Skin-like organ containing hair follicle structure and preparation method thereof

Abstract

The invention discloses a skin organoid containing a hair follicle structure and a preparation method thereof, and belongs to the field of tissue engineering, the method comprises the following steps: separating hair follicle stem cells from dermal papilla cells; hair follicle induced aggregate is formed through synergistic induction of a Wnt activator, a BMP inhibitor and FGF; embedding the aggregate into a biphase gradient collagen-hyaluronic acid hydrogel to construct a corium layer; after keratinocytes are inoculated, hair follicles are activated and promoted to mature through a gas-liquid interface staged signal channel, and the obtained organoid contains complete epidermal layer, corium layer and hair follicle-like structures and can be used for hair research and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and regenerative medicine technology, specifically relating to skin organoids containing hair follicle structures and their preparation methods. Background Technology

[0002] The skin, the largest organ in the human body, consists of three layers: the epidermis, dermis, and subcutaneous tissue. It contains various skin appendages such as hair follicles, sebaceous glands, and sweat glands. Hair follicles, as one of the most important skin appendages, have a highly complex structure and function, comprising multiple functional areas including the outer root sheath, inner root sheath, hair shaft, dermal sheath, and dermal papillae. They participate in key physiological processes such as hair growth cycle regulation, skin wound repair, and immune defense. Hair follicle-related diseases, such as androgenetic alopecia, alopecia areata, and cicatricial alopecia, severely impact patients' quality of life. Currently, there are no effective treatments available clinically, making the establishment of in vitro models that realistically simulate hair follicle development and function urgently needed for disease mechanism research and drug screening.

[0003] Existing in vitro skin models mainly fall into three categories: two-dimensional cell culture, three-dimensional full-thickness skin equivalents, and skin organoids. While two-dimensional cell culture is simple to operate, it cannot simulate the three-dimensional spatial structure of the skin and intercellular interactions. Commercially available full-thickness skin equivalents, such as EpiDerm and MatTek, while containing the basic structures of the epidermis and dermis, generally lack skin appendages such as hair follicles and sebaceous glands. They can only be used for evaluating skin barrier function and drug permeability, and cannot meet the needs of hair biology research and screening for hair loss treatments.

[0004] In recent years, breakthroughs have been made in skin organoid technology based on human pluripotent stem cells. Lee et al. (Nature, 2020, 582: 399-404) reported a method for inducing the generation of hair follicle-containing skin organoids from human pluripotent stem cells. These organoids can generate hair follicles after about 60 days of culture and reach full maturity in about 130 days, containing layered epidermis, pigmented hair follicles, sebaceous glands, Merkel cells, and sensory neurons. However, this method has significant limitations: First, the culture cycle is extremely long, requiring 4 to 5 months from stem cell induction to the generation of mature hair follicles, which is difficult to meet the time-sensitive requirements of high-throughput drug screening; second, the organoids have a vesicular spherical structure with hair follicles growing inward, which is opposite to the polarity direction of hair follicles extending from the epidermis to the dermis in natural skin, making it impossible to truly simulate the spatial positioning of hair follicles in the skin; third, the number and distribution of hair follicles are uncontrollable, with large batch-to-batch variations and low standardization.

[0005] There are also reports on strategies for constructing tissue-engineered skin using primary cells derived from adults. Chinese patent CN109172868A discloses a method for rapidly reconstructing skin and hair follicles, which involves preparing a tissue homogenate from full-thickness skin of adult mice and transplanting it into nude mice to reconstruct hair follicles. However, this method relies on in vivo transplantation and cannot form skin constructs containing hair follicles in vitro. International patent WO2024035684A1 reports a method for preparing hair follicle organoids by co-culturing iPSC-derived dermal papillary cells and epithelial stem cells. However, this method generates independent hair follicle units, rather than complete organoids containing full-thickness skin structures, lacking systematic integration of the epidermal barrier and dermal microenvironment.

[0006] In summary, existing technologies face the following technical bottlenecks in preparing full-thickness skin organoids containing functional hair follicle structures: excessively long culture cycles limit practicality, hair follicle polarity and spatial positioning cannot be precisely controlled, hair follicle density and distribution lack standardized methods, and there is a lack of short-cycle whole-body in vitro culture systems starting from adult primary cells. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide skin organoids containing hair follicle structures and their preparation methods. These skin organoids use adult-derived hair follicle stem cells and dermal papillary cells as core seed cells. Through a strategy of hair follicle-induced aggregate pre-programming, biphasic gradient hydrogel localization and embedding, and phased activation of signaling pathways at the gas-liquid interface, full-thickness skin organoids containing functional hair follicle structures can be constructed in vitro in a short period of time. This overcomes the technical defects of existing pluripotent stem cell methods, such as long culture cycles, uncontrollable hair follicle polarity, and low standardization.

[0008] To achieve the above objectives, this invention provides a skin organoid containing hair follicle structures, comprising a dermis, an epidermis, and hair follicle-like structures embedded within the dermis. The dermis is composed of a biphasic gradient hydrogel formed by dermal fibroblasts, type I collagen, and hyaluronic acid. The hair follicle-like structures develop from hair follicle-inducing aggregates formed by co-culturing hair follicle stem cells and dermal papilla cells in vitro. The hair follicle-inducing aggregates are synergistically induced by GSK-3β inhibitors, bone morphogenetic protein inhibitors, and fibroblast growth factor 2 to form hair follicle primordium-like structures. The hair follicle-like structures express hair follicle-specific markers keratin 17 and keratin 75, and transcription factor Sox9; the dermal papilla region expresses alkaline phosphatase and the proteoglycan versican. The skin organoid contains 30 to 80 hair follicle-like structures per square centimeter.

[0009] The present invention also provides a method for preparing the above-mentioned skin organoid containing hair follicle structure, comprising four steps: isolating and culturing hair follicle stem cells and dermal papillary cells, preparing hair follicle inducible aggregates, constructing a biphasic gradient dermal layer containing hair follicles, constructing an epidermal layer and inducing hair follicle maturation in stages.

[0010] The beneficial effects of this invention include: First, by using primary cells derived from adults instead of pluripotent stem cells as seed cells, the total culture cycle from preparing hair follicle-induced aggregates to obtaining mature skin organoids is 30 to 45 days, which is 3 to 4 times shorter than existing pluripotent stem cell methods, significantly improving practical efficiency. Second, through a hair follicle-induced aggregate pre-programming strategy, aggregates with hair follicle development potential are pre-formed in vitro using the synergistic effect of Wnt / BMP / FGF, and then embedded in dermal hydrogels, achieving precise control over hair follicle density and distribution. Third, by employing a biphasic gradient hydrogel system, the lower layer of dense collagen provides mechanical support and polarity anchoring for hair follicles, while the upper layer of loose collagen-hyaluronic acid provides a suitable microenvironment for nutrient penetration and growth factor diffusion for hair follicle development. The two layers synergistically simulate the structural gradient of the papillary and reticular layers in the natural dermis. Fourth, the phased signaling pathway activation strategy at the gas-liquid interface divides the hair follicle maturation process into a polarity establishment stage mediated by the Shh pathway and a dermal papilla maturation stage mediated by PDGF-BB, avoiding interference between signaling pathways and improving hair follicle maturation efficiency. Fifth, the resulting skin organoids exhibit a natural polarity direction extending from the epidermis to the dermis, and the hair follicles express a complete lineage of differentiation markers, which can be used for hair growth cycle research, screening of hair loss treatment drugs, and exploration of hair regeneration mechanisms. Instruction manual illustrations

[0011] Figure 1 This is a schematic flowchart of the method for preparing skin organoids containing hair follicle structures according to the present invention.

[0012] Figure 2 Bright field micrograph of hair follicle induced aggregates after 72 h of culture.

[0013] Figure 3 Immunofluorescence staining image of frozen sections of hair follicle-induced aggregates.

[0014] Figure 4 HE staining image of a cross section after immersion culture for 7 days following embedding of a biphasic gradient hydrogel into hair follicle aggregates.

[0015] Figure 5 This is an HE-stained tissue section of the finished skin organoid.

[0016] Figure 6 Immunofluorescence staining image of hair follicle-like structures in finished skin organoids.

[0017] Figure 7 A bar chart showing the effect of different mixing ratios of hair follicle stem cells and dermal papillary cells on hair follicle formation density.

[0018] Figure 8 A bar chart comparing the effects of a phased signaling pathway activation strategy at the gas-liquid interface on hair follicle maturity.

[0019] Figure 9 This is a dose-response curve for three hair loss treatment drugs. Detailed Implementation

[0020] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only for illustrating the technical solution of the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0021] The present invention provides a method for preparing skin organoids containing hair follicle structures, such as... Figure 1 As shown, the process includes four steps: isolating and culturing hair follicle stem cells and dermal papilla cells, preparing hair follicle-inducing aggregates, constructing a biphasic gradient dermal layer containing hair follicles, and constructing an epidermal layer and inducing hair follicle maturation in stages.

[0022] Regarding step one, the separation and culture of hair follicle stem cells and dermal papilla cells, the human scalp tissue used was obtained from discarded occipital scalp tissue during plastic surgery. Donors were aged 20 to 60 years, regardless of gender, and their use was only after informed consent and ethical approval. The scalp tissue was processed within 2 hours of collection. First, it was rinsed three times with a phosphate buffer solution containing 100 U / mL penicillin and 100 μg / mL streptomycin to remove blood and contaminants. The cleansed scalp tissue was placed under a stereomicroscope, and the dermis was longitudinally incised along the hair follicle growth direction using ophthalmic surgical scissors and microforceps to separate complete hair follicle units one by one. During separation, the integrity of the hair follicles must be preserved, ensuring that the entire structure from the bulge area to the hair bulb remains undamaged. Each approximately 2 cm × 3 cm piece of scalp tissue yielded 80 to 150 complete hair follicles.

[0023] The isolated intact hair follicles were transferred to serum-free DMEM / F12 medium containing a working concentration of 2–5 U / mL dispersant enzyme and digested at 37°C for 1–2 h. The dispersant enzyme mainly acts on the basement membrane, separating the outer root sheath of the hair follicle from the surrounding connective tissue without disrupting intercellular connections. After digestion, the outer root sheath was completely detached from the hair follicle using a fine needle under a stereomicroscope, focusing on collecting the outer root sheath tissue from the bulging region, i.e., the permanent upper segment of the hair follicle. The bulging region is located below the sebaceous gland opening and morphologically appears as a slightly swollen area of ​​the outer root sheath. The collected outer root sheath tissue from the bulging region was digested with 0.25% trypsin-EDTA at 37°C for 10–15 min to prepare a single-cell suspension.

[0024] Hair follicle stem cells (HFCs) were screened for CD34- and CD200-positive results using magnetic bead sorting. First, anti-human CD34 magnetic beads were used for the first round of positive sorting to enrich CD34-positive cells from the mixed cell population. Then, anti-human CD200 magnetic beads were used for the second round of positive sorting, ultimately obtaining a HFC population that was double-positive for both CD34 and CD200. Flow cytometry verification showed that the CD34 positivity rate in the sorted cell population should be no less than 90%, and the CD200 positivity rate should be no less than 85%. HFCs were seeded in culture dishes coated with type IV collagen using serum-free CnT-07 keratinocyte culture medium or serum-free keratinocyte culture medium containing 5% fetal bovine serum (K-SFM), supplemented with epidermal growth factor at 5 ng / mL and bovine pituitary extract at 50 μg / mL. The culture was incubated at 37°C in a 5% CO2 incubator. Hair follicle stem cells exhibit a typical cobblestone-like morphology. They are passaged when they reach 80% to 90% confluence, expanding to the second or third generation for future use. During passage, the expression of CD34 and CD200 markers needs to be monitored to ensure the maintenance of the stem cell phenotype.

[0025] For the isolation of dermal papilla cells, the hair follicle base (hair bulb) after dispersive enzyme digestion was further microscopically dissected under a stereomicroscope, and the dermal papilla was carefully detached from the base of the hair bulb using a fine needle. Morphologically, the dermal papilla appears as an oval-shaped cluster of mesenchymal cells embedded at the base of the hair bulb, slightly darker in color than the surrounding hair matrix cells. The isolated dermal papilla tissue was transferred to DMEM medium containing 0.2% to 0.5% type II collagenase and digested at 37°C for 2 to 4 hours until the tissue block was completely dissociated. The digested cell suspension was filtered through a 200-mesh cell sieve, centrifuged to collect the cell pellet, and seeded into uncoated standard cell culture dishes. The culture medium for dermal papilla cells was DMEM medium containing 10% fetal bovine serum (FBS) supplemented with basic fibroblast growth factor 2 at a concentration of 10 ng / mL. Dermal papillary cells initially appear spindle-shaped or triangular in vitro. After proliferation and confluence, they tend to spontaneously aggregate to form three-dimensional spherical structures. This characteristic is an important marker for maintaining the hair follicle-inducing activity of dermal papillary cells. Identification of dermal papillary cells is performed using alkaline phosphatase activity staining and α-smooth muscle actin immunofluorescence staining; the double-positive rate should be no less than 85%. Cells are then expanded to the second or third generation for future use. The number of passages should not exceed five to avoid loss of inducible activity.

[0026] Regarding step two, namely the preparation of hair follicle-induced aggregates, the hair follicle stem cells and dermal papillary cells obtained in step one are digested separately with 0.25% trypsin-EDTA to prepare single-cell suspensions. Cell viability is assessed using the trypan blue rejection method; viability must reach 95% or higher for use. The two cell types are mixed at a cell ratio of 1:1 to 2:1, with a preferred ratio of 1.5:1. The total cell density of the mixed cell suspension is adjusted to 1 × 10⁶ cells / mL. 6 Up to 5×10 6 indivual.

[0027] Aggregate culture was performed using 96-well U-bottom culture plates with ultra-low adsorption. 200 μL of culture medium containing 5000 to 10000 mixed cells was added to each well, and the plates were centrifuged at 200×g for 3 min to promote rapid aggregation of cells to the bottom of the wells, forming single aggregates. The culture plates were then incubated statically at 37°C in a cell culture incubator with 5% CO2. The culture medium was prepared using serum-free DMEM / F12 as the basal medium, supplemented with 2% B27 as a serum-free substitute, with L-glutamine at 2 mmol / L and penicillin-streptomycin at 100 U / mL. The following three key inducing factors were added to this basal culture medium: CHIR99021, a GSK-3β inhibitor, at a concentration of 3 μmol / L, acts as an activator of the classic Wnt / β-catenin signaling pathway. By inhibiting GSK-3β-mediated phosphorylation and degradation of β-catenin, it allows β-catenin to accumulate in the cytoplasm and translocate into the nucleus to activate the transcription of Wnt target genes, thereby promoting the proliferation of hair follicle stem cells and hair follicle fate determination; Noggin, a bone morphogenetic protein inhibitor, at a concentration of 100 ng / mL, directly binds to BMP2 / 4 / 7 ligands to block their binding to receptors, thereby relieving the inhibitory effect of BMP signaling on hair follicle induction, maintaining the self-renewal capacity of peduncle stem cells, and promoting epithelial-mesenchymal interaction; and fibroblast growth factor 22, or basic fibroblast growth factor 2, at a concentration of 20 ng / mL, activates the FGFR2 signaling pathway to promote the proliferation and aggregation of dermal papilla cells, and enhances their ability to secrete alkaline phosphatase and versican, among other hair follicle induction-related molecules.

[0028] During aggregate culture, the culture medium was replaced at half capacity every 48 hours. After 24 hours of culture, the mixed cells initially formed loose cell clusters; after 48 hours, the cell clusters gradually compacted, forming well-defined spherical aggregates; after 72 hours, the aggregates developed into dense hair follicle primordium-like structures with a diameter of 100 to 250 μm, preferably 150 to 200 μm. Successful hair follicle-induced aggregates are morphologically characterized by a dense core region and a radially arranged peripheral region, such as... Figure 2As shown, dermal papillary cells tend to aggregate in the core region to form mesenchymal aggregates, while hair follicle stem cells surround the periphery to form an epithelial-like shell. This self-assembled spatial distribution mimics the initial configuration of epithelial-mesenchymal interaction in the hair follicle primordium during early embryonic development. Figure 3 As shown, the aggregates were identified by frozen sectioning and immunofluorescence staining. Positive expression of versican and alkaline phosphatase should be detected in the core region, and positive expression of keratin 15 and P-cadherin should be detected in the peripheral region. The aggregate formation rate, i.e., the proportion of dense spherical aggregates with a diameter greater than 100 μm, should be no less than 80% of the total number of inoculated wells.

[0029] Alternatively, the hanging drop culture method can be used to prepare hair follicle-induced aggregates. A 20-30 μL droplet of culture medium containing 3000-8000 mixed cells is suspended on the inner surface of a culture dish lid. Phosphate buffer solution or sterile water is added to the bottom of the dish to maintain humidity. The cells in the droplet aggregate spontaneously to the bottom of the droplet under gravity, forming individual aggregates. The hanging drop culture method produces aggregates with better size uniformity, but has a lower throughput, making it suitable for small-batch, precise preparation.

[0030] Regarding step three, which involves constructing a biphasic gradient dermal layer containing hair follicles, the core innovation of this step lies in designing a biphasic gradient hydrogel system to simulate the layered structure of natural dermis. The dermis of natural skin is divided into the papillary layer near the epidermis and the deeper reticular layer, which differ significantly in collagen fiber density, orientation, and matrix composition. The papillary layer has fine collagen fibers rich in proteoglycans, providing a loose growth environment for the upper part of the hair follicle; the reticular layer has coarse and dense collagen fibers, providing mechanical anchoring for the bulb of the hair follicle and the dermal papilla. The biphasic gradient hydrogel of this invention is based on this natural structural feature and is designed biomimetously.

[0031] First, a dense collagen sublayer was prepared. A rat tail type I collagen solution (stock solution concentration 8 to 10 mg / mL, dissolved in 0.02 mol / L acetic acid solution), 10×DMEM concentrate, and 1 mol / L NaOH solution were rapidly mixed on ice at a volume ratio of 8:1:1. The pH was adjusted to 7.2 to 7.4 with NaOH, resulting in a final type I collagen concentration of 4 to 6 mg / mL, preferably 5 mg / mL. Pre-digested and collected dermal fibroblasts were then injected at a concentration of 1×10¹² mg / mL. 5 Up to 5×10 5The cells are uniformly mixed into the collagen solution. Dermal fibroblasts are obtained from the remaining dermal tissue after hair follicle separation in step one, and are digested with 0.2% type I collagenase at 37°C for 4 to 6 hours. They are then expanded to the third to fifth generation for use. The collagen solution containing cells is injected into Transwell culture dishes or custom-made silicone molds. Approximately 200 μL of collagen solution is injected into each 12 mm diameter culture dish to form a dense collagen layer 0.5 to 1 mm thick. The culture dishes are incubated at 37°C for 20 to 30 minutes until the collagen solution semi-gels. At this point, the collagen has begun to fibrose and cross-link but is not yet fully solidified, and the upper surface maintains a certain degree of adhesion to facilitate subsequent integration of the upper layer.

[0032] Subsequently, a loose collagen-hyaluronic acid superlayer containing hair follicle aggregates was prepared. Type I collagen solution was diluted to 1.5 to 3 mg / mL, preferably 2 mg / mL, while sodium hyaluronate with a molecular weight of 1000 to 1500 kDa was added to a final concentration of 0.5 to 2 mg / mL, preferably 1 mg / mL. The addition of hyaluronic acid reduces the mechanical stiffness of the superlayer hydrogel, thus providing space for the growth and extension of hair follicle aggregates. Furthermore, as an important matrix component of the natural dermal papillary layer of skin, hyaluronic acid can bind to CD44 receptors to promote the migration and proliferation of hair follicle stem cells, and maintain the moist microenvironment required for hair follicle development through its water-retention effect. The hair follicle-induced aggregates obtained in step two were gently aspirated from the ultra-low adsorption culture plate, collected by low-speed centrifugation (100×g, 2 min), and dispersed in the prepared loose collagen-hyaluronic acid solution. The aggregation density was 50 to 120 aggregates per square centimeter, preferably 70 to 90 aggregates per square centimeter. When adding aggregates, care should be taken to ensure that they are evenly distributed in the solution to avoid local aggregation or sedimentation to the bottom.

[0033] A loose collagen-hyaluronic acid solution containing hair follicle aggregates was carefully injected onto a semi-gelled, dense collagen layer, approximately 300 to 400 μL per culture dish to form a 1 to 2 mm thick top layer. Air bubbles should be avoided during injection, and the liquid level should be kept horizontal to ensure a uniform top layer thickness. The culture dishes were then incubated at 37°C for 30 to 45 minutes until the top layer was completely gelled. After gelation, the two layers bonded tightly together due to interfacial integration in the semi-gelled state, forming a continuous biphasic gradient hydrogel structure.

[0034] After gelation, add DMEM medium containing 10% fetal bovine serum to the culture dish for immersion culture. The medium should completely cover the hydrogel surface to a depth of approximately 2 to 3 mm. Immersion culture should last for 5 to 10 days, preferably 7 days. During immersion culture, if... Figure 4As shown, dermal fibroblasts proliferate within dense collagen and secrete endogenous collagen and fibronectin, further strengthening the mechanical properties of the dermis. Follicle-induced aggregates continue to develop in the loose collagen-hyaluronic acid microenvironment, gradually increasing in size to a diameter of 250-400 μm. Preliminary spatial polarity begins to emerge internally, with the mesenchymal core extending downwards towards the dense collagen layer and the epithelial shell expanding upwards towards the surface. The culture medium was completely replaced every two days during immersion culture.

[0035] Regarding step four, namely constructing the epidermis and inducing hair follicle maturation in stages, firstly, human primary keratinocytes are seeded onto the upper surface of the dermis. The keratinocytes can be obtained from the scalp epidermis or foreskin of the same donor, or commercially available human primary epidermal keratinocytes can be used. The keratinocytes are seeded at a rate of 2 × 10⁶ cells per square centimeter. 5 Up to 5×10 5 Keratinocytes were evenly seeded at a density on the upper surface of the dermis. After seeding, the culture dish was kept horizontal and incubated for 2 to 4 hours to allow the keratinocytes to fully adhere to the dermal surface. Submerged culture was then performed using DMEM / F12 medium containing 10% fetal bovine serum (FBS). The medium level was slightly above the dermal surface (approximately 1 to 2 mm) to cover the seeded keratinocytes. Submerged culture continued for 3 to 5 days until the keratinocytes proliferated and expanded to form a continuous monolayer covering the dermal surface. The growth status of the keratinocytes was observed daily during this period. When the coverage reached over 90%, the cells were transferred to an air-liquid interface for further culture.

[0036] Air-liquid interface culture is a crucial step in inducing epidermal stratification and hair follicle structure development. The constructs are transferred to a Transwell scaffold containing a microporous membrane (0.4 μm pore size) or directly in the original culture dish, with the culture medium level lowered to be flush with the lower surface of the dermis, exposing the epidermis to air while keeping the dermis immersed in the medium. The air-liquid interface medium is a serum-free keratinocyte culture medium containing 1.2 mmol / L calcium chloride; the high calcium concentration helps promote terminal differentiation of keratinocytes and stratum corneum formation. The medium is also supplemented with hydrocortisone at a concentration of 0.4 μg / mL to maintain the balance of keratinocyte proliferation and differentiation, insulin at a concentration of 5 μg / mL to promote cell metabolism, and magnesium ascorbate phosphate at a concentration of 50 μg / mL as an antioxidant to protect cells and promote collagen synthesis. During air-liquid interface culture, the culture medium level is monitored daily for evaporation; if necessary, an appropriate amount of medium is added to maintain the level exactly flush with the lower surface of the dermis, ensuring the epidermis is always exposed to air. The culture medium should be completely replaced every two days. Care should be taken during the replacement to avoid the culture medium overflowing the surface of the epidermis and causing water immersion.

[0037] The gas-liquid interface culture employs a staged signaling pathway activation strategy, which is another core innovation distinguishing this invention from existing technologies. The first stage is the Sonic hedgehog (Shh) pathway activation stage, in which the Smoothened agonist SAG is added to the gas-liquid interface culture medium at a concentration of 0.5 to 2 μmol / L, preferably 1 μmol / L. The Shh signaling pathway plays a crucial role in establishing polarity and hair bud elongation during embryonic hair follicle development. SAG bypasses the requirement of upstream Shh ligands by directly binding to and activating the Smoothened receptor, activating the downstream Gli transcription factor signaling cascade, promoting the directional proliferation of epithelial cells within the hair follicle aggregate and their elongation and invasion into the dermis, thus establishing the longitudinal axis polarity of the hair follicle. The first stage lasts for 7 to 14 days, preferably 10 days. At the end of the first stage, dark-field microscopy of whole-skin specimens shows that the hair follicle aggregate has developed from a spherical shape into a pear-shaped or rod-shaped structure, with the long axis perpendicular to the dermis, indicating that hair follicle polarity has been successfully established.

[0038] The second stage is the maturation of the dermal papilla and the refinement of the hair follicle sheath structure. SAG is removed, and platelet-derived growth factor BB (PDGF-BB) is added to the air-liquid interface culture medium at a concentration of 10 to 50 ng / mL, preferably 20 ng / mL. PDGF-BB promotes the functional maturation of dermal papilla cells by activating the PDGFRα signaling pathway, enhancing their alkaline phosphatase activity and the secretion of key signaling molecules such as versican, Wnt5a, and BMP6, thereby maintaining the continuous induction of the dermal papilla on the hair follicle epithelium. Simultaneously, PDGF-BB promotes the proliferation and collagen secretion of dermal sheath cells, driving the refinement of the hair follicle sheath structure. The second stage lasts 14 to 21 days, preferably 18 days. During the second stage, the hair follicle structure gradually matures, forming a complete multi-layered concentric structure of the outer root sheath, inner root sheath, and dermal sheath. The formation of keratinized hair shaft-like structures can be observed in some hair follicles.

[0039] The total culture time for the gas-liquid interface is 21 to 35 days, preferably 28 days. The total culture time for the entire fourth step (including immersion culture and gas-liquid interface culture) is 24 to 40 days. Including the preparation time for steps one to three, the total time from material collection to obtaining mature skin organoids with hair follicle structures is 30 to 45 days.

[0040] The following is a detailed description of specific embodiments.

[0041] The objective of Example 1 was to prepare a skin organoid containing hair follicle structures under standard conditions. Scalp tissue, approximately 2 cm × 3 cm in area, was obtained from discarded tissue from a 35-year-old male donor undergoing occipital reconstruction surgery. Following the method described in Step 1, 126 intact hair follicles were isolated. After digestion with dispersing enzyme (3 U / mL) at 37°C for 1.5 h, the outer root sheath bulge area was separated. Hair follicle stem cells, approximately 2.8 × 10⁸, were obtained through trypsin digestion and CD34 / CD200 double-positive magnetic bead sorting. 5 Flow cytometry analysis showed a CD34 positivity rate of 93.2% and a CD200 positivity rate of 89.7%. Dermal papillae were isolated from the base of hair follicles in the same batch and digested with 0.3% type II collagenase at 37°C for 3 hours to obtain approximately 1.5 × 10⁻⁶ dermal papilla cells. 5 The positive rates for alkaline phosphatase were 91.4% and for α-smooth muscle actin were 87.6%. Both cell types were expanded to the third generation.

[0042] Following step two, hair follicle stem cells and dermal papillary cells were mixed at a ratio of 1.5:1, and 8000 cells were seeded per well in 96-well U-shaped culture plates with ultra-low adsorption, yielding 96 aggregates per plate. The culture medium contained 3 μmol / L CHIR99021, 100 ng / mL Noggin, and 20 ng / mL FGF2. After 72 h of culture, follicle-induced aggregates with diameters ranging from 160 to 190 μm were obtained, with an aggregate formation rate of 88.5% (85 / 96). Immunofluorescence of frozen sections showed versican positivity in the core region and keratin 15 positivity in the peripheral region, confirming that the spatial distribution of epithelial-mesenchymal junction was as expected.

[0043] Construct a biphasic gradient dermal layer following step three. The collagen concentration in the dense collagen sublayer is 5 mg / mL, and the density of dermal fibroblasts mixed in is 3 × 10⁻⁶ per mL. 5 200 μL of each of the following 12-well transwell samples was injected and incubated at 37°C for 25 min until semi-gelatinized: A loose collagen-hyaluronic acid layer with a collagen concentration of 2 mg / mL and a hyaluronic acid concentration of 1 mg / mL was added, along with 80 hair follicle-inducing aggregates per square centimeter, injected into each well at 350 μL and incubated at 37°C for 40 min until fully gelatinized. The mixture was then immersed in the culture for 7 days.

[0044] Following step four, the density of keratinocytes inoculated should be 3 × 10⁶ per square centimeter. 5 After immersion culture for 4 days, the cells were transferred to gas-liquid interface culture. In the first stage, SAG was added at a concentration of 1 μmol / L for 10 days, and in the second stage, PDGF-BB was added at a concentration of 20 ng / mL for 18 days, for a total of 28 days of gas-liquid interface culture.

[0045] The results of the examination of the finished skin organoid are as follows. Gross observation shows that the construct is disc-shaped, approximately 12 mm in diameter, with a total thickness of about 2 to 3 mm. The surface is covered with a translucent stratum corneum, and uniformly distributed fine protrusions, i.e., the openings at the upper ends of hair follicles, are visible to the naked eye. Figure 5 As shown, HE staining revealed a clear layering of the epidermis, consisting of the basal layer, spinous layer (3 to 5 cell layers), granular layer (1 to 2 cell layers), and stratum corneum from the inside out, with a total epidermal thickness of approximately 80 μm. The dermis exhibited two distinct density gradient regions: a denser collagen layer in the lower layer and a looser collagen layer in the upper layer. Follicle-like structures extended from the epidermis into the dermis, with most follicles ranging from 400 to 800 μm in length, possessing identifiable outer and inner root sheaths. Dermal papillae were visible embedded at the base of some follicles. There were 47 follicle-like structures per square centimeter. Figure 6 As shown, immunofluorescence staining results revealed that the outer root sheath of the hair follicle expressed keratin 17 and keratin 75, the hair follicle stem cell region expressed Sox9 and CD34, and the dermal papilla region expressed alkaline phosphatase and versican. Keratin 31-positive hair shaft-like keratinized structures were observed within approximately 15% of the hair follicles.

[0046] The objective of Example 2 was to investigate the effect of the mixing ratio of hair follicle stem cells to dermal papilla cells on hair follicle formation efficiency. Using the same cell source and culture conditions as in Example 1, four experimental groups were set up with mixing ratios of 1:1, 1.5:1, 2:1, and 3:1 for hair follicle stem cells to dermal papilla cells. Forty-eight hair follicle-induced aggregates were prepared from each group, and after 72 h of culture, the morphology of the aggregates and the expression of hair follicle markers were evaluated. The results showed that the 1:1 group had an aggregate formation rate of 82.0%, with aggregate diameters ranging from 140 to 170 μm. The core mesenchymal aggregates were large, but the epithelial envelope was thin. The 1.5:1 group had an aggregate formation rate of 87.5%, with diameters ranging from 155 to 195 μm. The epithelial-mesenchymal ratio was balanced, and the structure was the most ideal. The 2:1 group had an aggregate formation rate of 85.4%, with diameters ranging from 160 to 210 μm. The epithelial layer thickness increased, but the core aggregates decreased in size. The 3:1 group had an aggregate formation rate that decreased to 72.9%, with some aggregates exhibiting irregular morphologies, suggesting that insufficient dermal papillary cells affected mesenchymal aggregation and epithelial-mesenchymal interaction. After embedding the four aggregate groups into the biphasic gradient dermis and completing the entire culture process, the number of mature hair follicles per square centimeter was counted. Figure 7As shown, the 1:1 ratio had 38 cells, the 1.5:1 ratio had 52 cells, the 2:1 ratio had 44 cells, and the 3:1 ratio had 29 cells. The 1.5:1 ratio achieved the highest follicle formation density and optimal follicle maturity, confirming that the epithelial-mesenchymal interaction between hair follicle stem cells and dermal papilla cells was most complete at this ratio. From a histological perspective, the 1.5:1 ratio resulted in a slightly higher number of epithelial cells than mesenchymal cells in each aggregate, allowing the epithelial cells to completely envelop the mesenchymal core to form a complete epithelial shell. Simultaneously, the number of dermal papilla cells in the mesenchymal core was sufficient to maintain high levels of alkaline phosphatase activity and inducible signal secretion. When the ratio was excessively biased towards the epithelial side (e.g., in the 3:1 group), the mesenchymal signal intensity was insufficient to support the full initiation of hair follicle fate.

[0047] The objective of Example 3 was to investigate the effect of the composition of the biphasic gradient hydrogel on the establishment of hair follicle polarity. Three control groups were set up: a homogeneous collagen group (using 3 mg / mL type I collagen throughout the entire layer, without the addition of hyaluronic acid), a homogeneous high-concentration collagen group (using 5 mg / mL type I collagen throughout the entire layer, without the addition of hyaluronic acid), and the biphasic gradient group of this invention (5 mg / mL collagen in the lower layer, 2 mg / mL collagen plus 1 mg / mL hyaluronic acid in the upper layer). Each group used the same batch of hair follicle-inducing aggregates, with a density of 80 aggregates per square centimeter. After completing the entire culture process in step four, hair follicle polarity was evaluated, with a correct polarity defined as an angle of less than 30° between the long axis of the hair follicle and the normal direction of the dermis. The results showed that only 31% of hair follicles in the homogeneous collagen group exhibited the correct polarity, with most follicles growing in random directions. In the homogeneous high-concentration collagen group, the correct polarity rate was 42%, but overall follicle development was limited, with diameters and lengths smaller than the other two groups. The correct polarity rate in the biphasic gradient group reached 78%, significantly better than the two control groups. This result indicates that the dense collagen in the lower layer of the biphasic gradient hydrogel provides mechanical guidance for the downward extension of hair follicles, while the loose collagen-hyaluronic acid in the upper layer provides sufficient space and nutritional support for the initial growth of hair follicle buds. Both work synergistically to promote the establishment of correct follicle polarity.

[0048] The objective of Example 4 was to investigate the necessity of a phased signaling pathway activation strategy at the gas-liquid interface. Three experimental groups were set up: a control group (28 days of gas-liquid interface culture without any exogenous factors), a single-stage group (28 days of gas-liquid interface culture with SAG 1 μmol / L added throughout), and the phased group of this invention (1 μmol / L SAG added for 10 days in the first stage, then removed; PDGF-BB 20 ng / mL added for 18 days in the second stage). Six skin organoid samples were prepared from each group. After culture, hair follicle maturity was comprehensively evaluated, with indicators including hair follicle length, hair follicle structural integrity score (0 to 4 points, corresponding to no structure, only epithelial invagination, presence of outer root sheath, presence of inner root sheath and dermal sheath, and presence of hair shaft-like structure, respectively), and dermal papilla alkaline phosphatase activity. The control group had an average hair follicle length of 210 μm, a structural score of 1.2, and alkaline phosphatase activity (expressed as the hydrolysis rate of 4-nitrophenyl phosphate) of 0.15 U / mg protein. The single-stage group had an average hair follicle length of 520 μm, a structural score of 2.4, and an alkaline phosphatase activity of 0.28 U / mg protein. While hair follicle elongation was significant, persistent Shh signaling in the later stages led to excessive epithelial proliferation, resulting in cystic dilatation in some follicles. The phased-stage group had an average hair follicle length of 680 μm, a structural score of 3.1, and an alkaline phosphatase activity of 0.52 U / mg protein. The phased-stage group significantly outperformed the other two groups in all three dimensions: hair follicle length, structural integrity, and dermal papilla function. Figure 8 As shown, this confirms that a phased signaling pathway activation strategy can provide the correct developmental signals at the appropriate time window, avoiding the side effects caused by continuous activation of a single pathway.

[0049] The objective of Example 5 was to validate the application of the prepared skin organoid containing hair follicle structures in drug screening. Three active ingredients commonly used clinically for treating hair loss were selected for drug screening: minoxidil at concentrations of 0, 1, 5, 10, and 50 μmol / L; finasteride at concentrations of 0, 0.1, 1, 5, and 10 μmol / L; and the prostaglandin F2α analog latanoprost at concentrations of 0, 0.01, 0.1, 1, and 10 μmol / L. Three replicates were prepared for each concentration. Drug treatment began in the second stage of gas-liquid interface culture and continued for 14 days with culture medium additions. After treatment, the proliferation index of the hair follicle matrix region was calculated using Ki67 immunofluorescence staining, and the hair shaft formation ratio was assessed using keratin 31 immunofluorescence staining. The results showed that minoxidil at a concentration of 10 μmol / L increased the follicular matrix proliferation index from 18% to 34% and the hair shaft formation rate from 15% to 28%; finasteride at a concentration of 5 μmol / L increased the proliferation index to 29% and the hair shaft formation rate to 24%; and latanoprost at a concentration of 1 μmol / L increased the proliferation index to 31% and the hair shaft formation rate to 26%. All three drugs exhibited dose-dependent follicular growth-promoting effects. Figure 9 As shown, the half-maximal effective concentration (MCC) results were largely consistent with the reported in vitro hair follicle organoid culture results, validating the feasibility and sensitivity of this skin organoid as a screening model for hair loss drugs. Further analysis of the hair follicle growth cycle was conducted. On day 35 of culture, immunofluorescence staining of whole skin specimens from the skin organoids revealed that approximately 62% of the follicle-like structures expressed Ki67 and proliferating cell nuclear antigen, indicating an anagen-like state; approximately 23% of the follicles showed TUNEL-positive signals indicating apoptosis of outer root sheath cells, suggesting a catagen-like state; and approximately 15% of the follicles exhibited a quiescent morphology with upward displacement of the dermal papilla, suggesting a telogen-like state. This heterogeneous distribution of the growth cycle preliminarily reflects the biological characteristics of the periodic transition of hair follicles. In the minoxidil 10 μmol / L treatment group, the proportion of anagen-like follicles increased to 76%, while the proportion of catagen-like follicles decreased to 14%, further confirming that this model can reflect the effect of drugs on the regulation of the hair follicle cycle. The expression levels of hair follicle-related genes in mature skin organoids were detected by real-time quantitative PCR. The results showed that the expression level of LEF1 (a Wnt pathway effector) was 0.7 times that of human scalp tissue, SHH was 0.5 times, FOXN1 (a hair follicle-specific transcription factor) was 0.6 times, and DKK1 (a Wnt pathway inhibitor) was 0.8 times. These results suggest that the hair follicle-related signaling pathways in the organoids have a certain level of activity, which can basically support functional studies on hair follicle development and drug response.

[0050] Comparative Example 1 replaced the three-factor induction system in step two with a single-factor system containing only CHIR99021 (3 μmol / L), while the remaining steps were the same as in Example 1. The results showed that the aggregate formation rate decreased to 62%, and most aggregates exhibited a loose structure lacking a dense mesenchymal core. After completing the entire process, only 12 follicle-like structures were formed per square centimeter, with a structure score of only 1.5. This indicates that activation of the Wnt pathway alone is insufficient to drive complete follicle fate pre-programming, and that BMP signaling inhibition and FGF signaling synergy are indispensable for maintaining the induction activity of dermal papilla cells and promoting epithelial-mesenchymal interaction.

[0051] Comparative Example 2 replaced the biphasic gradient hydrogel in step 3 with a Matrigel embedding system, directly embedding the follicle-inducing aggregates in 100% Matrigel and then culturing them. Matrigel is rich in laminin and type IV collagen, and its composition is similar to the basement membrane rather than dermal connective tissue. The results showed that the follicle aggregates swelled into spherical shapes in Matrigel rather than elongating polarly, with a polarity accuracy of only 18%, and an average follicle length of only 150 μm. This indicates that the homogeneous and soft properties of Matrigel cannot provide the mechanical gradient required for establishing follicle polarity, and its basement membrane-like components cannot simulate the microenvironment of dermal connective tissue.

[0052] In Comparative Example 3, the hair follicle stem cells in step one were replaced with ordinary epidermal keratinocytes (non-protruding region source), with the remaining steps identical to Example 1. Although aggregates formed after co-culturing ordinary keratinocytes and dermal papilla cells, these aggregates lacked a clear epithelial-mesenchymal spatial distribution, and the area of ​​the versican-positive core region was 60% smaller than that of the hair follicle stem cell group. After completing the entire process, only 8 follicle-like structures were formed per square centimeter, and most were superficial epithelial invaginations rather than complete hair follicle structures. This indicates that hair follicle stem cells have a stronger ability to interact with dermal papilla cells and a higher hair follicle differentiation potential compared to ordinary keratinocytes. This is closely related to the high expression of the Wnt receptor Frizzled7 and Notch ligand Jagged1 on the surface of hair follicle stem cells. These molecules mediate specific ligand-receptor recognition between hair follicle stem cells and dermal papilla cells, which is a necessary condition for initiating the hair follicle development process.

[0053] Regarding the storage and transportation conditions of the finished skin organoids, the prepared skin organoids containing hair follicle structures can maintain their viability for at least 14 days under standard cell culture conditions of 37°C and 5% CO2 (v / v), with the gas-liquid interface medium changed every two days during this period. For short-term storage, the organoids can be stored in a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) at 4°C for no more than 24 hours. For long-term storage, it is recommended to use a programmed freezing method, cooling from room temperature to -80°C at a rate of 1°C per minute before transferring to liquid nitrogen for storage. After thawing, the organoids are rapidly thawed in a 37°C water bath and serially diluted to remove DMSO. After 48 to 72 hours of recovery culture under standard conditions, the expression of hair follicle markers and cell viability can be restored. The survival rate of the follicle-like structures after cryopreservation and thawing is approximately 70% to 80%, which basically meets the requirements for transportation and long-distance use.

[0054] The synergistic mechanism of the various technical features in this invention can be explained from the following four levels. At the first level, the epithelial-mesenchymal interaction level, in the co-culture system of hair follicle stem cells (CD34⁺ / CD200⁺) and dermal papillary cells, the Wnt ligands (Wnt3a / Wnt10b), BMP inhibitors (Noggin / Gremlin), and FGF signals secreted by dermal papillary cells synergistically act on hair follicle stem cells, activating their hair follicle fate determination program; conversely, the SHH and EDAR ligands secreted by hair follicle stem cells act on dermal papillary cells to maintain their aggregated state and induce activity. The exogenously added CHIR99021, Noggin, and FGF2 three-factor system precisely simulates and enhances this natural bidirectional signal exchange, enabling the rapid formation of hair follicle primordium-like structures within 72 hours in vitro. At the second level, the biomimetic microenvironment level, the biphasic gradient hydrogel, through its dual gradient design of mechanical hardness gradient (hard lower layer, soft upper layer) and matrix composition gradient (pure collagen lower layer, collagen + hyaluronic acid upper layer), provides mechanical guidance for hair follicle aggregates to "anchor downwards and extend upwards," enabling hair follicles to establish a natural polar orientation from the epidermis to the dermis. At the third level, the temporal signal regulation level, the gas-liquid interface phased strategy decouples the hair follicle maturation process into two independent and controllable stages: Shh-mediated early polarity establishment (hair bud elongation and epithelial tubularization) and PDGF-BB-mediated late dermal papilla functional maturation (alkaline phosphatase upregulation and enhanced signal molecule secretion). This avoids cross-interference between signaling pathways and achieves more precise developmental temporal control. At the fourth level, the overall system integration level, the air-liquid interface culture of the epidermis not only promotes the layered keratinization of the epidermis itself, but also provides necessary localization signals from the epidermis (such as Wnt and Notch ligands) for the epidermal-follicle junction area at the upper end of the hair follicle, achieving coordinated coupling between epidermal homeostasis maintenance and hair follicle development. The technical features of these four levels are interconnected and progressive, jointly supporting the successful construction of full-thickness skin organoids containing functional hair follicle structures.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A skin organoid containing a hair follicle structure, characterized in that, The skin organoid comprises a dermis, an epidermis, and follicle-like structures embedded within the dermis. The dermis is composed of a biphasic gradient hydrogel formed by dermal fibroblasts, type I collagen, and hyaluronic acid. The follicle-like structures develop from follicle-inducible aggregates formed by co-culturing hair follicle stem cells and dermal papilla cells in vitro. These follicle-inducible aggregates are synergistically induced by GSK-3β inhibitors, bone morphogenetic protein inhibitors, and fibroblast growth factor 2 to form follicle primordium-like structures. These follicle-like structures express hair follicle-specific markers keratin 17, keratin 75, and transcription factor Sox9. The dermal papilla region expresses alkaline phosphatase and the proteoglycan versican. Each square centimeter of the skin organoid contains 30 to 80 follicle-like structures.

2. The skin organoid containing a hair follicle structure according to claim 1, characterized in that, The hair follicle stem cells were characterized by CD34 positivity and CD200 positivity, while the dermal papillary cells were characterized by alkaline phosphatase positivity and α-smooth muscle actin positivity.

3. The skin organoid containing a hair follicle structure according to claim 1, characterized in that, The biphase gradient hydrogel comprises a lower dense collagen region and an upper loose collagen region. The lower dense collagen region has a type I collagen concentration of 4 to 6 mg / mL, and the upper loose collagen region has a type I collagen concentration of 1.5 to 3 mg / mL and a hyaluronic acid concentration of 0.5 to 2 mg / mL.

4. The skin organoid containing a hair follicle structure according to claim 1, characterized in that, The hair follicle-like structure has a complete outer root sheath, inner root sheath, and dermal sheath structure, and some hair shaft-like keratinized structures can be seen in the hair follicle-like structure.

5. The skin organoid containing a hair follicle structure according to claim 1, characterized in that, The epidermis comprises a basal layer, a spinous layer, a granular layer, and a stratum corneum, and has a thickness of 50 to 120 μm.

6. A method for preparing a skin organoid containing a hair follicle structure as described in any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1: Separate and culture hair follicle stem cells and dermal papilla cells. Complete hair follicles are separated by microscopic dissection from human scalp tissue. The outer root sheath of the hair follicle is separated by dispersing enzyme digestion. CD34-positive and CD200-positive hair follicle stem cells are screened from the bulging region of the outer root sheath. Dermal papilla cells are obtained from the dermal papilla region at the bottom of the hair follicle by collagenase digestion and adherent culture. The two types of cells are expanded to the second and third generations, respectively. Step 2: Prepare hair follicle-induced aggregates. Mix hair follicle stem cells and dermal papillary cells at a cell ratio of 1:1 to 2:1 and co-culture them in three dimensions using an ultra-low adsorption culture plate or a hanging drop culture method. Add 2 to 5 μmol / L of GSK-3β inhibitor CHIR99021, 50 to 200 ng / mL of bone morphogenetic protein inhibitor Noggin, and 10 to 40 ng / mL of fibroblast growth factor 22 to the culture medium. Culture for 48 to 72 h to form hair follicle primordium-like aggregates with a diameter of 100 to 250 μm. Step 3: Construct a biphasic gradient dermal layer containing hair follicles. First, mix dermal fibroblasts with a high-concentration type I collagen solution and inject it into the bottom of the culture mold to form a dense collagen sublayer. After it semi-gels, evenly distribute hair follicle-induced aggregates at a density of 50 to 120 per square centimeter in a mixed solution of low-concentration collagen and hyaluronic acid in the upper layer and inject it above the dense collagen layer to complete the gelation, forming a biphasic gradient dermal layer embedded with hair follicle aggregates. Immerse and culture for 5 to 10 days. Step four involves constructing the epidermis and inducing hair follicle maturation in stages. Keratinocytes are seeded onto the dermal surface and immersed for 3 to 5 days to form a continuous epidermal monolayer. The monolayer is then transferred to an air-liquid interface culture system. In the first stage of the air-liquid interface culture, SAG (0.5 to 2 μmol / L) is added to the culture medium for 7 to 14 days to promote the establishment of hair follicle polarity and hair bud elongation. In the second stage of the air-liquid interface culture, SAG is removed and platelet-derived growth factor (BB) (10 to 50 ng / mL) is added for 14 to 21 days to promote dermal papilla maturation and hair follicle sheath structure improvement. The total air-liquid interface culture time is 21 to 35 days.

7. The preparation method according to claim 6, characterized in that, The working concentration of the dispersing enzyme in step one is 2 to 5 U / mL, the digestion temperature is 37°C, and the digestion time is 1 to 2 h. The collagenase is type II collagenase with a working concentration of 0.2 to 0.5% and a digestion time of 2 to 4 h.

8. The preparation method according to claim 6, characterized in that, In step three, the concentration of type I collagen in the dense collagen sublayer is 4 to 6 mg / mL, and the thickness is 0.5 to 1 mm. The concentration of type I collagen in the upper loose collagen region is 1.5 to 3 mg / mL, the concentration of hyaluronic acid is 0.5 to 2 mg / mL, and the thickness is 1 to 2 mm. The density of dermal fibroblasts in the dense collagen sublayer is 1 × 10⁻⁶ cells / mL. 5 Up to 5×10 5 indivual.

9. The preparation method according to claim 6, characterized in that, The seeding density of keratinocytes in step four is 2 × 10⁻⁶ per square centimeter. 5 Up to 5×10 5 The cells were immersed in DMEM / F12 medium containing 10% fetal bovine serum and the air-liquid interface medium was serum-free keratinocyte medium containing 1.2 mmol / L calcium chloride.

10. The preparation method according to claim 6, characterized in that, The culture medium for the hair follicle-inducing aggregates described in step two is based on serum-free DMEM / F12, supplemented with 2% B27 additive, 2 mmol / L L-glutamine, and 100 U / mL penicillin-streptomycin.

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