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CN-122012379-A - Skin organoid containing hair follicle structure and preparation method thereof

CN122012379ACN 122012379 ACN122012379 ACN 122012379ACN-122012379-A

Abstract

The invention discloses a skin organoid containing hair follicle structure and a preparation method thereof, belonging to the field of tissue engineering, wherein the method comprises the steps of separating hair follicle stem cells from dermal papilla cells; the hair follicle inducing aggregate is formed through synergistic induction of Wnt activator, BMP inhibitor and FGF, the aggregate is embedded into biphasic gradient collagen-hyaluronic acid hydrogel to constitute dermis, and after keratinocyte inoculation, the dermis is activated through the staged signal path of air-liquid interface to promote the maturation of hair follicle, and the obtained organoid contains complete epidermis layer, dermis layer and hair follicle-like structure and may be used in hair research and medicine screening.

Inventors

  • NI HONGWEI
  • XU YAN
  • DONG YUE
  • HAN YUE
  • LIU SHIGUANG
  • Qi Pengkun
  • ZHANG ZHIQIANG

Assignees

  • 沈阳泽尔检测服务有限公司

Dates

Publication Date
20260512
Application Date
20260306

Claims (10)

  1. 1. Skin organoid comprising a hair follicle structure, characterized in that the skin organoid comprises a dermis layer, a epidermis layer and a hair follicle-like structure embedded in the dermis layer, the dermis layer is composed of a biphasic gradient hydrogel formed by dermal fibroblasts, type I collagen and hyaluronic acid, the hair follicle-like structure is formed by hair follicle-induced aggregates formed by in vitro co-culture of hair follicle stem cells and dermal papilla cells, the hair follicle-induced aggregates are synergistically induced by GSK-3 beta inhibitor, bone morphogenic protein inhibitor and fibroblast growth factor 2 to form a hair follicle primordium-like structure, the hair follicle-like structure expresses hair follicle-specific markers keratin 17, keratin 75 and transcription factor Sox9, the dermis papilla region expresses alkaline phosphatase and proteoglycan versican, and the skin organoid contains 30 to 80 hair follicle-like structures per square centimeter.
  2. 2. The skin organoid comprising a hair follicle structure according to claim 1, wherein the hair follicle stem cells are phenotypically labeled with CD34 positive and CD200 positive and the dermal papilla cells are phenotypically labeled with alkaline phosphatase positive and alpha-smooth muscle actin positive.
  3. 3. The skin organoid comprising a hair follicle structure according to claim 1, wherein the biphasic gradient hydrogel comprises a lower dense collagen region having a type I collagen concentration of 4 to 6 mg/mL and an upper porous collagen region having 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. 4. The skin organoid comprising hair follicle structures of claim 1, wherein said hair follicle-like structure has intact hair follicle outer root sheath, inner root sheath and dermis sheath structures, hair shaft-like keratinization structures being visible within portions of the hair follicle-like structure.
  5. 5. The skin organoid comprising a hair follicle structure according to claim 1, wherein the epidermal layer comprises a basal layer, a stratum spinosum, a stratum granulosum and a stratum corneum, and the epidermal layer has a thickness of 50 to 120 μm.
  6. 6. A method of preparing a skin organoid comprising a hair follicle structure according to any of claims 1 to 5, comprising the steps of: Firstly, separating and culturing hair follicle stem cells and dermal papilla cells, micro-dissecting and separating complete hair follicle from human source skin tissues, adopting dispersive enzyme digestion to separate hair follicle outer root sheaths, screening CD34 positive and CD200 positive hair follicle stem cells from outer root sheath carina areas, obtaining the dermal papilla cells from the bottom dermal papilla areas of the hair follicle through collagenase digestion and adherent culture, and respectively amplifying the two types of cells to the second generation to the third generation; Preparing hair follicle induction aggregate, mixing hair follicle stem cells and dermal papilla cells according to a cell number ratio of 1:1 to 2:1, performing three-dimensional co-culture by adopting an ultralow adsorption culture plate or a hanging drop culture method, adding a GSK-3 beta inhibitor CHIR99021 with a concentration of 2 to 5 mu mol/L, a bone morphogenetic protein inhibitor Noggin with a concentration of 50 to 200 ng/mL and a fibroblast growth factor 22 with a concentration of 10 to 40 ng/mL into a culture medium, and culturing 48 to 72h to form a hair follicle primordial aggregate with a diameter of 100 to 250 mu m; Step three, constructing a biphasic gradient dermis layer containing hair follicles, firstly mixing dermal fibroblasts with a high-concentration type I collagen solution, injecting the mixture into the bottom of a culture mold to form a dense collagen lower layer, uniformly distributing hair follicle-induced aggregates in the mixed solution of upper-layer low-concentration collagen and hyaluronic acid according to the density of 50-120 per square centimeter after half gelation of the mixture, injecting the mixed solution into the upper-layer low-concentration collagen and hyaluronic acid into the upper part of the dense collagen layer to complete gelation, forming the biphasic gradient dermis layer embedded with hair follicle aggregates, and carrying out submerged culture for 5-10 days; Step four, constructing an epidermal layer and inducing hair follicle maturation in stages, inoculating keratinocytes on the surface of the dermis layer, immersing and culturing for 3 to 5 days to form a continuous epidermis monolayer, transferring to a gas-liquid interface culture system, adding an Sonic hedgehog signal pathway activator SAG with the concentration of 0.5 to 2 mu mol/L into the culture medium in the first stage of gas-liquid interface culture for 7 to 14 days to promote hair follicle polarity establishment and Mao Ya elongation, removing SAG in the second stage of gas-liquid interface culture, adding a platelet-derived growth factor BB with the concentration of 10 to 50 ng/mL for 14 to 21 days to promote dermis nipple maturation and hair follicle sheath structure perfection, and carrying out total gas-liquid interface culture for 21 to 35 days.
  7. 7. The method according to claim 6, wherein the working concentration of the dispersing enzyme in the first step is 2 to 5U/mL, the digestion temperature is 37 ℃, the digestion time is 1 to 2h, the collagenase is type II collagenase, the working concentration is 0.2 to 0.5%, and the digestion time is 2 to 4h.
  8. 8. The method according to claim 6, wherein the density of the type I collagen in the dense collagen lower layer in the third step is 4 to 6 mg/mL, the thickness is 0.5 to 1 mm, the density of the 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, the thickness is 1 to 2 mm, and the density of the dermal fibroblasts in the dense collagen lower layer is 1 x 10 5 to 5 x 10 5 per mL.
  9. 9. The method according to claim 6, wherein the keratinocytes in the fourth step are inoculated at a density of 2X 10 5 to 5X 10 5 cells per square centimeter, the submerged medium is DMEM/F12 medium containing 10% fetal bovine serum by volume fraction, and the gas-liquid interface medium is serum-free keratinocyte medium containing 1.2 mmol/L calcium chloride.
  10. 10. The method according to claim 6, wherein the culture medium of the hair follicle inducing aggregate in the second step is based on serum-free DMEM/F12 supplemented with 2% by volume of B27 supplement, L-glutamine at a concentration of 2 mmol/L and penicillin-streptomycin at a concentration of 100U/mL.

Description

Skin organoid containing hair follicle structure and preparation method thereof Technical Field The invention belongs to the technical field of tissue engineering and regenerative medicine, and particularly relates to a skin organoid containing a hair follicle structure and a preparation method thereof. Background The skin is the largest organ of the human body and consists of three layers of epidermis, dermis and subcutaneous tissue, wherein various skin appendages such as hair follicles, sebaceous glands, sweat glands and the like are distributed. Hair follicles are one of the most important appendages of skin, have highly complex structures and functions, and are composed of a plurality of functional areas such as an outer root sheath, an inner root sheath, hair shafts, a dermis sheath, a dermis nipple and the like, and participate in key physiological processes such as hair growth cycle regulation, skin wound repair, immune defense and the like. Hair follicle-related diseases such as androgenetic alopecia, alopecia areata, and cicatricial alopecia seriously affect the quality of life of patients, and currently, there is a clinical lack of effective treatment means, and it is urgently required to build an in vitro model capable of truly simulating hair follicle development and function for disease mechanism research and drug screening. The existing skin in-vitro model mainly comprises three types of two-dimensional cell culture, three-dimensional full-layer skin equivalent and skin organoids. Two-dimensional cell culture, although simple in operation, cannot mimic the three-dimensional structure of the skin and the interactions between cells. Commercial full-thickness skin equivalents such as EpiDerm and MatTek products contain basic structures of epidermis layers and dermis layers, but generally lack skin appendages such as hair follicles, sebaceous glands and the like, can only be used for evaluating skin barrier functions and drug permeability, and cannot meet the requirements of hair biological research and screening of hair loss treatment drugs. In recent years, skin organoid technology based on human pluripotent stem cells has made breakthrough progress. Lee et al (Nature, 2020,582:399-404) report a method of inducing the formation of hair follicle-containing skin organoids from human pluripotent stem cells, which are capable of producing hair follicles after about 60 days of culture, reaching full maturation around 130 days, including stratified epidermis, pigmented hair follicles, sebaceous glands, mercer cells and sensory neurons. The method has the obvious limitations that firstly, the culture period is extremely long, 4 to 5 months are required from the induction of stem cells to the generation of mature hair follicles, the aging requirement of high-flux drug screening is difficult to meet, secondly, the organoids are in vesicle-like spherical structures, the hair follicles grow inwards, the polarity direction of the hair follicles extending from epidermis to dermis in natural skin is opposite, the spatial positioning of the hair follicles in the skin cannot be truly simulated, thirdly, the number and distribution of the hair follicles are uncontrollable, the batch-to-batch difference is large, and the standardization degree is low. Strategies for constructing tissue engineering skin using primary cells of adult origin have also been reported. Chinese patent CN109172868a discloses a method for quickly reconstructing skin and hair follicle, in which adult mouse whole skin is transplanted into nude mouse to reconstruct hair follicle after being prepared into tissue homogenate, but the method relies on living transplantation, and skin construct containing hair follicle cannot be formed in vitro. International patent WO2024035684A1 reports a method for preparing a follicular organoid using coculture of iPSC-derived dermal papilla cells with epithelial stem cells, but the method results in an independent follicular unit, rather than a complete organoid comprising a full-thickness skin structure, lacking systemic integration of the epidermal barrier and dermal microenvironment. In summary, the prior art has the technical bottlenecks in preparing the full-layer skin organoids containing the functional hair follicle structure that the culture period is too long to restrict the practicability, the polarity and the spatial localization of the hair follicle cannot be accurately regulated, the hair follicle density and the distribution lack standardized means, and the short-period whole external culture system starting from the adult primary cells is lacking. Disclosure of Invention The invention aims to overcome the defects of the prior art, and provides a skin organoid containing a hair follicle structure and a preparation method thereof, wherein the skin organoid takes hair follicle stem cells and dermal papilla cells which are derived from an adult as core seed cells, by the strategies of hair follicl