WO2023113324A1 - Artificial skin mimicking papillae layer structure and manufacturing method therefor - Google Patents

Artificial skin mimicking papillae layer structure and manufacturing method therefor Download PDF

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WO2023113324A1
WO2023113324A1 PCT/KR2022/019488 KR2022019488W WO2023113324A1 WO 2023113324 A1 WO2023113324 A1 WO 2023113324A1 KR 2022019488 W KR2022019488 W KR 2022019488W WO 2023113324 A1 WO2023113324 A1 WO 2023113324A1
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layer
artificial skin
dermal
cells
epidermal
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PCT/KR2022/019488
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French (fr)
Korean (ko)
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한동욱
김기수
강문성
권미나
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부산대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
    • A61L27/3891—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types as distinct cell layers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/18—Materials or treatment for tissue regeneration for hair reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the present invention relates to artificial skin simulating a papillary layer structure and a manufacturing method thereof.
  • Hair follicle dermal papilla cells play a role in continuously supplying nutrients and growth factors to keratinocytes that form hair. Since male pattern baldness occurs when the number of hair follicle dermal papilla cells decreases and the hair forming ability decreases, a hair loss treatment method in which artificially cultured hair follicle dermal papilla cells are transplanted to the scalp is being studied. However, hair follicle dermal papilla cells cultured in vitro rapidly lose their hair-forming ability and proliferative ability, so a culture environment capable of maintaining their hair-forming ability is required. A technique for culturing hair follicle dermal papilla spheroids in an artificial skin layer including a keratinocyte layer and a dermal cell layer is known, but this is also known to easily lose hair forming ability.
  • methacrylic gelatin and methacrylated hyaluronic acid are used as main components, and the boundary between the epidermis-like layer and the dermis-like layer of artificial skin is produced in a form that mimics the curved shape of the papilla layer, thereby increasing the number of hair follicle dermal papilla cells.
  • an artificial skin in which growth and hair formation properties are maintained.
  • One aspect is a dermal mimic layer comprising a hydrogel and dermal cells in which methacrylated gelatin and methacrylated hyaluronic acid are crosslinked; In contact with one surface of the dermis-like layer, the epidermis-like layer containing epidermal cells; And artificial skin comprising a hair follicle spheroid, wherein one surface of the dermal-like layer to which the epidermis-like layer comes into contact has a curved shape, providing artificial skin.
  • the artificial skin includes methacrylic gelatin instead of collagen, and one side of the dermal mimic layer in contact with the epidermal mimic layer is made in a curved shape to mimic the shape of the papillary layer, thereby providing epidermal cells, dermal cells, and It was confirmed that cytocompatibility for hair follicle cells was significantly improved.
  • the methacrylic gelatin may have a methacrylation degree of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • the methacrylated gelatin may be type A or type B gelatin, and according to one embodiment, type A gelatin.
  • the methacrylated gelatin may be derived from bone, skin, tendon, or cartilage, for example, pig skin.
  • the molecular weight of the methacrylated gelatin may be a molecular weight suitable for 3D printing, and may be, for example, 12000 to 25000 Da, 14000 to 25000 Da, 16000 to 25000 Da, 18000 to 25000 Da, or 20000 to 25000 Da, but particularly It is not limited.
  • the methacrylic acid methacrylate may have a methacrylation substitution degree of 100 to 140 mol%, 110 to 130 mol%, or about 120 mol%.
  • the methacrylic acid methacrylate may have a molecular weight of 150 to 250 kDa, 160 to 240 kDa, 170 to 230 kDa, 180 to 220 kDa, 190 to 210 kDa, or about 200 kDa.
  • the curved shape of one surface of the dermal mimic layer contacting the epidermal mimic layer may be expressed as surface roughness.
  • Surface roughness can be measured by several measurement methods, specifically, using atomic force microscopy (AFM) for artificial skin specimens, non-contact mode using silicon cantilever, scan size 1 mm ⁇ 1 mm, It can be measured under the condition of height profile through 512 ⁇ 512 analysis points and 1.0 Hz/line scan rate.
  • the surface roughness may be expressed as center line average roughness (Ra), maximum height roughness (Rmax), and 10-point average roughness (Rz).
  • the center line average roughness (Ra) is the distance between the center line and the average line in the roughness cross-section curve
  • the maximum height roughness (Rmax) is the highest peak to the deepest valley ( valley)
  • ten point median height (Rz) is the difference between the average height of the five highest peaks and the average depth of the five deepest valleys in the cross-sectional curve.
  • the center line average roughness (Ra) of the dermal mimic layer in contact with the epidermal mimic layer is 10 to 60 ⁇ m, 15 to 60 ⁇ m, 20 to 60 ⁇ m, 25 to 60 ⁇ m, 30 to 60 ⁇ m, 35 to 60 ⁇ m, 40 to 60 ⁇ m, 45 to 60 ⁇ m, 10 to 55 ⁇ m, 15 to 55 ⁇ m, 20 to 55 ⁇ m, 25 to 55 ⁇ m, 30 to 55 ⁇ m, 35 to 55 ⁇ m, 40 to 55 ⁇ m, 45 to 55 ⁇ m, 10 to 50 ⁇ m, 15 to 50 ⁇ m, 20 to 50 ⁇ m, 25 to 50 ⁇ m, 30 to 50 ⁇ m, 35 to 50 ⁇ m, 40 to 50 ⁇ m, or 45 to 60 ⁇ m.
  • the curved shape may be a shape in which a plurality of ridges are repeated.
  • the height of each protrusion may be 50 to 100 ⁇ m or 80 to 90 ⁇ m.
  • the width of each protrusion may be 50 to 150 ⁇ m, 60 to 140 ⁇ m, 70 to 130 ⁇ m, 80 to 120 ⁇ m, 90 to 110 ⁇ m, or about 100 ⁇ m.
  • the distance between each of the protrusions may be 100 to 200 ⁇ m.
  • the inclination angle of the protrusion may be 50 to 70 °, 55 to 65 °, 57 to 63 °, 59 to 61 ° or 60 °.
  • the height, width, and spacing of each of the protrusions are measured using an atomic force microscope (AFM) for artificial skin specimens, in a non-contact mode using a silicon cantilever, scan size 1 mm ⁇ 1 mm, a height profile through 512 ⁇ 512 analysis points and a scan rate of 1.0 Hz/line.
  • AFM atomic force microscope
  • the hair follicle spheroid may be located inside the dermis-like layer or at the interface between the epidermis-like layer and the dermal-like layer.
  • the mass ratio of the methacrylated gelatin and the methacrylated hyaluronic acid is 320:1 to 500:1, 330:1 to 480:1, 340:1 to 460:1, 350:1 to 440: 1, 360:1 to 420:1, 380:1 to 410:1, or about 400:1.
  • the epidermal cells may be HaCaT, NHK (Normal Human Keratinocyte), or NHEK (Normal Human Epidermal Keratinocyte).
  • the hair follicle spheroid may be a spheroid obtained by three-dimensionally culturing dermal papilla cells (DPC), and the dermal papilla cells may be, for example, DPC or hair follicle DPC (HFDPC) cells.
  • DPC dermal papilla cells
  • HFDPC hair follicle DPC
  • a spheroid refers to a cell aggregate having a circular or elliptical cross section.
  • the diameter of the spheroid is 30 to 300 ⁇ m, 30 to 250 ⁇ m, 30 to 200 ⁇ m, 30 to 150 ⁇ m, 30 to 100 ⁇ m, 40 to 300 ⁇ m, 40 to 250 ⁇ m, 40 to 200 ⁇ m, 40 to 150 ⁇ m , 40 to 100 ⁇ m, 50 to 300 ⁇ m, 50 to 250 ⁇ m, 50 to 200 ⁇ m, 50 to 150 ⁇ m, or 50 to 100 ⁇ m.
  • the three-dimensional culture can be cultured by a method known to those skilled in the art, for example, hanging drop culture, spinner flask culture, concave microwell plate culture, or commercially available It can be cultured with a 3D culture kit.
  • the dermal cells may be human dermal fibroblasts (Normal Human Dermal Fibroblast, NHDF) cells.
  • Another aspect is crosslinking a pregel composition in which methacrylic gelatin, methacrylic hyaluronic acid, dermal cells, and hair follicle spheroids are mixed on a mold having a curved surface; Obtaining a dermis-like layer having a curved surface by removing the mold; And seeding and culturing epidermal cells on the curved surface of the dermis-like layer to form an epidermal-like layer, providing an artificial skin manufacturing method.
  • the mass ratio of methacrylated gelatin and methacrylated hyaluronic acid in the pregel composition is 320:1 to 500:1, 330:1 to 480:1, 340:1 to 460:1, 350:1 to 440: 1, 360:1 to 420:1, 380:1 to 410:1, or about 400:1.
  • the content of methacrylic gelatin and methacrylic hyaluronic acid in the pregel composition is 1 to 10 (w/w)%, 2 to 8 (w/w)%, 3 to 6 (w/w)%, 4 to 6 (w/w)% 6 (w/w)%, or 5 (w/w)%.
  • the solvent of the pregel composition may be phosphate buffered saline (PBS) or Dulbecco's phosphate-buffered saline (DPBS).
  • PBS phosphate buffered saline
  • DPBS Dulbecco's phosphate-buffered saline
  • the pregel composition may further include a photoinitiator, for example, PI113 (Wako Pure Chemical), PAG-281 (Wako Pure Chemical), PAG-336 (Wako Pure Chemical), PAG-337 (Wako Pure Chemical), At least one selected from the group consisting of Darocur 1173 (BASF), Irgacure 2959 (BASF), Irgacure 500 (BASF), Irgacure 754 (BASF), and combinations thereof may be used, but is not particularly limited.
  • a photoinitiator for example, PI113 (Wako Pure Chemical), PAG-281 (Wako Pure Chemical), PAG-336 (Wako Pure Chemical), PAG-337 (Wako Pure Chemical), At least one selected from the group consisting of Darocur 1173 (BASF), Irgacure 2959 (BASF), Irgacure 500 (BASF), Irgacure 754 (BASF), and combinations thereof may be used, but is not particularly limited.
  • BASF
  • Another aspect is crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, and dermal cells are mixed on a mold having microcolumns on a curved surface; And removing the mold to obtain a dermal mimic layer having a curved surface in which micropores are formed; Injecting hair follicle spheroids into the micropores; And seeding and culturing epidermal cells on the curved surface of the dermis-like layer to form an epidermal-like layer, providing an artificial skin manufacturing method.
  • micropores are formed by removing the microcolumns, and the width and depth of the micropores can be appropriately adjusted according to the thickness of the dermis-like layer, the size of the hair follicle spheroid, and the injection depth of the hair follicle spheroid.
  • crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, and dermal cells are mixed on a mold having a curved surface; And removing the mold to obtain a dermis-like layer having a curved surface; Forming an epidermal mimic layer by seeding and culturing epidermal cells on the curved surface of the dermal mimic layer; and injecting the hair follicle spheroid into the dermis-like layer or the interface between the dermal-like layer and the epidermis-like layer, providing an artificial skin manufacturing method.
  • the injection method is not particularly limited, and may be injected by injection, microneedle, or 3D dot printing method.
  • it can be used by mounting an Electromagnetic Droplet (EMD) printhead module on a 3D printer.
  • EMD Electromagnetic Droplet
  • Artificial skin has GelMA and HAMA as main components, and the boundary between the epidermis-like layer and the dermis-like layer is produced in a form that mimics the curved shape of the papillary layer, thereby proliferating and functioning epidermal cells, dermal cells, and hair follicle cells. expression can be improved.
  • Figure 2 shows a method of manufacturing artificial skin having a curved interface (papilla layer) between the epidermis layer and the dermis layer.
  • Figure 3 shows the shape of the mold used in the production of the dermal mimic layer in one embodiment.
  • FIG. 6 is a result of confirming cross-sections of artificial skin having a flat interface between an epidermal-like layer and a dermal-like layer and a curved artificial skin.
  • Figure 7a shows a mold having a microcolumn for producing a dermal mimic layer having a hair follicle spheroid injection hole
  • Figure 7b shows a method of injecting hair follicle spheroids into artificial skin by a dot printing method.
  • HFDPC spheroids were prepared by 3D culture of HFDPC cells (Human Follicle Dermal Papilla cells), which are skin papilla cells, by the following method.
  • HFDPC cells were added to DPCBM (human follicle dermal papillar cell basal medium low serum, Promocell) supplementMIX (ready-to-use supplement, 4% FCS, 0.4% bovine pituitary extract, 1 ng/ml bFGF, 5 ⁇ g/ml insulin, Promocell) and 1% Antibiotic-antimycotic solution (Abs) were mixed medium (hereinafter referred to as DPCBM medium) and subcultured for 3 to 5 passages.
  • DPCBM medium human follicle dermal papillar cell basal medium low serum, Promocell
  • MIX ready-to-use supplement
  • FCS 4% FCS
  • bovine pituitary extract 1 ng/ml bFGF
  • 5 ⁇ g/ml insulin Promocell
  • Abs Antibiotic-antimycotic
  • NHDF cells and HaCaT cells were prepared with 10% fetal bovine serum (FBS, Welgene) and 1% antibiotic antifungal (Abs) solution (containing 10,000 units penicillin per mL, 10 mg streptomycin and 25 ⁇ g amphotericin B, Sigma-Aldrich, USA). This was added to DMEM medium (Welgene, Korea) at 37° C., 5% CO 2 and cultured in a humid environment. Subculture was performed using a trypsin-EDTA solution (Welgene) at about 80% confluence and the culture medium was replaced every 48 hours. NHDF cells and HaCaT cells were used after subculture of 3 to 5 passages.
  • FBS fetal bovine serum
  • Abs antibiotic antifungal
  • Example 2 Establishment of a hydrogel composition with excellent cytocompatibility for skin cells
  • hydrogel bioink with a composition similar to the extracellular matrix of human skin. It is known that the composition of the extracellular matrix of human skin is that the mass ratio of collagen to hyaluronic acid is 400:1 (see Table 1 below).
  • HaCaT cells were cultured for 7 days using a hydrogel containing collagen (Type I atelocollagen of pig skin (Dalim Tissen, Korea) and hyaluronic acid at a mass ratio of 400: 1), but there were the following problems.
  • a hydrogel containing collagen at a low concentration ( ⁇ 1%) could induce cell spreading, but was easily degraded and was not suitable for long-term cell culture.
  • the hydrogel containing collagen at a high concentration (>1%) could culture cells for a long period of time, but it did not promote cell spreading and suppressed the normal expression of cell phenotype.
  • Methacryloyl functional group attached gelatin (GelMA, porcine skin type A, DOM ⁇ 90% (H-NMR), powder, 12,000 ⁇ 14,000 Da or more and 25,000 Da or less, 3D material) and methacryloyl functional group attached hyaluronic acid ( HAMA, DS (Degree of substitution) 120 ⁇ 10 mol%, powder, 200kDa, SNvia) was used, and their mixing ratio was 4 to 5% (w/v): 0.01 to 0.0125% (w/v). did
  • HaCaT cells cultured in Col/HAMA hydrogel for 7 days showed insignificant spreading, but HaCaT cells cultured in GelMA/HAMA showed active spreading.
  • composition of the hydrogel to be used for artificial skin was 4 to 5% (w/v) of GelMA and 0.01 to 0.0125% (w/v) of HAMA.
  • Example 3 Fabrication of artificial skin simulating the structure of the papillary layer
  • FIG. 2 a method for manufacturing artificial skin simulating a papillary layer in which an interface between an epidermal layer and a dermal layer is curved will be described.
  • a pre-gel solution was prepared by dissolving 2 mL of a DPBS solution containing 5 (w/w)% of GelMA/HAMA (mass ratio 400:1) at 37° C. for 4 hours.
  • NHDF cell pellet and HFDPC spheroid pellet were resuspended in a pre-gel solution at a density of 5x10 5 cells/ml. 40 to 50 mg/ml (about 1 w/v%) of the photoinitiator Irgacure 2959 (10-fold dilution) was added to the pre-gel solution. Before 3D printing, light was blocked and stored in a refrigerator at 4°C for 10 minutes to increase viscosity.
  • a mold having a curved surface having a plurality of ridges formed thereon was prepared. According to FIG. 3, the distance between ridges on the surface of the mold was about 100 to 200 ⁇ m, the width of the ridges was about 100 ⁇ m, and the height was about 80 to 90 ⁇ m. The inclination angle of the ridge was about 60 degrees.
  • Nozzle gauge Temp Bed/Dispenser Moving speed Flow rate Distance (between dispenser and bed) UV intensity 21G 5°C 10cm/s 1ml/h 0.1mm 365nm/4W
  • the ink containing the prepared NHDF cells and HFDPC spheroids was stacked in four layers on a mold while applying UV radiation (365 nm).
  • the printed constructs were transferred to a 6-well plate and subjected to an additional 10 minutes of UV irradiation.
  • the mold was removed after cross-linking was completed by stabilizing at room temperature for 5 minutes.
  • the co-culture medium for promoting hair follicle formation helps the growth and differentiation of skin cells and hair follicle dermal papilla cell spheroids, and its composition is Fetal Bovine Serum 10%, Fetal Calf Serum 4%, basic Fibroblast Growth Factor 1ng/ml, Insulin 5 It is DMEM (high glucose) medium supplemented with ug/ml, Bovine Pituitary Extract 0.4%, and 1% Antibiotic-Antimycotic Solution.
  • HaCaT cells were spot seeded three times at a concentration of 1 X 10 6 cells/200 ⁇ l on the curved surface of the gel.
  • the spot-seeded gel was transferred to a dedicated culture container (24mm Transwell® with 0.4 ⁇ m Pore Polycarbonate, Corning), and air-liquid culture was performed at 37° C. for 7 to 21 days.
  • a dedicated culture container 24mm Transwell® with 0.4 ⁇ m Pore Polycarbonate, Corning
  • air-liquid culture was performed at 37° C. for 7 to 21 days.
  • the upper epidermal layer (keratinocytes) of the artificial skin structure was not immersed in the medium, and the medium was replaced every 24 hours.
  • artificial skin having a flat interface between the epidermal layer and the dermal layer was fabricated in the same manner as in the manufacturing method, except that a cover glass having a flat surface was used instead of a mold having a rough surface.
  • Example 4 Characteristic analysis of papillary layer simulated artificial skin
  • the artificial skin of the present invention (in which the interface between the epidermal layer and the dermal layer is curved like the papillary layer) was cultured for 21 days and the shape was observed by immunofluorescence staining.
  • the artificial skin of the present invention (the interface between the epidermal layer and the dermis layer is curved like the papillary layer) and the artificial skin of the control group (the interface between the epidermal layer and the dermis layer is flat) were cultured for 21 days, and TRITC, DAPI, and FITC immunofluorescence staining was performed. The difference in cytocompatibility for epidermal cells, dermal cells, and hair follicle cells was confirmed.
  • the artificial skin of the present invention maintained full confluency without HaCaT cell aggregation.
  • control artificial skin showed aggregation of HaCaT cells.
  • the artificial skin of the present invention had active F-actin expression and proliferated smoothly, but the artificial skin of the control group did not express F-actin actively.
  • the artificial skin of the present invention showed high expression of Keratin 5, one of epidermis differentiation markers, but the artificial skin of the control group had relatively low expression of Keratin 5.
  • the artificial skin of the present invention After cryosectioning the cultured artificial skin, the cross section was observed under an optical microscope. According to the sectioned epidermis-dermis boundary of FIG. 6, the artificial skin of the present invention has a curved papillary layer-like structure. According to the sectioned dermis of FIG. 6, NHDF cells included in the artificial skin of the present invention proliferated in a mesh-like morphology along the interface similar to real skin.
  • the artificial skin structure of the present invention has a curved contact surface between the epidermal layer and the dermal layer, which is similar to the structure of the papillary layer, and the contact area increases and cell-cell interaction occurs more actively, resulting in epidermal cells and dermis It has been found to promote cell growth and differentiation.
  • Example 5 HFDPC spheroid injection through 3D dot printing or 3D printed mold
  • An ink containing NHDF cells was prepared in the same manner as in Example 3-1 above, except for the HFDPC spheroid.
  • Artificial skin with micropores was prepared with ink containing NHDF cells and HFDPC spheroids were injected, or artificial skin was prepared and HFDPC spheroids were injected by a dot printing method. This is to precisely control the HFDPC spheroid to be located on the inside of the dermal-like layer, or at the interface between the dermal-like layer and the dermal epidermal layer.
  • the ink containing NHDF cells was placed on a curved surface in a mold having a plurality of micropillars (see Fig. 7a, mold radius of about 16 mm, pillar diameter of 0.5 mm, pillar height of 4 mm, distance between pillars of 0.5 mm, and the bottom of the mold was curved). with a true surface).
  • the mold was removed to obtain a dermal-like layer having a plurality of fine holes formed at intervals of about 0.5 to 1 mm on one surface and having a curved surface.
  • HFDPC spheroids resuspended in 200 ⁇ l of the co-culture medium were spot seeded in the micropores of the dermal mimic layer, and 1 mL of the co-culture medium was injected so that the upper surface of the skin structure was not submerged.
  • an additional 3-4 mL of co-culture medium was injected and incubation was performed for 24 h. Thereafter, 1 ⁇ 10 5 cells / 200 ⁇ l of HaCaT cell suspension was spot-seeded on the dermal mimic layer three times, and 1 mL of co-culture medium was injected so that the upper surface of the skin structure was not submerged.
  • an additional 3-4 mL of co-culture medium was injected and cultured in a cell culture environment.
  • Ink containing NHDF cells was printed on a mold, HaCaT cells were seeded on the surface from which the mold was removed, and cultured for 7 days to prepare artificial skin (see Example 3-3).
  • HFDPC spheroids were resuspended in the GelMA/HAMA pregel composition (see Example 3-1).
  • An Electromagnetic Droplet (EMD) printhead module was installed on Celink's BIO X model 3D printer, and the HFPDC spheroid-containing pregel composition was dot-printed 25 times at 1 mm intervals, 1 to 4 mm depth, and 2 ⁇ l per time on the artificial skin.
  • HFPDC spheroids were positioned at the dermal layer or at the interface between the dermal and epidermal layers (see FIG. 7b).
  • the artificial skin injected with HFPDC spheroids was irradiated with UV for 3 minutes and then stabilized at room temperature for 10 minutes.
  • a co-culture medium was injected and cultured in a cell culture environment.
  • Immunofluorescence staining reagents were performed in the following manner. Between all reagent treatments, a DPBS washing process was performed three times. First, pretreatment was performed in the order of 10% formalin treatment followed by 10-minute incubation, 0.1% TRITON-X treatment followed by 5-minute incubation, and 2% Bovine serum albumin treatment followed by 30-minute incubation. Thereafter, each sample was treated with an anti-versican primary antibody (abcam) or an anti-ki67 primary antibody (abcam) according to the manufacturer's protocol, and an overnight reaction was performed at 4°C.
  • abcam anti-versican primary antibody
  • abcam anti-ki67 primary antibody
  • FITC secondary antibody FITC-labelled IgG antibody 1:1000 PBS, Abcam
  • DAPI 4,6-diamidino-2-phenylindole
  • rhodamine phalloidin Molecular probe, 165 nM
  • Example 8 is an observation result of the artificial skin of Example 5-1. F-actin expression was active in both epidermal cells (HaCaT) and dermal cells (NHDF) in the artificial skin injected with HFDPC into the micropores. In addition, versican expression of hair follicle cells (HFDPC) was confirmed in the upper part of the dermis, which is the interface between the epidermal layer and the dermis layer, and it was shown that the hair forming ability of the hair follicle cells was maintained.
  • HFDPC hair follicle cells
  • FIG. 9 and 10 are observation results of the artificial skin of Example 5-2. As in FIG. 8, F-actin expression was active in epidermal cells and dermal cells, and expression of versican and Ki-67 in HFDPC cells was confirmed, indicating that proliferation and hair formation ability were maintained.
  • the artificial skin of the present invention has GelMA/HAMA as a main component and mimics the shape of the papillary layer, thereby promoting the spreading and proliferation of keratinocytes and fibroblasts, and improving the proliferation ability and hair forming ability of hair follicle spheroids.
  • the artificial skin of the present invention is produced by printing ink in which hair follicle spheroids are mixed, or artificial skin having micropores is manufactured and hair follicle spheroids are injected, or hair follicle spheroids are injected into artificial skin by a dot printing method. can also be manufactured.
  • the hair forming ability can be maintained by positioning the HFDPC spheroid to span the dermal mimic layer and the dermal epidermal layer.

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Abstract

The present invention relates to artificial skin including gelatin methacrylate and hyaluronic acid methacrylate as main components and fabricated to have the interface between the epidermis-mimicked layer and the dermis-mimicked layer in a curved shape. With improved cytocompatibility for epidermal cells, dermal cells, and hair follicle cells, the artificial skin can allow for improvement in proliferating the cells and developing functions of the cells.

Description

유두층 구조를 모사한 인공피부 및 그의 제조방법Artificial skin mimicking papillae layer structure and manufacturing method thereof
본 발명은 유두층 구조를 모사한 인공피부 및 그의 제조방법에 관한 것이다.The present invention relates to artificial skin simulating a papillary layer structure and a manufacturing method thereof.
모낭모유두 세포는 모발을 형성하는 각질세포에 지속적으로 영양분과 성장인자를 공급해주는 역할을 한다. 남성형 탈모는 모낭모유두 세포의 수가 감소하여 모발형성능이 저하됨으로써 발생하므로, 인공배양된 모낭모유두 세포를 두피에 이식하는 탈모치료 방법이 연구되고 있다. 그러나 체외 배양된 모낭모유두 세포는 모발형성능과 증식능을 빠르게 상실하므로 이의 모발형성능을 유지할 수 있는 배양환경이 요구된다. 각질세포층과 진피세포층을 포함하는 인공피부층에서 모낭모유두 스페로이드를 배양하는 기술이 알려져 있으나, 이 역시 모발형성능을 쉽게 상실하는 것으로 알려져 있다.Hair follicle dermal papilla cells play a role in continuously supplying nutrients and growth factors to keratinocytes that form hair. Since male pattern baldness occurs when the number of hair follicle dermal papilla cells decreases and the hair forming ability decreases, a hair loss treatment method in which artificially cultured hair follicle dermal papilla cells are transplanted to the scalp is being studied. However, hair follicle dermal papilla cells cultured in vitro rapidly lose their hair-forming ability and proliferative ability, so a culture environment capable of maintaining their hair-forming ability is required. A technique for culturing hair follicle dermal papilla spheroids in an artificial skin layer including a keratinocyte layer and a dermal cell layer is known, but this is also known to easily lose hair forming ability.
일 구체예에 따르면, 메타크릴화 젤라틴 및 메타크릴화 히알루론산을 주성분으로 하고, 인공피부의 표피 모사층과 진피 모사층의 경계를 유두층의 굴곡진 형태를 모사한 형태로 제작함으로써 모낭모유두 세포의 증식 및 모발형성능이 유지된 인공피부를 제공한다.According to one embodiment, methacrylic gelatin and methacrylated hyaluronic acid are used as main components, and the boundary between the epidermis-like layer and the dermis-like layer of artificial skin is produced in a form that mimics the curved shape of the papilla layer, thereby increasing the number of hair follicle dermal papilla cells. Provided is an artificial skin in which growth and hair formation properties are maintained.
일 양상은 메타크릴화 젤라틴 및 메타크릴화 히알루론산이 가교된 하이드로겔 및 진피세포를 포함하는 진피 모사층; 상기 진피 모사층의 일면에 접하고, 표피세포를 포함하는 표피 모사층; 및 모낭 스페로이드를 포함하는 인공피부로서, 상기 표피 모사층이 접하는 진피 모사층의 일면은 굴곡진 형태인, 인공피부를 제공한다.One aspect is a dermal mimic layer comprising a hydrogel and dermal cells in which methacrylated gelatin and methacrylated hyaluronic acid are crosslinked; In contact with one surface of the dermis-like layer, the epidermis-like layer containing epidermal cells; And artificial skin comprising a hair follicle spheroid, wherein one surface of the dermal-like layer to which the epidermis-like layer comes into contact has a curved shape, providing artificial skin.
일 실시예에 따르면, 상기 인공피부는 콜라겐 대신 메타크릴화 젤라틴을 포함하고, 표피 모사층이 접하는 진피 모사층의 일면을 굴곡진 형태로 제작하여 유두층의 형태를 모사함으로써 표피세포, 진피세포, 및 모낭세포에 대한 세포적합성이 현저히 향상됨을 확인하였다. According to one embodiment, the artificial skin includes methacrylic gelatin instead of collagen, and one side of the dermal mimic layer in contact with the epidermal mimic layer is made in a curved shape to mimic the shape of the papillary layer, thereby providing epidermal cells, dermal cells, and It was confirmed that cytocompatibility for hair follicle cells was significantly improved.
상기 메타크릴화 젤라틴(Gelatin Methacrylate, GelMA)은 메타크릴화 정도가 70% 이상, 75% 이상, 80% 이상, 85% 이상, 90% 이상, 또는 95% 이상일 수 있다. The methacrylic gelatin (Gelatin Methacrylate, GelMA) may have a methacrylation degree of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
상기 메타크릴화 젤라틴은 타입 A 또는 타입 B 젤라틴일 수 있으며, 일 실시예에 따르면 타입 A 젤라틴일 수 있다. The methacrylated gelatin may be type A or type B gelatin, and according to one embodiment, type A gelatin.
상기 메타크릴화 젤라틴은 뼈, 피부, 힘줄, 연골에서 유래한 것일 수 있으며, 예를 들면 돼지 피부에서 유래한 것일 수 있다.The methacrylated gelatin may be derived from bone, skin, tendon, or cartilage, for example, pig skin.
상기 메타크릴화 젤라틴의 분자량은 3D 프린팅에 적절한 분자량을 선택할 수 있으며, 예를 들면 12000 내지 25000 Da, 14000 내지 25000 Da, 16000 내지 25000 Da, 18000 내지 25000 Da, 또는 20000 내지 25000 Da 일 수 있으나 특별히 한정되는 것은 아니다. The molecular weight of the methacrylated gelatin may be a molecular weight suitable for 3D printing, and may be, for example, 12000 to 25000 Da, 14000 to 25000 Da, 16000 to 25000 Da, 18000 to 25000 Da, or 20000 to 25000 Da, but particularly It is not limited.
상기 메타크릴화 히알루론산(Hyaluronic acid Methacrylate, HAMA)은 메타크릴화 치환도가 100 내지 140 mol%, 110 내지 130 mol%, 또는 약 120 mol% 일 수 있다.The methacrylic acid methacrylate (HAMA) may have a methacrylation substitution degree of 100 to 140 mol%, 110 to 130 mol%, or about 120 mol%.
상기 메타크릴화 히알루론산(Hyaluronic acid Methacrylate, HAMA)은 분자량이 150 내지 250 kDa, 160 내지 240 kDa, 170 내지 230 kDa, 180 내지 220 kDa, 190 내지 210 kDa, 또는 약 200kDa 일 수 있다. The methacrylic acid methacrylate (HAMA) may have a molecular weight of 150 to 250 kDa, 160 to 240 kDa, 170 to 230 kDa, 180 to 220 kDa, 190 to 210 kDa, or about 200 kDa.
일 구체예에 따르면, 상기 표피 모사층이 접하는 진피 모사층의 일면의 굴곡진 형태는 표면 거칠기(Surface Roughness)로 표현될 수 있다. 표면 거칠기는 여러 측정 방법으로 측정될 수 있으며, 구체적으로 인공피부 시편을 대상으로 원자힘현미경(Atomic force microscopy, AFM)을 사용하여, 실리콘 캔틸리버를 이용한 비접촉모드, 스캔 사이즈 1 mm × 1 mm, 512 × 512 분석 포인트를 통한 높이 프로파일 그리고 1.0 Hz/line 스캔 속도의 조건에서 측정될 수 있다. 예를 들면 상기 표면 거칠기는 중심선 평균 거칠기(Ra), 최대높이 거칠기(Rmax), 10점 평균 거칠기(Rz)로 표현될 수 있다. 한편, 중심선 평균 거칠기(arithmetical average roughness; Ra)는 거칠기 단면곡선에서 중심선이 평균선으로부터 떨어진 거리이고, 최대높이 거칠기(maximum height roughness; Rmax)는 단면곡선의 가장 높은 봉우리(peak)에서 가장 깊은 골짜기(valley)까지의 수직 거리이고, 10점 평균 거칠기(ten point median height; Rz)는 단면곡선에서 가장 높은 봉우리 5개의 평균 높이와 가장 깊은 골짜기 5개의 평균 깊이의 차이다. 예를 들면 상기 표피 모사층이 접하는 진피 모사층의 중심선 평균 거칠기(Ra)는 10 내지 60 ㎛, 15 내지 60 ㎛, 20 내지 60 ㎛, 25 내지 60 ㎛, 30 내지 60 ㎛, 35 내지 60 ㎛, 40 내지 60 ㎛, 45 내지 60 ㎛, 10 내지 55 ㎛, 15 내지 55 ㎛, 20 내지 55 ㎛, 25 내지 55 ㎛, 30 내지 55 ㎛, 35 내지 55 ㎛, 40 내지 55 ㎛, 45 내지 55 ㎛, 10 내지 50 ㎛, 15 내지 50 ㎛, 20 내지 50 ㎛, 25 내지 50 ㎛, 30 내지 50 ㎛, 35 내지 50 ㎛, 40 내지 50 ㎛, 또는 45 내지 60 ㎛ 일 수 있다.According to one embodiment, the curved shape of one surface of the dermal mimic layer contacting the epidermal mimic layer may be expressed as surface roughness. Surface roughness can be measured by several measurement methods, specifically, using atomic force microscopy (AFM) for artificial skin specimens, non-contact mode using silicon cantilever, scan size 1 mm × 1 mm, It can be measured under the condition of height profile through 512 × 512 analysis points and 1.0 Hz/line scan rate. For example, the surface roughness may be expressed as center line average roughness (Ra), maximum height roughness (Rmax), and 10-point average roughness (Rz). On the other hand, the center line average roughness (Ra) is the distance between the center line and the average line in the roughness cross-section curve, and the maximum height roughness (Rmax) is the highest peak to the deepest valley ( valley), and ten point median height (Rz) is the difference between the average height of the five highest peaks and the average depth of the five deepest valleys in the cross-sectional curve. For example, the center line average roughness (Ra) of the dermal mimic layer in contact with the epidermal mimic layer is 10 to 60 μm, 15 to 60 μm, 20 to 60 μm, 25 to 60 μm, 30 to 60 μm, 35 to 60 μm, 40 to 60 μm, 45 to 60 μm, 10 to 55 μm, 15 to 55 μm, 20 to 55 μm, 25 to 55 μm, 30 to 55 μm, 35 to 55 μm, 40 to 55 μm, 45 to 55 μm, 10 to 50 μm, 15 to 50 μm, 20 to 50 μm, 25 to 50 μm, 30 to 50 μm, 35 to 50 μm, 40 to 50 μm, or 45 to 60 μm.
일 구체예에 따르면, 상기 굴곡진 형태는 다수의 돌기부(ridge)가 반복된 형태일 수 있다. According to one embodiment, the curved shape may be a shape in which a plurality of ridges are repeated.
일 구체예에 따르면, 상기 각각의 돌기부의 높이는 50 내지 100 ㎛ 또는 80 내지 90㎛일 수 있다. According to one embodiment, the height of each protrusion may be 50 to 100 μm or 80 to 90 μm.
일 구체예에 따르면, 상기 각각의 돌기부의 폭은 50 내지 150 ㎛, 60 내지 140 ㎛, 70 내지 130 ㎛, 80 내지 120 ㎛, 90 내지 110 ㎛, 또는 약 100 ㎛ 일 수 있다. According to one embodiment, the width of each protrusion may be 50 to 150 μm, 60 to 140 μm, 70 to 130 μm, 80 to 120 μm, 90 to 110 μm, or about 100 μm.
일 구체예에 따르면, 상기 각각의 돌기부 사이의 간격은 100 내지 200 ㎛일 수 있다. According to one embodiment, the distance between each of the protrusions may be 100 to 200 μm.
일 구체예에 따르면, 상기 돌기부의 경사각은 50 내지 70°, 55 내지 65°, 57 내지 63°, 59 내지 61° 또는 60°일 수 있다.According to one embodiment, the inclination angle of the protrusion may be 50 to 70 °, 55 to 65 °, 57 to 63 °, 59 to 61 ° or 60 °.
예를 들어, 상기 각각의 돌기부의 높이, 폭, 및 간격은 인공피부 시편을 대상으로 원자힘현미경(Atomic force microscopy, AFM)을 사용하여, 실리콘 캔틸리버를 이용한 비접촉모드, 스캔 사이즈 1 mm × 1 mm, 512 × 512 분석 포인트를 통한 높이 프로파일 그리고 1.0 Hz/line 스캔 속도의 조건에서 측정될 수 있다.For example, the height, width, and spacing of each of the protrusions are measured using an atomic force microscope (AFM) for artificial skin specimens, in a non-contact mode using a silicon cantilever, scan size 1 mm × 1 mm, a height profile through 512 × 512 analysis points and a scan rate of 1.0 Hz/line.
일 구체예에 따르면, 상기 모낭 스페로이드는 상기 진피 모사층 내부, 또는 상기 표피 모사층과 진피 모사층의 경계면에 위치한 것일 수 있다.According to one embodiment, the hair follicle spheroid may be located inside the dermis-like layer or at the interface between the epidermis-like layer and the dermal-like layer.
일 구체예에 따르면, 상기 메타크릴화 젤라틴 및 메타크릴화 히알루론산의 질량비는 320:1 내지 500:1, 330:1 내지 480:1, 340:1 내지 460:1, 350:1 내지 440:1, 360:1 내지 420:1, 380:1 내지 410:1, 또는 약 400:1일 수 있다. According to one embodiment, the mass ratio of the methacrylated gelatin and the methacrylated hyaluronic acid is 320:1 to 500:1, 330:1 to 480:1, 340:1 to 460:1, 350:1 to 440: 1, 360:1 to 420:1, 380:1 to 410:1, or about 400:1.
일 구체예에 따르면 상기 표피세포는 HaCaT, NHK(Normal Human Keratinocyte), 또는 NHEK(Normal Human Epidermal Keratinocyte)일 수 있다. According to one embodiment, the epidermal cells may be HaCaT, NHK (Normal Human Keratinocyte), or NHEK (Normal Human Epidermal Keratinocyte).
일 구체예에 따르면, 상기 모낭 스페로이드는 모유두세포(dermal papilla cell,DPC)를 3차원 배양한 스페로이드일 수 있으며, 모유두세포는 예를 들면 DPC, 또는 hair follicle DPC(HFDPC) 세포일 수 있다. 스페로이드는 단면이 원형 또는 타원형인 세포응집체를 의미한다. 상기 스페로이드의 직경은 30 내지 300 ㎛, 30 내지 250 ㎛, 30 내지 200 ㎛, 30 내지 150 ㎛, 30 내지 100 ㎛, 40 내지 300 ㎛, 40 내지 250 ㎛, 40 내지 200 ㎛, 40 내지 150 ㎛, 40 내지 100 ㎛, 50 내지 300 ㎛, 50 내지 250 ㎛, 50 내지 200 ㎛, 50 내지 150 ㎛, 또는 50 내지 100 ㎛ 일 수 있다. 상기 3차원 배양은 통상 기술자에게 알려진 방법으로 배양할 수 있으며, 예를 들면 현적배양(hanging drop culture), 스피너 플라스크 배양(spinner flask culture), 반구형 마이크로웰 플레이트(concave microwell plate) 배양, 또는 시판되는 3D 배양 키트로 배양할 수 있다.According to one embodiment, the hair follicle spheroid may be a spheroid obtained by three-dimensionally culturing dermal papilla cells (DPC), and the dermal papilla cells may be, for example, DPC or hair follicle DPC (HFDPC) cells. . A spheroid refers to a cell aggregate having a circular or elliptical cross section. The diameter of the spheroid is 30 to 300 ㎛, 30 to 250 ㎛, 30 to 200 ㎛, 30 to 150 ㎛, 30 to 100 ㎛, 40 to 300 ㎛, 40 to 250 ㎛, 40 to 200 ㎛, 40 to 150 ㎛ , 40 to 100 μm, 50 to 300 μm, 50 to 250 μm, 50 to 200 μm, 50 to 150 μm, or 50 to 100 μm. The three-dimensional culture can be cultured by a method known to those skilled in the art, for example, hanging drop culture, spinner flask culture, concave microwell plate culture, or commercially available It can be cultured with a 3D culture kit.
일 구체예에 따르면, 상기 진피세포는 인간 진피 섬유아세포 (Normal Human Dermal Fibroblast, NHDF) 세포일 수 있다.According to one embodiment, the dermal cells may be human dermal fibroblasts (Normal Human Dermal Fibroblast, NHDF) cells.
다른 양상은, 메타크릴화 젤라틴, 메타크릴화 히알루론산, 진피세포, 및 모낭 스페로이드가 혼합된 프리겔 조성물을 굴곡진 표면의 몰드 위에서 가교시키는 단계; 상기 몰드를 제거하여 굴곡진 일면을 갖는 진피 모사층을 얻는 단계; 및 상기 진피 모사층의 굴곡진 일면 위에 표피세포를 파종하고 배양하여 표피 모사층을 형성하는 단계를 포함하는, 인공피부 제조방법을 제공한다. Another aspect is crosslinking a pregel composition in which methacrylic gelatin, methacrylic hyaluronic acid, dermal cells, and hair follicle spheroids are mixed on a mold having a curved surface; Obtaining a dermis-like layer having a curved surface by removing the mold; And seeding and culturing epidermal cells on the curved surface of the dermis-like layer to form an epidermal-like layer, providing an artificial skin manufacturing method.
상기 프리겔 조성물의 메타크릴화 젤라틴과 메타크릴화 히알루론산의 질량비는 질량비는 320:1 내지 500:1, 330:1 내지 480:1, 340:1 내지 460:1, 350:1 내지 440:1, 360:1 내지 420:1, 380:1 내지 410:1, 또는 약 400:1일 수 있다.The mass ratio of methacrylated gelatin and methacrylated hyaluronic acid in the pregel composition is 320:1 to 500:1, 330:1 to 480:1, 340:1 to 460:1, 350:1 to 440: 1, 360:1 to 420:1, 380:1 to 410:1, or about 400:1.
상기 프리겔 조성물의 메타크릴화 젤라틴 및 메타크릴화 히알루론산의 함량은 1 내지 10(w/w)%, 2 내지 8(w/w)%, 3 내지 6(w/w)%, 4 내지 6(w/w)%, 또는 5(w/w)% 일 수 있다. The content of methacrylic gelatin and methacrylic hyaluronic acid in the pregel composition is 1 to 10 (w/w)%, 2 to 8 (w/w)%, 3 to 6 (w/w)%, 4 to 6 (w/w)% 6 (w/w)%, or 5 (w/w)%.
상기 프리겔 조성물의 용매는 PBS(phosphate buffered saline) 또는 DPBS(Dulbecco's phosphate-buffered saline) 일 수 있다. The solvent of the pregel composition may be phosphate buffered saline (PBS) or Dulbecco's phosphate-buffered saline (DPBS).
상기 프리겔 조성물은 광개시제를 더 포함할 수 있으며, 예를 들면 PI113(Wako Pure Chemical), PAG-281(Wako Pure Chemical), PAG-336(Wako Pure Chemical), PAG-337(Wako Pure Chemical), Darocur 1173(BASF), Irgacure 2959(BASF), Irgacure 500(BASF), Irgacure 754(BASF), 이들의 조합으로 이루어진 군에서 선택된 하나 이상을 사용할 수 있으나 특별히 한정되는 것은 아니다.The pregel composition may further include a photoinitiator, for example, PI113 (Wako Pure Chemical), PAG-281 (Wako Pure Chemical), PAG-336 (Wako Pure Chemical), PAG-337 (Wako Pure Chemical), At least one selected from the group consisting of Darocur 1173 (BASF), Irgacure 2959 (BASF), Irgacure 500 (BASF), Irgacure 754 (BASF), and combinations thereof may be used, but is not particularly limited.
또 다른 양상은 메타크릴화 젤라틴, 메타크릴화 히알루론산, 및 진피세포가 혼합된 프리겔 조성물을 굴곡진 표면 위에 미세기둥을 갖는 몰드 위에서 가교시키는 단계; 및 상기 몰드를 제거하여 미세구멍이 형성된 굴곡진 일면을 갖는 진피 모사층을 얻는 단계; 모낭 스페로이드를 상기 미세구멍에 주입하는 단계; 및 상기 진피 모사층의 굴곡진 일면 위에 표피세포를 파종하고 배양하여 표피 모사층을 형성시키는 단계를 포함하는, 인공피부 제조방법을 제공한다. Another aspect is crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, and dermal cells are mixed on a mold having microcolumns on a curved surface; And removing the mold to obtain a dermal mimic layer having a curved surface in which micropores are formed; Injecting hair follicle spheroids into the micropores; And seeding and culturing epidermal cells on the curved surface of the dermis-like layer to form an epidermal-like layer, providing an artificial skin manufacturing method.
상기 미세구멍은 미세기둥이 제거되어 형성된 것으로, 미세구멍의 넓이와 깊이는 진피 모사층의 두께, 모낭 스페로이드의 크기, 모낭 스페로이드 주입 깊이에 따라 적절히 조절될 수 있다. The micropores are formed by removing the microcolumns, and the width and depth of the micropores can be appropriately adjusted according to the thickness of the dermis-like layer, the size of the hair follicle spheroid, and the injection depth of the hair follicle spheroid.
또 다른 양상은, 메타크릴화 젤라틴, 메타크릴화 히알루론산, 및 진피세포가 혼합된 프리겔 조성물을 굴곡진 표면의 몰드 위에서 가교시키는 단계; 및 상기 몰드를 제거하여 굴곡진 일면을 갖는 진피 모사층을 얻는 단계; 상기 진피 모사층의 굴곡진 일면 위에 표피세포를 파종하고 배양하여 표피 모사층을 형성시키는 단계; 및 모낭 스페로이드를 진피 모사층 또는 진피 모사층과 표피 모사층의 경계면에 주입하는 단계를 포함하는, 인공피부 제조방법을 제공한다. In another aspect, crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, and dermal cells are mixed on a mold having a curved surface; And removing the mold to obtain a dermis-like layer having a curved surface; Forming an epidermal mimic layer by seeding and culturing epidermal cells on the curved surface of the dermal mimic layer; and injecting the hair follicle spheroid into the dermis-like layer or the interface between the dermal-like layer and the epidermis-like layer, providing an artificial skin manufacturing method.
상기 주입 방법은 특별히 한정되는 것은 아니며, 주사, 마이크로니들, 또는 3D 닷 프린팅 방법으로 주입할 수 있다. 예를 들면 3D 프린터에 Electromagnetic Droplet(EMD) printhead 모듈을 장착하여 사용할 수 있다. The injection method is not particularly limited, and may be injected by injection, microneedle, or 3D dot printing method. For example, it can be used by mounting an Electromagnetic Droplet (EMD) printhead module on a 3D printer.
일 구체예에 따른 인공피부는 GelMA 및 HAMA를 주성분으로 하고, 표피 모사층과 진피 모사층의 경계를 유두층의 굴곡진 형태를 모사한 형태로 제작함으로써 표피세포, 진피세포, 모낭세포의 증식 및 기능 발현이 향상될 수 있다.Artificial skin according to one embodiment has GelMA and HAMA as main components, and the boundary between the epidermis-like layer and the dermis-like layer is produced in a form that mimics the curved shape of the papillary layer, thereby proliferating and functioning epidermal cells, dermal cells, and hair follicle cells. expression can be improved.
도 1은 Col/HAMA 하이드로겔에서 배양된 HaCaT 세포와 GelMA/HAMA 하이드로겔에서 배양된 HaCaT 세포의 퍼짐(spreading) 및 형상(morphology)을 비교한 결과이다. 1 is a result of comparing the spreading and morphology of HaCaT cells cultured in Col/HAMA hydrogel and HaCaT cells cultured in GelMA/HAMA hydrogel.
도 2는 표피층과 진피층 사이에 굴곡진 경계면(유두층)을 갖는 인공피부 제작방법을 나타낸 것이다. Figure 2 shows a method of manufacturing artificial skin having a curved interface (papilla layer) between the epidermis layer and the dermis layer.
도 3은 일 실시예에서 진피 모사층 제작에 사용된 몰드의 형태를 나타낸 것이다. Figure 3 shows the shape of the mold used in the production of the dermal mimic layer in one embodiment.
도 4는 일 실시예에서 제작된 인공피부의 면역형광염색 및 형태 관찰 결과를 나타낸 것이다. 4 shows the results of immunofluorescence staining and morphological observation of artificial skin prepared in one example.
도 5는 표피 유사층과 진피 유사층 사이의 경계면이 평평한 인공피부와 굴곡진 형태의 인공피부가 세포의 증식 및 기능 발현에서 차이를 나타냄을 확인한 결과이다. 5 is a result confirming that the interface between the epidermal-like layer and the dermal-like layer shows a difference in cell proliferation and function expression between artificial skin with a flat surface and artificial skin with a curved shape.
도 6은 표피 유사층과 진피 유사층 사이의 경계면이 평평한 인공피부와 굴곡진 형태의 인공피부의 단면을 확인한 결과이다. 6 is a result of confirming cross-sections of artificial skin having a flat interface between an epidermal-like layer and a dermal-like layer and a curved artificial skin.
도 7a는 모낭 스페로이드 주입구를 갖는 진피 모사층을 제작하기 위한 미세기둥을 가진 몰드를 나타낸 것이고, 도 7b는 인공피부에 닷 프린팅 방법으로 모낭 스페로이드를 주입 방법을 나타낸 것이다. Figure 7a shows a mold having a microcolumn for producing a dermal mimic layer having a hair follicle spheroid injection hole, and Figure 7b shows a method of injecting hair follicle spheroids into artificial skin by a dot printing method.
도 8은 미세기둥을 가진 몰드로 제작한 인공피부에 주입된 모낭 스페로이드가 모발형성능을 유지하는 것을 확인한 결과이다.8 is a result confirming that the hair follicle spheroid injected into the artificial skin made of a mold with fine columns maintains the hair forming ability.
도 9 및 도 10은 닷 프린팅 방법으로 주입한 모낭스페로이드가 증식능 및 모발형성능을 유지함을 확인한 결과이다.9 and 10 are results confirming that the hair follicle spheroid injected by the dot printing method maintains the proliferative ability and hair forming ability.
이하 하나 이상의 구체예를 실시예를 통해 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.
실시예 1: 세포 준비Example 1: Cell preparation
1-1. HFDPC 스페로이드 준비1-1. Preparing HFDPC spheroids
하기 방법으로 피부 유두 세포인 HFDPC 세포(Human Follicle Dermal Papilla cell)를 3D 배양하여 HFDPC 스페로이드를 제작하였다. HFDPC 세포를 DPCBM(human follicle dermal papillar cell basal medium low serum, Promocell사)에 supplementMIX(ready-to-use supplement, 4% FCS, 0.4% bovine pituitary extract, 1 ng/ml bFGF, 5 ㎍/ml insulin, Promocell사) 및 1% Antibiotic-antimycotic solution(Abs)이 혼합된 배지(이하 DPCBM 배지로 지칭함)에서 3 내지 5 passage 계대배양하였다. Lab to lab 사의 랩스페로우 배양 용기(Type-P 35mm)를 멸균 후 70% EtoH로 1회, DPBS로 3회 세척하고, DPCBM 1mL 주입 후 HFDPC 세포를 3x105 cells/3mL DPCBM으로 주입하여 seeding하였다. 세포 aggregation 속도에 따라 7 내지 14일간 스페로이드 형태로 배양 후 사용하였다. 스페로이드의 크기는 약 50 내지 100 ㎛ 였다.HFDPC spheroids were prepared by 3D culture of HFDPC cells (Human Follicle Dermal Papilla cells), which are skin papilla cells, by the following method. HFDPC cells were added to DPCBM (human follicle dermal papillar cell basal medium low serum, Promocell) supplementMIX (ready-to-use supplement, 4% FCS, 0.4% bovine pituitary extract, 1 ng/ml bFGF, 5 μg/ml insulin, Promocell) and 1% Antibiotic-antimycotic solution (Abs) were mixed medium (hereinafter referred to as DPCBM medium) and subcultured for 3 to 5 passages. After sterilizing Lab to lab's Labsparrow culture vessel (Type-P 35mm), it was washed once with 70% EtoH and three times with DPBS, and after injecting 1mL of DPCBM, HFDPC cells were injected and seeded with 3x10 5 cells/3mL DPCBM. It was used after culturing in the form of spheroids for 7 to 14 days according to the cell aggregation rate. The size of the spheroids was about 50 to 100 μm.
1-2. NHDF(Normal Human Dermal Fibroblasts) 및 HaCaT 세포 준비 1-2. Preparation of Normal Human Dermal Fibroblasts (NHDF) and HaCaT cells
NHDF 세포 및 HaCaT cell은 10% 소태아혈청(FBS, Welgene) 및 1% 항생제 항진균(Abs) 용액(mL 당 10,000 단위 페니실린, 10mg 스트렙토마이신 및 25 ㎍ 암포테리신 B 포함, Sigma-Aldrich, USA)이 첨가된 DMEM 배지(Welgene, Korea)에서 37℃, 5% CO2 습윤 환경에서 배양하였다. 계대배양은 약 80% confluence에서 트립신-EDTA 용액(Welgene)을 사용하여 수행하였고 배양 배지는 48시간마다 교체하였다. NHDF 세포 및 HaCaT 세포는 3 내지 5 passage 계대배양 후 사용하였다.NHDF cells and HaCaT cells were prepared with 10% fetal bovine serum (FBS, Welgene) and 1% antibiotic antifungal (Abs) solution (containing 10,000 units penicillin per mL, 10 mg streptomycin and 25 μg amphotericin B, Sigma-Aldrich, USA). This was added to DMEM medium (Welgene, Korea) at 37° C., 5% CO 2 and cultured in a humid environment. Subculture was performed using a trypsin-EDTA solution (Welgene) at about 80% confluence and the culture medium was replaced every 48 hours. NHDF cells and HaCaT cells were used after subculture of 3 to 5 passages.
실시예 2: 피부 세포에 대해 세포적합성이 우수한 하이드로겔 조성 확립 Example 2: Establishment of a hydrogel composition with excellent cytocompatibility for skin cells
인체 피부의 세포외기질(Extracellular Matrix)와 유사한 조성의 하이드로겔 바이오잉크를 제작하고자 하였다. 인체 피부의 세포외기질 조성은 콜라겐과 히알루론산의 질량비가 400:1로 알려져 있다(하기 표 1 참고). It was intended to produce a hydrogel bioink with a composition similar to the extracellular matrix of human skin. It is known that the composition of the extracellular matrix of human skin is that the mass ratio of collagen to hyaluronic acid is 400:1 (see Table 1 below).
ComponentComponent Subtype Subtype %% ReferenceReference
CollagenCollagen ⅠI 6060 Oikarinen, A. "Connective tissue and aging." International Journal of Cosmetic Science 26.2 (2004): 107-107.Oikarinen, A. "Connective tissue and aging." International Journal of Cosmetic Science 26.2 (2004): 107-107.
ⅢIII 11.2511.25
Other typesOther types 3.753.75
ElastinElastin 1-21-2
GAGGAG HAHA 0.150.15 Salbach, Juliane, et al. "Regenerative potential of glycosaminoglycans for skin and bone." Journal of molecular medicine 90.6 (2012): 625-635.Salbach, Juliane, et al. "Regenerative potential of glycosaminoglycans for skin and bone." Journal of molecular medicine 90.6 (2012): 625-635.
Chondroitin sulfateChondroitin sulfate 0.050.05
EtcEtc 22.822.8
이에 따라 콜라겐(돼지 피부의 Type I 아텔로콜라겐(Dalim Tissen, Korea) 및 히알루론산을 400:1 질량비로 포함하는 하이드로겔을 이용하여 7일간 HaCaT 세포를 배양하였으나 다음과 같은 문제점이 있었다. Accordingly, HaCaT cells were cultured for 7 days using a hydrogel containing collagen (Type I atelocollagen of pig skin (Dalim Tissen, Korea) and hyaluronic acid at a mass ratio of 400: 1), but there were the following problems.
콜라겐을 저농도(<1%)로 포함한 하이드로겔은 세포의 spreading을 유도할 수 있었지만 쉽게 분해되어 장기간 세포배양에 적합하지 않았다. 반면, 콜라겐을 고농도(>1%)로 포함한 하이드로겔은 세포를 장기간 세포 배양할 수 있었으나 세포의 spreading을 촉진시키지 못해 cell phenotype의 정상적인 발현을 억제하였다. A hydrogel containing collagen at a low concentration (<1%) could induce cell spreading, but was easily degraded and was not suitable for long-term cell culture. On the other hand, the hydrogel containing collagen at a high concentration (>1%) could culture cells for a long period of time, but it did not promote cell spreading and suppressed the normal expression of cell phenotype.
따라서 콜라겐 대신 젤라틴(gelatin)으로 변경하였다. 메타크릴로일 작용기 부착 젤라틴 (GelMA, porcine skin type A, DOM ≥ 90% (H-NMR), powder, 12,000 ~ 14,000 Da 이상 25,000Da 이하, 3D material사)과 메타크릴로일 작용기 부착 히알루론산(HAMA, DS(Degree of substitution) 120±10 mol%, powder, 200kDa, SNvia사)을 사용하고, 이들의 혼합 비율은 4 내지 5 %(w/v): 0.01 내지 0.0125 %(w/v)으로 하였다.Therefore, it was changed to gelatin instead of collagen. Methacryloyl functional group attached gelatin (GelMA, porcine skin type A, DOM ≥ 90% (H-NMR), powder, 12,000 ~ 14,000 Da or more and 25,000 Da or less, 3D material) and methacryloyl functional group attached hyaluronic acid ( HAMA, DS (Degree of substitution) 120±10 mol%, powder, 200kDa, SNvia) was used, and their mixing ratio was 4 to 5% (w/v): 0.01 to 0.0125% (w/v). did
도 1에 따르면, Col/HAMA 하이드로겔에서 7일간 배양된 HaCaT 세포는 spreading이 미미하였으나, GelMA/HAMA에서 배양된 HaCaT 세포는 spreading이 활발한 것으로 확인되었다.According to FIG. 1, HaCaT cells cultured in Col/HAMA hydrogel for 7 days showed insignificant spreading, but HaCaT cells cultured in GelMA/HAMA showed active spreading.
이에 따라 인공피부에 사용할 하이드로겔의 조성은 GelMA 4 ~ 5 %(w/v) 및 HAMA 0.01 ~ 0.0125 %(w/v)으로 하였다.Accordingly, the composition of the hydrogel to be used for artificial skin was 4 to 5% (w/v) of GelMA and 0.01 to 0.0125% (w/v) of HAMA.
실시예 3: 유두층(papillary layer) 구조를 모사한 인공피부 제작 Example 3: Fabrication of artificial skin simulating the structure of the papillary layer
도 2를 참고하여 표피층과 진피층의 경계면이 굴곡진 유두층 모사 인공피부 제작방법을 설명한다. Referring to FIG. 2, a method for manufacturing artificial skin simulating a papillary layer in which an interface between an epidermal layer and a dermal layer is curved will be described.
3-1. 진피 세포 및 모낭 스페로이드를 포함하는 GelMA/HAMA 바이오잉크 준비3-1. Preparation of GelMA/HAMA bioink containing dermal cells and hair follicle spheroids
GelMA/HAMA(질량비 400:1)를 5(w/w)% 포함하는 DPBS 용액 2mL를 37℃에서 4시간 동안 용해시켜 프리겔 용액(pre-gel solution)을 준비하였다. A pre-gel solution was prepared by dissolving 2 mL of a DPBS solution containing 5 (w/w)% of GelMA/HAMA (mass ratio 400:1) at 37° C. for 4 hours.
NHDF cell pellet 및 HFDPC spheroid pellet을 5x105 cells/ml 밀도로 pre-gel solution에 재현탁하였다. 광개시제 Irgacure 2959(10배 희석)을 40 내지 50 mg/ml(약 1 w/v%)를 pre-gel solution에 첨가하였다. 3D 프린팅 전까지 빛을 차단하고 10분 간 4℃ 냉장고에 보관하여 점성을 증가시켰다. NHDF cell pellet and HFDPC spheroid pellet were resuspended in a pre-gel solution at a density of 5x10 5 cells/ml. 40 to 50 mg/ml (about 1 w/v%) of the photoinitiator Irgacure 2959 (10-fold dilution) was added to the pre-gel solution. Before 3D printing, light was blocked and stored in a refrigerator at 4°C for 10 minutes to increase viscosity.
3-2. 몰드 준비 3-2. mold preparation
표면에 다수의 릿지(ridge)가 형성된 굴곡진 표면을 가진 몰드를 준비하였다. 도 3에 따르면, 몰드 표면의 릿지(ridge) 사이 거리는 약 100 내지 200 ㎛이며, 릿지의 폭(width)은 약 100 ㎛, 높이는 약 80 내지 90 ㎛였다. 릿지(ridge)의 경사각은 약 60도였다.A mold having a curved surface having a plurality of ridges formed thereon was prepared. According to FIG. 3, the distance between ridges on the surface of the mold was about 100 to 200 μm, the width of the ridges was about 100 μm, and the height was about 80 to 90 μm. The inclination angle of the ridge was about 60 degrees.
3-3. NHDF 세포 및 HFDPC 스페로이드 함유 인공피부 제작3-3. Fabrication of artificial skin containing NHDF cells and HFDPC spheroids
압출형 3D 바이오프린터(BIO X, Cellink, Boston, MA)를 이용하여 인공피부 (2 cm x 2 cm x 0.2 cm)를 제작하였다. 인공피부 모델은 HeartOS 소프트웨어(Cellink)를 사용하여 g-코드 파일로 코딩되었다. 노즐 게이지, 프린팅 베드/디스펜서의 온도, 프린팅 베드의 이동 속도, 유속, 노즐과 베드 사이의 거리, UV 강도 등 3D 프린팅 조건은 하기 표 2와 같다.Artificial skin (2 cm x 2 cm x 0.2 cm) was fabricated using an extrusion-type 3D bioprinter (BIO X, Cellink, Boston, MA). Artificial skin models were coded into g-code files using HeartOS software (Cellink). 3D printing conditions such as nozzle gauge, temperature of printing bed/dispenser, moving speed of printing bed, flow rate, distance between nozzle and bed, and UV intensity are shown in Table 2 below.
Nozzle gaugeNozzle gauge Temp
(Bed/Dispenser)Temp
(Bed/Dispenser) 		Moving speedMoving speed Flow rateFlow rate Distance
(between dispenser and bed)Distance
(between dispenser and bed) 		UV intensityUV intensity
21G21G 5℃5℃ 10 cm/s10cm/s 1 ml/h1ml/h 0.1mm0.1mm 365nm/4W365nm/4W
상기 제작한 NHDF 세포 및 HFDPC 스페로이드가 함유된 잉크를 UV 복사(365 nm)를 가하면서 몰드 위에 4층으로 적층하였다. 인쇄된 구조물을 6-웰 플레이트로 옮기고 추가로 10분의 UV 복사를 수행했다. 실온에서 5분 동안 안정화시켜 가교결합이 완료된 후 몰드를 제거하였다.The ink containing the prepared NHDF cells and HFDPC spheroids was stacked in four layers on a mold while applying UV radiation (365 nm). The printed constructs were transferred to a 6-well plate and subjected to an additional 10 minutes of UV irradiation. The mold was removed after cross-linking was completed by stabilizing at room temperature for 5 minutes.
몰드가 제거된 굴곡진 표면을 위로 향하도록 겔을 위치시킨 후에, 모낭형성 촉진 공배양배지를 이용하여 7일간 인큐베이션하였다. 모낭형성 촉진 공배양배지는 피부세포 및 모낭모유두 세포 스페로이드의 성장 및 분화를 돕는 역할을 하며, 이의 조성은 Fetal Bovine Serum 10%, Fetal Calf Serum 4%, basic Fibroblast Growth Factor 1ng/ml, Insulin 5 ㎍/ml, Bovine Pituitary Extract 0.4%, 1% Antibiotic-Antimycotic Solution이 첨가된 DMEM(high glucose) 배지이다.After the gel was placed with the curved surface from which the mold was removed facing upward, it was incubated for 7 days using a co-culture medium for promoting folliculogenesis. The co-culture medium for promoting hair follicle formation helps the growth and differentiation of skin cells and hair follicle dermal papilla cell spheroids, and its composition is Fetal Bovine Serum 10%, Fetal Calf Serum 4%, basic Fibroblast Growth Factor 1ng/ml, Insulin 5 It is DMEM (high glucose) medium supplemented with ug/ml, Bovine Pituitary Extract 0.4%, and 1% Antibiotic-Antimycotic Solution.
7일간 인큐베이션 후에 HaCaT 세포를 겔의 굴곡진 표면위에 1 X 106 cells/200 ㎕ 농도로 3회에 걸쳐 스팟 파종(spot seeding)하였다. 스팟 파종된 겔을 전용 배양용기(24mm Transwell® with 0.4 ㎛ Pore Polycarbonate, Corning)에 옮기고, 7 내지 21일간 37℃에서 Air-liquid culture를 실시하였다. 각질세포의 분화 촉진을 위해 인공피부 구조체의 상단 표피층(각질세포)는 배지에 잠기지 않도록 하고, 24시간마다 배지를 교체하였다. After incubation for 7 days, HaCaT cells were spot seeded three times at a concentration of 1 X 10 6 cells/200 μl on the curved surface of the gel. The spot-seeded gel was transferred to a dedicated culture container (24mm Transwell® with 0.4 μm Pore Polycarbonate, Corning), and air-liquid culture was performed at 37° C. for 7 to 21 days. To promote the differentiation of keratinocytes, the upper epidermal layer (keratinocytes) of the artificial skin structure was not immersed in the medium, and the medium was replaced every 24 hours.
대조군으로서, 상기 제조방법에서 거친 표면을 가진 몰드 대신 평평한 표면의 커버유리를 사용한 것을 제외하고 동일한 방법으로 표피층과 진피층이 평평한 경계면을 갖는 인공피부를 제작하였다.As a control, artificial skin having a flat interface between the epidermal layer and the dermal layer was fabricated in the same manner as in the manufacturing method, except that a cover glass having a flat surface was used instead of a mold having a rough surface.
실시예 4: 유두층 모사 인공피부의 특성 분석Example 4: Characteristic analysis of papillary layer simulated artificial skin
본 발명의 인공피부(표피층과 진피층 사이의 경계면이 유두층과 같이 굴곡진 형태)를 21일간 배양하고 면역형광염색으로 형태를 관찰하였다. The artificial skin of the present invention (in which the interface between the epidermal layer and the dermal layer is curved like the papillary layer) was cultured for 21 days and the shape was observed by immunofluorescence staining.
도 4의 Ph(Phase contrast)에 따르면, 상기 인공피부는 세포배양환경에서 장기간 배양하였음에도 몰드에 의해 형성된 하이드로겔의 굴곡진 구조가 유지되었다. 도 4의 TRITC 염색 결과에 따르면, 스팟 파종한 HaCaT 세포는 F-actin 발현이 활발하여 굴곡진 하이드로겔 표면에 정상적으로 adhesion 및 spreading 한 것으로 나타났다. 도 4의 진피 Optical microscopy 결과에 따르면, HFDPC 스페로이드는 응집(aggregation)된 구형의 구조를 유지하였다.According to Ph (Phase contrast) of FIG. 4, the curved structure of the hydrogel formed by the mold was maintained even though the artificial skin was cultured for a long time in a cell culture environment. According to the TRITC staining results in FIG. 4, the spot-seeded HaCaT cells showed active F-actin expression and normally adhered and spread on the curved hydrogel surface. According to the dermal optical microscopy results of FIG. 4, HFDPC spheroids maintained an aggregated spherical structure.
본 발명의 인공피부(표피층과 진피층 사이의 경계면이 유두층과 같이 굴곡진 형태)와 대조군 인공피부(표피층과 진피층 사이의 경계면이 flat)를 21일간 배양하고, TRITC, DAPI, FITC 면역형광염색을 실시하여 표피세포, 진피세포, 모낭세포에 대한 세포적합성(cytocompatibity) 차이를 확인하였다. The artificial skin of the present invention (the interface between the epidermal layer and the dermis layer is curved like the papillary layer) and the artificial skin of the control group (the interface between the epidermal layer and the dermis layer is flat) were cultured for 21 days, and TRITC, DAPI, and FITC immunofluorescence staining was performed. The difference in cytocompatibility for epidermal cells, dermal cells, and hair follicle cells was confirmed.
도 5의 Phase contrast에 따르면, 본 발명의 인공피부는 HaCaT 세포가 aggregation 되지 않고 완전히 밀집된 상태(full confluency)를 유지하였다. 반면 대조군 인공피부는 HaCaT 세포의 응집이 나타났다. 도 5의 TRITC 염색 결과에 따르면, 본 발명의 인공피부는 F-actin 발현이 활발하여 원활히 증식하였으나, 대조군 인공피부는 F-actin의 발현이 활발하지 않았다. 도 5의 FITC 염색 결과에 따르면, 본 발명의 인공피부는 표피 세포의 분화 마커(epidermis differentiation marker) 중 하나인 Keratin 5 발현이 높게 나타났으나, 대조군 인공피부는 Keratin 5 발현이 상대적으로 낮았다.According to the phase contrast of FIG. 5, the artificial skin of the present invention maintained full confluency without HaCaT cell aggregation. On the other hand, control artificial skin showed aggregation of HaCaT cells. According to the TRITC staining result of FIG. 5, the artificial skin of the present invention had active F-actin expression and proliferated smoothly, but the artificial skin of the control group did not express F-actin actively. According to the FITC staining results of FIG. 5, the artificial skin of the present invention showed high expression of Keratin 5, one of epidermis differentiation markers, but the artificial skin of the control group had relatively low expression of Keratin 5.
상기 배양된 인공피부를 cryosection한 후에 광학현미경으로 단면을 관찰하였다. 도 6의 sectioned epidermis-dermis boundary에 따르면, 본 발명의 인공피부는 굴곡진 형태의 유두층 모사 구조가 관찰되었다. 도 6의 Sectioned dermis에 따르면, 본 발명의 인공피부에 포함된 NHDF 세포는 실제 피부와 유사하게 경계면을 따라 그물 구조(mesh-like morphology)로 증식한 것으로 나타났다. After cryosectioning the cultured artificial skin, the cross section was observed under an optical microscope. According to the sectioned epidermis-dermis boundary of FIG. 6, the artificial skin of the present invention has a curved papillary layer-like structure. According to the sectioned dermis of FIG. 6, NHDF cells included in the artificial skin of the present invention proliferated in a mesh-like morphology along the interface similar to real skin.
상기 관찰결과를 종합하면, 본 발명의 인공피부 구조체는 표피층과 진피층 의 접촉면이 굴곡진 형태 형태를 가짐으로써 유두층 구조와 유사하며, 접촉 면적이 증가하고 cell-cell interaction이 더 활발히 일어나 표피세포 및 진피세포의 성장 및 분화를 촉진하는 것으로 확인되었다.Summarizing the above observations, the artificial skin structure of the present invention has a curved contact surface between the epidermal layer and the dermal layer, which is similar to the structure of the papillary layer, and the contact area increases and cell-cell interaction occurs more actively, resulting in epidermal cells and dermis It has been found to promote cell growth and differentiation.
실시예 5: 3D dot printing 또는 3D printed mold를 통한 HFDPC 스페로이드 주입 Example 5: HFDPC spheroid injection through 3D dot printing or 3D printed mold
HFDPC 스페로이드를 제외한 것을 제외하고 앞서 3-1 실시예와 동일한 방법으로 NHDF 세포를 포함하는 잉크를 제작하였다. NHDF 세포를 포함하는 잉크로 미세구멍이 형성된 인공피부를 제작하고 HFDPC 스페로이드를 주입하거나, 또는 인공피부를 제작하고 닷 프린팅 방법으로 HFDPC 스페로이드를 주입하였다. 이는 HFDPC 스페로이드가 진피모사층 내부, 또는 진피모사층 및 진피표피층의 경계면에 위치하도록 정교하게 조절하기 위함이다.An ink containing NHDF cells was prepared in the same manner as in Example 3-1 above, except for the HFDPC spheroid. Artificial skin with micropores was prepared with ink containing NHDF cells and HFDPC spheroids were injected, or artificial skin was prepared and HFDPC spheroids were injected by a dot printing method. This is to precisely control the HFDPC spheroid to be located on the inside of the dermal-like layer, or at the interface between the dermal-like layer and the dermal epidermal layer.
5-1. 미세구멍이 형성된 인공피부에 HFDPC 스페로이드 주입 5-1. Injection of HFDPC spheroids into artificial skin with micropores
NHDF 세포를 포함하는 잉크를 굴곡진 표면 위에 다수의 미세기둥을 가진 몰드(도 7a 참고, 몰드 반경 약 16mm, 기둥의 직경 0.5mm, 기둥의 높이 4mm, 기둥 사이 거리 0.5 mm, 몰드의 바닥은 굴곡진 표면을 가짐) 위에서 가교시켰다. 하이드로겔이 형성된 후 몰드를 제거하여 일면에 다수의 미세 구멍이 약 0.5 내지 1 mm 간격으로 형성되고 굴곡진 표면을 갖는 진피 모사층을 얻었다. 200 ㎕ 공배양배지에 재현탁한 HFDPC 스페로이드를 진피 모사층의 미세 구멍에 spot seeding 하고, 공배양배지 1mL를 피부구조체의 윗면이 잠기지 않도록 주입하였다. 4h 이후 공배양배지를 추가적으로 3~4 mL 주입하고 24h 인큐베이션을 진행하였다. 이후, 3회에 걸쳐 1 X 105 cells/200 ㎕의 HaCaT 세포 현탁액을 진피 모사층 위에 spot seeding 하고 공배양배지 1mL을 피부 구조체의 윗면이 잠기지 않도록 주입하였다. 4h 이후 공배양배지를 추가적으로 3~4 mL 주입하고 세포배양환경에서 배양하였다.The ink containing NHDF cells was placed on a curved surface in a mold having a plurality of micropillars (see Fig. 7a, mold radius of about 16 mm, pillar diameter of 0.5 mm, pillar height of 4 mm, distance between pillars of 0.5 mm, and the bottom of the mold was curved). with a true surface). After the hydrogel was formed, the mold was removed to obtain a dermal-like layer having a plurality of fine holes formed at intervals of about 0.5 to 1 mm on one surface and having a curved surface. HFDPC spheroids resuspended in 200 μl of the co-culture medium were spot seeded in the micropores of the dermal mimic layer, and 1 mL of the co-culture medium was injected so that the upper surface of the skin structure was not submerged. After 4 h, an additional 3-4 mL of co-culture medium was injected and incubation was performed for 24 h. Thereafter, 1 × 10 5 cells / 200 μl of HaCaT cell suspension was spot-seeded on the dermal mimic layer three times, and 1 mL of co-culture medium was injected so that the upper surface of the skin structure was not submerged. After 4h, an additional 3-4 mL of co-culture medium was injected and cultured in a cell culture environment.
5-2. 닷 프린팅 방법으로 HFDPC 스페로이드 주입 5-2. Injection of HFDPC spheroids by dot printing method
NHDF 세포를 포함하는 잉크를 몰드 위에 프린팅하고, 몰드가 제거된 표면에 HaCaT 세포를 seeding하고, 7일간 배양하여 인공피부를 제작하였다(실시예 3-3 참고).Ink containing NHDF cells was printed on a mold, HaCaT cells were seeded on the surface from which the mold was removed, and cultured for 7 days to prepare artificial skin (see Example 3-3).
HFDPC 스페로이드를 GelMA/HAMA 프리겔 조성물(실시예 3-1 참고)에 재현탁시켰다. Celink사의 BIO X 모델의 3D 프린터에 Electromagnetic Droplet(EMD) printhead 모듈을 장착하고, HFPDC 스페로이드 함유 프리겔 조성물을 상기 인공피부에 1mm 간격, 1 내지 4mm 깊이, 회당 2 ㎕ 용량으로 25회 닷 프린팅하여 HFPDC 스페로이드가 진피층 또는 진피층과 표피층의 경계면에 위치하도록 하였다(도 7b 참고). HFPDC 스페로이드가 주입된 인공피부에 3분간 UV를 조사한 후 10분간 상온에서 안정화시켰다. 공배양배지를 주입하고 세포배양환경에서 배양하였다.HFDPC spheroids were resuspended in the GelMA/HAMA pregel composition (see Example 3-1). An Electromagnetic Droplet (EMD) printhead module was installed on Celink's BIO X model 3D printer, and the HFPDC spheroid-containing pregel composition was dot-printed 25 times at 1 mm intervals, 1 to 4 mm depth, and 2 μl per time on the artificial skin. HFPDC spheroids were positioned at the dermal layer or at the interface between the dermal and epidermal layers (see FIG. 7b). The artificial skin injected with HFPDC spheroids was irradiated with UV for 3 minutes and then stabilized at room temperature for 10 minutes. A co-culture medium was injected and cultured in a cell culture environment.
실시예 6: HFDPC 스페로이드를 주입한 인공피부의 세포적합성Example 6: Cytocompatibility of artificial skin injected with HFDPC spheroids
몰드 방법으로 HFDPC 스페로이드를 주입한 인공피부(실시예 5-1) 및 닷 프린팅 방법으로 HFDPC 스페로이드를 주입한 인공피부(실시예 5-2)를 21일 배양 후 면역형광염색을 진행하고 공초점레이저주사현미경으로 관찰하여 각 세포의 형상, versican, Ki-67 발현을 확인하였다. Versican은 세포외기질을 구성하는 proteoglycan로서 HFDPC 세포의 모발형성능과 관련된 마커이며, Ki-67은 HFDPC 세포의 세포 증식에 대한 마커이다. Artificial skin injected with HFDPC spheroids by the mold method (Example 5-1) and artificial skin injected with HFDPC spheroids by the dot printing method (Example 5-2) were cultured for 21 days, and immunofluorescence staining was performed. The shape of each cell and the expression of versican and Ki-67 were confirmed by observation under a focusing laser scanning microscope. Versican is a proteoglycan constituting the extracellular matrix and is a marker related to the hair forming ability of HFDPC cells, and Ki-67 is a marker for cell proliferation of HFDPC cells.
면역형광염색 시약은 다음 방법으로 실시하였다. 모든 시약 처리 과정 사이에는 DPBS 3회 washing 과정을 실시하였다. 먼저, 10% formalin 처리 후 10 분 인큐베이션, 0.1% TRITON-X 처리 후 5분 인큐베이션, 2% Bovine serum albumin 처리 후 30분 인큐베이션 순서로 전처리하였다. 이후 제조사의 프로토콜에 따라 샘플 별로 anti-versican 1차 항체 (abcam) 또는 anti-ki67 1차 항체 (abcam)를 처리하고, 4℃에서 overnight reaction을 진행하였다. Washing 이후 각 host에 맞는 FITC 2차 항체(FITC-labelled IgG antibody 1:1000 PBS, Abcam)를 1시간 처리하고, DAPI(4',6-diamidino-2-phenylindole, Sigma-Aldrich Co., 300 nM) 및 rhodamine phalloidin (Molecular probe, 165 nM)을 30분간 처리하였다. Olympus IX81 (형광현미경) 및 ZEISS LSM 800 (공초점 형광현미경)을 이용하여 형광이미징을 실시하였다.Immunofluorescence staining reagents were performed in the following manner. Between all reagent treatments, a DPBS washing process was performed three times. First, pretreatment was performed in the order of 10% formalin treatment followed by 10-minute incubation, 0.1% TRITON-X treatment followed by 5-minute incubation, and 2% Bovine serum albumin treatment followed by 30-minute incubation. Thereafter, each sample was treated with an anti-versican primary antibody (abcam) or an anti-ki67 primary antibody (abcam) according to the manufacturer's protocol, and an overnight reaction was performed at 4°C. After washing, FITC secondary antibody (FITC-labelled IgG antibody 1:1000 PBS, Abcam) for each host was treated for 1 hour, and DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich Co., 300 nM ) and rhodamine phalloidin (Molecular probe, 165 nM) for 30 minutes. Fluorescence imaging was performed using an Olympus IX81 (fluorescence microscope) and a ZEISS LSM 800 (confocal fluorescence microscope).
도 8은 실시예 5-1의 인공피부를 관찰한 결과이다. 미세구멍에 HFDPC를 주입한 인공피부는 표피 세포(HaCaT) 및 진피 세포(NHDF) 모두 F-actin 발현이 활발하였다. 또한 표피층과 진피층의 경계면인 Dermis upper part에서 모낭 세포(HFDPC)의 versican 발현이 확인되어 모낭 세포의 모발형성능이 유지되는 것으로 나타났다.8 is an observation result of the artificial skin of Example 5-1. F-actin expression was active in both epidermal cells (HaCaT) and dermal cells (NHDF) in the artificial skin injected with HFDPC into the micropores. In addition, versican expression of hair follicle cells (HFDPC) was confirmed in the upper part of the dermis, which is the interface between the epidermal layer and the dermis layer, and it was shown that the hair forming ability of the hair follicle cells was maintained.
도 9 및 도 10은 실시예 5-2의 인공피부를 관찰한 결과이다. 도 8과 마찬가지로 표피세포 및 진피세포의 F-actin 발현이 활발하였으며, HFDPC 세포의 versican 및 Ki-67발현이 확인되어 증식능 및 모발형성능이 유지되는 것으로 나타났다. 9 and 10 are observation results of the artificial skin of Example 5-2. As in FIG. 8, F-actin expression was active in epidermal cells and dermal cells, and expression of versican and Ki-67 in HFDPC cells was confirmed, indicating that proliferation and hair formation ability were maintained.
상기 실험결과를 종합하면, 본 발명의 인공피부는 GelMA/HAMA를 주성분으로 하고 유두층의 형태를 모사함으로써 각질세포 및 섬유아세포의 spreading 및 증식을 촉진할 수 있으며, 모낭 스페로이드의 증식능 및 모발형성능을 유지시킬 수 있다. 그리고 본 발명의 인공피부는 모낭 스페로이드가 혼합된 잉크를 프린팅하여 제작하거나, 미세구멍을 갖는 인공피부를 제작하고 모낭 스페로이드를 주입하거나, 또는 인공피부에 닷 프린팅 방법으로 모낭 스페로이드를 주입하여 제작할 수도 있다. 또한 HFDPC 스페로이드가 진피모사층과 진피표피층에 걸치도록 위치시킴으로써 모발형성능이 유지될 수 있다.Summarizing the above experimental results, the artificial skin of the present invention has GelMA/HAMA as a main component and mimics the shape of the papillary layer, thereby promoting the spreading and proliferation of keratinocytes and fibroblasts, and improving the proliferation ability and hair forming ability of hair follicle spheroids. can be maintained In addition, the artificial skin of the present invention is produced by printing ink in which hair follicle spheroids are mixed, or artificial skin having micropores is manufactured and hair follicle spheroids are injected, or hair follicle spheroids are injected into artificial skin by a dot printing method. can also be manufactured. In addition, the hair forming ability can be maintained by positioning the HFDPC spheroid to span the dermal mimic layer and the dermal epidermal layer.

Claims (9)

  1. 메타크릴화 젤라틴 및 메타크릴화 히알루론산이 가교된 하이드로겔 및 진피세포를 포함하는 진피 모사층; a dermal mimic layer comprising methacrylic gelatin and methacrylic hyaluronic acid cross-linked hydrogel and dermal cells;
    상기 진피 모사층의 일면에 접하고, 표피세포를 포함하는 표피 모사층; 및 In contact with one surface of the dermis-like layer, the epidermis-like layer containing epidermal cells; and
    모낭 스페로이드를 포함하는 인공피부로서, As an artificial skin containing hair follicle spheroids,
    상기 표피 모사층이 접하는 진피 모사층의 일면은 굴곡진 형태인, One side of the dermis-like layer in contact with the epidermis-like layer is a curved shape,
    인공피부.artificial skin.
  2. 제1항에 있어서,According to claim 1,
    상기 표피 모사층이 접하는 진피 모사층의 일면은 중심선 평균 거칠기(Arithmetic average roughness; Ra)가 10 내지 60 ㎛인, One side of the dermis-like layer in contact with the epidermal-like layer has a center line average roughness (Arithmetic average roughness; Ra) of 10 to 60 μm,
    인공피부.artificial skin.
  3. 제1항에 있어서, According to claim 1,
    상기 굴곡진 형태는 다수의 돌기부가 반복된 형태인, The curved form is a form in which a plurality of protrusions are repeated,
    인공피부.artificial skin.
  4. 제3항에 있어서, According to claim 3,
    상기 각각의 돌기부는 높이가 50 내지 100 ㎛이고, 간격이 100 내지 200 ㎛인, Each of the protrusions has a height of 50 to 100 μm and an interval of 100 to 200 μm.
    인공피부.artificial skin.
  5. 제1항에 있어서, According to claim 1,
    상기 모낭 스페로이드는 상기 진피 모사층의 내부, 또는 상기 표피 모사층과 진피 모사층의 경계면에 위치한 것인, The hair follicle spheroid is located on the inside of the dermis-like layer, or at the interface between the epidermis-like layer and the dermal-like layer,
    인공피부.artificial skin.
  6. 제1항에 있어서, According to claim 1,
    상기 메타크릴화 젤라틴 및 메타크릴화 히알루론산의 질량비는 350:1 내지 450:1인, The mass ratio of the methacrylated gelatin and the methacrylated hyaluronic acid is 350: 1 to 450: 1,
    인공피부.artificial skin.
  7. 메타크릴화 젤라틴, 메타크릴화 히알루론산, 진피세포, 및 모낭 스페로이드가 혼합된 프리겔 조성물을 굴곡진 표면의 몰드 위에서 가교시키는 단계; crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, dermal cells, and hair follicle spheroids are mixed on a mold having a curved surface;
    상기 몰드를 제거하여 굴곡진 일면을 갖는 진피 모사층을 얻는 단계; 및Obtaining a dermis-like layer having a curved surface by removing the mold; and
    상기 진피 모사층의 굴곡진 일면 위에 표피세포를 파종하고 배양하여 표피 모사층을 형성하는 단계를 포함하는, Forming an epidermal mimic layer by seeding and culturing epidermal cells on the curved surface of the dermal mimic layer,
    인공피부 제조방법.Artificial skin manufacturing method.
  8. 메타크릴화 젤라틴, 메타크릴화 히알루론산, 및 진피세포가 혼합된 프리겔 조성물을 굴곡진 표면 위에 미세기둥을 갖는 몰드 위에서 가교시키는 단계; 및 crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, and dermal cells are mixed on a mold having microcolumns on a curved surface; and
    상기 몰드를 제거하여 미세구멍이 형성된 굴곡진 일면을 갖는 진피 모사층을 얻는 단계; Obtaining a dermal mimic layer having a curved surface in which fine holes are formed by removing the mold;
    모낭 스페로이드를 상기 미세구멍에 주입하는 단계; 및 Injecting hair follicle spheroids into the micropores; and
    상기 진피 모사층의 굴곡진 일면 위에 표피세포를 파종하고 배양하여 표피 모사층을 형성시키는 단계를 포함하는, Forming an epidermal mimic layer by seeding and culturing epidermal cells on the curved surface of the dermal mimic layer,
    인공피부 제조방법.Artificial skin manufacturing method.
  9. 메타크릴화 젤라틴, 메타크릴화 히알루론산, 및 진피세포가 혼합된 프리겔 조성물을 굴곡진 표면의 몰드 위에서 가교시키는 단계; 및 crosslinking a pregel composition in which methacrylated gelatin, methacrylated hyaluronic acid, and dermal cells are mixed on a mold having a curved surface; and
    상기 몰드를 제거하여 굴곡진 일면을 갖는 진피 모사층을 얻는 단계; Obtaining a dermis-like layer having a curved surface by removing the mold;
    상기 진피 모사층의 굴곡진 일면 위에 표피세포를 파종하고 배양하여 표피 모사층을 형성시키는 단계; 및 Forming an epidermal mimic layer by seeding and culturing epidermal cells on the curved surface of the dermal mimic layer; and
    모낭 스페로이드를 진피 모사층 또는 진피 모사층과 표피 모사층의 경계면에 주입하는 단계를 포함하는, Including the step of injecting the hair follicle spheroid into the dermis-like layer or the interface between the dermal-like layer and the epidermal-like layer,
    인공피부 제조방법.Artificial skin manufacturing method.
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