Manipulated skin equivalent, manufacturing method thereof and product derived therefrom

Abstract

To provide a manipulated skin equivalent, a manufacturing method thereof, and a product derived therefrom.SOLUTION: A synthetic leather manufacturing method is disclosed in the specification. The method in some embodiments may include a step to form an artificial dermal layer including fibroblast. The method in some embodiments may include a step to form a synthetic leather by tanning at least a portion of a dermal layer. Fibroblast may be differentiated from artificial pluripotent stem cell in some embodiments.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Application No. 62 / 325,819, filed April 21, 2016, which is incorporated by reference in its entirety.

[0002] Government support This invention was made with support from the National Institutes of Health (NIH) grant numbers R21 ARO61583 and R01 AR051930, the Medical Research Council (UK) grant number G0801061, the Research Service of the Department of Veterans Affairs and Dystrophic Epidermolysis Bullosa Research Association. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein is a method of making synthetic leather. In some embodiments, the method may include forming an artificial dermal layer comprising fibroblasts. In some embodiments, the method may include tanning at least a portion of the dermal layer, thereby forming the synthetic leather. In some embodiments, the fibroblasts may be differentiated from induced pluripotent stem cells. In some embodiments, the method may further include forming an artificial epidermal layer. In some embodiments, the epidermal layer may include keratinocytes. In some embodiments, the method may include disposing the epidermal layer on the dermal layer, thereby forming a layered structure. In some embodiments, the keratinocytes may be mammalian keratinocytes. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the layered structure may further comprise melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the synthetic leather may comprise a pigment. In some embodiments, the melanocytes may be mammalian melanocytes. In some embodiments, the mammal may be human. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the non-human mammal may be one of a primate, a cow, a sheep, a pig, an equine, a dog, a cat, a rodent, or a rabbit.In some embodiments, the fibroblasts may be non-mammalian fibroblasts. In some embodiments, the non-mammalian may be fish, bird or reptile. In some embodiments, the epidermal layer may further comprise collagen. In some embodiments, the epidermal layer may be subjected to further processing. In some embodiments, the layered structure may further comprise collagen. In some embodiments, the layered structure may be subjected to further processing. In some embodiments, the dermal layer may further comprise collagen. In some embodiments, the dermal layer may be subjected to further processing. In some embodiments, the treatment is preserving, soaking, baiting, pickling, depickling, thinning, retanning, lubricating, crusting, moistening, sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixing. The method may be selected from the group consisting of fixing, setting, drying, conditioning, milling, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, tumbling, and any combination thereof. In some embodiments, the collagen may be at least partially produced by collagen-producing cells, may be added separately, or any combination thereof. In some embodiments, the collagen-producing cells may include epithelial cells, keratinocytes, fibroblasts, comeocytes, melanocytes, Langerhans cells, basal cells, or combinations thereof.In some embodiments, the collagen-producing cells may include epithelial cells, which may include squamous cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may include keratinocytes, which may include epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, the collagen-producing cells may include smooth muscle cells. In some embodiments, the synthetic leather may further include one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the thickness of the dermis layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermal layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the synthetic leather may further include a first dermal layer and a second dermal layer. In some embodiments, the first dermal layer may be disposed on the second dermal layer. In some embodiments, the thickness of the epidermal layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermal layer may range from about 0.1 mm to about 0.2 mm. In some embodiments, the synthetic leather may further include a first epidermal layer and a second epidermal layer. In some embodiments, the synthetic leather may further include a basement membrane substitute. In some embodiments, the basement membrane substitute may be between the epidermal layer and the dermal layer. In some embodiments, the basement membrane substitute may include dried acellular amniotic membrane. In some embodiments, the dermal layer may be formed on a scaffold. In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may include silk. In some embodiments, the scaffold may comprise chitosan. In some embodiments, the scaffold may comprise a natural tissue adhesive. In some embodiments, the natural tissue adhesive may comprise fibrin glue. In some embodiments, the scaffold may be partially comprised in synthetic leather. In some embodiments, the dermal or epidermal layer may be cultured in vitro.In some embodiments, the dermal layer can be cultured in vitro. In some embodiments, the dermal layer can be cultured with a supplement. In some embodiments, the supplement can include one or more of collagen, fibrin, growth factors, ascorbic acid, dextran sulfate, or carrageenan. In some embodiments, the supplement can be a natural supplement. In some embodiments, the supplement can be a synthetic supplement. In some embodiments, the induced pluripotent stem cells can be produced through inducible gene expression of Oct3, Oct4, Sox2, Klf4, c-Myc, or combinations thereof in adult somatic cells. In some embodiments, at least a portion of the leather product can be formed from the methods disclosed herein. In some embodiments, the leather product may include one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, a garment, a bag, a pouch, a clutch, a wallet, a backpack, a billfold, a saddle, a harness, a whip, an interior, an exterior, an upholstery, a book binding, a furniture, a lamp, a lamp shade, a table covering, a wall covering, a floor covering, a ceiling covering, an automobile interior, an automobile exterior, a boat interior, a boat exterior, an airplane interior, a yacht interior, a yacht exterior, a pillowcase, a sheet, a duvet cover, jewelry, an accessory, an eyeglass, a sunglasses, or a consumer electronics product. In some embodiments, the leather product may be a watch band. In some embodiments, the leather product may be a belt. In some embodiments, the leather product may be a pouch. At least about 2% of the cells in the synthetic leather may be differentiated from induced pluripotent stem cells. At least about 10% of the cells in the synthetic leather may be differentiated from induced pluripotent stem cells.At least about 50% of the cells in the synthetic leather may be differentiated from induced pluripotent stem cells.

[0004] Disclosed herein is a method of making synthetic leather. In some embodiments, the method may include disposing an artificial epidermal layer on an artificial dermal layer, thereby forming a layered structure. In some embodiments, the epidermal layer may include keratinocytes and the dermal layer may include fibroblasts. In some embodiments, the method may include tanning at least a portion of the layered structure, thereby forming the synthetic leather. In some embodiments, the fibroblasts or keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the layered structure may further include melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes can express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the synthetic leather can include a pigment. In some embodiments, the melanocytes can be mammalian melanocytes. In some embodiments, the mammal can be human. In some embodiments, the fibroblasts can be differentiated from induced pluripotent stem cells. In some embodiments, the fibroblasts can express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts can be mammalian fibroblasts. In some embodiments, the mammal can be a non-human mammal. In some embodiments, the non-human mammal can be one of a primate, bovine, ovine, porcine, equinine, canine, feline, rodent, or rabbit. In some embodiments, the fibroblasts can be non-mammalian fibroblasts. In some embodiments, the non-mammal can be a fish, a bird, or a reptile. In some embodiments, the epidermal layer may further comprise collagen.In some embodiments, the epidermis layer may be subjected to further processing. In some embodiments, the layered structure may further comprise collagen. In some embodiments, the layered structure may be subjected to further processing. In some embodiments, the dermis layer may be subjected to further processing. In some embodiments, the processing may be preserving, soaking, baiting, pickling, depickling, thinning, retanning, lubricating, crusting, moistening, sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixing. The method may be selected from the group consisting of fixing, setting, drying, conditioning, milling, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, tumbling, and any combination thereof. In some embodiments, the collagen may be at least partially produced by collagen-producing cells, may be added separately, or any combination thereof. In some embodiments, the collagen-producing cells may include epithelial cells, and the epithelial cells may include squamous cells, cuboidal cells, columnar cells, basal cells, or any combination thereof.In some embodiments, the collagen-producing cells may include keratinocytes, which may include epidermal keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, the collagen-producing cells may include smooth muscle cells. In some embodiments, the synthetic leather may further include one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the thickness of the dermal layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermal layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the synthetic leather may further include a first dermal layer and a second dermal layer. In some embodiments, the first dermal layer can be disposed on the second dermal layer. In some embodiments, the thickness of the epidermal layer can range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermal layer can range from about 0.1 mm to about 0.2 mm. In some embodiments, the synthetic leather can further include a first epidermal layer and a second epidermal layer. In some embodiments, the synthetic leather can further include a basement membrane substitute. In some embodiments, the basement membrane substitute can be between the epidermal layer and the dermal layer. In some embodiments, the basement membrane substitute can include dried acellular amniotic membrane. In some embodiments, the dermal layer can be formed on a scaffold. In some embodiments, the scaffold can be natural or synthetic. In some embodiments, the scaffold can include silk. In some embodiments, the scaffold can include chitosan. In some embodiments, the scaffold can include a natural tissue adhesive. In some embodiments, the natural tissue adhesive can include fibrin glue. In some embodiments, the scaffold can be partially included in the synthetic leather. In some embodiments, the dermal layer or epidermal layer can be cultured in vitro. In some embodiments, the dermal layer can be cultured in vitro. In some embodiments, the dermal layer can be cultured with supplements.In some embodiments, the supplement may include one or more of collagen, fibrin, growth factors, ascorbic acid, dextran sulfate, or carrageenan. In some embodiments, the supplement may be a natural supplement. In some embodiments, the supplement may be a synthetic supplement. In some embodiments, the induced pluripotent stem cells may be produced through inducible gene expression of Oct3, Oct4, Sox2, Klf4, c-Myc, or combinations thereof in adult somatic cells. In some embodiments, at least a portion of the leather product may be formed from the methods disclosed herein. In some embodiments, the leather product may include one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, a garment, a bag, a pouch, a clutch, a wallet, a backpack, a billfold, a saddle, a harness, a whip, an interior, an exterior, an upholstery, a book binding, a furniture, a lamp, a lamp shade, a table covering, a wall covering, a floor covering, a ceiling covering, an automobile interior, an automobile exterior, a boat interior, a boat exterior, an airplane interior, a yacht interior, a yacht exterior, a pillowcase, a sheet, a duvet cover, jewelry, an accessory, an eyeglass, a sunglasses, or a consumer electronics product. In some embodiments, the leather product may be a watch band. In some embodiments, the leather product may be a belt. In some embodiments, the leather product may be a pouch. At least about 2% of the cells in the synthetic leather may be differentiated from induced pluripotent stem cells. At least about 10% of the cells in the synthetic leather may be differentiated from induced pluripotent stem cells.At least about 50% of the cells in the synthetic leather may be differentiated from induced pluripotent stem cells.

[0005] Disclosed herein is a method of making synthetic leather. In some embodiments, the method may include disposing an artificial epidermal layer on an artificial dermal layer, thereby forming a layered structure. In some embodiments, the epidermal layer may include keratinocytes and the dermal layer may include fibroblasts. In some embodiments, the method may include removing at least a portion of the epidermal layer from the layered structure to form a removal product. In some embodiments, the method may include tanning at least a portion of the removal product, thereby forming synthetic leather. In some embodiments, the fibroblasts or keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the layered structure may further include melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the synthetic leather may include pigments. In some embodiments, the melanocytes may be mammalian melanocytes. In some embodiments, the mammal may be human. In some embodiments, the fibroblasts may be differentiated from induced pluripotent stem cells. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the removal product may further include collagen. In some embodiments, the removal product may be subjected to further processing. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the mammal may be a non-human mammal.In some embodiments, the non-human mammal may be one of a primate, a cow, a sheep, a pig, an equine, a dog, a cat, a rodent, or a rabbit. In some embodiments, the fibroblasts may be non-mammalian fibroblasts. In some embodiments, the non-mammal may be a fish, a bird, or a reptile. In some embodiments, the epidermal layer may further comprise collagen. In some embodiments, the epidermal layer may be subjected to further processing. In some embodiments, the layered structure may further comprise collagen. In some embodiments, the layered structure may be subjected to further processing. In some embodiments, the dermal layer may be subjected to further processing. In some embodiments, the treatment is preserving, soaking, baiting, pickling, depickling, thinning, retanning, lubricating, crusting, moistening, sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixing. The method may be selected from the group consisting of fixing, setting, drying, conditioning, milling, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, tumbling, and any combination thereof. In some embodiments, the collagen may be at least partially produced by collagen-producing cells, may be added separately, or any combination thereof. In some embodiments, the collagen-producing cells may include epithelial cells, keratinocytes, fibroblasts, comeocytes, melanocytes, Langerhans cells, basal cells, or combinations thereof.In some embodiments, the collagen-producing cells may include epithelial cells, which may include squamous cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may include keratinocytes, which may include epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, the collagen-producing cells may include smooth muscle cells. In some embodiments, the synthetic leather may further include one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the thickness of the dermis layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermal layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the synthetic leather may further include a first dermal layer and a second dermal layer. In some embodiments, the first dermal layer may be disposed on the second dermal layer. In some embodiments, the thickness of the epidermal layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermal layer may range from about 0.1 mm to about 0.2 mm. In some embodiments, the synthetic leather may further include a first epidermal layer and a second epidermal layer. In some embodiments, the synthetic leather may further include a basement membrane substitute. In some embodiments, the basement membrane substitute may be between the epidermal layer and the dermal layer. In some embodiments, the basement membrane substitute may include dried acellular amniotic membrane. In some embodiments, the dermal layer may be formed on a scaffold. In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may include silk. In some embodiments, the scaffold may comprise chitosan. In some embodiments, the scaffold may comprise a natural tissue adhesive. In some embodiments, the natural tissue adhesive may comprise fibrin glue. In some embodiments, the scaffold may be partially comprised in synthetic leather. In some embodiments, the dermal or epidermal layer may be cultured in vitro.In some embodiments, the dermal layer can be cultured in vitro. In some embodiments, the dermal layer can be cultured with a supplement. In some embodiments, the supplement can include one or more of collagen, fibrin, growth factors, ascorbic acid, dextran sulfate, or carrageenan. In some embodiments, the supplement can be a natural supplement. In some embodiments, the supplement can be a synthetic supplement. In some embodiments, the induced pluripotent stem cells can be produced through inducible gene expression of Oct3, Oct4, Sox2, Klf4, c-Myc, or combinations thereof in adult somatic cells. In some embodiments, at least a portion of the leather product can be formed from the methods disclosed herein. In some embodiments, the leather product may include one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, a garment, a bag, a pouch, a clutch, a wallet, a backpack, a billfold, a saddle, a harness, a whip, an interior, an exterior, an upholstery, a bookbinding material, a furniture, a lamp, a lampshade, a table covering, a wallpaper, a floor covering, a ceiling covering, an automobile interior, an automobile exterior, a boat interior, a boat exterior, an airplane interior, a yacht interior, a yacht exterior, a pillowcase, a sheet, a duvet cover, jewelry, an accessory, an eyeglass, a sunglasses, or a consumer electronics. In some embodiments, the leather product may be a watch band. In some embodiments, the leather product may be a belt. In some embodiments, the leather product may be a pouch. At least about 2% of the cells included in the synthetic leather may be differentiated from induced pluripotent stem cells. At least about 10% of the cells included in the synthetic leather may be differentiated from induced pluripotent stem cells. At least about 50% of the cells included in the synthetic leather may be differentiated from induced pluripotent stem cells.

[0006] Disclosed herein is a synthetic tanned leather. In some embodiments, the synthetic tanned leather may include an artificial dermal layer including fibroblasts prior to tanning. In some embodiments, the fibroblasts may be differentiated from artificial pluripotent stem cells. In some embodiments, the synthetic tanned leather may further include an artificial epidermal layer prior to tanning. In some embodiments, the epidermal layer may further include keratinocytes. In some embodiments, the epidermal layer may be on the dermal layer, thereby forming a layered structure. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the keratinocytes may be differentiated from artificial pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the layered structure may further include melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the synthetic leather may further comprise a pigment. In some embodiments, the melanocytes may be mammalian melanocytes. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the non-human mammal may be one of a primate, bovine, ovine, porcine, equinine, canine, feline, rodent, or rabbit. In some embodiments, the fibroblasts may be non-mammalian fibroblasts. In some embodiments, the non-mammal may be a fish, a bird, or a reptile. In some embodiments, the epidermal layer may further comprise collagen. In some embodiments, the layered structure may further comprise collagen.In some embodiments, the dermal layer may further comprise collagen. In some embodiments, the collagen may be at least partially produced by collagen-producing cells, may be added separately, or any combination thereof. In some embodiments, the collagen-producing cells may comprise epithelial cells, keratinocytes, fibroblasts, comeocytes, melanocytes, Langerhans cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may comprise epithelial cells, which may comprise squamous cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may comprise keratinocytes, which may comprise epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, the collagen-producing cells may comprise smooth muscle cells. In some embodiments, the synthetic leather may further comprise one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the thickness of the dermis layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermis layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the synthetic leather may further comprise a first dermis layer and a second dermis layer. In some embodiments, the first dermis layer may be on the second dermis layer. In some embodiments, the thickness of the epidermis layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermis layer may range from about 0.1 mm to about 0.2 mm. In some embodiments, the synthetic leather may further include a first epidermal layer and a second epidermal layer. In some embodiments, the synthetic leather may include a basement membrane substitute. In some embodiments, the basement membrane substitute may be between the epidermal layer and the dermal layer. In some embodiments, the basement membrane substitute may include dried acellular amniotic membrane. In some embodiments, the dermal layer may be formed on a scaffold. In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may include silk.In some embodiments, the scaffold may comprise chitosan. In some embodiments, the scaffold may comprise a natural tissue adhesive. In some embodiments, the natural tissue adhesive may comprise fibrin glue. In some embodiments, the scaffold may be partially comprised in synthetic tanned leather. In some embodiments, the dermis layer or epidermis layer may be cultured in vitro. In some embodiments, the dermis layer may be cultured in vitro. In some embodiments, the synthetic tanned leather may be comprised in one or more of watch bands, belts, packaging, shoes, boots, footwear, gloves, clothing, bags, sacks, clutches, wallets, backpacks, billfolds, saddles, harnesses, whips, interiors, exteriors, upholstery, bookbinding materials, furniture, lamps, lampshades, table coverings, wallpaper, flooring, ceiling coverings, automobile interiors, automobile exteriors, boat interiors, boat exteriors, airplane interiors, yacht interiors, yacht exteriors, pillowcases, sheets, duvet covers, jewelry, accessories, eyeglasses, sunglasses, or consumer electronics. In some embodiments, the synthetic tanned leather may be included in a watch band. In some embodiments, the synthetic tanned leather may be included in a belt. In some embodiments, the synthetic tanned leather may be included in a bag. At least about 2% of the cells included in the synthetic tanned leather may be differentiated from induced pluripotent stem cells. At least about 10% of the cells included in the synthetic tanned leather may be differentiated from induced pluripotent stem cells. At least about 50% of the cells included in the synthetic tanned leather may be differentiated from induced pluripotent stem cells.

[0007] Disclosed herein is a synthetic tanned leather. In some embodiments, the synthetic tanned leather may include a layered structure prior to tanning. In some embodiments, the layered structure may include an artificial dermal layer. In some embodiments, the dermal layer may include fibroblasts. In some embodiments, the layered structure may include an artificial epidermal layer. In some embodiments, the epidermal layer may include keratinocytes. In some embodiments, the fibroblasts or keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the fibroblasts may be differentiated from induced pluripotent stem cells. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the layered structure may further include melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the synthetic leather may further include a pigment. In some embodiments, the synthetic leather may further include a pigment. In some embodiments, the melanocytes may be mammalian melanocytes. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the non-human mammal may be one of a primate, bovine, ovine, porcine, equinine, canine, feline, rodent, or rabbit. In some embodiments, the fibroblasts may be non-mammalian fibroblasts. In some embodiments, the epidermal layer may further comprise collagen.In some embodiments, the layered structure may further comprise collagen. In some embodiments, the dermal layer may further comprise collagen. In some embodiments, the collagen may be at least partially produced by collagen-producing cells, may be added separately, or any combination thereof. In some embodiments, the collagen-producing cells may comprise epithelial cells, keratinocytes, fibroblasts, comeocytes, melanocytes, Langerhans cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may comprise epithelial cells, which may comprise squamous cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may comprise keratinocytes, which may comprise epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, the collagen-producing cells may comprise smooth muscle cells. In some embodiments, the synthetic leather may further comprise one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the thickness of the dermis layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermis layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the synthetic leather may further comprise a first dermis layer and a second dermis layer. In some embodiments, the first dermis layer may be on the second dermis layer. In some embodiments, the thickness of the epidermis layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermis layer may range from about 0.1 mm to about 0.2 mm. In some embodiments, the synthetic leather may further include a first epidermal layer and a second epidermal layer. In some embodiments, the synthetic leather may include a basement membrane substitute. In some embodiments, the basement membrane substitute may be between the epidermal layer and the dermal layer. In some embodiments, the basement membrane substitute may include a dried acellular amniotic membrane. In some embodiments, the dermal layer may be formed on the scaffold.In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may comprise silk. In some embodiments, the scaffold may comprise chitosan. In some embodiments, the scaffold may comprise a natural tissue adhesive. In some embodiments, the natural tissue adhesive may comprise fibrin glue. In some embodiments, the scaffold may be partially comprised in synthetic tanned leather. In some embodiments, the dermal or epidermal layer may be cultured in vitro. In some embodiments, the dermal layer may be cultured in vitro. In some embodiments, the synthetic tanned leather may be included in one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, clothing, a bag, a pouch, a clutch, a wallet, a backpack, a billfold, a saddle, a harness, a whip, an interior, an exterior, an upholstery, a bookbinding material, a furniture, a lamp, a lampshade, a table covering, a wallpaper, a floor covering, a ceiling covering, an automobile interior, an automobile exterior, a boat interior, a boat exterior, an airplane interior, a yacht interior, a yacht exterior, a pillowcase, a sheet, a duvet cover, jewelry, an accessory, an eyeglass, a sunglasses, or a consumer electronics product. In some embodiments, the synthetic tanned leather may be included in a watch band. In some embodiments, the synthetic tanned leather may be included in a belt. In some embodiments, the synthetic tanned leather may be included in a pouch. At least about 2% of the cells included in the synthetic tanned leather may be differentiated from induced pluripotent stem cells. At least about 10% of the cells contained in the synthetic tanned leather may be differentiated from induced pluripotent stem cells.At least about 50% of the cells contained in the synthetic tanned leather may be differentiated from induced pluripotent stem cells.

[0008] Disclosed herein is a synthetic tanned leather. In some embodiments, the synthetic tanned leather may include a removal product including a layered structure prior to tanning. In some embodiments, the layered structure may include an artificial dermal layer. In some embodiments, the dermal layer may include fibroblasts. In some embodiments, the layered structure may include an artificial epidermal layer. In some embodiments, the epidermal layer may include keratinocytes. In some embodiments, a portion of the epidermal layer may be removed. In some embodiments, the fibroblasts or keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the removal product may further include collagen. In some embodiments, the fibroblasts may be differentiated from induced pluripotent stem cells. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes can express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the layered structure can further include melanocytes. In some embodiments, the melanocytes can be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes can express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the synthetic leather can further include a pigment. In some embodiments, the synthetic leather can further include a pigment. In some embodiments, the melanocytes can be mammalian melanocytes. In some embodiments, the mammal can be a non-human mammal. In some embodiments, the fibroblasts can express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts can be mammalian fibroblasts. In some embodiments, the mammal may be a non-human mammal, hi some embodiments, the non-human mammal may be one of a primate, cow, sheep, pig, equine, dog, cat, rodent, or rabbit.In some embodiments, the fibroblasts may be non-mammalian fibroblasts. In some embodiments, the epidermal layer may further comprise collagen. In some embodiments, the layered structure may further comprise collagen. In some embodiments, the dermal layer may further comprise collagen. In some embodiments, the collagen may be at least partially produced by collagen-producing cells, may be added separately, or any combination thereof. In some embodiments, the collagen-producing cells may comprise epithelial cells, keratinocytes, fibroblasts, comeocytes, melanocytes, Langerhans cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may comprise epithelial cells, which may comprise squamous cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. In some embodiments, the collagen-producing cells may comprise keratinocytes, which may comprise epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, the collagen-producing cells may comprise smooth muscle cells. In some embodiments, the synthetic leather may further comprise one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the thickness of the dermis layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermis layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the synthetic leather may further comprise a first dermis layer and a second dermis layer. In some embodiments, the first dermis layer may be on the second dermis layer. In some embodiments, the thickness of the epidermis layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermis layer may range from about 0.1 mm to about 0.2 mm. In some embodiments, the synthetic leather may further include a first epidermis layer and a second epidermis layer. In some embodiments, the synthetic leather may include a basement membrane substitute. In some embodiments, the basement membrane substitute may be between the epidermis layer and the dermis layer.In some embodiments, the basement membrane substitute may comprise dried acellular amniotic membrane. In some embodiments, the dermal layer may be formed on a scaffold. In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may comprise silk. In some embodiments, the scaffold may comprise chitosan. In some embodiments, the scaffold may comprise a natural tissue adhesive. In some embodiments, the natural tissue adhesive may comprise fibrin glue. In some embodiments, the scaffold may be partially comprised in a synthetic tanned leather. In some embodiments, the dermal or epidermal layer may be cultured in vitro. In some embodiments, the dermal layer may be cultured in vitro. In some embodiments, the synthetic tanned leather may be included in one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, clothing, a bag, a pouch, a clutch, a wallet, a backpack, a billfold, a saddle, a harness, a whip, an interior, an exterior, an upholstery, a bookbinding material, a furniture, a lamp, a lampshade, a table covering, a wallpaper, a floor covering, a ceiling covering, an automobile interior, an automobile exterior, a boat interior, a boat exterior, an airplane interior, a yacht interior, a yacht exterior, a pillowcase, a sheet, a duvet cover, jewelry, an accessory, an eyeglass, a sunglasses, or a consumer electronics product. In some embodiments, the synthetic tanned leather may be included in a watch band. In some embodiments, the synthetic tanned leather may be included in a belt. In some embodiments, the synthetic tanned leather may be included in a pouch. At least about 2% of the cells included in the synthetic tanned leather may be differentiated from induced pluripotent stem cells. At least about 10% of the cells contained in the synthetic tanned leather may be differentiated from induced pluripotent stem cells.At least about 50% of the cells contained in the synthetic tanned leather may be differentiated from induced pluripotent stem cells.

[0009] Disclosed herein is an artificial epidermal layer. In some embodiments, the artificial epidermal layer may include hair follicle cells and melanocytes. In some embodiments, the artificial epidermal layer may include hair follicle cells. In some embodiments, the artificial epidermal layer may include melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the hair follicle cells may include dermal papilla cells, outer root sheath cells, or a combination thereof. In some embodiments, the melanocytes may be mammalian melanocytes. In some embodiments, the epidermal layer may further include keratinocytes. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the keratinocytes may be mammalian keratinocytes. In some embodiments, the mammal may be a non-human mammal. In some embodiments, the mammal may be a human mammal. In some embodiments, the non-human mammal may be one of a primate, a cow, an sheep, a pig, a horse, a dog, a cat, a rodent, or a rabbit. In some embodiments, the fibroblasts may be non-mammalian fibroblasts. In some embodiments, the non-mammal may be a fish, a bird, or a reptile. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the epidermal layer may include hair follicles. At least about 2% of the cells included in the artificial epidermal layer may be differentiated from induced pluripotent stem cells. At least about 10% of the cells included in the artificial epidermal layer may be differentiated from induced pluripotent stem cells. At least about 50% of the cells included in the artificial epidermal layer may be differentiated from induced pluripotent stem cells.

[0010] Disclosed herein is a layered structure. In some embodiments, the layered structure may include an artificial epidermal layer. In some embodiments, the epidermal layer may include hair follicle cells. In some embodiments, the layered structure may include an artificial dermal layer. In some embodiments, the dermal layer may include fibroblasts. In some embodiments, the fibroblasts may be differentiated from induced pluripotent stem cells. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the epidermal layer may further include keratinocytes. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the hair follicle cells may be dermal papilla cells, outer root sheath cells, or a combination thereof. In some embodiments, the epidermal layer may further comprise melanocytes. In some embodiments, the melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the layered structure may be pigmented. In some embodiments, the epidermal layer may be stratified. In some embodiments, the layered structure may comprise a basement membrane substitute. In some embodiments, the basement membrane substitute may be between the epidermal layer and the dermal layer. In some embodiments, the basement membrane substitute may comprise dried acellular amniotic membrane. In some embodiments, the layered structure may further comprise a scaffold. In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may comprise silk. In some embodiments, the scaffold may comprise chitosan. In some embodiments, the dermal layer may be on a scaffold.In some embodiments, the layered structure may include one or more components selected from the group consisting of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. In some embodiments, the layered structure may further include two or more dermal layers. In some embodiments, the layered structure may further include a hair follicle. In some embodiments, the layered structure may further include a soft skin. At least about 2% of the cells included in the layered structure may be differentiated from induced pluripotent stem cells. At least about 10% of the cells included in the layered structure may be differentiated from induced pluripotent stem cells. At least about 50% of the cells included in the layered structure may be differentiated from induced pluripotent stem cells.

[0011] Disclosed herein is a method of producing a layered structure. In some embodiments, the method may include disposing an artificial epidermal layer comprising hair follicle cells on an artificial dermal layer comprising cells differentiated from induced pluripotent stem cells, thereby forming a layered structure. In some embodiments, the cells differentiated from induced pluripotent stem cells may be fibroblasts, melanocytes, keratinocytes, or a combination thereof. In some embodiments, the cells differentiated from induced pluripotent stem cells may be fibroblasts. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the epidermal layer may further comprise keratinocytes. In some embodiments, the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the keratinocytes can express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the hair follicle cells can include dermal papilla cells, outer root sheath cells, or a combination thereof. In some embodiments, the epidermal layer can further include melanocytes. In some embodiments, the melanocytes can be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes can express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the dermal layer can be cultured with a supplement. In some embodiments, the supplement can include collagen, fibrin, growth factors, ascorbic acid, dextran sulfate, carrageenan, or a combination thereof. In some embodiments, the supplement can be a natural supplement. In some embodiments, the supplement can be a synthetic supplement. In some embodiments, the dermal layer can be cultured on a scaffold. In some embodiments, the scaffold may be natural or synthetic. In some embodiments, the scaffold may comprise silk. In some embodiments, the scaffold may comprise chitosan. In some embodiments, a dermal layer may be disposed on the scaffold.In some embodiments, the epidermal layer may be stratified. In some embodiments, the dermal layer may be cultured on a second dermal layer. In some embodiments, the dermal layer may be cultured in vivo. In some embodiments, the dermal layer may not be cultured on a collagen matrix. In some embodiments, the thickness of the dermal layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermal layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the thickness of the epidermal layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermal layer may range from about 0.1 mm to about 0.2 mm. At least about 2% of the cells included in the layered structure may be differentiated from induced pluripotent stem cells. At least about 10% of the cells included in the layered structure may be differentiated from induced pluripotent stem cells. At least about 50% of the cells included in the layered structure may be differentiated from induced pluripotent stem cells.

[0012] A layered structure is disclosed herein. In some embodiments, the layered structure may include an artificial epidermal layer comprising hair follicle cells and keratinocytes or melanocytes; an artificial dermal layer comprising fibroblasts, and the fibroblasts, keratinocytes or melanocytes may be differentiated from artificial pluripotent stem cells, the melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof, the fibroblasts express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta or a combination thereof, and the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. In some embodiments, the layered structure may include an artificial epidermal layer. In some embodiments, the epidermal layer may include hair follicle cells. In some embodiments, the epidermal layer may include keratinocytes or melanocytes. In some embodiments, the layered structure may include an artificial dermal layer. In some embodiments, the dermal layer may include fibroblasts. In some embodiments, the fibroblasts, keratinocytes, or melanocytes may be differentiated from induced pluripotent stem cells. In some embodiments, the melanocytes may express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA, or a combination thereof. In some embodiments, the fibroblasts may express CD10, CD73, CD44, CD90, collagen type I, collagen type III, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the fibroblasts may be mammalian fibroblasts. In some embodiments, the keratinocytes may express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. In some embodiments, the thickness of the dermis layer may range from about 0.02 mm to about 5 mm. In some embodiments, the thickness of the dermis layer may range from about 0.1 mm to about 0.5 mm. In some embodiments, the thickness of the epidermis layer may range from about 0.01 mm to about 2 mm. In some embodiments, the thickness of the epidermis layer may range from about 0.1 mm to about 0.2 mm.At least about 2% of the cells contained in the layered structure may be differentiated from induced pluripotent stem cells. At least about 10% of the cells contained in the layered structure may be differentiated from induced pluripotent stem cells. At least about 50% of the cells contained in the layered structure may be differentiated from induced pluripotent stem cells.

[0013] Disclosed herein is an artificial epidermal layer. The artificial epidermal layer may include a stratum corneum. The artificial epidermal layer may include a stratum granulosum. The artificial epidermal layer may include a stratum spinosum. The artificial epidermal layer may include a stratum basale. In some embodiments, the stratum corneum, stratum granulosum, stratum spinosum, or stratum basale may be organized as shown in FIG. 6A or FIG. 8A. The thickness of the stratum corneum may range from about 0.01 mm to about 0.05 mm. The thickness of the stratum granulosum may range from about 0.01 mm to about 0.15 mm. The thickness of the stratum spinosum may range from about 0.01 mm to about 0.15 mm. The thickness of the stratum basale may range from about 0.01 mm to about 0.15 mm. The thickness of the stratum corneum may range from about 4% to about 20% of the artificial epidermal layer. The thickness of the stratum granulosum may range from about 4% to about 60% of the artificial epidermal layer. The thickness of the spinous layer may range from about 4% to about 40% of the artificial epidermal layer. The thickness of the basal layer may range from about 4% to about 40% of the artificial epidermal layer. At least about 2% of the cells in the artificial epidermal layer may be differentiated from induced pluripotent stem cells. At least about 10% of the cells in the artificial epidermal layer may be differentiated from induced pluripotent stem cells. At least about 50% of the cells in the artificial epidermal layer may be differentiated from induced pluripotent stem cells.

[0014] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0015] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages thereof described herein will be obtained by reference to the following detailed description and accompanying drawings, which set forth illustrative examples in which the principles of the features described herein are utilized. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows a schematic diagram of synthetic leather production.

[0017] [Diagram 2] 2A-2F show layered structures. FIG. 2A shows a layered structure including an epidermal layer and a dermal layer on a scaffold. FIG. 2B shows a layered structure including an epidermal layer, a basement membrane substitute, and a dermal layer on a scaffold. FIG. 2C shows a layered structure including an epidermal layer and multiple dermal layers on a scaffold. FIG. 2D shows a layered structure including an epidermal layer, a basement membrane substitute, and multiple dermal layers on a scaffold. FIG. 2E shows a layered structure including an epidermal layer, a basement membrane substitute, and multiple dermal layers. FIG. 2F shows a layered structure including an epidermal layer and multiple dermal layers.

[0018] [Diagram 3] FIG. 3 shows the development of a layered structure.

[0019] [Figure 4] 4A-4C show a comparative analysis of leather (FIG. 4A), natural skin (FIG. 4B), and an epidermis equivalent (FIG. 4C).

[0020] [Diagram 5] Figures 5A-5C show a comparative analysis of the stratum corneum of natural skin and epidermal equivalents. Figure 5A shows an epidermal surface image. Figure 5B shows a corneo-desmosome image. Figure 5C shows a CDSN / Hoechst image.

[0021] [Figure 6]Figures 6A-6E show comparative analysis of the granular layer of native skin and epidermal equivalents. Figure 6A shows loricrin (LOR) staining. Figure 6B shows epidermal Ca++ gradients photographed by transmission electron microscopy as electron-dense precipitates. Figure 6C shows assessment of permeability barrier integrity by lanthanum perfusion. Figure 6D shows that tight junction protein 1 / zonula occludens-1 (TJP1 / ZO-1) anchors tight junction chain proteins, which may be fibril-like structures within the lipid bilayer, to the actin cytoskeleton. Figure 6E shows that filaggrin (FLG) monomers, which cluster in tandem to become the large 350 kDa protein precursor known as profilaggrin, are present in keratohyalin granules within the cells of SGs.

[0022] [Figure 7] Figures 7A-7C show lipid bilayer formation in native skin and epidermal equivalents assessed by TEM. Figure 7A shows normal lipid secretion at the border of SC and SG. Figure 7B shows the lamellar body of SG. Figure 7C shows normal lipid bilayer (LB) morphology in native skin.

[0023] [Figure 8] Figures 8A-8C show a comparative analysis of suprabasal markers of native skin and epidermal equivalents, including suprabasal markers keratin 10 (KRT10; Figure 8A), keratin 1 (KRT1; Figure 8B), and desmocollin 1 (DCL1; Figure 8C). Figure 8D shows desmosomes of both in vivo native skin and in vitro generated epidermal equivalents.

[0024] [Figure 9]Figures 9A-9C show a comparative analysis of the basal layer of natural skin and epidermal equivalents. The proliferation markers MKI67 (Figure 9A), keratin 14 (KRT14; Figure 9B), and transcription factor TP63 (Figure 9C) show a typical basal layer distribution of both in vivo natural skin (left panel) and in vitro generated epidermal equivalents. Figure 9D shows hemidesmosomes of both in vivo natural skin and in vitro generated epidermal equivalents.

[0025] [Figure 10] 10A-10F show comparative analysis of extracellular matrix components of basement membrane. FIG. 10A shows integrin β1 expression. FIG. 10B shows fibronectin expression. FIG. 10C shows collagen IV expression. FIG. 10D shows collagen VI expression. FIG. 10E shows collagen VII expression. FIG. 10F shows laminin 5 expression.

[0026] [Figure 11] Figures 11A-11I show the structural analysis of a full thickness skin equivalent (FSE). Figures 11A and 11B show that the cross section of the FSE shows the distinct cell layers of the epidermis at a magnification of 2600x (Figure 11A) and 5200x (Figure 11B). Figure 11C shows the surface of the FSE at a magnification of 900x. Figures 11D-1F show the longitudinal section of a dermal scaffold with the presence of dermal fibroblasts and abundant extracellular matrix at a magnification of 91x (Figure 11D), 162x (Figure 11E) and 405x (Figure 11F). Figures 11G-11I show the dermal scaffold with the presence of dermal fibroblasts and abundant extracellular matrix at a magnification of 80x (Figure 11G), 695x (Figure 11H) and 2700x (Figure 11I).

[0027] [Figure 12-1]Figures 12A-12R show the time course of engineering the dermal equivalent, with Figures 12A-12I showing 2 days after seeding of dermal fibroblasts onto the scaffold at 36x magnification (Figure 12A), 695x magnification (Figure 12B), 1470x magnification (Figure 12C), 7750x magnification (Figure 12D), 2320x magnification (Figure 12E), 2420x magnification (Figure 12F), 6560x magnification (Figure 12G), 17000x magnification (Figure 12H) and 22000x magnification (Figure 12I). Figures 12J-12R show the scaffolds 7 days after seeding with dermal fibroblasts at a magnification of 64x (Figure 12J), 100x (Figure 12K), 364x (Figure 12L), 82x (Figure 12M), 253x (Figure 12N), 3940x (Figure 12O), 5550x (Figure 12P), 9440x (Figure 12Q) and 21680x (Figure 12R). [Figure 12-2] Figures 12A-12R show the time course of engineering the dermal equivalent, with Figures 12A-12I showing 2 days after seeding of dermal fibroblasts onto the scaffold at 36x magnification (Figure 12A), 695x magnification (Figure 12B), 1470x magnification (Figure 12C), 7750x magnification (Figure 12D), 2320x magnification (Figure 12E), 2420x magnification (Figure 12F), 6560x magnification (Figure 12G), 17000x magnification (Figure 12H) and 22000x magnification (Figure 12I). Figures 12J-12R show the scaffolds 7 days after seeding with dermal fibroblasts at a magnification of 64x (Figure 12J), 100x (Figure 12K), 364x (Figure 12L), 82x (Figure 12M), 253x (Figure 12N), 3940x (Figure 12O), 5550x (Figure 12P), 9440x (Figure 12Q) and 21680x (Figure 12R). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Some aspects are described below with reference to application examples for illustration. It should be understood that numerous specific details, relationships, and methods are shown to provide a full understanding of the features described herein. However, those skilled in the art will easily recognize that the features described herein can be implemented without one or more specific details, or by using other methods. Unless specifically indicated, the features described herein are not limited to the illustrated ordering of acts or events, since some acts may occur in different orders and / or simultaneously with other acts or events. Moreover, not all illustrated acts or events are required to perform a method according to the features described herein.

[0029] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "include," "having," "having," "involving," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0030] In this disclosure, the term "about" or "approximately" can mean a range of up to 10% of a given value. In this disclosure, the term "substantially" refers to something that can be done to a great extent or degree.

[0031] As used herein, the term "pluripotent stem cell" may refer to any precursor cell that has the potential to form any adult cell.

[0032] As used herein, the term "embryonic stem cell" or "ES cell" or "ESC" may refer to a precursor cell that has the potential to form any adult cell.

[0033] As used herein, the term "induced pluripotent stem cell" or "iPS cell" or "iPSC" may refer to a type of pluripotent stem cell artificially derived from non-pluripotent cell (e.g. adult somatic cell). Induced pluripotent stem cell may be identical to embryonic stem cell in the ability to form any adult cell, but is not derived from an embryo. Optionally, the IPSC cell disclosed herein may be an IPSC cell.

[0034] As used herein, the term "synthetic leather" may mean that the skin equivalents described herein may serve as skin equivalents for any mammal or non-mammal. The embodiments may be practiced with non-human mammals such as humans, as well as non-human primates and members of bovine, ovine, porcine, equine, canine, and feline species, as well as rodents such as mice, rats, and guinea pigs, members of the family Leporidae, including rabbits; and fish, including sharks and stingrays, birds, including ostriches, and reptiles, including lizards, snakes, and crocodiles. The particular mammalian synthetic leather formed may depend on the source of cells, e.g., keratinocytes and fibroblasts, used in the embodiments described herein; for example, if bovine keratinocytes and fibroblasts are used to form the skin equivalent, a bovine synthetic leather may be formed.

[0035] overview Disclosed herein are synthetic leather, artificial epidermal layer, artificial dermal layer, layered structures, products made therefrom, and methods of producing the same. Disclosed herein in certain cases are synthetic leather. Optionally, the synthetic leather comprises one or more layers. Optionally, the one or more layers comprise cells, and the cells are cultured in vitro. Optionally, the methods described herein provide high-throughput methods for reliably, accurately, and reproducibly scaling up the production of synthetic leather to commercial levels. Advantages of the synthetic leather, engineered epidermal equivalent, engineered full-thickness skin equivalent, and methods of producing the same disclosed herein include, but are not limited to, reproducibly, high-throughput, and easily scalable production of customized tissues of attractive appearance, texture, thickness, and durability. As used herein, a full-thickness skin equivalent may comprise at least one dermal layer and at least one epidermal layer. As used herein, full-thickness skin equivalent and full-thickness skin equivalent can be used interchangeably.

[0036] The synthetic leather disclosed herein may include a layer called an artificial dermis layer, which contains fibroblasts, and an artificial epidermis layer, which contains keratinocytes. The dermis layer and the epidermis layer may form a layered structure. The synthetic leather may include one or more layered structures. The synthetic leather may be tanned and further processed. The cells forming the synthetic layer may be differentiated from stem cells, for example, induced pluripotent stem cells (iPSCs). To achieve the thickness and texture of natural leather, the dermis layer may be placed on a scaffold, such as silk.

[0037] Also disclosed herein is a method for producing synthetic leather. The method may include forming a layered structure including an artificial dermis layer and an artificial epidermis layer, and tanning the layered structure. The method may also include further processing the artificial dermis layer and the epidermis layer, for example, to achieve the thickness and texture of natural leather.

[0038] synthetic leather The synthetic leather may include one or more cell layers. For example, the synthetic leather may include one or more of a dermis layer, an epidermis layer, and a basement membrane or a basement membrane substitute. The synthetic leather may further include a subcutaneous tissue, a scale, a lamina, an osteoderm, or a combination thereof. Optionally, the synthetic layer includes a full thickness skin equivalent. Such a full thickness skin equivalent may include any one or combination of the layers disclosed herein. A portion of one or more cell layers in the synthetic leather may be removed, for example, by shaving. Optionally, the synthetic leather may be tanned. Tanning may be performed after the formation of one or more of the cell layers or layered structures. Tanning may be performed after at least a portion of the cell layer can be removed from the synthetic leather. Optionally, the synthetic leather may be further processed. Optionally, the synthetic leather may include hair follicle cells and melanocytes. The hair follicle cells and / or melanocytes may be differentiated from stem cells (e.g., iPSCs).

[0039] In some embodiments, the synthetic tanned leather may include a layered structure. The layered structure may include an artificial dermal layer containing fibroblasts. The layered structure may also include an artificial epidermal layer containing keratinocytes. Optionally, the layered structure may include an artificial dermal layer containing fibroblasts and an artificial epidermal layer containing keratinocytes. Optionally, the fibroblasts or keratinocytes may be differentiated from induced pluripotent stem cells.

[0040] Optionally, the synthetic leather may include at least a portion of the dermis layer. Optionally, the synthetic leather does not include the dermis layer. Optionally, the dermis layer may be removed.

[0041] dermal layer The synthetic leather may include a dermal layer (e.g., an artificial dermal layer). The dermal layer may be an engineered dermal equivalent, for example, an artificial dermal layer formed in vitro.

[0042] Dermal layer may include cells of connective tissue. For example, dermal layer may include fibroblasts. Fibroblasts in dermal layer may express one or more markers, including but not limited to, cluster of differentiation 10 (CD10), cluster of differentiation 73 (CD73), cluster of differentiation 44 (CD44), cluster of differentiation 90 (CD90), type I collagen, type III collagen, and prolyl-4-hydroxylase beta fibroblast. Optionally, dermal layer also includes other types of cells, such as immune cells, macrophages, adipocytes, or combinations thereof.

[0043] In addition to cells, the dermal layer may further comprise matrix components, including, but not limited to, any one or more of collagen, elastin, and extrafibrillar matrix, an extracellular gel-like substance composed primarily of glycosaminoglycans (e.g., hyaluronan), proteoglycans, and glycoproteins.

[0044] The dermal layer may include a matrix support. The matrix support may be a scaffold. The matrix support may include a contracted collagen gel. Alternatives to pure collagen matrix may be, for example, a polyglygolic acid mesh as described in Hansbrough et al., J. Burn Care Rehabil., 15:346-53 (1994), or a collagen and glycosaminoglycan matrix (C-GAG) covered with a silastic membrane as described in Burke et al., Ann. Surg., 194:413-420 (1981), or various biopolymers, such as chitosan as described in Kellouche et al., Biochem Biophys Res Commun., 363:472-478 (2007). Optionally, the matrix may be seeded with fibroblasts, for example to generate an organotypic model. Naturally derived dermis from allogeneic cadaveric skin may also be used with keratinocyte sheets. A variation of this technique can use freeze-dried devitalized dermis from cadaver skin to support the keratinocyte sheets.

[0045] The thickness of leather may be reported in millimeters, ounces, or irons. (1 ounce is equal to 1 / 64 inch or 0.0156 inch or 0.396 mm. 1 iron is equal to 1 / 48 inch or 0.0208 inch or 0.53 mm.)

[0046] The thickness of the dermis layer may be manipulated to suit the function or use of the synthetic leather. The dermis layer may have a thickness of about 0.01 mm to about 50 mm. For example, the dermis layer may have a thickness of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the dermis layer may have a thickness of about 0.02 mm to 5 mm. For example, the dermis layer may have a thickness of about 0.1 mm to 0.5 mm. For example, the dermis layer may have a thickness of about 0.2 mm to 0.5 mm. In some cases, the thickness of the dermal layer may be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the thickness of the dermal layer may be up to 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the dermal layer may have a thickness of at least about 50 mm.

[0047] The length of the dermis layer may be manipulated to suit the function or use of the synthetic leather. The dermis layer may have a length of about 0.01 mm to about 50 m. For example, the dermis layer may have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the dermis layer may have a length of about 0.02 mm to 5 mm. For example, the dermis layer may have a length of about 0.1 mm to 0.5 mm. For example, the dermis layer may have a length of about 0.2 mm to 0.5 mm. In some cases, the length of the dermal layer may be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the length of the dermal layer may be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the dermal layer may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the dermal layer may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the dermal layer may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0048] The width of the dermis layer may be manipulated to suit the function or use of the synthetic leather. The dermis layer may have a width of about 0.01 mm to about 50 mm. For example, the dermis layer may have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the dermis layer may have a width of about 0.02 mm to 5 mm. For example, the dermis layer may have a width of about 0.1 mm to 0.5 mm. For example, the dermis layer may have a width of about 0.2 mm to 0.5 mm. In some cases, the width of the dermal layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the width of the dermal layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the dermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the dermal layer may have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the dermal layer may have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0049] Synthetic leather may include one or more dermal layers. For example, synthetic leather may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 40, 60, 80, or 100 dermal layers. When synthetic leather includes more than one dermal layer, a dermal layer may be disposed on another dermal layer. For example, synthetic leather may include two dermal layers, such as a first dermal layer and a second dermal layer. The first dermal layer may be disposed on the second dermal layer.

[0050] The dermal layer may be stratified, e.g., with multiple sublayers. The sublayers may have different compositions, e.g., different concentrations of fibers. The sublayers of the dermal layer may have different thicknesses and densities. For example, the dermal layer may have a papillary dermal layer, a reticular dermal layer, or any combination thereof. The papillary dermal layer may include loose areolar connective tissue and / or loosely arranged fibers, e.g., collagen fibers. The reticular dermal layer may include dense irregular connective tissue including collagen fibers and dermal elastic fibers.

[0051] The dermal layer may comprise a free collagen matrix or lattice, which may be contractile and homogeneous in all directions. Fibroblasts, and where appropriate other types of cells of the dermis, may be distributed in a continuous collagen gel. The dermal equivalent may comprise at least one collagen type I matrix in which fibroblasts are distributed. It may also contain other extracellular matrix components. The extracellular matrix components may include collagen, for example collagen IV, laminin, entactin, fibronectin, proteoglycans, glycosaminoglycans or hyaluronic acid. The dermal layer may contain collagen type IV and laminin, entactin, or a combination thereof. The concentrations of these various components may be adjusted. For example, the concentration of laminin may be about 1% to about 15% of the final volume. For example, the concentration of collagen IV may be about 0.3% to about 4.5% of the final volume. For example, the concentration of entactin may be about 0.05% to about 1% of the final volume. The collagen used may be of bovine origin, rat tail or fish origin, or any other source of natural collagen or collagen produced by genetic engineering that allows contraction in the presence of fibroblasts. In some embodiments, the collagen may be from a non-natural source. The matrix may be a collagen gel that may not be taut, obtained by both horizontal and vertical contraction that does not impose preferential organization of fibroblasts. Such a matrix, also called "free", may not adhere to a support and may be given various thicknesses and diameters to modify its volume indefinitely. The thickness of the dermis equivalent may be at least 0.05 cm, and possibly approximately 0.05 to 2 cm. The thickness may also be increased without compromising the advantageous properties of the skin equivalent or synthetic leather. In some cases, the thickness may be from about 3 mm to about 20 cm or more.

[0052] epidermal layer The synthetic leather may include a skin layer (e.g., an artificial skin layer). The skin layer may be an engineered skin equivalent, e.g., an artificial skin layer formed in vitro.

[0053] The epidermis layer may comprise one or more types of cells, including keratinocytes, melanocytes, Langerhans cells, Merkel cells, and inflammatory cells.For example, the epidermis layer may comprise keratinocytes.The keratinocytes in the epidermis layer may comprise epidermal keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or any combination thereof.

[0054] In some cases, the epidermal layer comprises at least basal keratinocytes, e.g., undifferentiated keratinocytes. The epidermal layer may further comprise partially differentiated keratinocytes and fully differentiated keratinocytes. In one or more epidermal layers in synthetic leather, there may be a transition from undifferentiated basal keratinocytes to fully differentiated keratinocytes as the keratinocytes advance from the dermal-epidermal junction where the basal keratinocytes are located.

[0055] Basal keratinocytes can express hemidesmosomes, which serve to help hold the epidermal layer and dermal layer together. Basal keratinocytes can also serve to regenerate skin. The epidermal layer in the synthetic leather herein may have basal keratinocytes that serve these functions. Thus, synthetic leather containing such basal keratinocytes may have regenerative capabilities. Another difference between basal keratinocytes and differentiated keratinocytes in one or more epidermal layers in synthetic leather may be that both E-cadherin and P-cadherin are present in epidermal keratinocytes along the basal membrane zone (BMZ), while differentiated keratinocytes that are localized away from the BMZ express only E-cadherin.

[0056] Basal keratinocytes of the epidermis layer may be aligned in a layer that directly contacts the dermis layer, and serve as a boundary between differentiated keratinocytes and fibroblasts. In other cases, there is a gap between the basal keratinocytes and the dermis layer. Still further, there may be a gap between the basal keratinocytes and other basal keratinocytes, leaving a gap between the differentiated keratinocytes and the dermis layer. In these latter cases, where there is a gap between the basal keratinocytes or the differentiated keratinocytes and the dermis layer, the dermis layer and the epidermis layer are adjacent to each other, not in uniform contact with each other. They are adjacent in that there may be fluid between the dermis layer and the epidermis layer, but there may be substantially no other intervening material, such as cells, collagen, matrix or other support layers.

[0057] Keratinocytes in the epidermal layer can express one or more markers, including, but not limited to, keratin 14 (KRT14), tumor protein p63 (p63), desmoglein 3 (DSG3), integrin beta 4 (ITGB4), laminin alpha 5 (LAMA5), keratin 5 (KRT5), isoforms of tumor protein p63 (e.g., TAp63), laminin beta 3 (LAMB3), and keratin 18 (KRT18).

[0058] The thickness of the skin layer may be manipulated to suit the function or use of the synthetic leather. The skin layer may have a thickness of about 0.001 mm to about 10 mm. For example, the skin layer can have a thickness of about 0.005 mm to about 10 mm, about 0.005 mm to about 5 mm, about 0.005 mm to about 2 mm, about 0.01 mm to about 10 mm, about 0.01 mm to about 5 mm, about 0.01 mm to about 2 mm, about 0.01 mm to about 1, about 0.01 mm to about 0.8 mm, about 0.01 mm to about 0.4 mm, about 0.01 mm to about 0.2 mm, about 0.01 mm to about 0.1 mm, about 0.05 mm to about 0.4 mm, about 0.05 mm to about 0.2 mm, about 0.05 mm to about 0.1 mm, about 0.1 mm to about 0.4 mm, about 0.1 mm to about 0.2 mm, about 0.08 mm to about 1 mm, or about 0.05 mm to about 1.5 mm. For example, the epidermal layer may have a thickness of about 0.01 mm to about 2 mm. For example, the epidermal layer may have a thickness of about 0.1 mm to about 0.22 mm. In some cases, the thickness of the epidermal layer may be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the thickness of the dermal layer may be up to 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some cases, the thickness values ​​described herein may be the thickness of the epidermal layer and basement membrane substitute.

[0059] The length of the skin layer may be manipulated to suit the function or use of the synthetic leather. The skin layer may have a length of about 0.01 mm to about 50 m. For example, the skin layer may have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the skin layer may have a length of about 0.02 mm to 5 mm. For example, the skin layer may have a length of about 0.1 mm to 0.5 mm. For example, the skin layer may have a length of about 0.2 mm to 0.5 mm. In some cases, the length of the skin layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the length of the skin layer can be up to 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the skin layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the skin layer may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the skin layer may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0060] The width of the skin layer may be manipulated to suit the function or use of the synthetic leather. The skin layer may have a width of about 0.01 mm to about 50 mm. For example, the skin layer may have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the skin layer may have a width of about 0.02 mm to 5 mm. For example, the skin layer may have a width of about 0.1 mm to 0.5 mm. For example, the skin layer may have a width of about 0.2 mm to 0.5 mm. In some cases, the width of the skin layer can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the width of the skin layer can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the skin layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the skin layer may have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the skin layer may have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0061] Synthetic leather may include one or more skin layers. For example, synthetic leather may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 40, 60, 80, or 100 skin layers. When synthetic leather includes more than one skin layer, one skin layer may be disposed on another skin layer. For example, synthetic leather may include two skin layers, such as a first skin layer and a second skin layer. The first skin layer may be disposed on the second skin layer.

[0062] The epidermal layer may be stratified, for example, with multiple sublayers. The sublayers may have different cell compositions, for example, different types of keratinocytes. The sublayers of the epidermal layer may have different thicknesses and / or densities. For example, the epidermal layer may have one or more of the following: stratum corneum, clear / translucent layer, stratum granulosum, stratum spinosum, stratum basale / germinativum, or any combination thereof. In some cases, the epidermal layer includes a functional epidermal permeability barrier (e.g., an organized lipid bilayer in the stratum corneum). In some cases, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / germinativum may have a thickness of about 0.0001 mm to about 5 mm. In some cases, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / blastema layer may have a thickness of at least about 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.15 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / blastema layer may have a thickness of at most about 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.15 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm.

[0063] The epidermis layer may further include cells that produce pigments, such as melanin. Such pigment-producing cells may be melanocytes. The melanocytes in the epidermis layer may express one or more markers. Such markers may include, but are not limited to, SRY box-containing gene 10 (Sox-10), microphthalmia-associated transcription factor (MITF-M), pre-melanosomal protein (gp-100), dopachrome tautomerase (DCT), tyrosinase (TYR), and melan A (MLANA).

[0064] Cells in synthetic leather The synthetic leather may include cells in the dermis and epidermis layers disclosed herein. Optionally, the synthetic leather also includes hair follicle cells, endothelial cells, dermal papilla cells, immune system cells (such as lymphocytes, dendritic cells, macrophages, or Langerhans cells), adipocytes, neuronal cells, and mixtures thereof.

[0065] One or more cells in the synthetic leather may be genetically engineered cells. The term "genetically engineered" may refer to an artificial change to the nucleic acid content of a cell. Thus, genetically engineered cells may include cells that contain an insertion, deletion, and / or substitution of one or more nucleotides in the genome of the cell, as well as cells that contain changes that include the introduction of a self-replicating extrachromosomal nucleic acid inserted into the cell. Genetically engineered cells also include cells in which the transcription of one or more genes has been altered, e.g., increased or decreased.

[0066] In some cases, synthetic leather has at least one of the components of natural skin, such as melanocytes, hair follicles, sweat glands, and nerve endings. In certain cases, synthetic leather can be distinguished from normal, natural skin by its lack of at least one of these components. In some cases, synthetic leather can contain all of these components, including at least one cell that exhibits an abnormal phenotype or has an altered genotype.

[0067] In some cases, additional components may be added to the synthetic leather. Such additional components may include myoepithelial cells, ductal cells, secretory cells, alveolar cells, Langerhans cells, Merkel cells, adhesion, mammary gland, or any mixture thereof. In some cases, the synthetic leather includes one or more of nerve cells, connective tissue (including bone, cartilage, osteogenic cells and cells that are differentiated into chondrocytes, and lymphatic tissue), epithelial cells (including endothelial cells that form the lining or channels of cavities and ducts, exocrine epithelial cells, epithelial absorptive cells, cornified epithelial cells, and extracellular matrix secreting cells), and undifferentiated cells (such as embryonic cells, stem cells, and other progenitor cells).

[0068] The synthetic leather may include hair follicles. The hair follicles may include one or more structures including papilla, matrix, root sheath, ridge, infundibulum, arrector pili muscle, sebaceous gland, and apocrine sweat gland. The hair follicles may include one or more hair follicle cells including dermal papilla cells, outer root sheath cells, or any combination thereof. In some cases, the hair follicles may be in the epidermis layer. In some cases, the hair follicles may be in the dermis layer. The hair follicle cells may be differentiated from precursors, for example stem cells, such as iPSCs. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the hair follicle cells may be differentiated from induced pluripotent stem cells.

[0069] In some embodiments, the synthetic leather may lack hair, blood vessels, sebaceous glands, hair follicles, sebaceous glands, nerves, or combinations thereof.

[0070] In some cases, the synthetic leather may include hair, for example in one or more layered structures. For example, the synthetic leather may include ruff. The hair (e.g., ruff) may be natural, synthetic, or a combination thereof. The hair (e.g., ruff) may be grown from cells in the synthetic leather or added to the synthetic leather from an exogenous source. In other cases, the synthetic leather may not have hair at all.

[0071] stem cells One or more cells in the synthetic leather may be differentiated from a precursor cell, such as a stem cell. For example, the fibroblasts in the synthetic leather may be differentiated from a stem cell. For example, the keratinocytes in the synthetic leather may be differentiated from a stem cell. For example, the melanocytes in the synthetic leather may be differentiated from a stem cell. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the cells disclosed herein may be differentiated from a stem cell. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the fibroblasts may be differentiated from induced pluripotent stem cells. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the keratinocytes may be differentiated from induced pluripotent stem cells. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the melanocyte cells may be differentiated from induced pluripotent stem cells.

[0072] The stem cells can be embryonic stem cells (ESCs), adult stem cells (i.e. somatic stem cells) or induced pluripotent stem cells (iPSCs). In some embodiments, the stem cells can be totipotent, pluripotent or multipotent, e.g. adult stem cells and umbilical cord blood stem cells). Embryonic stem cells can be derived from fertilized embryos less than one week old. Induced pluripotent stem cells can be obtained through the induced expression of one or more of Oct3, Oct4, Sox2, Klf4 and c-Myc genes in any somatic cell (e.g. adult somatic cell), such as a fibroblast. Optionally, one or more other genes can be induced to reprogram the somatic cell into an induced pluripotent stem cell. Examples of such genes include NANOG, UTF1, LIN28, SALL4, NR5A2, TBX3, ESSRB, DPPA4, SV40LT, REM2, MDM2 and cyclin D1.

[0073] Various delivery methods may be used to modulate gene expression to reprogram somatic cells into iPSCs. Exemplary delivery methods include naked DNA delivery, adenovirus, electrical delivery, chemical delivery, mechanical delivery, polymer-based systems, microinjection, retrovirus (e.g., MMLV-derived retrovirus), and lentivirus (e.g., excisable lentivirus). Optionally, induced pluripotent stem cells can be obtained according to the protocol described by Takahashi et al., Cell. 2007-11-30;131(5):861-72 (2007), or Yu et al., Science 318,1917-1920 (2007) (2007). Optionally, somatic cells (e.g., adult somatic cells) are transfected with a viral vector, such as a retroviral vector that includes Oct3, Oct4, Sox2, Klf4, and c-Myc genes. In some cases, Sendai virus, for example, Sendai virus manufactured by ID Pharma Co., Ltd. (Japan), is used as a delivery system.

[0074] Cell Source The synthetic leather may include cells derived from one or more species of animals. For example, the cells in the synthetic leather may be derived from a mammal, a bird, a reptile, an amphibian, a fish, an invertebrate, or any combination thereof.

[0075] The synthetic leather may include a mammal, e.g., a mammalian cell, or a cell derived from a non-mammal. The mammal may be a non-human mammal. The non-human mammal may be an antelope, bear, beaver, bison, wild boar, camel, caribou, cat, cow, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, bull, peccary, pig, rabbit, rhino, seal, sheep, squirrel, tiger, whale, wolf, yak, or zebra. In some cases, the mammal may be a primate, cow, sheep, pig, horse, dog, cat, rodent, or rabbit. The non-mammal may be a fish, bird, or reptile. In some cases, the mammal may be a human. In some embodiments, the human may be a celebrity. As used herein, the term "celebrity" may be defined as a person who has come into the public eye due to the reputation of previous activities or general fame. "Celebrities" may be associated with industries including, but not limited to, professional and amateur sports, entertainment, music, film, business, print and electronic media, politics, etc.

[0076] The synthetic leather may contain cells from other species. Optionally, the cells are from birds such as chickens, ducks, emus, geese, grouse, ostriches, pheasants, pigeons, quails, or turkeys. Optionally, the cells are from reptiles such as turtles, snakes, crocodiles, or alligators. Optionally, the cells are from amphibians such as frogs, toads, salamanders, or newts. In some cases, the cells are derived from fish such as anchovy, bass, catfish, carp, cod, eel, flounder, pufferfish, grouper, haddock, halibut, herring, mackerel, mahi-mahi, manta ray, marlin, orange roughy, perch, pike, pollack, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, or walleye.

[0077] In some cases, all the cells in the synthetic leather are from the same species. For example, all the cells in the synthetic leather can be bovine cells. In other cases, the synthetic leather contains cells from multiple species. For example, the synthetic leather can contain bovine cells and alligator cells. In some cases, the synthetic leather contains cells from at least 2, 3, 4, 5, 6, 7, 8, or 10 species.

[0078] The precursors of cells in synthetic leather can also be derived from sources described herein.For example, stem cells (e.g., iPSCs), somatic cells (e.g., for reprogramming into iPSCs), primary cells used for synthetic cells, dermal layer cells, epidermal layer cells, or any cells in synthetic cells and their precursors can be derived from sources described herein.

[0079] Any cell may be a living or dead cell. When multiple cells are present, the cells may be living, dead, or any combination thereof.

[0080] Layered structure The synthetic leather may include one or more layered structures. The layered structure may be formed by placing a first type of layer on a second type of layer. The first type of layer and the second type of layer may be the same or different. In some cases, the layered structure may be formed by placing an epidermis layer on a dermis layer. For example, the layered structure may be formed by placing an epidermis layer on a dermis layer with a basement membrane substitute therebetween.

[0081] The layered structure may include two or more layers. In some cases, the layered structure includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 layers. In some cases, the layered structure includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 layers of a first type and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 layers of a second type. For example, the layered structure may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 dermal layers and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 epidermal layers.

[0082] The layered structure may comprise one or more types of cells as described herein.For example, the layered structure may comprise cells in the dermis layer, such as fibroblasts, cells in the epidermis layer, such as keratinocytes, or any combination thereof.Optionally, the layered structure may further comprise cells other than fibroblasts and keratinocytes.For example, the layered structure may comprise melanocytes.

[0083] The layered structure may have a thickness of about 0.001 mm to about 100 mm. For example, the layered structure may have a thickness of about 0.005 mm to about 50 mm, about 0.005 to about 10, about 0.01 mm to about 10 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, or about 0.1 to about 0.5 mm. In some cases, the thickness of the layered structure may be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, or 100 mm. In some cases, the thickness of the layered structure can be up to 100 mm, 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the layered structure can have a thickness of at least about 100, 200, 300, 400, 500, 600, 700, 800 mm.

[0084] The length of the layered structure may be manipulated to suit the function or use of the synthetic leather. The layered structure may have a length of about 0.01 mm to about 50 m. For example, the layered structure may have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the layered structure may have a length of about 0.02 mm to 5 mm. For example, the layered structure may have a length of about 0.1 mm to 0.5 mm. For example, the layered structure may have a length of about 0.2 mm to 0.5 mm. In some cases, the length of the layered structure may be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the length of the layered structure may be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the layered structure may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the layered structure may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the layered structure may have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0085] The width of the layered structure may be manipulated to suit the function or use of the synthetic leather. The layered structure may have a width of about 0.01 mm to about 50 mm. For example, the layered structure may have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the layered structure may have a width of about 0.02 mm to 5 mm. For example, the layered structure may have a width of about 0.1 mm to 0.5 mm. For example, the layered structure may have a width of about 0.2 mm to 0.5 mm. In some cases, the width of the layered structure can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the width of the layered structure can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the layered structure can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the layered structure may have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the layered structure may have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0086] The layered structure may comprise fibroblasts and keratinocytes in any ratio of at least about 50:1, 40:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:10, or 1:100. Optionally, the ratio of fibroblasts to keratinocytes can be from about 20:1 to about 3:1, from about 20:1 to about 4:1, from about 20:1 to about 5:1, from about 20:1 to about 10:1, or from about 20:1 to about 15:1.

[0087] The layered structure may comprise fibroblasts and melanocytes in any ratio of at least about 50:1, 40:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:10, or 1:100. Optionally, the ratio of fibroblasts to melanocytes can be from about 20:1 to about 3:1, from about 20:1 to about 4:1, from about 20:1 to about 5:1, from about 20:1 to about 10:1, or from about 20:1 to about 15:1.

[0088] The layered structure may comprise keratinocytes and melanocytes in any ratio of at least about 50:1, 40:1, 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:10, or 1:100. Optionally, the ratio of keratinocytes to melanocytes can be from about 20:1 to about 3:1, from about 20:1 to about 4:1, from about 20:1 to about 5:1, from about 20:1 to about 10:1, or from about 20:1 to about 15:1.

[0089] A type of cell in a layered structure may comprise up to 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, 5%, or 1% of the total cell population in the layered structure. A type of cell in a layered structure may comprise at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total cell population in the layered structure. For example, the fibroblasts in the layered structure may constitute at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the total cell population in the layered structure.

[0090] synthetic leather Synthetic leather may be formed by one or more layered structures. For example, synthetic leather may be formed by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 layered structures.

[0091] Synthetic leather can be of various thicknesses. For example, synthetic leather can have a thickness similar to natural leather. In some cases, synthetic leather can have a thickness of about 0.001 mm to about 100 mm. For example, the layered structure can have a thickness of about 0.005 mm to about 50 mm, about 0.005 to about 10, about 0.01 mm to about 10 mm, about 0.1 to about 5 mm, about 0.5 mm to about 5 mm, about 0.5 mm to about 3 mm, about 0.8 mm to about 3 mm, about 0.8 mm to about 2 mm, about 0.8 mm to about 1.8 mm, about 0.8 mm to about 1.6 mm, about 0.9 mm to about 1.4 mm, about 1 mm to about 1.5 mm, about 1 mm to about 1.4 mm, or about 1 mm to about 1.3 mm. In some cases, the thickness of the synthetic leather may be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, or 100 mm. In some cases, the thickness of the synthetic leather may be up to 100 mm, 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some cases, the thickness of the synthetic leather may be about 1.2 mm.

[0092] The synthetic leather may have a length of about 0.01 mm to about 50 m. For example, the synthetic leather may have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the synthetic leather may have a length of about 0.02 mm to 5 mm. For example, the synthetic leather may have a length of about 0.1 mm to 0.5 mm. For example, the synthetic leather may have a length of about 0.2 mm to 0.5 mm. In some cases, the length of the synthetic leather can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the length of the synthetic leather can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the synthetic leather can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the synthetic leather can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the synthetic leather can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0093] The synthetic leather may have a width of about 0.01 mm to about 50 mm. For example, the synthetic leather may have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 to about 5 mm, about 0.02 to about 5 mm, about 0.05 to about 5 mm, about 0.1 to about 5 mm, about 0.1 to about 2 mm, about 0.1 to about 1 mm, about 0.1 to about 0.8 mm, or about 0.1 to about 0.5 mm. For example, the synthetic leather may have a width of about 0.02 mm to 5 mm. For example, the synthetic leather may have a width of about 0.1 mm to 0.5 mm. For example, the synthetic leather may have a width of about 0.2 mm to 0.5 mm. In some cases, the width of the synthetic leather can be at least 0.001 mm, 0.01 mm, 0.02 mm, 0.04 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.4 mm, 0.8 mm, 1 mm, 2 mm, 4 mm, 8 mm, or 10 mm. In some cases, the width of the synthetic leather can be at most 50 mm, 40 mm, 20 mm, 10 mm, 8 mm, 4 mm, 2 mm, 1 mm, 0.8 mm, 0.4 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.04 mm, 0.02 mm, or 0.01 mm. In some embodiments, the synthetic leather can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 1000 mm. In some embodiments, the synthetic leather can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700 cm. In some embodiments, the synthetic leather can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0094] basement membrane substitute The synthetic leather may further comprise a basement membrane substitute. The basement membrane substitute may be between two cell layers, for example, between the dermis layer and the epidermis layer. The basement membrane substitute may be a dermal-epidermal junction similar to that present in vivo from a structural and / or biochemical point of view. From a biochemical point of view, the basement membrane substitute may comprise components of basement membrane, lamina densa, lamina lucida and sub-basal zone, such as collagen IV, collagen VII, laminin 5, entactin, fibronectin, or any combination thereof.

[0095] The basement membrane substitute in the synthetic leather can be urinary basement membrane (UBM), liver basement membrane (LBM), amnion, chorion, allograft pericardium, allograft acellular dermis, amniotic membrane, Wharton's gel, or any combination thereof. For example, the basement membrane substitute can be dry acellular amniotic membrane. In certain cases, the basement membrane substitute can be a polymer, such as a nanopolymer. For example, the basement membrane substitute can be nanofibrous polyhydroxybutyrate-co-hydroxyvalerate (PHBV) as described by Bye et al., Journal of Biomaterials and Tissue Engineering, Vol. 4, pp. 1-7, 2014.

[0096] scaffold A cell layer (e.g., dermis layer), a layered structure, or synthetic leather may be disposed on the scaffold. The scaffold may provide a certain firmness (e.g., tear resistance), elasticity, or both. In some cases, a part or the whole of the scaffold may be included in the synthetic leather. In other cases, the scaffold may not be included in the synthetic leather. After assisting in the formation of the layers in the synthetic leather, the scaffold may be removed from the final synthetic leather product. In certain cases, the scaffold included in the synthetic leather may degrade after a period of time. The scaffolds described herein may include a trabecular pattern.

[0097] Scaffolds may be made from natural materials, synthetic materials, or a combination thereof. Examples of scaffolds include scaffolds formed using a net made from a bioabsorbable synthetic polymer, scaffolds formed by attaching a nylon net to a thin silicone membrane, scaffolds having a bilayer structure of collagen sponge and silicone sheet, scaffolds formed using an atelocollagen sponge made into a sheet, scaffolds formed by matching collagen sponges with different pore sizes, and acellular dermal matrix (ADM) formed using fibrin glue or allogeneic skin made without cells.

[0098] The scaffold may include natural substances such as collagen (e.g., collagen matrix), natural adhesives (e.g., fibrin glue, cold glue, animal glue, blood albumen glue, casein glue, or vegetable glues such as starch and dextrin glue). Optionally, the scaffold includes silk. For example, the scaffold may be made from silk. In some embodiments, the scaffold may include silk fibroin, cellulose, cotton, acetate, acrylic, latex fibers, linen, nylon, rayon, velvet, modacrylic, olefin polyester, saran, vinyon, wool, jute, hemp, bamboo, flax, or combinations thereof. In some embodiments, the scaffold may include fibers. In some embodiments, the fibers may be silk, cotton, wool, linen, cellulose, especially extracted from wood, plants or algae, polyamide, modified cellulose (rayon, viscose, acetate, especially acetate rayon), poly-p-phenylene terephthalamide, acrylic fibers, such as polymethyl methacrylate or poly-2-hydroxyethyl methacrylate fibers, polyolefin fibers, such as polyethylene or polypropylene fibers, glass, silica, aramid, carbon, for example in the form of graphite, poly(tetrafluoroethylene), insoluble collagen, polyester, polyvinyl chloride or polyvinylidene chloride, polyvinyl alcohol, polyacrylonitrile, chitosan, polyurethane, poly(urethane-urea) or polyethylene phthalate, and fibers formed from blends of polymers such as the above fibers, for example polyamide / polyester fibers or any combination thereof.

[0099] The scaffold may include a polymer. The polymer may be a biopolymer. The biopolymer may include, but is not limited to, chitin, chitosan, elastin, collagen, keratin, or polyhydroxyalkanoates. The polymer may be biodegradable, biostable, or a combination thereof. The polymer in the scaffold may be a natural polymer. Exemplary natural polymers include polysaccharides, such as alginate, cellulose, dextran, pullulan, polyhyaluronic acid, chitin, poly(3-hydroxyalkanoates), poly(3-hydroxyoctanoates), or poly(3-hydroxy fatty acids). In some cases, the scaffold also includes a chemical derivative of the natural polymer. Such chemical derivatives may include substitution and / or addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, and other modifications familiar to those skilled in the art. The natural polymer may also be selected from proteins such as collagen, zein, casein, gelatin, gluten, and serum albumin. The polymer in the scaffold can be a biodegradable synthetic polymer including polyalpha-hydroxy acids, such as poly-L-lactic acid (PLA), polyglycolic acid (PGA) or copolymers thereof (eg, poly-D,L-lactic-co-glycolic acid (PLGA)), and hyaluronic acid.

[0100] The scaffold may be bioabsorbable. A bioabsorbable scaffold may be a non-cytotoxic structure or material that may be capable of containing or supporting living cells and holding them in a desired configuration for a period of time. The term "bioresorbable" may refer to any material that the body can break down into non-toxic by-products that can be excreted from the body or metabolized within the body. Exemplary bioabsorbable materials for the scaffold include poly(lactic acid), poly(glycolic acid), poly(trimethylene carbonate), poly(dimethyltrimethylene carbonate), poly(amino acids), tyrosine-derived poly(carbonates), poly(carbonates), poly(caprolactone), poly(para-dioxanone), poly(esters), poly(ester-amides), poly(anhydrides), poly(orthoesters), collagen, gelatin, serum albumin, proteins, polysaccharides, mucopolysaccharides, carbohydrates, glycosaminoglycans, poly(ethylene glycol), poly(propylene glycol), poly(acrylic acid esters), poly(methacrylic acid esters), poly(vinyl alcohol), hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, versican, copolymers, blends and mixtures of polymers, and oligomers containing bioabsorbable linkages.

[0101] The scaffold may have various thicknesses. For example, the scaffold may have a thickness that may be suitable for forming a cell layer. For example, the scaffold may have a thickness of about 0.1 mm to about 10 mm, for example, about 0.1 mm to about 5 mm, about 0.1 mm to about 4 mm, about 0.1 mm to about 3 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.2 mm to about 1 mm, about 0.3 mm to about 1 mm, about 0.4 mm to about 1 mm, about 0.5 mm to about 1 mm, 0.3 mm to about 1.5 mm, about 0.4 mm to about 1.2 mm, about 0.6 mm to about 1.2 mm, or about 0.7 mm to about 1.5 mm. For example, the scaffold may have a thickness of about 0.5 mm to 1 mm. In some cases, the scaffold may be at least 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm thick. In some cases, the scaffold may be at most 0.5 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm thick. In some embodiments, the scaffold may have the length and / or width of the cell layer to be placed and / or grown on the scaffold. In some embodiments, the scaffold may have the length and / or width of the cell layer described herein.

[0102] The scaffold may have a surface area on the side of the synthetic leather. For example, the scaffold may have a surface area of ​​about 0.1 mm 2 From about 100 mm 2 , about 0.1mm 2 From about 95 mm 2 , about 0.1mm 2 From about 90 mm 2 , about 0.1mm 2 From about 85 mm 2 , about 0.1mm 2 From about 80 mm 2 , about 0.1mm 2 From about 75 mm 2 , about 0.1mm 2 From about 70 mm 2 , about 0.1mm 2 From about 65 mm 2 , about 0.1mm 2 From about 60 mm 2 , about 0.1mm 2 From about 55 mm2 , about 0.1mm 2 From about 50 mm 2 , about 0.1mm 2 From about 45 mm 2 , about 0.1mm 2 From about 40 mm 2 , about 0.1mm 2 From about 35 mm 2 , about 0.1mm 2 From about 30 mm 2 , about 0.1mm 2 From about 25 mm 2 , about 0.1mm 2 From about 20 mm 2 , about 0.1mm 2 From about 15 mm 2 , about 0.1mm 2 From about 10 mm 2 , about 0.1mm 2 From about 5 mm 2 , or about 0.1 mm 2 From about 1 mm 2 Optionally, the scaffold may have a surface area of ​​about 0.1 cm 2 About 100cm from 2 , about 0.1cm 2 Approximately 95cm from 2 , about 0.1cm 2 About 90cm from 2 , about 0.1cm 2 About 85cm from 2 , about 0.1cm 2 About 80cm from 2 , about 0.1cm 2 About 75cm from 2 , about 0.1cm 2 About 70cm from 2 , about 0.1cm 2 About 65cm from 2 , about 0.1cm 2 About 60cm from 2 , about 0.1cm 2 About 55cm from 2 , about 0.1cm 2 About 50cm from 2 , about 0.1cm 2 About 45cm from 2 , about 0.1cm 2 About 40cm from 2 , about 0.1cm2 About 35cm from 2 , about 0.1cm 2 About 30cm from 2 , about 0.1cm 2 Approximately 25cm from 2 , about 0.1cm 2 About 20cm from 2 , about 0.1cm 2 About 15cm from 2 , about 0.1cm 2 About 10cm from 2 , about 0.1cm 2 About 5cm from 2 , or about 0.1 cm 2 About 1 cm from 2 In some cases, the scaffold may have a surface area of ​​about 0.1 m. 2 About 100m from 2 , about 0.1m 2 Approximately 95m from 2 , about 0.1m 2 Approximately 90m from 2 , about 0.1m 2 Approximately 85m from 2 , about 0.1m 2 Approximately 80m from 2 , about 0.1m 2 Approximately 75m from 2 , about 0.1m 2 Approximately 70m from 2 , about 0.1m 2 Approximately 65m from 2 , about 0.1m 2 Approximately 60m from 2 , about 0.1m 2 Approximately 55m from 2 , about 0.1m 2 About 50m from 2 , about 0.1m 2 Approximately 45m from 2 , about 0.1m 2 Approximately 40m from 2 , about 0.1m 2 Approximately 35m from 2 , about 0.1m 2 Approximately 30m from 2 , about 0.1m 2 Approximately 25m from 2 , about 0.1m 2 Approximately 20m from 2 , about 0.1m 2Approximately 15m from 2 , about 0.1m 2 About 10m from 2 , about 0.1m 2 Approximately 5m from 2 , or about 0.1 m 2 About 1m from 2 may have a surface area of

[0103] In some cases, the scaffold is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 mm. 2 In some cases, the scaffold may have a surface area of ​​at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 cm. 2 In some cases, the scaffold may have a surface area of ​​at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, or 100 m. 2 may have a surface area of

[0104] Alternatively, the cell layer may not form on the scaffold. For example, the dermis layer may not form on the scaffold (e.g., collagen matrix). In certain cases, the synthetic leather does not include a scaffold.

[0105] pigment The synthetic leather may include one or more pigments. One or more layer structures of the synthetic leather may be pigmented. The pigments in the synthetic leather may be natural pigments produced in the cells forming the synthetic leather. For example, the pigments may be melanins, including eumelanins (e.g., brown eumelanins and black eumelanins), pheomelanins, neuromelanins, or any combination thereof. The pigments in the synthetic leather may be exogenous pigments, such as leather pigment dyes.

[0106] collagen The synthetic leather may include collagen. Collagen may refer to any member of a family of at least 28 different collagen types. Collagen may be characterized by a repeating triplet of amino acids -(Gly-XY)n-, such that approximately one-third of the amino acid residues in collagen are glycine. X may be proline and Y may be hydroxyproline. Thus, collagen structures may have paired triplet units of peptide chains of different lengths. The synthetic leather may include collagen from one or more species. In some cases, the synthetic leather includes collagen from different animals. Different animals may produce collagen of different amino acid composition, which may result in different properties (and resulting differences in leather). Collagen fiber monomers may be produced from alpha chains of about 1050 amino acids in length, resulting in a triple helix that assumes a rod-like shape of about 300 nm in length with a diameter of about 1.5 nm.

[0107] Synthetic leather may include one or more types of collagen. The collagen included in synthetic leather may include fibrillar collagen, nonfibrillar collagen, or a combination thereof. Fibrillar collagen includes collagen type I, type II, type III, type V, and type XI. Nonfibrillar collagen includes fibril-associated collagen with interrupted triple helices (e.g., type IX, type XII, type XIV, type XVI, and type XIX), short-chain collagen (e.g., type VIII and type X), basement membrane collagen (type IV), multiplexin (multiple triple helical domains with interruptions) (e.g., type XV and type XVIII), MACIT collagen (membrane-associated collagen with interrupted triple helices) (e.g., type XIII and type XVII).

[0108] Collagen can be included in one or more parts of synthetic leather.For example, collagen can be included in one or more dermis layers, one or more epidermis layers, or a combination thereof in synthetic leather.For example, collagen can be included in one or more layered structures in synthetic leather.Optionally, when a part of synthetic leather can be removed during the process, collagen can also be included in the removed product.

[0109] The collagen in the synthetic leather can be from one or more sources. For example, the collagen may be produced by collagen-producing cells in the synthetic leather. For example, the collagen can be added separately to the leather. In some cases, the synthetic leather includes collagen produced by collagen-producing cells, and the collagen is added separately.

[0110] At least a portion of the collagen in the synthetic leather may be produced by collagen-producing cells. Such collagen-producing cells may be included in the synthetic leather. Exemplary collagen-producing cells include epithelial cells, fibroblasts, keratinocytes, keratinocytes, melanocytes, Langerhans cells, basal cells, smooth muscle cells, or combinations thereof. Epithelial cells may include squamous epithelial cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. Fibroblasts may include dermal fibroblasts. Keratinocytes may include epidermal keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. Collagen in the synthetic leather may be produced by one or more types of collagen-producing cells.

[0111] Additives The synthetic leather may further include one or more additives. Such additives may enhance commercial appeal (e.g., appearance, color, or odor). Exemplary additives include minerals, fibers, fatty acids, and amino acids, proteins. The additive may be an odorant.

[0112] Additives may include one or more of matrix proteins, proteoglycans, antioxidants, perfluorocarbons, and growth factors. Growth factors may be proteins, polypeptides, or complexes of polypeptides, including cytokines (e.g., those produced by cells and that may affect themselves and / or a variety of other adjacent or distant cells). Growth factors can affect the growth and / or differentiation of specific types of cells developmentally or in response to a number of physiological or environmental stimuli. Some, but not all, growth factors are hormones. Exemplary growth factors include insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), such as basic FGF (bFGF), platelet-derived growth factor (PDGF), such as PDGF-AA and PDGF-AB, hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), such as TGFpi and TGFP3, epidermal growth factor (EGF), granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, etc. Other polypeptides or molecules (e.g., healing agents; enzymes such as matrix-degrading enzymes and matrix-degrading enzyme inhibitors (e.g., TIMPs), antibiotics, and antifungals) can also be added to the synthetic leather.

[0113] Additives may also include preservatives known in the art. Exemplary preservatives include antimicrobial preservatives such as calcium propionate, sodium nitrate, sodium nitrite, sulfites (e.g., sulfur dioxide, sodium bisulfate, potassium hydrogen sulfite, etc.), disodium ethylenediaminetetraacetic acid (disodium ethylenediaminetetraacetic acid), and the like. and antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

[0114] In certain cases, the synthetic leather may further include extracellular matrix or connective tissue, for example, collagen, keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipids, fatty acids, carbohydrates, and combinations thereof.

[0115] Synthetic leather pattern The synthetic leather can be patterned. For example, the synthetic leather can be patterned with antelope, bear, beaver, bison, wild boar, camel, caribou, cat, cow, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, bull, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, zebra, turtle, snake, crocodile, alligator, dinosaur, frog, toad, salamander, newt, chicken, duck, lemur ... In some embodiments, the fish may be patterned to resemble the skin of an animal selected from the group consisting of goose, goose, grouse, ostrich, pheasant, pigeon, quail, turkey, anchovy, bass, catfish, carp, cod, eel, flounder, pufferfish, grouper, haddock, halibut, herring, mackerel, mahi-mahi, manta ray, marlin, orange roughy, perch, pike, pollack, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, walleye, and combinations thereof. The pattern may be a fantasy animal skin pattern selected from a dragon, unicorn, griffin, siren, phoenix, sphinx, cyclops, satyr, medusa, pegasus, cerberus, typhoeus, gorgon, charybdis, empusa, chimera, minotaur, cetus, hydra, centaur, fairy, mermaid, nessie, sasquatch, thunderbird, yeti, chupacabra, and combinations thereof.

[0116] Synthetic leather can be manufactured to resemble traditional animal skins, hides, or leather products and design parameters (e.g., cell type, additives, size, shape). Optionally, synthetic leather includes a cell layer characterized by a composition that can be substantially similar to traditional animal skins, hides, or leather products. For example, such a layer can be characterized by a composition that can be substantially about 60% to 80% aqueous fluid, about 14%-35% protein, and about 1%-25% fat. Optionally, the keratinocytes of the cell layer are aligned. For example, the keratinocytes can be aligned by application of an electric field. For example, the keratinocytes can be aligned by application of a mechanical stimulus, such as cyclically stretching and relaxing the substratum. Optionally, the aligned (e.g., electrically oriented and mechanically oriented) keratinocytes have substantially the same orientation with respect to each other as can be found in many animal skin tissues.

[0117] Leather products The synthetic leather herein can be at least a part of a leather product. For example, synthetic leather can be used as a substitute for natural leather in leather products. Exemplary leather products include watch bands, belts, suspenders, packaging, shoes, boots, footwear, gloves, clothing (e.g., tops, bottoms, and outerwear), bags, pouches (e.g., handbags with or without shoulder straps), clutches, wallets, coin purses, billfolds, key pouches, credit card cases, pencil cases, backpacks, cases, billfolds, saddles, harnesses, whips, travel items (e.g., trunks, suitcases, travel bags, cosmetic cases, or toiletries), rucksacks, briefcases, document bags, briefcases, attache cases, pet items (e.g., leashes or collars), hunting and fishing items (e.g., gun cases, knife cases, or firm weapons), and the like. The leather product may be a leather holster for a leather arm, a stationery item (e.g., a letter paper, a book cover, a camera case, an eyeglass case, a cigarette case, a cigar case, a jewelry box, or a cell phone case), a sports equipment item (e.g., a ball such as a basketball, a soccer ball, or a football), a building interior, a building exterior, upholstery, bookbinding material, furniture, a lamp, a lamp shade, a table covering, wallpaper, flooring, ceiling covering, an automobile interior, an automobile exterior, a boat interior, a boat exterior, an airplane interior, a yacht interior, a yacht exterior, a pillowcase, a sheet, a duvet cover, jewelry, an accessory, eyeglasses, sunglasses, or a consumer electronics product. For example, the leather product may be a watch case. For example, the leather product may be a belt. For example, the leather product may be a bag.

[0118] skin graft The synthetic leather or a portion thereof can also be used as a skin graft, for example, an allograft or xenograft for transplantation into a subject. For example, the synthetic leather, dermis layer, epidermis layer and / or layered structure can be a source of a skin graft for allograft or xenograft. In some cases, the synthetic leather, dermis layer, epidermis layer and / or layered structure can be produced using cells genetically modified to reduce immune rejection in the recipient of the graft.

[0119] method Also disclosed herein is a method for producing synthetic leather. The method may include forming an artificial dermis layer, forming an artificial epidermis layer, or a combination thereof. The method may further include tanning at least a portion of the artificial dermis layer and / or the artificial epidermis layer. The cells in the synthetic leather, for example, the cells in the dermis layer and / or the epidermis layer, may be differentiated from stem cells (e.g., iPSCs). The method herein may further include differentiating stem cells (e.g., iPSCs) into cells in the synthetic leather, for example, the cells in the dermis layer and / or the epidermis layer. In certain cases, the method includes disposing a first cell layer (e.g., epidermis layer) on a second cell layer (e.g., dermis layer), thereby forming a layered structure, and tanning at least a portion of the layered structure. In some cases, the method may further include removing at least a portion of the first cell layer (e.g., epidermis layer).

[0120] Cell layer formation A cell layer can be formed by preparing a plurality of multicellular bodies comprising one or more types of cells and arranging such multicellular bodies to form a cell layer. For example, a cell layer may be formed by arranging a plurality of multicellular bodies side by side, where the multicellular bodies fuse to form a planar layer.

[0121] The step of forming a cell layer may require a scaffold. The cell layer may be formed by arranging a plurality of multicellular bodies on a scaffold. For example, the step of forming may include arranging or disposing the multicellular bodies on a support carrier, which fuses the multicellular bodies to form a layer (e.g., a substantially planar layer). In some cases, the multicellular bodies or layers are arranged next to each other horizontally and / or vertically. Alternatively, the step of forming a cell layer may not require a scaffold.

[0122] The cell layer can be formed by embedding the cells in a medium or gel. Optionally, the dermis layer can be formed using fibroblasts embedded in collagen I or fibrin gel. Other types of medium can also be used. For example, the medium can encourage fibroblasts to secrete a sufficient amount of extracellular matrix to allow long-term maintenance of epidermis without the need for collagen gel.

[0123] Formation of multicellular bodies There are various methods for producing multicellular bodies having the characteristics described herein. In some cases, the multicellular bodies can be fabricated from a cell paste containing a plurality of cells, for example, with a desired cell density and viscosity. In further cases, the cell paste can be molded into a desired shape, and the multicellular body can form through maturation (e.g., incubation). In some cases, the elongated multicellular body can be produced by molding the cell paste containing a plurality of cells into an elongated shape (e.g., cylindrical). In further cases, the cell paste can be incubated in a controlled environment to allow the cells to adhere and / or adhere to each other to form the elongated multicellular body. For example, the multicellular body may be produced by molding the cell paste containing a plurality of live cells in a device that holds the cell paste in a three-dimensional shape. In some cases, the cell paste can be incubated in a controlled environment while the cell paste can be held in a three-dimensional shape for a sufficient time to produce a multicellular body that is sufficiently adherent to the support itself on a flat surface as described herein.

[0124] A cell paste can be provided by (A) mixing cells or cell aggregates (of one or more cell types) with cell culture medium (e.g., in a predetermined ratio) to produce a cell suspension, and (B) compressing the cell suspension to produce a cell paste with a desired cell density and viscosity. Compression can be achieved by several methods, such as by concentrating a particular cell suspension resulting from cell culture to achieve the desired cell concentration (density), viscosity, and consistency required for the cell paste. Optionally, a relatively dilute cell suspension from cell culture can be centrifuged for a determined time to achieve a cell concentration in a pellet that can be molded in a mold. Tangential flow filtration ("TFF") is another suitable method of concentrating or compressing cells. Optionally, a compound is combined with the cell suspension to provide the required extrusion properties. Suitable compounds include collagen, hydrogels, matrigel, nanofibers, self-assembling nanofibers, gelatin, and fibrinogen. One or more ECM components (or derivatives of ECM components) may also be included by resuspending the cell pellet in one or more physiologically acceptable buffers containing the ECM components (or derivatives of ECM components) and centrifuging the resulting cell suspension again to form a cell paste.

[0125] Various methods can be used to shape the cell paste. For example, in certain embodiments, the cell paste can be manually molded or compressed (e.g., after concentration / compression) to achieve a desired shape. As a further example, the cell paste can be introduced (e.g., aspirated) into an instrument such as a micropipette (e.g., a capillary pipette) that molds the cell paste to fit the inner surface of the preformed instrument. The cross-sectional shape of the micropipette (e.g., a capillary pipette) can alternatively be circular, square, rectangular, triangular, or other non-circular cross-sectional shapes. In some embodiments, the cell paste can be molded by depositing it into a preformed mold, such as a plastic mold, a metal mold, or a gel mold. In some embodiments, the cell paste can be molded using centrifugal casting or continuous casting.

[0126] The cell paste can be further matured. Optionally, the cell paste can be incubated at about 37° C. for a period of time (which may depend on the type of cell) to promote adhesion and / or adhesion. Alternatively or additionally, the cell paste can be kept in the presence of cell culture medium containing factors and / or ions to promote adhesion and / or adhesion.

[0127] Arrangement of multicellular bodies on a support to form a layer The multicellular bodies can be arranged on a support to produce a desired three-dimensional structure (e.g., a substantially planar layer). For example, the multicellular bodies can be placed in contact with one another manually, deposited in place by extrusion from a pipette, nozzle, or needle, or placed in contact with one another by an automated machine, such as a biofabricator.

[0128] The support carrier may be permeable to fluids, gases, and nutrients, allowing cell culture medium to contact all surfaces of the multicellular bodies and / or layers during alignment and subsequent fusion. Optionally, the support carrier may be made of natural biomaterials such as collagen, fibronectin, laminin, and other extracellular matrices. Optionally, the support carrier may be made of synthetic biomaterials such as hydroxyapatite, alginate, agarose, polyglycolic acid, polylactic acid, and copolymers thereof. Optionally, the support carrier may be solid, semi-solid, or a combination of solid and semi-solid support elements. Optionally, the support carrier may be planar to facilitate the production of planar layers. Optionally, the support carrier may be elevated or elevated from a non-permeable surface such as the cell culture environment (e.g., Petri dish, cell culture flask, etc.) or part of a bioreactor. The increased permeability of the support carrier may contribute to the prevention of premature cell death and increase cell proliferation, facilitating the fusion of the multicellular bodies to form a layer.

[0129] After the assembly of the layers is complete, tissue culture medium can be poured on top of the construct. Optionally, the tissue culture medium enters the spaces between the multicellular bodies to support the cells in the multicellular bodies. The multicellular bodies in the three-dimensional construct can be fused together to produce a layer (e.g., a substantially planar layer) for use in forming synthetic leather. The terms "fuse," "fused," or "fusion" can mean that the cells of the continuous multicellular bodies become attached and / or intimately attached to each other either directly through interactions between cell surface proteins or indirectly through interactions between the cells and ECM components or derivatives of ECM components. The fused layer can be completely fused, and the multicellular bodies become substantially continuous. Alternatively, the fused layer can be substantially fused or partially fused, and the cells of the multicellular bodies become attached and / or intimately attached to the extent necessary to allow the layer to be moved and manipulated intact.

[0130] The multicellular bodies can fuse to form a layer in a cell culture environment (e.g., a petri dish, a cell culture flask, or a bioreactor). In some cases, the multicellular bodies can fuse to form a layer in an environment under conditions suitable for facilitating the growth of the cell types contained in the multicellular bodies. In some cases, the fusion occurs over about 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes, and increments therein. In other cases, the fusion occurs over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and 48 hours, and increments therein. In still other cases, fusion occurs over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 14 days, and increments therein. In further cases, fusion occurs over about 2 hours to about 24 hours. Factors related to fusion time can include cell type, ratio of cell types, culture conditions, and the presence of additives such as growth factors.

[0131] After fusion of the layer is complete, the layer and the support carrier can be separated. In other cases, the layer and the support carrier are separated when fusion of the layer is substantially or partially complete, but the cells of the layer are still adhered and / or intimately attached to each other to the extent necessary to allow the layer to be moved, manipulated, and stacked without breaking apart. The layer and the support carrier can be separated via standard procedures to melt, dissolve, or decompose the support carrier. In some cases, the support carrier can be dissolved, for example, by a temperature change, light, or other stimulus that does not adversely affect the layer. In certain cases, the support carrier can be made of a flexible material and peeled off from the layer. The separated layer can be transferred to a bioreactor for further maturation. In some cases, the separated layer is further fused after maturation and incorporation into an engineered animal hide, leather, or leather product.

[0132] Alternatively, the layers and the support carrier may not be separated, and the support carrier decomposes or biodegrades prior to packaging, freezing, sale or consumption of the assembled and manipulated animal hide, leather, or leather product.

[0133] The cell layer can be formed over a period of time. In some cases, the cell layer, for example, the epidermal layer or the dermal layer, can be formed within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 120, 300 days. In some cases, the dermal layer can be formed in about 1 to 15 days, for example, 5 to 10 days, or 10 to 12 days. In some cases, the dermal layer can be formed in about 5 to 25 days, for example, 14 to 15 days.

[0134] The present disclosure provides a method for producing synthetic leather with improved barrier function. Optionally, the method includes providing a culture medium containing keratinocytes, ascorbic acid and linoleic acid; and culturing the keratinocytes under conditions such that a synthetic leather with improved barrier function can be formed. Optionally, the culture conditions include culturing at about 50 to 95% humidity, for example about 75% humidity. Optionally, the ascorbic acid can be provided at a concentration of about 10 to 100 micrograms / ml. In yet further instances, the linoleic acid can be provided at a concentration of about 5 to 80 micromolar. The present disclosure is not limited to synthetic leather formed from keratinocytes of a particular source. Indeed, synthetic leather can be formed from a variety of primary and immortalized keratinocytes, including, but not limited to, near-diploid immortalized keratinocyte (NIKS) cells. In yet further cases, the keratinocytes express exogenous wild-type or variant Krüppel-like factor (GKLF). In yet further cases, the keratinocytes are derived from two different sources. In other cases, the synthetic leather has a surface capacitance of about 40 to about 240 pF. In some preferred cases, the skin equivalent has a surface capacitance of about 80 to about 120 pF. In other preferred cases, the content of ceramides 5, 6, and 7 in the skin equivalent can be about 20 to about 50% of the total ceramide content. In still other preferred cases, the content of ceramide 2 in the skin equivalent can be about 10 to about 40% of the total ceramide content. In still further cases, the present disclosure provides a skin equivalent produced by the method just described.

[0135] Arrangement of layers to form a layered structure A plurality of cell layers can be arranged to form a layered structure to produce the synthetic leather described herein. In some cases, the dermis layer and epidermis layer are formed separately and assembled by placing the epidermis layer on the dermis layer (e.g., when both the epidermis layer and the dermis layer are fully formed). In some cases, the epidermis layer can be grown on the dermis layer. In certain cases, a basement membrane or basement membrane substitute can be placed between the dermis layer and the epidermis layer. For example, the cell layers can be manually placed in contact with each other or deposited in place according to a computer script by an automated computer-assisted machine such as a biofabricator.

[0136] Prior to assembling multiple cell layers, one or more quality control steps can be performed. For example, transepithelial electrical resistance (TEER) can be performed on the epidermis prior to placement on the dermis (e.g., day 0), followed by histology (e.g., a minimum of 3-5 days). Using the methods provided herein, the risk of improperly formed layered structures or full thickness skin equivalents can be low.

[0137] The multiple cell layers can be assembled in various ways. Optionally, the epidermal layer and the multiple dermal layers (with or without a basement membrane substitute) are placed on a scaffold (e.g., silk) to achieve the thickness and tensile strength of, for example, natural leather. Optionally, the epidermal layer and the multiple dermal layers (with or without a basement membrane substitute) are assembled without the use of a scaffold. Such an assembly can achieve a thickness and tensile strength similar to natural leather. Optionally, the epidermal layer and the multiple dermal layers (with or without a basement membrane substitute) are placed on a scaffold (e.g., silk) to achieve a thickness and tensile strength similar to natural leather.

[0138] In some embodiments, chemical tests, mechanical tests, performance tests, strength tests, durability tests, moisture tests, dimensional tests, or combinations thereof can be performed on one or more multicellular layers, synthetic leather, artificial epidermal layers, artificial dermal layers, layered structures, products produced therefrom. In some embodiments, chemical tests, mechanical tests, performance tests, strength tests, durability tests, moisture tests, dimensional tests, or combinations thereof can be performed using non-standard tests. In some embodiments, chemical tests, mechanical tests, performance tests, strength tests, durability tests, moisture tests, dimensional tests, or combinations thereof can be performed using standard tests. In some embodiments, tests can be performed as directed and / or adopted and / or approved and / or developed by the International Organization for Standardization (ISO), the European Committee for Standardization (CEN), ASTM International, or the International Union of Leather Technologists and Chemists (IULTCS). In some embodiments, the tests in any one of Tables 1-11, or any variations thereof, can be performed using any one or more corresponding methods, or any variations thereof. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

[0139] In some embodiments, the leather products may have physical properties similar to real leather. In some embodiments, the synthetic leather disclosed herein or leather products made therefrom may have a mechanical strength of at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 lb / in 2 In some embodiments, the synthetic leather disclosed herein or leather products made therefrom may have a tensile strength as measured by ASTM D-2209-95 of about 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 lb / in 2 The composition may have a tensile strength as measured by ASTM D-2209-95 of less than 0.15 mm.

[0140] In some embodiments, the synthetic leather disclosed herein or leather products produced therefrom can have a slit of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 lbs as measured by ASTM-D2212-94. In some embodiments, the synthetic leather disclosed herein or leather products produced therefrom may have a slit weight of less than about 200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 lb as measured by ASTM-D2212-94. In some embodiments, the synthetic leather disclosed herein or leather products produced therefrom can have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 stitches as measured according to ASTM-D4705-93. In some embodiments, the synthetic leather disclosed herein or leather products made therefrom may have less than about 200, 150, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 stitches.In some embodiments, the synthetic leather disclosed herein or leather products produced therefrom can have slit and stitch values ​​that are at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 lb when measured according to the respective slit and stitch tests. In some embodiments, the synthetic leather disclosed herein or leather products produced therefrom may have a Bally flex as measured by ASTM D6182 of at least about 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 45000, 50,000, 55000, 60000, 65000, 70000, 80000.

[0141] Multiple cell layers can be assembled to form synthetic leather (e.g., full-thickness skin equivalent). The synthetic leather can include an upper portion, a middle portion, and a lower portion. The upper portion can include an epidermal layer. For example, the upper portion can be a single epidermal layer. The middle portion can include a basement membrane substitute. Optionally, the middle portion does not have a basement membrane substitute. For example, the middle portion can have a layer of negligible thickness. The lower portion can have one or more dermal layers. Optionally, the lower portion has a single dermal layer disposed on a scaffold (e.g., silk). Optionally, the lower portion has multiple dermal layers (e.g., up to 5 layers) without a scaffold. Optionally, the lower portion has multiple dermal layers stacked on top of each other and disposed on a scaffold (e.g., silk).

[0142] The adhesion between the epidermis layer and the dermis layer may be strong enough to withstand the splitting of the layers. Optionally, the cell layers may be assembled by gluing onto the scaffold. Natural or synthetic adhesives may be used for assembly. The natural adhesive may be fibrin glue, cold glue, animal glue (e.g., bone glue, fish glue, hide glue, hoof glue, rabbit skin glue, meat glue), blood albumin glue, casein glue, vegetable glue (e.g., starch, dextrin glue, Canada balsam, rosin-based glue, cocconia, gum arabic, postage stamp glue, latex, library paste, methylcellulose, mucus, resorcinol resin, or urea-formaldehyde resin), or any combination thereof. Synthetic adhesives include acrylonitrile, cyanoacrylate (e.g., n-butyl-2-cyanoacrylate glue), acrylic, resorcinol glue, epoxy resin, epoxy putty, ethylene vinyl acetate, phenol formaldehyde resin, polyamide, polyester resin, polyethylene, polypropylene, polysulfide, polyurethane, polyvinyl acetate (white glue (e.g., Elmer's Glue)), and polyvinyl acetate (polyvinyl acetate). The adhesive may be selected from the group consisting of acrylic glue, yellow carpenter's glue (containing aliphatic resins), polyvinyl alcohol, polyvinyl chloride (PVC), polyvinyl chloride emulsion (PVCE), polyvinylpyrrolidone, rubber glue, silicone, and styrene acrylic copolymers. For example, the assembly may be performed using fibrin glue. For example, the assembly may be performed using n-butyl-2-cyanoacrylate glue.

[0143] In some cases, cell layers (e.g., substantially planar layers) are stacked to form synthetic leather. The cell layers may have an orientation defined by the arrangement, pattern, or orientation of the multicellular bodies. In some cases, each layer may be stacked in a specific orientation relative to the support carrier and / or one or more other layers. For example, one or more layers may be stacked in an orientation that includes a rotation relative to the support carrier and / or the layer below, where the rotation may be between 0.1 and 180 degrees, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, and 180 degrees, or increments therein. In other cases, all of the layers are oriented in substantially the same way.

[0144] After the layer stacking is completed, the layers in the three-dimensional construct can be fused together to produce synthetic leather. In some cases, the layers are fused in a cell culture environment (e.g., a petri dish, a cell culture flask, a bioreactor, etc.). In some cases, the fusion occurs for about 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes, and increments therein. In other cases, the fusion occurs for 1 to 48 hours, for example, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, and 48 hours, and increments therein. For example, the fusion can occur for about 2 hours to about 24 hours.

[0145] Culture conditions The cells and cell layers can be cultured under various cell culture conditions. The cells or cell layers can be cultured in vitro. For example, the dermis layer and / or the epidermis layer can be cultured in vitro. Alternatively, the cells or cell layers can be cultured in vivo. For example, the dermis layer and / or the epidermis layer can be cultured in vivo.

[0146] The cells and cell layer can be cultured with one or more supplements. The one or more supplements can be natural supplements, synthetic supplements, or a combination thereof. Optionally, the supplements can be additives. Optionally, one or more of the supplements induce the production and assembly of extracellular matrix from iPSC-derived fibroblasts, thereby enhancing the natural appearance of the synthetic leather. Exemplary supplements can include ECM components such as collagen and fibrin, growth factors, small molecules such as ascorbic acid, macromolecules such as dextran sulfate, carrageenan, etc.

[0147] The cell layer can be cultured at a certain air humidity. For example, the cell layer (e.g., the dermis layer or the epidermis layer) can be cultured at a humidity of about 20% to about 100%. For example, the humidity can be about 40% to about 100%, about 50% to about 95%, about 45% to about 90%, about 55% to about 95%, about 60% to about 90%, about 70% to about 80%, about 71% to about 79%, about 72% to about 78%, about 73% to about 77%, about 74% to about 76%, about 60% to about 70%, about 65% to about 75%, about 70% to about It can be 80%, about 75% to about 85%, about 80% to about 90%, about 85% to about 95%, or about 90% to about 100%, about 40% to about 60%, about 45% to about 55%, about 46% to about 54%, about 47% to about 53%, about 48% to about 52%, about 48% to about 53%, about 49% to about 54%, or about 47% to about 51%.

[0148] Leather Treatment Tanning The methods herein may include tanning at least a portion of the synthetic leather, for example, at least a portion of the corium and / or epidermis layers in the synthetic leather. Tanning makes the synthetic leather resemble natural leather, which can be a durable, flexible material created by tanning raw animal hides, often adult cowhides. Tanning herein may refer to the process of treating animal hides to produce leather. Tanning can be done in a variety of ways, including vegetable tanning (e.g., using tannins), chrome tanning (chromium salts including chromium sulfate), aldehyde tanning (using glutaraldehyde or oxazolidine compounds), syntan (using synthetic tannins, aromatic polymers), bacterial staining, and the like.

[0149] Tanning can be done to convert the proteins in leather / hide into a stable material that will not spoil, while allowing the material to remain flexible. Chromium can be used as a tanning material. The pH of the cell layer or layered structure can be adjusted (e.g., lowered; e.g., to about pH 2.8-3.2) to enhance tanning; after tanning, the pH can be raised ("basified" to a slightly higher level, e.g., to about pH 3.8-4.2).

[0150] Tanning can be performed on a cell layer, for example, the dermis layer and the epidermis layer. Tanning can also be performed on a layered structure, for example, a layered structure including at least the dermis layer and at least the epidermis layer. In certain cases, tanning can also be performed on synthetic leather. For example, tanning can be performed after forming a cell layer, for example, the dermis layer or the epidermis layer. For example, tanning can be performed after forming a layered structure.

[0151] Tanning can be achieved by modifying the extracellular matrix (ECM) material. Tanning can be achieved by modifying collagen in the ECM. Tanning can be achieved using tanning agents such as chromium(III) sulfate ([Cr(H2O)6]2(SO4)3). Chromium(III) sulfate converts the hexaaquachromium(III) cation [Cr(H2O)6]2(SO4)3, which at higher pH undergoes a process called olation to give polychromium(III) compounds. 3+ Polychromium(III) compounds are active in tanning and cross-linking of collagen subunits. Some ligands include sulfate anions, carboxyl groups of collagen, amine groups from amino acid side chains, and masking agents. Masking agents can be carboxylic acids such as acetic acid, which are used to suppress the formation of polychromium(III) chains. Masking agents can allow the tanner to further increase the pH to increase collagen reactivity without inhibiting the permeation of chromium(III) complexes. Tanning can increase the spacing between protein chains in collagen (e.g., 10 to 17 Å), consistent with cross-linking by polychromium species of the kind resulting from olation and oxolation. Chromium can be cross-linked with collagen. Chrome-tanned leather can contain between about 4% and 5% chromium. This efficiency can be characterized by the leather's increased hydrothermal stability and its resistance to shrinkage in hot water. Other tanning agents can be used to tan the layered body and modify the collagen.

[0152] Tanning can also be carried out using other minerals. Optionally, tanning can be carried out using agents based on alum, zirconium, titanium, iron salts, or combinations thereof.

[0153] Further Processing The cell layers, layered structures, and synthetic leathers produced herein can be further processed after tanning. Optionally, the methods provided herein further include one or more leather processing steps (e.g., steps used in the formation of traditional leather). Examples of processing steps include curing, soaking, liming, unhairing, fleshing, splitting, deliming, reliming, baching, degreasing, frizing, bleaching, coloring, pickling, deoxidizing, tanning, retanning (e.g., if the color has run during processing), thinning, retanning, lubricating, crusting, wetting, wringing, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixing, setting, drying, conditioning, blank beating (e.g., dry blank beating), staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, and tumbling.

[0154] Synthetic leather can be shaped, for example, by controlling the number, size, and arrangement of the multicellular bodies and / or layers used to construct the animal hide, leather, or hide. In other cases, the animal hide, leather, or hide can be shaped, for example, by cutting, pressing, molding, or stamping. The shape of the synthetic leather can be made to resemble a traditional animal hide, leather, or leather product.

[0155] The method herein may include a step of removing a portion of the synthetic leather produced herein. Optionally, the method includes a step of removing at least a portion of the skin layer to form a removed product. For example, the removing may be shaving.

[0156] Pigmentation The method herein may include a step of pigmenting the synthetic leather. Optionally, pigmentation may be performed by introducing pigment-producing cells (e.g., melanocytes) into the synthetic leather. Optionally, the synthetic leather includes functional live melanocytes. The melanocytes may have a location similar to that of human skin. Optionally, melanin may be constitutively produced by the melanocytes. Optionally, melanin may be transported to the keratinocytes. Optionally, the melanocytes are produced upon stimulation, for example, UV irradiation, or by pigmentation-promoting activators such as alpha melanocyte stimulating hormone (aMSH), endothelin 1 (ET1), stem cell factor (SCF), prostaglandin E2 and F2α (PGE2, PGF2α), basic fibroblast growth factor (bFGF) or nerve growth factor (NGF).

[0157] Differentiation of precursor cells into cells in synthetic leather Cells in the epidermis layer, such as keratinocytes and melanocytes, and cells in the dermis layer, such as fibroblasts, can be derived, e.g., differentiated, from precursor cells, such as iPSCs. In other cases, synthetic leather can be produced using primary cells or cultured cells derived from primary cells to form cell layers.

[0158] Various methods of differentiating iPSCs into cells in synthetic leather, such as keratinocytes, melanocytes, or fibroblasts, can be used. In some cases, differentiation of iPSCs into keratinocytes and construction of a 3D epidermis from iPSC-derived keratinocytes can be performed using the methods described by Petrova et al., 3D In vitro model of a functional epidermal permeability barrier from human embryonic stem cell and induced pluripotent stem cells. Stem Cell Reports. 2014 Apr. 24; Vol. 2(5): pp. 675-89. In other cases, the cell layer can be formed using primary cells. For example, construction of a 3D epidermis from primary keratinocytes can be performed using the methods described by Sun R et al., Lowered humidity produces human epidermal equivalents with enhanced barrier properties. Tissue Eng Part C Methods. 2015 Jan.; Vol. 21(1): pp. 15-22.

[0159] In some cases, the methods described herein provide a high-throughput method for reliably, accurately, and reproducibly scaling up the production of synthetic leather to commercial levels. Advantages of synthetic leather, engineered epidermis equivalents, engineered full-thickness skin equivalents, and methods for producing the same disclosed herein include, but are not limited to, reproducibly, high-throughput, and easily scalable production of customized tissues with attractive appearance, texture, thickness, and durability. In some embodiments, the methods described herein can produce increased yields of one or more of epidermal layers, dermal layers, layered structures, or synthetic leather. In some embodiments, the increased yields can be at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or about 15 times the yield of comparable methods. In some embodiments, the methods disclosed herein can reduce the manufacturing cost of synthetic leather, artificial epidermal layer, artificial dermal layer, layered structure, and products produced therefrom. In some embodiments, the methods disclosed herein can produce synthetic leather, artificial epidermal layer, artificial dermal layer, layered structure, and products produced therefrom of uniform thickness. In some embodiments, the synthetic leather, artificial epidermal layer, artificial dermal layer, layered structure, and products produced therefrom can have a substantially uniform thickness, length, and / or width. In some embodiments, the cells in any one or more of the epidermal layer, dermal layer, layered structure can be homogeneously distributed. In some embodiments, the cells in any one or more of the epidermal layer, dermal layer, layered structure can be heterogeneously distributed.

[0160] Comparative analysis of epidermal equivalents Figures 4A-4C show a comparative analysis of quality leather, natural skin and epidermis equivalents. Figure 4A shows FESEM of longitudinal sections of natural skin and quality leather. Figure 4A shows the distinct morphological structure of the epidermis (e) and dermis (d). Tanning has permanently altered the structure of the leather. The boundaries between individual cells in the epidermis have become indistinguishable. Removal of water has made the collagen bundles in the dermis more compact and durable. Magnification: 1000x.

[0161] Figure 4B shows an FESEM image. As an example, Figure 4B shows that both the surface of good quality leather and the surface of the epidermis equivalent have a similar smooth appearance, indicating that they are likely to induce a comparable haptic (touch) experience. Magnification: 2000x.

[0162] Figure 4C shows FESEM of a longitudinal section of a good quality leather and epidermis equivalent. Prior to tanning, distinct cell layers are discernible in the epidermis equivalent, similar to the epidermis of natural skin, Figure 4A. Since collagen in the dermis may be primarily responsible for the tensile strength of skin, collagen bundles may give leather thickness and durability (inset), but may not provide a sensory experience that may be entirely dependent on the outer layer of the epidermis.

[0163] Figures 5A-5C show a comparative analysis of the stratum corneum (SC; cornified layer) of natural skin and epidermal equivalents. Figure 5A shows an FESEM image showing that the surface of the epidermal equivalent appears smoother than that of the natural skin, which may be due to the controlled environment of cell culture. Figure 5B shows that using TEM, corneodesmosomes (arrows), the main "mechanical" junctions of the SC, could be detected as more electron-dense areas in the epidermal equivalent. Figure 5C shows that corneodesmosins (CDSNs) can be synthesized and dumped into the extracellular space by cells in SGs just before the onset of keratinization. CDSNs may embed within the intercellular portions of SG desmosomes that are occupied by cadherins, and thus can form corneodesmosomes. Arrows point to punctate accumulations of similarly aligned CDSNs in the SC of natural skin and epidermal equivalents, which likely represent corneodesmosomes. SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum.

[0164] Figures 6A-6E show comparative analysis of the granular layer (SG; stratum granulosum) of native skin and epidermal equivalent. Figure 6A shows its loricrin (LOR) staining. LOR is a major protein component of the keratinocyte envelope and can be expressed in the granular layer of keratinizing epithelium. A similar LOR expression pattern in SG (dark brown pigment in H&E stained tissue section pointed to by arrow) was detected in both the epidermis of native skin (left side of panel) and epidermal equivalent (right side of panel). d, dermis; H&E, hematoxylin & eosin; SB, stratum basale; SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; *, transwell filter membrane. In Figure 6B, epidermal Ca ++ The gradient can be captured by transmission electron microscopy as electron-dense precipitates. ++Deposits of lanthanum were present in the SG and absent in the SC in both in vivo native skin (left side of panel) and in the in vitro generated epidermal equivalent (right side of panel). SG, stratum granulosum; SS, stratum spinosum. In Figure 6C, the integrity of the permeability barrier was assessed by lanthanum perfusion. Lanthanum was visualized as electron-dense deposits in the extracellular space of viable SG, demonstrating that lanthanum, and by extension water and other small ions, can pass between keratinocytes in this layer. In contrast, lanthanum cannot penetrate further into the SC, as a functioning lipid barrier blocks the upward movement of lanthanum. The in vitro generated epidermal equivalent demonstrated a permeability barrier equally functional as native skin. SG, stratum granulosum; SS, stratum spinosum. FIG. 6D shows that tight junction protein 1 / zonula occludens-1 (TJP1 / ZO-1) anchors tight junction chain proteins, fibril-like structures within the lipid bilayer, to the actin cytoskeleton. Arrows point to similarly aligned bright green cell membrane-associated accumulation of TJP1 / ZO-1 in SGs of native skin in vivo (left panel) and in vitro-generated epidermal equivalents (right panel). SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum. FIG. 6E shows that filaggrin (FLG) monomers clustered vertically to a large 350 kDa protein precursor known as profilaggrin are present in keratohyalin granules in cells of SGs. Arrows point to similarly aligned bright red granules and cell membrane-associated accumulation of FLG in SGs of native skin in vivo (left panel) and in vitro-generated epidermal equivalents (right panel). SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum.

[0165] Figures 7A-7C show lipid bilayer formation in native skin and epidermal equivalents assessed by TEM. In Figure 7A, the white arrows indicate normal lipid secretion at the border between SC and SG in both in vivo native skin (left side of panel) and in vitro-generated epidermal equivalents (right side of panel). In Figure 7B, lamellar bodies (white arrowheads) are seen in the SGs of both in vivo native skin (left side of panel) and in vitro-generated epidermal equivalents (right side of panel). Figure 7C shows normal lipid bilayer (LB) morphology in in vivo native skin (left side of panel). The lipid bilayer in the in vitro-generated epidermal equivalent (right side of panel) had a similar appearance. SC, stratum corneum.

[0166] Figures 8A-8D show comparative analysis of suprabasal markers of native skin and epidermal equivalents. As demonstrated by immunohistochemistry (KRT10; dark brown pigment in H&E stained tissue sections pointed to by arrows) and immunofluorescence (red cytoplasmic / KRT1 / and red cell membrane / DCL1 / staining pointed to by arrows), suprabasal, spinous (SS) and granular (SG) markers Keratin 10 (KRT10), Keratin 1 (KRT1), Desmocollin 1 (DCL1) have similar expression patterns in native skin in vivo (left side of panel) and in vitro generated epidermal equivalents (right side of panel). d, dermis; H&E, hematoxylin & eosin; SB, basal layer; SC, stratum corneum; SG, granular layer; SS, spinous layer; *, transwell filter membrane. In Figure 8D, desmosomes (arrows) are clearly defined in both native skin in vivo and in the in vitro generated epidermal equivalent. SS, stratum spinosum.

[0167] Figures 9A-9D show a comparative analysis of the basal layer (SB; basal layer) of natural skin and epidermal equivalents. With respect to Figures 9A, 9B, 9C, and 9D, the proliferation marker MKI67, keratin 14 (KRT14), and transcription factor TP63 show a typical basal layer distribution in both in vivo natural skin (left side of panel) and in vitro generated epidermal equivalents (right side of panel) as demonstrated by immunohistochemistry (MKI67; dark brown pigment in H&E stained tissue sections pointed to by arrows) and immunofluorescence (green cytoplasmic / KRT14 / and white nuclear / TP63 / staining indicated by arrows). In both in vivo natural skin and in vitro generated epidermal equivalents, hemidesmosomes (arrows) are clearly defined. BM, basement membrane; Cy, cytoplasm; d, dermis; H&E, hematoxylin & eosin; SB, stratum basale; SS, stratum spinosum; TM, transwell filter membrane.

[0168] Figures 10A-10F show a comparative analysis of extracellular matrix components of basement membranes. Basement membranes (BMs) can be formed from a condensed network of extracellular matrix (ECM) proteins that can provide an essential structural scaffold on the dermal-epidermal junction. Integrin β1 regulates multiple epithelial cell functions by linking cells with the ECM, which may be important for the maintenance of the BM at the dermal-epidermal junction. In Figure 10A, integrin β1 shows a typical basal layer distribution in both in vivo native skin (left side of panel) and in vitro generated epidermal equivalents (right side of panel), as indicated by arrows (cell membrane-associated staining) and arrowheads (on the tips of cells protruding through holes in the transwell membrane). BM, basement membrane; d, dermis; SB, basal layer; SS, spinous layer; SG, granular layer; TM, transwell membrane.

[0169] Fibronectin may play a role in cell adhesion. It shows that in native skin in vivo (left panel), fibronectin can be expressed mostly in the dermis, with relatively little detectable in the BM region. Similarly, in the in vitro generated epidermal equivalent (right panel), fibronectin can be detected at the leading edge of cells protruding through holes in the transwell membrane (red arrowheads). d, dermis; SB, basal layer; SS, spinous layer; SG, granular layer; TM, transwell membrane.

[0170] The mechanical support provided by the BM can be determined by collagen IV, or in the case of the epidermal equivalent, the scaffold. As indicated by the arrowhead in Figure 10C, collagen IV expression has a patchy pattern in native skin in vivo (left side of panel) similar to that of the in vitro generated epidermal equivalent (right side of panel). BM, basement membrane; d, dermis; SB, stratum basale; SS, stratum spinosum; SG, stratum granulosum; TM, transwell membrane.

[0171] Collagen VI may play a role in cell adhesion and may associate with fibronectin. As in Figure 10D, in native skin in vivo (left panel), collagen VI may be expressed mostly in the dermis, with relatively little detected in the BM region. Similarly, in epidermal equivalents generated in vitro (right panel), collagen VI may be detected at the tips of cells protruding through holes in the transwell membrane (arrowheads), Figure 10D. BM, basement membrane; d, dermis; SB, basal layer; SS, spinous layer; SG, granular layer; TM, transwell membrane.

[0172] Collagen VII can anchor the basement membrane to collagen I and III fibrils in the dermis. As indicated by the arrowheads in Figure 10E, collagen VII expression has a patchy pattern in native skin in vivo (left panel) similar to that of the epidermal equivalent generated in vitro (right panel). BM, basement membrane; d, dermis; SB, basal layer; SS, spinous layer; SG, granular layer; TM, transwell membrane.

[0173] Laminin 5 (chain composition α3β3γ2) may be the main component of anchoring filaments and may be essential for the initial assembly of BM in vivo. As indicated by the arrowheads in FIG. 10F, expression of laminin 5 has a similar pattern in native skin in vivo (left panel) to the in vitro generated epidermal equivalent (right panel). In addition to laminin 5 being particularly abundant in the epidermal equivalent, traces of laminin 5 can be seen on the plasma membrane of basal layer cells (arrows). BM, basement membrane; d, dermis; SB, basal layer; SS, spinous layer; SG, granular layer; TM, transwell membrane.

[0174] Figures 11A-11I show the structural analysis of the full thickness skin equivalent (FSE). Figures 11A and 11B show that the cross-section of the FSE reveals distinct cell layers of the epidermis at a magnification of 2600x (Figure 11A) and 5200x (Figure 11B). Figure 11C shows the surface of the FSE at a magnification of 900x, which has a smooth appearance similar to that of good quality leather, indicating that the FSE can induce a comparable haptic (touch) experience. Figures 11D-1F show the longitudinal section of the dermal scaffold with present dermal fibroblasts and abundant extracellular matrix at a magnification of 91x (Figure 11D), 162x (Figure 11E) and 405x (Figure 11F). 11G-11I show dermal scaffolds with present dermal fibroblasts and abundant extracellular matrix at magnifications of 80× (FIG. 11G), 695× (FIG. 11H) and 2700× (FIG. 11I).

[0175] Figures 12A-12R show the time course of the engineering of the dermal equivalent. Figures 12A-12I show the results at 36x magnification (Figure 12A), 695x magnification (Figure 12B), 1470x magnification (Figure 12C), 7750x magnification (Figure 12D), 2320x magnification (Figure 12E), 2420x magnification (Figure 12F), 6560x magnification (Figure 12G), 17000x magnification (Figure 12H) and 22000x magnification (Figure 12I) on the second day after seeding dermal fibroblasts on the scaffold. The cells can migrate into the hollow structure of the scaffold and start to secrete extracellular matrix. Figures 12J-12R show the scaffolds at 7 days after seeding with dermal fibroblasts at a magnification of 64x (Figure 12J), 100x (Figure 12K), 364x (Figure 12L), 82x (Figure 12M), 253x (Figure 12N), 3940x (Figure 12O), 5550x (Figure 12P), 9440x (Figure 12Q) and 21680x (Figure 12R). The longitudinal (Figures 12J-12L) and transverse sections (Figures 12M-12R) can show denser cells and more abundant extracellular matrix, with some areas showing almost complete occlusion of the hollow structure of the scaffolds (Figures 12M-12P). EXAMPLES

[0176] Example 1 Differentiation of iPSCs into Keratinocytes To induce differentiation, undifferentiated iPSCs are transferred to a 20% O2 atmospheric environment and treated with mTESR1 or other pluripotent stem cell basal medium supplemented with 1 mM ATRA (Sigma-Aldrich) and 25 ng / ml BMP4 (R&D) for 7 days (Induction).

[0177] To select for cells that exhibit early acquisition of an ectodermal fate, cells were harvested and cultured on freshly prepared 3D HDF ECM or other types of ECM for 1 cm. 2 Winning 5~10×10 3 Cells are replated at a density of 100x and grown for an additional 7 days in Dulbecco's modified Eagle's medium / Ham's F12 (3:1; Life Technologies) or Keratinocyte medium supplemented with a serum substitute such as human platelet lyste, as well as 1 mM ATRA and 25 ng / ml BMP4 (selection).

[0178] To enrich for putative epidermal progenitor cells, rapid attachment to collagen type IV coated dishes can be used, and the rapidly attached cells are cultured in defined Keratinocyte-SFM or other keratinocyte medium supplemented with 1 mM ATRA for 7 days (enrichment), after which the cells are cultured in EpiLife medium (Life Technologies) or other keratinocyte medium for an additional 7 days (expansion), before finally being harvested and analyzed.

[0179] Example 2 Differentiation of induced pluripotent stem cells into keratinocyte lineage Coating tissue culture dishes with Geltrex and Col This procedure may be performed using aseptic technique in a biological safety cabinet. Geltrex matrix, like Matrigel, solidifies rapidly at room temperature (RT). Aliquot each new batch of matrix upon arrival and use pre-chilled pipette tips, racks, and tubes when handling this reagent. Make 50, 100, and 200 μL aliquots and store at -80°C. Use Geltrex at a 1:100 dilution.

[0180] The coating procedure below may be described for 60 mm tissue culture dishes. If larger dishes are used, adjust the volume of coating solution accordingly. 1. Remove a 50 μL aliquot of Geltrex from the -80° C. freezer and place on ice in a biological safety cabinet. 2. Add 5 mL of chilled sterile DMEM / F12 to the 15 mL conical tube. 3. Using a 1 mL glass pipette, take 1 mL of chilled DMEM / F12 from the 15 mL conical tube prepared in step 2 and add to the frozen Geltrex. Thaw and dissolve the Geltrex by gently pipetting up and down. Transfer the dissolved Geltrex to the remainder of the DMEM / F12 in the 15 mL conical tube prepared in step 2. Pipette to mix the diluted Geltrex. 4. Add 50 μL of the 3 mg / mL ColI stock solution to the diluted Geltrex from step 3. Pipette and mix the diluted Geletrex with the ColI. Add 4 mL of Coating Solution to the 60 mm dish. Tap or swirl the plate to ensure the entire surface is coated. 5. Incubate the dishes with the Geltrex / ColI coating solution in a 37° C. tissue culture incubator for at least 1 hour. 6. Once coating is complete, leave the coating solution in the dish and proceed to plating iPSCs as described in the next subsection. Alternatively, aspirate the coating solution and add 2 mL of fresh DMEM / F12 to the coated dish to prevent the dish from drying out before plating the cells.

[0181] Plating iPSCs for differentiation Prepare one 60 mm tissue culture dish of feeder-free iPSCs grown to approximately 70% confluence. Examine the cells under a microscope to ensure there is no contamination and that the undifferentiated phenotype is maintained. When cells are under stress or dying, they will begin to differentiate and show "cobblestone" areas with larger pleomorphic cells. These cells should not be used for differentiation into keratinocytes.

[0182] iPSCs at a 1:8 split ratio to differentiate iPSCs into keratinocytes. 1. Pre-warm N2B27 medium and dispase in a 37°C water bath. 2. Use a microscope to verify that the colonies are ready for passaging. Gently aspirate the medium from the dish. Add 2 mL of 1x PBS, swirl the plate to wash the cells, and gently aspirate the PBS. 3. 1 mL of dispase was added and the plate was returned to the 37° C. tissue culture incubator for 3-5 minutes. 4. While the cells are incubating with dispase, gently aspirate the Geltrex / ColI coating solution (or DMEM / F12) from step 6 of the Geltrex / ColI coating procedure and add 4 mL of complete N2B27 medium to the coating dish. 5. After the 3-5 minute incubation with dispase, verify that the cells are ready to be harvested by examining the edges around the colonies for curling or folding back. 6. Transfer the plate to a biological safety cabinet and carefully aspirate off the dispase. After treatment with dispase, the colonies are very loosely attached to the surface of the dish and can be detached without using much force. 7. Gently add 2 mL of plain DMEM / F12. Aspirate the medium and repeat the wash 3 times. 8. Add 2 mL of complete N2B27 to the dish and gently scrape the colonies off the plate. Transfer the cells from the dish to a 15 mL conical tube and add 6 mL of complete N2B27 to bring the total volume of the cell suspension to 8 mL. 9. Gently mix the cell suspension to loosen any large cell clumps. Transfer 1 mL of the cell suspension to the coated dish prepared in step 3 of this subsection. Discard or replate the remaining cells using the conditions prescribed by the given laboratory. 10. Transfer the freshly plated cells to an incubator and gently rock the plate back and forth to evenly distribute the cells. Incubate the cells overnight in a 37°C tissue culture incubator.

[0183] Differentiation of iPSCs with RA and BMP4 Differentiation and passaging of iPSC-derived keratinocytes is performed using sterile technique in a biological safety cabinet. Inspect new plates the day after passaging to ensure successful attachment of iPSCs. If iPSCs begin to form colonies, proceed with the differentiation protocol below.

[0184] 1. Pre-warm complete DKSFM (with antibiotics and DKSFM supplements) in a 37° C. water bath. 2. Add 5 mL of DKSFM (pre-warmed from the previous step) to a 15 mL conical tube, add 5 μL of 1 mM RA to achieve a final working concentration of 1 μM, and 5 μL of 25 μg / mL BMP4 to achieve a final working concentration of 25 ng / mL, and mix well. 3. Aspirate the N2B27 medium from the dish with plated iPSCs, wash once with 4 mL of 1x PBS, and add 4 mL of DKSFM containing 1 µM RA and 25 ng / mL BMP4 from the above step. This is day 1 of the differentiation procedure. 4. Transfer the cells to an incubator and incubate for 48 hours. 5. After 48 hours of incubation, the medium is replaced with fresh DKSFM containing 1 μM RA and 25 ng / mL BMP4. The cells are transferred to the incubator for another 48 hours. 6. After the second round of 48 hour induction (day 4 of differentiation), replace the medium with complete DKSFM without RA and BMP4. Incubate the cells in complete DKSFM for 10 days in the incubator, changing the medium every other day. 7. On day 14 of differentiation, prepare complete CnT-07 medium by adding antibiotics and supplements as provided and pre-warm the medium. By this day, the majority of cells in the grown iPSC colonies begin to exhibit an epithelial-like phenotype. 8. Aspirate the DKSFM from the differentiating cells and replace with 4 mL of complete CnT-07. Incubate the cells for an additional 10 days in a tissue culture incubator, changing the complete CnT-07 every other day.

[0185] Rapid attachment and culture of iPSC-derived keratinocytes At day 24 of differentiation, many cells that migrate away from the growing iPSC colony display a keratinocyte-like phenotype and begin to express p63, a key regulator required for ectoderm commitment to a keratinocyte fate, and Krt14. By this day, the 60 mm dishes used for iPSC differentiation are fully confluent and need to be passaged. To enrich for iPSC-derived keratinocytes during passaging, the differentiated iPSC culture is rapidly attached to ColI / ColIV-coated plates. Up to four 100 mm ColI / ColIV-coated tissue culture dishes are used to perform the rapid attachment procedure of one 60 mm dish containing differentiated iPSCs. If only one 100 mm dish is used, plate ¼ of the differentiated iPSC culture for the rapid attachment procedure.

[0186] Coating of plates with ColI and ColIV This procedure may be performed using aseptic technique in a biological safety cabinet. 1. Reconstitute ColIV powder to a concentration of 2 mg / mL in 0.25% sterile glacial acetic acid. Allow to dissolve at 2-8 °C for several hours, swirling occasionally. Make aliquots and store them at -20 °C. 2. Thaw an aliquot of ColIV stock solution (2 mg / mL) very slowly by placing the vial in an ice bucket and keeping at 4°C for several hours. 3. Resuspend ColIV stock solution in an appropriate volume (5 mL for each 100 mm dish) of 0.25% sterile glacial acetic acid to a final working concentration of 7 μg / mL. Add an appropriate volume of ColI stock solution to achieve a final working ColI concentration of 30 μg / mL. Coat the plate by using 5 mL of working solution to cover the 100 mm dish. Incubate the plate at room temperature for 1 hour in a biological safety cabinet. 4. Aspirate the liquid from the coated plates and rinse the dishes once with 5 mL of sterile 1x PBS and once with 5 mL of ddH2O. 5. Allow the washed dishes to air dry in a biological safety cabinet. Use the plates as is or seal them with parafilm and store at 4°C for up to 6 months. To use previously stored ColIV-coated plates, warm the plates to room temperature in a biological safety cabinet for at least 1 hour before plating the cells.

[0187] Rapid attachment of iPSC-derived keratinocytes 1. On day 24 of differentiation, pre-warm complete CnT-07, Accutase, and ColI / ColIV coated dishes. 2. Wash cells with 1x PBS, add 2mL Accutase, and incubate in a tissue culture incubator for 5 minutes. Check under a microscope that cells have begun to detach. 3. Add 3mL complete Cnt-07, detach cells by pipetting up and down, and collect cell suspension in a 15mL conical tube. Centrifuge cells at 260xg for 5 minutes and aspirate supernatant. Resuspend pellet in 10mL complete Cnt-07 medium, repeat centrifugation at 260xg for 5 minutes, and aspirate supernatant. 4. Resuspend the pellet in 4 mL of complete CnT-07 and pipette up and down to break up clumps into single cells. 5. Add 9 mL of complete CnT-07 medium to each ColI / ColIV coated dish and transfer 1 mL of the cell suspension from step 4 above to each ColI / ColIV coated dish. Allow the cells to adhere to the coated dishes for 15-30 minutes at room temperature. 6. Carefully aspirate the medium with the floating cells (these are undifferentiated or partially differentiated iPSCs). Leave the attached cells (these are iPSC-derived Krt14 positive cells) to rest. Add 10 mL of fresh complete CnT-07 to the plate with the attached cells. Expand the cells in a tissue culture incubator at 37°C, changing the medium every other day. Passage the cells in CnT-07 or EpiLife (with EDGS supplement) in ColI-coated dishes, with Accutase as needed. After passage 2 or 3, and after the rapid attachment step, the cultures should be composed of approximately 90% Krt14 positive cells that display a keratinocyte-like phenotype. The keratinocyte-like phenotype of the resulting cultures can be verified by standard immunofluorescence analysis of Krt14 expression and by the ability to reconstitute a normal stratified epidermis in organotypic culture.

[0188] Example 3 Preparation of epidermal layer from primary keratinocytes Primary keratinocytes were isolated from single neonatal foreskins and cultured in 0.07 mM Ca medium supplemented with human keratinocyte growth supplement. 2+ Grown in 154CF medium (Life Technologies). A suspension of first passage keratinocytes (2.21 × 10 5 / cm 2 Inserts) are seeded onto Cellstart CTS (Life Technologies) (or other ECM substrate) coated PET, 0.4 mm inserts (EMD Millipore) in CnT-07 medium (CELLnTEC) or CnT-Prime medium (CELLnTEC) according to the manufacturer's protocol.

[0189] On day 3 (D3) after seeding, the medium is switched to CnT-02-3D (CELLnTEC) or CnT-3D Barrier (CELLnTEC). On day 4, the HEE are exposed to air by feeding CnT-02-3D or CnT-3D Barrier to the bottom of the insert. From day 4 onwards, the HEE are fed daily with CnT-02-3D or CnT-3D Barrier until harvest. The HEE are grown in a high humidity (100% RH) or dry (50% RH) incubator at 37°C and 5% CO2. A dial hydrometer (Fisher Scientific) is used to measure incubator humidity. Low incubator humidity is maintained by removing the water dish.

[0190] To control for possible changes in osmolarity, medium is refreshed daily. Using this protocol, no significant changes in osmolarity are detected as measured by a microosmometer (Precision Systems). 12-well inserts are used for transepithelial electrical resistance (TEER) measurements, light microscopy, and electron microscopy, and 6-well inserts are used for transepidermal water loss (TEWL) measurements and immunoblotting.

[0191] Example 4: Cultivation of the epidermal layer Keratinocytes were plated at 2.0–2.5 × 10 cells / well on polyethylene terephthalate (PET) membranes with 0.4 μm pore inserts (EMD Millipore; catalog number: MCHT12H48) in CnT-07 medium (CELLnTEC) or CnT-Prime medium (CELLnTEC). 5 cells / cm 2 Sow at a density of 1000 x 1000 mm.

[0192] On day 3 (D3) after seeding, the medium is switched to CnT-02-3D (CELLnTEC) or CnT-3D Barrier (CELLnTEC). On day 4, the cells are exposed to air by feeding CnT-02-3D or CnT-3D Barrier to the bottom of the insert. From day 4 onwards, the epidermal layer is fed daily with CnT-02-3D or CnT-3D Barrier until harvest on day 14.

[0193] Example 5 Preparation of Support Carrier To prepare a 2% agarose solution, 2 g of ultrapure low melting point (LMP) agarose is dissolved in 100 mL of ultrapure water / buffer solution (1:1, v / v). The buffer solution may optionally be PBS (Dulbecco's Phosphate Buffered Saline 1x) or HBSS (Hank's Balanced Salt Solution 1x). The agarose solution may be placed in a beaker containing warm water (hotter than 80°C) and kept on a hot plate until the agarose is completely dissolved. The agarose solution will remain liquid as long as the temperature is above 36°C. Below 36°C, a phase transition occurs, the viscosity increases, and the agarose will eventually form a gel.

[0194] To prepare the agarose support, 10 mL of 2% liquid agarose (temperature >40° C.) may be placed in a 10 cm diameter Petri dish and evenly distributed to form a uniform layer. The agarose is allowed to form a gel at 4° C. in a refrigerator.

[0195] Example 6: Production of synthetic leather containing fibroblasts, keratinocytes, and melanocytes The protocol may be outlined as follows: a) fibroblasts are contacted with a solution of collagen, followed by incubation for a sufficient period of time to obtain a contracted collagen matrix onto which the fibroblasts are distributed and constituting the dermis equivalent; b) a mixture of keratinocytes and melanocytes is seeded onto the dermis equivalent obtained in a) and cultured submerged in liquid medium; c) the entire culture obtained in b) (keratinocytes and melanocytes seeded onto the dermis equivalent) is submerged and culture is continued at the air-liquid interface until a multi-stratified epidermis equivalent containing melanocytes on a dermis equivalent containing fibroblasts in a collagen matrix is ​​obtained, constituting the skin equivalent.

[0196] Step a) can be carried out with collagen type I, in particular of bovine origin, and a mixture of collagens I and III (approximately 30% relative to the final volume of the lattice). It is advantageous to add thereto other components such as laminin (in particular from 1% to 15% relative to the final volume), collagen IV (in particular from 0.3% to 4.5% relative to the final volume) and / or entactin (in particular from 0.05% to 1% relative to the final volume) in order to obtain a homogeneous suspension. Fibroblasts are obtained from the skin. The fibroblasts are cultured in a suitable medium and then suspended and mixed with the suspension of collagen and growth factors. The mixture is generally incubated at a temperature of 36°C to 37.5°C, approximately 37°C, for 1 to 6 days, preferably 4 or 5 days. Advantageously, the mixture is incubated on a support that does not allow its attachment, in particular that prevents the attachment of the mixture to the edges of the support. Such a support can in particular be obtained by previously treating its surface, for example by coating it with bovine albumin or serum. In this way, a collagen gel is obtained in which the fibroblasts are embedded, freely contracting in several directions while releasing nutrient medium.

[0197] For carrying out step b), keratinocytes derived from skin, preferably from adult skin, can be used. Before seeding according to the technique of Rheinwald and Green (Cell, vol. 6, pp. 331-344, 1975), the keratinocytes are expanded by culturing on a feeder support composed of 3T3 fibroblasts in a suitable medium known to those skilled in the art in the presence of growth factors, in particular in the presence of amino acids, serum, cholera toxin, insulin, triiodothyronine and a pH buffer solution. In particular, such a culture medium may contain at least one mitogenic growth factor, in particular for keratinocytes (e.g. epidermal growth factor (EGF) and / or keratinocyte growth factor (KGF), in particular KGF), insulin, hydrocortisone and optionally antibiotics (e.g. gentamicin, amphotericin B).

[0198] The melanocytes may be melanocytes derived from young or adult animal skin. The melanocytes are expanded by culturing in a suitable medium consisting of a basal medium such as DMEM / F12 or MCDB153, supplemented with melanocyte-specific growth factors (such as bFGF, SCF, ET-1, ET3 or αMSH) in the absence of phorbol esters, in particular in other medium such as M2 medium (Promocell) or M254 (Cascades Biologics™).

[0199] From these cultures, a cell suspension of melanocytes and a cell suspension of keratinocytes are prepared and mixed to obtain a mixed keratinocyte / melanocyte suspension. The melanocyte / keratinocyte ratio may be from 1:10 to 2:1, and is generally approximately 1:1. This mixed suspension is deposited on a dermis equivalent. The dermis equivalent is advantageously attached to a support via a biological material such as collagen. The melanocyte / keratinocyte suspension is deposited in a ring or any equivalent means for maintaining the suspension on a delimited surface area. A liquid nutrient medium is added in such a manner as to cover the mixture of cells. This medium contains growth factors known to those skilled in the art, in particular EGF and / or KGF. The medium will be replaced periodically and the culture is continued as an immersion, generally for a period of 2 to 10 days, in particular for a period of 5 to 8 days and for a period of approximately 7 days. The medium contains KGF from the second day of immersion, ideally from the fourth day of immersion.

[0200] The skin is then immersed in a manner known per se to obtain the differentiation of keratinocytes and the formation of a stratified epidermis equivalent. This step c), which corresponds to culturing as an immersion at an air-liquid interface, is continued until a differentiated structure is obtained, generally for approximately 7 days. However, step c) may also be continued for a longer period, for example for approximately 28 days, while at the same time preserving the skin equivalent with the advantageous characteristics specified in the above description. The nutrient culture medium will be renewed periodically. The skin equivalent is then removed and the necessary tests are carried out.

[0201] Example 7 Induction of follicle formation in cultured skin specimens The expanded DP cells are mixed with the cultured ORS cells, washed, and carefully resuspended in 20 ml of sterile phosphate-buffered saline (PBS, Sigma) at a suitable cell density. The cultured DP and ORS cells used in each experiment are obtained from different donors, because the culture periods of DP and ORS cells are different and it is not possible to prepare two cell types derived from the same donor. The cell suspension is slowly injected into the dermis of the cultured skin pieces one day after the culture is established.

[0202] Example 8 Cultivation of hair follicle cell populations Hair follicles are obtained from the occipital region of the scalp. Dermal papilla (DP) cells are prepared and cultured as described by Randall et al., A comparison of the culture and growth of dermal papilla cells from hair follicles from non-balding and balding (androgenetic alopecia) scalp., Br J Dermatol, 1996:134:437-444).

[0203] Briefly, the DP of hair follicles are isolated under a dissecting microscope and transferred individually to 24-well tissue culture plates (Sarstedt). Cell culture is performed in DMEM supplemented with 15% FCS (Sigma). Once cell proliferation is initiated, the cells are cultured until confluent and expanded for two passages. For the isolation of outer root sheath (ORS) cells, the central part of the hair follicle, including the bulge region, is excised and subjected to gentle trypsinization. At least 10 hair follicle cells are used for each culture. The obtained cells are washed twice with RPMI-1640 medium (Sigma) and subjected to cell culture in standard keratinocyte medium (Epilife, Sigma). After one week of culture, the cells are harvested.

[0204] Example 9 Tanning of Full Thickness Skin Equivalents Full thickness skin equivalents are tanned using a chrome tanning process. The first step is an ice and sulfuric acid treatment. This opens up the tissue so that it can accept chromium. The chromium is then added along with magnesium oxide.

[0205] This process reduces the pH level of the full thickness skin equivalent to about 3. Once the chrome has been worked into the full thickness skin equivalent, the tanning liquor is introduced, which raises the pH to about 4. It is then placed in a hot water bath and roll pressed to remove excess liquid. The final step is then any surface treatment required, after which the full thickness skin equivalent is stretched and dried, and then pressed again.

[0206] Example 10 X-tan tanning protocol The full thickness skin equivalent may be tanned using the X-tan procedure. Prior to tanning, the full thickness skin equivalent is limed. Liming includes the steps of soaking the skin equivalent, adding a substrate, adjusting the pH, and washing. The skin equivalent is then demineralized by washing the skin equivalent, adding a pre-demineralization buffer, demineralizing the skin equivalent, and washing. The skin equivalent is then tanned by rehydrating, adding a tanning substrate, adjusting the pH to the tanning pH, performing a fixation cycle and fixation, and emulsion fatliquoring to obtain a tanned skin equivalent.

[0207] Example 11 Full Thickness Skin Equivalent Type I collagen matrix (0.5 × 10 6 The matrix (containing 1×10 iPSC-derived fibroblasts) may be deposited on a polyethylene terephthalate membrane (BD Biosciences) and allowed to polymerize. After incubation for about 7 days, the polymerized matrix is ​​incubated with 1×10 6 0.1 x 10 iPSC-derived keratinocytes and 0.1 x 10 6iPSC-derived melanocytes may be seeded onto the matrix and incubated for an additional 7 days. The composite culture may be elevated to an air-liquid interface and fed from below to induce epidermal differentiation. After approximately 14 days, full-thickness skin equivalents may be harvested and either snap frozen in LN2 or embedded in wax for melanin quantification.

[0208] Example 12 Immunostaining of Frozen Sections Fixation: Tissues may be fixed in 3.8% paraformaldehyde / phosphate buffered saline (PBS), pH 7.2-7.6, for 30 minutes. Samples may be washed 3 times for 5 minutes in PBS. Tissue samples may be infiltrated with rotation at 4°C through a series of sterile sucrose gradients (10% sucrose overnight, 15% sucrose for 6-8 hours, 30% sucrose overnight, and finally 30% sucrose mixed 1:1 with optimal cutting temperature (OCT) compound overnight). Samples may be embedded in OCT and frozen in liquid nitrogen vapor. Frozen blocks may be stored at -80°C.

[0209] Cutting sections: The day before sectioning, frozen blocks may be transferred to -20°C overnight. Sections (10 μm) may be prepared using a standard cryostat. Sections may be kept at -20°C until processing.

[0210] Treatment: may include control incubations. Pre-immune serum or isotype-matched non-immune antibodies may be used instead of primary antibodies. Sections may be immersed in either 90% chilled acetone for 10 min or 0.2% triton X-100 / PBS for 5 min to expose antigens. Samples may be washed three times for 5 min with PBS. Non-specific antibody reactivity was blocked by immersing sections in 5% BSA with 0.1% triton X-100 for 1 h. Sections may then be incubated overnight at 4°C with a mixture of two antibodies: i) 2.5 μg / ml ChromPure donkey total IgG (for blocking; secondary antibodies may all be produced in donkey); ii) 1 μg / ml of the appropriate primary antibody. Sections may be rinsed three times for 5 min in PBS. Sections may be incubated for 30-60 min at room temperature with the appropriate species-specific secondary antibody produced in donkey and conjugated to either a red or green fluorophore. Sections may be washed 3 times for 5 minutes with PBS. Sections may then be incubated with 10 μg / ml Hoechst 33342 at room temperature for 10 minutes. Sections may be washed 3 times for 5 minutes with PBS.

[0211] Visualization: Samples may be mounted with Vectashield medium (Vector). Samples may be visualized with an epifluorescence microscope (Zeiss) equipped with appropriate filters.

[0212] Example 13 Immunostaining of Paraffin-embedded Sections Fixation: Tissues may be fixed in 3.8% paraformaldehyde / phosphate buffered saline (PBS), pH 7.2-7.6, for 30 min. Samples may be washed three times for 5 min in PBS. Tissue samples may be dehydrated in an ascending ethanol series (50%, 70%, 2x100%; 20 min each) and clearing agent (xylene, 2x20 min). Samples may be perfused twice with paraffin wax for 1 h at 65°C and embedded in paraffin blocks. Paraffin blocks can be stored at room temperature until further use.

[0213] Section cutting: Tissues were sectioned at 5 μm thickness using a standard microtome. Sections can be kept at room temperature until processing.

[0214] Treatment: Control incubations may be included. Instead of the primary antibody, pre-immune serum or isotype-matched non-immune antibodies may be used. Sections may be rewetted with an ascending series of xylene / ethanol series, 2x xylene, 2x 100% ethanol, and 1x 70% and 50%; 10 minutes each. Sections may then be rinsed briefly with tap water. Sections may then be stained with hematoxylin for 5 minutes. Sections may then be washed with dH2O until the solution is clear. Sections may then be stained with 0.5% eosin for 10 minutes. Sections may then be rinsed briefly with tap water. Non-specific antibody reactivity was blocked by immersing sections in 5% BSA for 1 hour. Sections may then be incubated overnight at 4°C with a mixture of two antibodies: i) ChromPure donkey total IgG at 2.5 μg / ml (for blocking; secondary antibodies may all be produced in donkey); ii) 1 μg / ml of the appropriate primary antibody. Sections may be rinsed three times for 5 minutes with PBS. Sections may be incubated with an appropriate species-specific secondary antibody raised in donkey and conjugated to horseradish peroxidase (HRP) for 30 minutes at room temperature. Sections may be washed three times for 5 minutes with PBS.

[0215] Visualization: Samples may be incubated with 3,3'-diaminobenzidine (DAB) substrate kit (Vector Laboratories) according to the manufacturer's protocol. DAB results in a brown staining. If nickel chloride is added to the substrate solution, a gray-black staining may result. Samples may be dehydrated in an ascending ethanol series (50%, 70%, 2 x 100%; 10 min each) and clearing agent (xylene, 2 x 10 min). Samples may be mounted with mounting medium and visualized with a phase-contrast microscope equipped with a digital camera (Zeiss).

[0216] Example 14: Field Emission Scanning Electron Microscopy (FESEM) Fixation: Samples may be fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 24 hours at 4° C., placed in 0.1 M sodium cacodylate buffer, and kept at 4° C. before further processing.

[0217] Processing: Samples may be post-fixed in 1% OsO4 in water for 1 hour. After dehydration in an ascending ethanol series (50%, 70%, 2x100%; 10 min each), samples may be critical point dried in liquid CO2 using a Tousimis Autosamdri-815B apparatus, mounted on 15 mm aluminum mounts with double-sided copper tape, and sputter-coated with 40 Å of gold-palladium using a Denton DeskII Sputter Coater.

[0218] Visualization: Cross sections of double overlapping specimens may be mounted in low-profile 45 / 90 degree SEM mounts for internal morphology analysis. Visualization may be performed on a Zeiss Sigma FESEM (Carl Zeiss Microscopy, Thornwood, NY) operated at 2-3 kV using inLens secondary electron (SE) detection as well as mixed signal InLens / SE2 (75 / 25%) detection at a working distance of 3-5 mm. Images may be captured in TIFF with a storage resolution of 2048 x 1536 and a line averaging noise reduction algorithm.

[0219] Processing: The previously dried samples (i.e., leather) were cut to size and 40 Å of gold-palladium may be sputter coated using a DeskII Sputter Coater.

[0220] Visualization: Cross sections of double overlapping specimens may be mounted in low-profile 45 / 90 degree SEM mounts for internal morphology analysis. Visualization may be performed on a Zeiss Sigma FESEM (Carl Zeiss Microscopy, Thornwood, NY) operated at 2-3 kV using inLens secondary electron (SE) detection as well as mixed signal InLens / SE2 (75 / 25%) detection at a working distance of 3-5 mm. Images may be captured in TIFF with a storage resolution of 2048 x 1536 and a line-average noise reduction algorithm.

[0221] Example 15 Transmission Electron Microscopy (TEM) Fixation: Samples may be fixed with 2% glutaraldehyde and 2% paraformaldehyde with 0.06% calcium chloride in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 minutes at 4° C. Samples may then be placed in 0.1 M sodium cacodylate buffer and kept at 4° C. before further processing.

[0222] Processing: Samples may then be washed and placed in either 0.2% ruthenium tetroxide (for visualization of lipid bilayers) or 1.5% osmium tetroxide with 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate, pH 7.4, in the dark for 45 minutes at room temperature. After rinsing in buffer, samples may be dehydrated in a graded ethanol series (50%, 70%, 2 x 100%; 10 minutes each) and then embedded in low viscosity epoxy resin.

[0223] Visualization: Semi-thin sections may be stained with 1% toluidine blue with 1% azure II in 1% borax solution and viewed under a phase contrast microscope (Zeiss). Ultrathin sections may be collected and stained with water-saturated 3% uranyl acetate and / or imaged with 2.5% lead citrate on uncoated nickel grids. Ultrathin sections may be viewed under a Zeiss 10A electron microscope operated at 60 kV. Images may be captured in TIFF.

[0224] Ion capture cytochemistry (Ca ++ gradient):

[0225] Fixation: Ultrafine Ca ++ For localization, samples may be fixed in 2% paraformaldehyde, 2% glutaraldehyde, 0.09M potassium oxalate containing 0.04M sucrose. Samples may then be fixed overnight at 4°C.

[0226] Processing: Samples may be post-fixed in 1% osmium tetroxide containing 2% potassium pyroantimonate, pH 7.4, for 2 hours in the dark at 4° C. Tissue samples may then be washed in alkalized water (pH 10), transferred to ethanol solutions (50%, 70%, 2×100%; 10 min each) for dehydration, and embedded in low viscosity epoxy resin.

[0227] Visualization: Ultrathin sections may be collected and stained with water-saturated 3% uranyl acetate and / or imaged with 2.5% lead citrate onto uncoated nickel grids. Ultrathin sections may be viewed on a Zeiss 10A electron microscope operated at 60 kV. Images may be captured in TIFF.

[0228] Lanthanum perfusion:

[0229] Fixation: Perfusion route was assessed in all subjects by immersing specimens in 4% lanthanum nitrate in 0.05 M Tris buffer, pH 7.4, containing 2% glutaraldehyde, 1% paraformaldehyde for 1 hour at room temperature.

[0230] Processing: Samples may be washed and placed in 1.5% osmium tetroxide with 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate, pH 7.4, for 45 minutes in the dark at room temperature. After rinsing in cacodylate buffer, samples may be dehydrated in a graded ethanol series (50%, 70%, 2x100%; 10 minutes each) and then embedded in low viscosity epoxy resin.

[0231] Visualization: Ultrathin sections may be collected and stained with water-saturated 3% uranyl acetate and / or imaged with 2.5% lead citrate onto uncoated nickel grids. Ultrathin sections may be viewed on a Zeiss 10A electron microscope operated at 60 kV. Images may be captured in TIFF.

[0232] While some embodiments have been shown and described herein, such embodiments are provided by way of example only. Those skilled in the art will now appreciate that numerous variations, modifications, and substitutions can be made without departing from the disclosure provided herein. It should be understood that various alternatives to the embodiments described herein can be used. The present invention provides, for example, the following items. (Item 1) A method for producing synthetic leather, comprising the steps of: a. forming an artificial dermis layer containing fibroblasts; b. tanning at least a portion of said corium layer, thereby forming said synthetic leather. Including, The method, wherein the fibroblasts are differentiated from induced pluripotent stem cells. (Item 2) Item 14. The method of item 1, further comprising forming an artificial epidermal layer. (Item 3) 3. The method of claim 2, wherein the epidermal layer comprises keratinocytes. (Item 4) 4. The method of claim 3, further comprising the step of disposing the epidermis layer on the dermis layer, thereby forming a layered structure. (Item 5) A method for producing synthetic leather, comprising the steps of: a. placing an artificial epidermal layer on an artificial dermal layer, thereby forming a layered structure, the epidermal layer comprising keratinocytes and the dermal layer comprising fibroblasts; and b. tanning at least a portion of said layered structure, thereby forming said synthetic leather. wherein the fibroblasts or the keratinocytes are differentiated from induced pluripotent stem cells. (Item 6) A method for producing synthetic leather, comprising the steps of: a. disposing an artificial epidermal layer on an artificial dermal layer, thereby forming a layered structure, the epidermal layer comprising keratinocytes and the dermal layer comprising fibroblasts; b. removing at least a portion of the epidermal layer to form an ablation product; and c. tanning at least a portion of said removal product, thereby forming said synthetic leather. wherein the fibroblasts or the keratinocytes are differentiated from induced pluripotent stem cells. (Item 7) 7. The method according to any one of items 3 to 6, wherein the keratinocytes are mammalian keratinocytes. (Item 8) 8. The method of claim 7, wherein the mammal is a non-human mammal. (Item 9) 8. The method according to any one of items 3 to 7, wherein the keratinocytes are differentiated from induced pluripotent stem cells. (Item 10) 10. The method of claim 9, wherein the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. (Item 11) 11. The method according to any one of items 4 to 10, wherein the layered structure further comprises melanocytes. (Item 12) Item 12. The method according to item 11, wherein the melanocytes are differentiated from induced pluripotent stem cells. (Item 13) 13. The method of claim 12, wherein the melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. (Item 14) The method according to any one of items 11 to 13, wherein the synthetic leather further comprises a pigment. 15. The method according to any one of items 11 to 14, wherein the melanocytes are mammalian melanocytes. (Item 16) 8. The method of claim 7, wherein the mammal is a human. (Item 17) 7. The method according to any one of items 5 to 6, wherein the fibroblasts are differentiated from induced pluripotent stem cells. (Item 18) 18. The method of item 1 or 17, wherein the fibroblasts express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta, or a combination thereof. (Item 19) 19. The method of any one of items 1 to 18, wherein the fibroblasts are mammalian fibroblasts. (Item 20) 20. The method of claim 19, wherein the mammal is a non-human mammal. (Item 21) 21. The method of claim 8 or 20, wherein the non-human mammal is one of a primate, cow, sheep, pig, horse, dog, cat, rodent, or rabbit. (Item 22) The method according to any one of claims 2 to 6, wherein the epidermal layer further comprises collagen. 23. The method of claim 22, wherein the epidermal layer is subjected to further processing. (Item 24) 7. The method according to any one of items 4 to 6, wherein the layered structure further comprises collagen. (Item 25) 25. The method of claim 24, wherein the layered structure is subjected to further processing. (Item 26) 7. The method of claim 6, wherein the removal product further comprises collagen. (Item 27) 27. The method of claim 26, wherein the removal product is subjected to further processing. (Item 28) 27. The method according to any one of the preceding claims, wherein the dermal layer further comprises collagen. (Item 29) 29. The method of claim 28, wherein the dermis layer is subjected to further processing. (Item 30) 30. The method according to item 23, 25, 27 or 29, wherein said treatment is selected from the group consisting of curing, soaking, baching, pickling, deacidifying, thinning, retanning, lubricating, crusting, wetting, wringing, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixing, setting, drying, conditioning, blank beating, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, tumbling and any combination thereof. (Item 31) 29. The method of claim 22, 24, 26 or 28, wherein the collagen is at least partially produced by collagen-producing cells, is added separately, or any combination thereof. (Item 32) 32. The method of claim 31, wherein the collagen-producing cells comprise epithelial cells, keratinocytes, fibroblasts, corneocytes, melanocytes, Langerhans cells, basal cells, or a combination thereof. (Item 33) 33. The method of claim 32, wherein the collagen-producing cells comprise epithelial cells, and the epithelial cells comprise squamous cells, cuboidal cells, columnar cells, basal cells, or a combination thereof. (Item 34) 33. The method of claim 32, wherein the collagen-producing cells comprise the keratinocytes, and the keratinocytes comprise epidermal keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or a combination thereof. (Item 35) 32. The method of claim 31, wherein the collagen-producing cells comprise smooth muscle cells. (Item 36) 36. The method of any one of items 1 to 35, wherein the synthetic leather further comprises one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combination thereof. (Item 37) 37. The method according to any one of the preceding claims, wherein the thickness of the dermis layer ranges from about 0.02 mm to about 5 mm. (Item 38) Item 38. The method of item 37, wherein the thickness of the dermis layer ranges from about 0.1 mm to about 0.5 mm. (Item 39) 39. The method of any one of the preceding claims, wherein the synthetic leather further comprises a first dermal layer and a second dermal layer. (Item 40) 40. The method of claim 39, wherein the first dermal layer is disposed on the second dermal layer. (Item 41) 7. The method according to any one of items 2 to 6, wherein the thickness of the epidermis layer ranges from about 0.01 mm to about 2 mm. (Item 42) Item 42. The method of item 41, wherein the thickness of the epidermis layer ranges from about 0.1 mm to about 0.2 mm. (Item 43) 43. The method of claim 41 or 42, wherein the synthetic leather further comprises a first skin layer and a second skin layer. (Item 44) 44. The method according to any one of items 41 to 43, wherein the synthetic leather further comprises a basement membrane substitute. (Item 45) Item 46. The method according to item 44, wherein the basement membrane substitute is between the epidermis layer and the dermis layer. 46. ​​The method of claim 45, wherein the basement membrane substitute comprises a dried acellular amniotic membrane. (Item 47) 47. The method according to any one of the preceding claims, wherein the dermal layer is formed on a scaffold. (Item 48) 48. The method of claim 47, wherein the scaffold is natural or synthetic. (Item 49) 48. The method of claim 47, wherein the scaffold comprises silk. (Item 50) 50. The method of claim 49, wherein the scaffold comprises a natural tissue adhesive. (Item 51) 51. The method of claim 50, wherein the natural tissue adhesive comprises fibrin glue. (Item 52) 52. The method according to any one of items 47 to 51, wherein the scaffold is partially contained in the synthetic leather. (Item 53) 52. The method according to any one of items 1 to 51, wherein the dermal layer or the epidermal layer is cultured in vitro. (Item 54) 54. The method of claim 53, wherein the dermal layer is cultured in vitro. (Item 55) 55. The method of claim 54, wherein the dermal layer is cultured with a supplement. (Item 56) 56. The method of claim 55, wherein the supplement comprises one or more of collagen, fibrin, growth factors, ascorbic acid, dextran sulfate, or carrageenan. (Item 57) 57. The method of item 55 or 56, wherein the supplement is a natural supplement. (Item 58) 57. The method of item 55 or 56, wherein the supplement is a synthetic supplement. (Item 59) 59. The method of any one of items 1 to 58, wherein the induced pluripotent stem cells are produced through inducible gene expression of Oct3, Oct4, Sox2, Klf4, c-Myc or a combination thereof in adult somatic cells. (Item 60) 1. A method for producing at least a portion of a leather product, comprising: 60. A method comprising forming said at least a part of said leather product from synthetic leather produced by the method according to any one of the preceding claims. (Item 61) Item 61. The method of item 60, wherein the leather product comprises one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, a garment, a bag, a sack, a clutch, a wallet, a backpack, a billfold, a saddle, a harness, or a whip. (Item 62) Item 62. The method of item 61, wherein the leather product is a watch band. (Item 63) Item 62. The method according to item 61, wherein the leather product is a belt. (Item 64) Item 62. The method according to item 61, wherein the leather product is a bag. (Item 65) 60. Synthetic tanned leather produced by the method according to any one of items 1 to 59. (Item 66) Before tanning, Artificial dermis layer containing fibroblasts 1. A synthetic tanned leather comprising: a fibroblast differentiated from an artificial pluripotent stem cell. (Item 67) 67. Synthetic tanned leather according to item 66, further comprising an artificial skin layer prior to tanning. (Item 68) Item 68. The synthetic tanned leather of item 67, wherein the epidermal layer further comprises keratinocytes. (Item 69) 69. Synthetic leather according to item 67 or 68, wherein the epidermis layer is on the dermis layer, thereby forming a layered structure. (Item 70) Before tanning, An artificial dermis layer containing fibroblasts, and Artificial epidermal layer containing keratinocytes A synthetic tanned leather comprising a layered structure comprising: A synthetic tanned leather, wherein the fibroblasts or the keratinocytes are differentiated from induced pluripotent stem cells. (Item 71) Before tanning, Removal products containing layered structures A synthetic tanned leather comprising the layered structure: An artificial dermis layer containing fibroblasts, and Artificial epidermal layer containing keratinocytes wherein a portion of the epidermal layer has been removed, and the fibroblasts or keratinocytes are differentiated from induced pluripotent stem cells. (Item 72) 72. Synthetic tanned leather according to any one of items 68 to 71, wherein the keratinocytes are mammalian keratinocytes. (Item 73) Item 73. Synthetic tanned leather according to item 72, wherein the mammal is a non-human mammal. (Item 74) 74. The synthetic tanned leather according to any one of items 68 to 73, wherein the keratinocytes are differentiated from induced pluripotent stem cells. (Item 75) 75. The synthetic tanned leather of item 74, wherein the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. (Item 76) 76. Synthetic leather according to any one of items 69 to 75, wherein the layered structure further comprises melanocytes. (Item 77) Item 77. The synthetic tanned leather according to item 76, wherein the melanocytes are differentiated from induced pluripotent stem cells. (Item 78) Item 78. The synthetic tanned leather according to item 77, wherein the melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. (Item 79) 79. Synthetic leather according to item 77 or 78, further comprising a pigment. (Item 80) 80. Synthetic tanned leather according to any one of items 76 to 79, wherein the melanocytes are mammalian melanocytes. (Item 81) Item 81. The synthetic tanned leather according to item 80, wherein the mammal is a non-human mammal. (Item 82) Item 72. Synthetic tanned leather according to item 70 or 71, wherein the fibroblasts are differentiated from induced pluripotent stem cells. (Item 83) 83. The synthetic tanned leather according to item 66 or 82, wherein the fibroblasts express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta, or a combination thereof. (Item 84) 83. Synthetic tanned leather according to any one of items 66 to 82, wherein the fibroblasts are mammalian fibroblasts. (Item 85) Item 85. The synthetic tanned leather according to item 84, wherein the mammal is a non-human mammal. (Item 86) The synthetic tanned leather according to item 73, 81 or 85, wherein the non-human mammal is one of a primate, a cow, an sheep, a pig, a horse, a dog, a cat, a rodent, or a rabbit. (Item 87) 72. Synthetic leather according to any one of items 67 to 71, wherein the epidermis layer further comprises collagen. (Item 88) 72. Synthetic leather according to any one of items 69 to 71, wherein the layered structure further comprises collagen. (Item 89) Item 72. The synthetic tanned leather of item 71, wherein the removal product further comprises collagen. (Item 90) 90. Synthetic leather according to any one of items 66 to 89, wherein the dermis layer further comprises collagen. (Item 91) 91. The synthetic tanned leather according to any one of items 87 to 90, wherein the collagen is at least partially produced by collagen-producing cells, added separately, or any combination thereof. (Item 92) Item 92. The synthetic tanned leather of item 91, wherein the collagen-producing cells comprise epithelial cells, keratinocytes, fibroblasts, corneocytes, melanocytes, Langerhans cells, basal cells, or a combination thereof. (Item 93) Item 93. The synthetic tanned leather of item 92, wherein the collagen-producing cells comprise the epithelial cells, and the epithelial cells comprise squamous cells, cuboidal cells, columnar cells, basal cells, or a combination thereof. (Item 94) Item 93. The synthetic tanned leather of item 92, wherein the collagen-producing cells comprise the keratinocytes, the keratinocytes comprising epidermal keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or a combination thereof. (Item 95) Item 93. The synthetic tanned leather of item 92, wherein the collagen-producing cells include smooth muscle cells. (Item 96) 96. The synthetic tanned leather of any one of items 66 to 95, further comprising one or more of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combination thereof. (Item 97) 97. Synthetic leather according to any one of items 66 to 96, wherein the thickness of the dermis layer ranges from about 0.02 mm to about 5 mm. (Item 98) Item 98. Synthetic leather according to item 97, wherein the thickness of the dermis layer ranges from about 0.1 mm to about 0.5 mm. (Item 99) 99. The synthetic leather of any one of items 66 to 98, wherein the synthetic leather further comprises a first dermis layer and a second dermis layer. (Item 100) Item 101. The synthetic leather according to item 99, wherein the first dermis layer is on the second dermis layer. 72. The synthetic leather according to any one of items 67 to 71, wherein the thickness of the skin layer ranges from about 0.01 mm to about 2 mm. (Item 102) Item 102. The synthetic leather according to item 101, wherein the thickness of the skin layer ranges from about 0.1 mm to about 0.2 mm. (Item 103) Item 103. The synthetic leather according to item 101 or 102, wherein the synthetic leather further comprises a first skin layer and a second skin layer. (Item 104) The synthetic tanned leather according to any one of items 101 to 103, which contains a basement membrane substitute. (Item 105) Item 105. The synthetic leather of item 104, wherein the basement membrane substitute is between the epidermis layer and the dermis layer. (Item 106) Item 106. The synthetic tanned leather of item 105, wherein the basement membrane substitute comprises a dried acellular amniotic membrane. (Item 107) 107. Synthetic leather according to any one of items 66 to 106, wherein the dermal layer is formed on a scaffold. (Item 108) Item 108. Synthetic leather according to item 107, wherein the scaffold is natural or synthetic. (Item 109) Item 108. Synthetic leather according to item 107, wherein the scaffold comprises silk. (Item 110) Item 110. Synthetic leather according to item 109, wherein the scaffold comprises a natural tissue adhesive. (Item 111) Item 111. The synthetic leather of item 110, wherein the natural tissue adhesive comprises fibrin glue. (Item 112) 112. Synthetic leather according to any one of items 107 to 111, wherein the scaffold is partially contained in the synthetic leather. (Item 113) 113. Synthetic leather according to any one of items 66 to 112, wherein the dermis layer or the epidermis layer is cultured in vitro. (Item 114) Item 115. The synthetic tanned leather according to item 113, wherein the dermis layer is cultured in vitro. 115. The synthetically tanned leather of any one of paragraphs 66 to 114 in one or more of a watch band, a belt, packaging, a shoe, a boot, footwear, a glove, a garment, a bag, a sack, a clutch, a wallet, a backpack, a billfold, a saddle, a harness or a whip. (Item 116) Synthetic leathers as described in item 115, contained in watch bands. (Item 117) Synthetic leather, as described in item 115, contained in belts. (Item 118) Synthetic leather as described in item 115, included in bags. (Item 119) Hair follicle cells and melanocytes wherein the melanocytes are differentiated from induced pluripotent stem cells. (Item 120) Item 120. The epidermis layer of item 119, wherein the hair follicle cells comprise dermal papilla cells, outer root sheath cells, or a combination thereof. (Item 121) 121. The epidermis layer according to item 119 or 120, wherein the melanocytes are mammalian melanocytes. (Item 122) 122. The epidermis layer according to items 119 to 121, further comprising keratinocytes. (Item 123) 123. The epidermal layer according to item 122, wherein the keratinocytes are differentiated from induced pluripotent stem cells. (Item 124) 124. The epidermal layer of item 123, wherein the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. (Item 125) 125. The epidermis layer according to item 122, 123, or 124, wherein the keratinocytes are mammalian keratinocytes. (Item 126) 126. The epidermis layer according to item 121 or 125, wherein the mammal is a non-human mammal. (Item 127) 127. The epidermal layer according to item 126, wherein the non-human mammal is one of a primate, cow, sheep, pig, horse, dog, cat, rodent, or rabbit. (Item 128) 128. The epidermis layer of any one of items 119 to 127, wherein said melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. (Item 129) 128. The epidermis layer according to any one of items 119 to 127, further comprising hair follicles. (Item 130) a. an artificial epidermal layer containing hair follicle cells; and b. An artificial dermis layer containing fibroblasts wherein the fibroblasts are differentiated from induced pluripotent stem cells. (Item 131) 131. The layered structure of item 130, wherein the fibroblasts express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta, or a combination thereof. (Item 132) Item 131. The layered structure of item 130, wherein the epidermal layer further comprises keratinocytes. (Item 133) 133. The layered structure according to item 132, wherein the keratinocytes are differentiated from induced pluripotent stem cells. (Item 134) 134. The layered structure of item 133, wherein the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. (Item 135) Item 133. The layered structure of item 132, wherein the hair follicle cells are dermal papilla cells, outer root sheath cells or a combination thereof. (Item 136) 131. The layered structure of item 130, wherein the epidermal layer further comprises melanocytes. (Item 137) Item 137. The layered structure according to item 136, wherein the melanocytes are differentiated from induced pluripotent stem cells. (Item 138) 138. The layered structure of item 137, wherein said melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. (Item 139) 139. The layered structure according to item 136, 137 or 138, which is pigmented. (Item 140) Item 138. The layered structure of item 137, wherein the epidermis layer is stratified. (Item 141) Item 142. The layered structure according to any one of items 130 to 140, further comprising a basement membrane substitute. Item 142. The layered structure according to item 141, wherein the basement membrane substitute is between the epidermis layer and the dermis layer. (Item 143) Item 143. The layered structure according to item 142, wherein the basement membrane substitute comprises a dried acellular amniotic membrane. (Item 144) 131. The layered structure according to item 130, further comprising a scaffold. (Item 145) 145. The layered structure according to item 144, wherein the scaffold is natural or synthetic. (Item 146) Item 145. The layered structure according to item 144, wherein the scaffold comprises silk. (Item 147) Item 145. The layered structure according to item 144, wherein the dermal layer is on the scaffold. (Item 148) 148. The lamellar structure of any one of items 130 to 147, further comprising one or more components selected from the group consisting of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or combinations thereof. (Item 149) 149. The layered structure according to any one of items 130 to 148, further comprising two or more dermal layers. (Item 150) 150. The layered structure according to any one of items 130 to 149, further comprising a hair follicle. (Item 151) 150. The layered structure of any one of items 130 to 149, further comprising a soft skin. (Item 152) 1. A method for producing a layered structure, comprising: Placing an artificial epidermal layer containing hair follicle cells on an artificial dermal layer containing cells differentiated from induced pluripotent stem cells, thereby forming the layered structure. A method comprising: (Item 153) Item 153. The method of item 152, wherein the cells differentiated from induced pluripotent stem cells are fibroblasts, melanocytes, keratinocytes, or a combination thereof. (Item 154) Item 154. The method of item 153, wherein the cells differentiated from induced pluripotent stem cells are fibroblasts. (Item 155) 155. The method of claim 154, wherein the fibroblasts express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta, or a combination thereof. (Item 156) 155. The method of any one of items 152 to 154, wherein the epidermal layer further comprises keratinocytes. (Item 157) Item 157. The method of item 156, wherein the keratinocytes are differentiated from induced pluripotent stem cells. (Item 158) 158. The method of claim 157, wherein the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18 or a combination thereof. (Item 159) 159. The method of any one of items 152 to 158, wherein the hair follicle cells comprise dermal papilla cells, outer root sheath cells, or a combination thereof. (Item 160) 160. The method of any one of items 152 to 159, wherein the epidermal layer further comprises melanocytes. (Item 161) Item 161. The method of item 160, wherein the melanocytes are differentiated from induced pluripotent stem cells. (Item 162) Item 162. The method of item 161, wherein the melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof. (Item 163) 164. The method according to any one of items 152 to 163, wherein the dermal layer is cultured with a supplement. (Item 164) Item 164. The method of item 163, wherein the supplement comprises collagen, fibrin, growth factors, ascorbic acid, dextran sulfate, carrageenan or a combination thereof. (Item 165) Item 164. The method of item 163, wherein the supplement is a natural supplement. (Item 166) Item 164. The method of item 163, wherein the one or more supplements are synthetic supplements. (Item 167) 167. The method according to any one of items 152 to 166, wherein the dermal layer is cultured on a scaffold. (Item 168) 168. The method of claim 167, wherein the scaffold is natural or synthetic. (Item 169) 168. The method of claim 167, wherein the scaffold comprises silk. (Item 170) 167. The method of any one of items 152 to 166, wherein the dermal layer is disposed on a scaffold. (Item 171) 171. The method of claim 170, wherein the scaffold is natural or synthetic. (Item 172) 171. The method of claim 170, wherein the scaffold comprises silk. (Item 173) 173. The method of any one of items 152 to 172, wherein the epidermal layer is stratified. (Item 174) Item 153. The method of item 152, further comprising culturing the dermal layer on a second dermal layer. (Item 175) 175. The method according to any one of items 152 to 174, wherein the dermal layer is cultured in vivo. (Item 176) Item 176. The method of item 175, wherein the dermal layer is not cultured on a collagen matrix. (Item 177) 177. The method of any one of items 152 to 176, wherein the thickness of the dermis layer ranges from about 0.02 mm to about 5 mm. (Item 178) Item 178. The method of item 177, wherein the thickness of the dermis layer ranges from about 0.1 mm to about 0.5 mm. (Item 179) 179. The method of any one of items 152 to 178, wherein the thickness of the epidermis layer ranges from about 0.01 mm to about 2 mm. (Item 180) Item 179. The method of item 179, wherein the thickness of the epidermis layer ranges from about 0.1 mm to about 0.2 mm. (Item 181) an artificial epidermal layer containing hair follicle cells and keratinocytes or melanocytes; Artificial dermis layer containing fibroblasts wherein the fibroblasts, the keratinocytes or the melanocytes are differentiated from induced pluripotent stem cells, the melanocytes express Sox-10, MITF-M, gp-100, DCT, TYR, MLANA or a combination thereof, the fibroblasts express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta, or a combination thereof, and the keratinocytes express KRT14, p63, DSG3, ITGB4, LAMA5, KRT5, TAp63, Lamb3, KRT18, or a combination thereof. (Item 182) Item 182. The layered structure according to item 181, wherein the thickness of the dermis layer ranges from about 0.02 mm to about 5 mm. (Item 183) Item 183. The layered structure of item 182, wherein the thickness of the dermis layer ranges from about 0.1 mm to about 0.5 mm. (Item 184) 184. The layered structure of any one of items 181 to 183, wherein the thickness of the skin layer ranges from about 0.01 mm to about 2 mm. (Item 185) Item 185. The layered structure of item 184, wherein the thickness of the skin layer ranges from about 0.1 mm to about 0.2 mm. (Item 186) a. stratum corneum; b. granular layer; c. stratum spinosum; and d. Basal layer wherein the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale are organized as shown in Figures 6A and 8A. (Item 187) Item 187. The artificial epidermis layer of item 186, wherein the thickness of the stratum corneum ranges from about 0.01 mm to about 0.05 mm. (Item 188) 188. The artificial epidermis layer according to any one of items 186 to 187, wherein the thickness of the granular layer ranges from about 0.01 mm to about 0.15 mm. (Item 189) 189. The artificial epidermal layer of any one of items 186 to 188, wherein the thickness of the spinous layer ranges from about 0.01 mm to about 0.15 mm. (Item 190) 189. The artificial epidermis layer of any one of items 186 to 189, wherein the thickness of the basal layer ranges from about 0.01 mm to about 0.15 mm. (Item 191) a. stratum corneum; b. granular layer; c. stratum spinosum; and d. Basal layer An artificial epidermis layer comprising: (Item 192) Item 192. The artificial epidermis layer according to item 191, wherein the thickness of the stratum corneum ranges from about 4% to about 20% of the artificial epidermis layer. (Item 193) 193. The artificial epidermis layer according to any one of items 191 to 192, wherein the thickness of the granular layer ranges from about 4% to about 60% of the artificial epidermis layer. (Item 194) 194. The artificial epidermal layer of any one of items 191 to 193, wherein the thickness of the spinous layer ranges from about 4% to about 40% of the artificial epidermal layer. (Item 195) 195. The artificial epidermis layer according to any one of items 191 to 194, wherein the thickness of the basal layer ranges from about 4% to about 40% of the artificial epidermis layer. (Item 196) 196. The artificial epidermal layer according to any one of items 186 to 195, wherein at least about 2% of the cells contained in the artificial epidermal layer are differentiated from induced pluripotent stem cells. (Item 197) 197. The artificial epidermal layer according to any one of items 186 to 196, wherein at least about 10% of the cells contained in the artificial epidermal layer are differentiated from induced pluripotent stem cells. (Item 198) 197. The artificial epidermal layer according to any one of items 186 to 196, wherein at least about 50% of the cells contained in the artificial epidermal layer are differentiated from induced pluripotent stem cells.

Claims

[Claim 1] The invention described in the specification.

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