial applicability.

Claims

1. A method for preparing organoids from skin lesions, characterized in that, Includes the following steps: (1) Culture organoids derived from pluripotent stem cells in the presence of Wnt agonists; (2) Culture the culture from step (1) in skin organoid maturation medium; (3) The culture from step (2) is cut and cultured at the gas-liquid interface to prepare skin organoids; and (4) Irradiate the skin organoids with ultraviolet light that simulates sunlight. Wnt agonist is added when non-neuroectoderm is induced to differentiate into cranial neuralcrest cells (CNCC).

2. The method according to claim 1, wherein, The skin injury organoids refer to hair follicle injury organoids, which are accessory organs of the skin.

3. The method according to claim 1, wherein, The Wnt agonist is selected from the group consisting of CHIR-99021, WNT3A, WNT5A, and RSPO1.

4. The method according to claim 1, wherein, The Wnt agonist was added on days 5 to 7 of pluripotent stem cell culture.

5. The method according to claim 1, wherein, In step (3), the culture from step (2) is cut into four uniformly sized pieces and cultured at the gas-liquid interface on a transmembrane culture insert coated with collagen, so that the dermis faces the collagen side and the epidermis is exposed to air.

6. The method according to claim 1, characterized in that, The skin-damaged organoids cause skin damage through NF-κB activation.

7. A skin injury organoid prepared by the method according to claim 1.

8. A method for screening skin injury treatment agents, preventive agents, or improvers, comprising the step of treating skin injury treatment agent, preventive agent, or improver candidate material on the skin injury organoid according to claim 7.

9. The method according to claim 2, characterized in that, The hair follicle damage organoids cause hair follicle damage through NF-κB activation.

10. The method according to claim 2, characterized in that, The follicle-damaged organoids were selected from the group consisting of dermal sheath marker α-SMA, outer root sheath marker KRT5 or KRT15, ​​follicle stem cell marker KRT15 or LHX2, and dermal papilla marker SOX2, in which the expression of one or more genes was reduced.

11. The method according to claim 2, characterized in that, In the aforementioned follicular damaged organoids, the gene expression of inflammatory cytokines COX-2, TNF-α, or IL-1β is increased.

12. A hair follicle injury organoid, prepared by the method according to claim 2.

13. A method for screening hair growth promoters, hair loss preventive agents, hair loss alleviating agents, or hair loss treatment agents, comprising the step of treating a candidate substance for a hair growth promoter, hair loss preventive agent, hair loss alleviating agent, or hair loss treatment agent in the hair follicle damaged organoid as described in claim 12.

14. The method according to claim 13, characterized in that, The candidate substance inhibits the activation of NF-κB in damaged hair follicle organoids.

15. A biomarker composition for detecting hair follicle damage, comprising NF-κB as an active ingredient.

16. A kit for detecting hair follicle damage, comprising a formulation capable of detecting NF-κB.

17. The kit according to claim 16, characterized in that, The formulation is selected from one or more of the group consisting of NF-κB specific primers, probes, antisense oligonucleotides, aptamers, and antibodies.

18. A method for providing information needed for detecting hair follicle damage, comprising the following steps: (1) Measure the expression level of NF-κB in the isolated samples; (2) The expression level of the NF-κB was compared with that of the control group; and (3) If the expression level of NF-kB is higher than that of the control group sample, the hair follicle is considered to be damaged.

19. A pharmaceutical composition for use as a hair growth promoter, hair loss preventive agent, hair loss reliever, or hair loss treatment agent, comprising an inhibitor of NF-κB activity or expression as an active ingredient.

20. The pharmaceutical composition according to claim 19, characterized in that, The NF-κB activity inhibitor is selected from any one of the following groups: small molecule compounds, peptides, peptide mimics, aptamers, antibodies, and natural products that specifically bind to the NF-κB protein.

21. The pharmaceutical composition according to claim 19, characterized in that, The NF-κB expression inhibitor is selected from any one of the following groups: antisense nucleotides that bind complementary to the mRNA of the NF-κB gene, small interfering RNA (siRNA), and short hairpin RNA (shRNA).

22. A pharmaceutical composition for hair removal, comprising an NF-κB activity or expression promoter as an active ingredient.

23. The pharmaceutical composition according to claim 22, characterized in that, The NF-κB activity enhancer is selected from any one of the following groups: small molecule compounds that specifically bind to NF-κB protein, peptides, peptide mimics, aptamers, antibodies, and natural products.

24. The pharmaceutical composition according to claim 22, characterized in that, The NF-kB expression promoter is selected from any one of the following groups: adeno-associated virus (AAV), adenovirus, lentivirus, and retrovirus, which contain an NF-kB encoding gene that increases the expression of the NF-kB gene mRNA.

25. A cosmetic composition for hair removal, comprising an NF-κB activity or expression promoter as an active ingredient.

26. Hair growth promotion, hair loss prevention, hair loss relief or hair loss treatment methods, including the steps of administering an NF-κB activity or expression inhibitor in an effective amount to an individual in need of the treatment.

27. A hair removal method comprising the step of applying an NF-κB activity or expression promoter in an effective amount to an individual requiring the treatment.

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