Immortalized cell lines for artificial synthetic leather

Abstract

The methods, compositions, kits and systems disclosed herein may be directed to modified cells for producing synthetic leather, artificial epidermal layers, artificial dermal layers, layered structures, products produced therefrom and methods of producing the same.

Description

[Technical Field]

[0001]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 152,256, filed February 22, 2021, which application is incorporated herein by reference. Summary of the Invention

[0002]

[0002] In one aspect, disclosed herein is a method comprising the steps of seeding transfected or transduced isolated fibroblasts or fibroblast-like cells onto a scaffold to form an artificial dermal layer, and contacting the transfected or transduced isolated cells with a culture medium, wherein the transfected or transduced isolated cells can comprise an exogenous polynucleotide, wherein the exogenous polynucleotide encodes: (i) a protein that interacts with a tumor suppressor protein or a fragment thereof and inhibits tumor suppression; (ii) a polynucleotide encoding a polypeptide that alters the activity of a tumor suppressor protein or fragment thereof, wherein the activity of the tumor suppressor protein or fragment thereof can be measured by an in vitro assay; or (ii) a polynucleotide encoding a polypeptide that interacts with a tumor suppressor protein or fragment thereof; wherein the transfected or transduced isolated cells, upon contact with a culture medium, have: (i) increased collagen production compared to otherwise identical cells not contacted with the culture medium, (ii) at least partially increased differentiation compared to otherwise identical cells not contacted with the culture medium, or (iii) any combination of (i) and (ii). In some embodiments, the method can further include at least partially decellularizing the artificial dermal layer to form an at least partially decellularized artificial dermal layer. In some embodiments, the method can include tanning the at least partially decellularized artificial dermal layer to form synthetic leather. In some embodiments, the exogenous polynucleotide can encode an SV40 large T antigen (SV40-TAg) polypeptide, a telomerase (TERT) protein, a Bmi-1 protein, a cyclin D1 protein, a biologically active fragment of any of these, or any combination thereof. In certain embodiments, the exogenous polynucleotide may comprise the SV40-TAg gene, the TERT gene, the BMI1 gene, the CCND1 gene, a transcription product of any of these, an exon of any of these, or any combination thereof.In some embodiments, the exogenous polynucleotide may encode the SV40 large T antigen (SV40-TAg) protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode the TERT protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode the Bmi-1 protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode the cyclin D1 protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the tumor suppressor protein or biologically active fragment thereof may be cyclin-dependent kinase 4, retinoblastoma, p53, a biologically active fragment of any of these, or any combination thereof. In some embodiments, after transfection or transduction, the cell growth cycle of the transfected or transduced isolated cells may not be at least partially inhibited. In some embodiments, after transfection or transduction, the transfected or transduced isolated cells can be grown for about 50 cell divisions, about 70 cell divisions, about 90 cell divisions, or about 100 cell divisions. In some embodiments, the medium can include a growth medium, a tissue formation medium, or a combination thereof. In some embodiments, after transfection or transduction with an exogenous polynucleotide, the transfected or transduced isolated cells (a) proliferate, (b) avoid senescence, or (c) both when present in a growth medium. In some embodiments, prior to seeding, the transfected or transduced isolated cells can be expanded in a growth medium to form multiple transfected or transduced cells. In some embodiments, prior to seeding, the transfected or transduced isolated cells can be expanded in a container that at least partially inhibits cell adhesion. In some embodiments, after seeding, the transfected or transduced isolated cells can be contacted with a tissue formation medium.In some embodiments, the growth medium can include growth factors, buffers, salts, sugars, amino acids, lipids, vitamins, ECM proteins, fragments of any of these, or any combination thereof. In some embodiments, disclosed herein are isolated modified fibroblasts or fibroblast-like cells comprising an exogenous molecule, wherein the exogenous molecule at least partially alters the activity of pRB or p53, and wherein the isolated modified fibroblasts or fibroblast-like cells can include isolated immortalized bovine fibroblasts or fibroblast-like cells as disclosed herein. In some embodiments, the isolated modified fibroblasts or fibroblast-like cells can be in contact with a scaffold. In some embodiments, the scaffold can include silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, polylactic acid (PLA), saran, vignon, wool, jute, hemp, bamboo, flax, or a combination thereof.

[0003]

[0003] Disclosed herein, in one embodiment, is a composition comprising: (i) a transfected or transduced isolated fibroblast or fibroblast-like cell comprising an exogenous polynucleotide, wherein after transfection or transduction, the activity of at least one of pRB or P53 can be at least partially altered in the transfected or transduced isolated fibroblast or fibroblast-like cell, (ii) a scaffold, and (iii) a medium, wherein the transfected or transduced isolated fibroblast or fibroblast-like cell can be at least partially contained on, in, or around the scaffold. In one embodiment, the transfected or transduced isolated fibroblast or fibroblast-like cell can be in contact with the scaffold. In some embodiments, the scaffold can comprise silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, polylactic acid (PLA), saran, vinyon, wool, jute, hemp, bamboo, flax, or any combination thereof.

[0004]

[0004] Disclosed herein, in one aspect, is an artificial dermal layer comprising: (i) transfected or transduced isolated fibroblasts or fibroblast-like cells comprising an exogenous polynucleotide; and (ii) a scaffold, wherein after transfection or transduction, the activity of at least one of pRB or P53 can be at least partially altered in the transfected or transduced isolated fibroblasts or fibroblast-like cells, and wherein the transfected or transduced isolated fibroblasts or fibroblast-like cells can be at least partially contained on, in, or around the scaffold. Disclosed herein, in one aspect, is a method comprising at least partially decellularizing an artificial dermal layer as disclosed herein to produce an at least partially decellularized artificial dermal layer. Disclosed herein, in one aspect, is a method comprising tanning an at least partially decellularized artificial dermal layer as disclosed herein to form synthetic leather. Disclosed herein, in some embodiments, is an artificial dermal layer, wherein the scaffold can comprise silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, polylactic acid (PLA), saran, vinyon, wool, jute, hemp, bamboo, flax, or any combination thereof.

[0005]

[0005] Disclosed herein, in one aspect, is a composition comprising isolated immortalized fibroblasts or fibroblast-like cells and a medium comprising an effective amount of: FBS, L-ascorbic acid 2-phosphate (AA2P) or a salt thereof, transforming growth factor beta 1 (TGFB1) or a biologically active fragment thereof, or any combination thereof, wherein the effective amount may be sufficient to induce reporter cells comprising the transfected or transduced polynucleotide as follows: lacking an effective amount of FBS, L-ascorbic acid 2-phosphate (AA2P) or a salt thereof, transforming growth factor beta 1 (TGFB1), a biologically active fragment thereof, or a combination thereof. (i) collagen production; (ii) collagen secretion; or (iii) both, increased relative to an otherwise comparable medium, and, if reporter cells are present in the medium, arresting cell growth in the reporter cells, as determined by: transfecting or transducing the cells with a polynucleotide encoding SV40 large T antigen, a biologically active fragment thereof, TERT, a biologically active fragment thereof, or any combination thereof; growing the cells in the medium and the otherwise comparable medium; comparing the growth rate of the cells grown in the medium relative to the otherwise comparable medium; and comparing the production of collagen produced in the medium relative to the otherwise comparable medium. In some embodiments, the cells can be in contact with a scaffold. In some embodiments, the transfected or transduced isolated fibroblast or fibroblast-like cells disclosed herein, wherein the scaffold can comprise silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, polylactic acid (PLA), saran, vinyon, wool, jute, hemp, bamboo, flax, or any combination thereof.

[0006]

[0006] In one embodiment, a method is disclosed herein that includes the following steps: 1) seeding isolated immortalized animal fibroblasts or fibroblast-like cells onto a scaffold to form an artificial dermal layer; 2) at least partially decellularizing the artificial dermal layer to form an at least partially decellularized artificial dermal layer; and 3) tanning the at least partially decellularized artificial dermal layer to form synthetic leather. In one embodiment, prior to seeding, the isolated immortalized animal fibroblasts or fibroblast-like cells can be expanded in culture to form a plurality of isolated immortalized animal fibroblasts or fibroblast-like cells. In one embodiment, the plurality of isolated immortalized animal fibroblasts or fibroblast-like cells can be propagated beyond the Hayflick limit. In one embodiment, the plurality of isolated immortalized animal fibroblasts or fibroblast-like cells can be propagated past about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. In one embodiment, after expansion, the plurality of isolated immortalized animal fibroblasts or fibroblast-like cells can be stored at a temperature below 0°C. In some embodiments, after storage, a plurality of isolated immortalized animal fibroblasts or fibroblast-like cells can be grown in culture before seeding. In some embodiments, the scaffold can comprise silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, polylactic acid (PLA), saran, vinyon, wool, jute, hemp, bamboo, flax, or any combination thereof. In some embodiments, tanning can include crosslinking collagen in the artificial dermal layer. In some embodiments, seeding isolated immortalized animal fibroblasts or fibroblast-like cells onto the scaffold can further comprise culturing the isolated immortalized animal fibroblasts or fibroblast-like cells on the scaffold to form an artificial dermal layer. In some embodiments, at least partially decellularizing can include contacting the artificial dermal layer with a salt solution, a crystalline salt, or a combination thereof. In some embodiments, contacting can include immersing the artificial dermal layer in a salt solution. In some embodiments, the salt solution may include sodium chloride.In some embodiments, the sodium chloride solution can include about 30-40% sodium chloride. In some embodiments, the animal fibroblasts or fibroblast-like cells can include bovine fibroblasts or fibroblast-like cells. In some embodiments, the tanning can include vegetable tanning, chrome tanning, aldehyde tanning, syntan tanning, bacterial dyeing, or any combination thereof.

[0007]

[0007] In one embodiment, a method is disclosed herein that includes the following steps: 1) seeding isolated immortalized animal fibroblasts or fibroblast-like cells onto a scaffold to form an artificial dermal layer; 2) removing at least a portion of a cell layer from the artificial dermal layer; and 3) tanning the artificial dermal layer to form synthetic leather. In one embodiment, prior to seeding, the isolated immortalized animal fibroblasts or fibroblast-like cells can be expanded in culture to form a plurality of immortalized animal fibroblasts or fibroblast-like cells. In one embodiment, the plurality of immortalized animal fibroblasts or fibroblast-like cells can be propagated beyond the Hayflick limit. In one embodiment, the plurality of immortalized animal fibroblasts or fibroblast-like cells can be propagated past about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. In one embodiment, after expansion, the plurality of immortalized animal fibroblasts or fibroblast-like cells can be stored at a temperature below 0°C. In some embodiments, after storage, the plurality of immortalized animal fibroblasts or fibroblast-like cells can be expanded in culture before seeding onto the scaffold. In some embodiments, culturing the isolated immortalized animal fibroblasts or fibroblast-like cells can include expanding the immortalized animal fibroblasts or fibroblast-like cells. In some embodiments, tanning can include cross-linking collagen in the artificial dermal layer. In some embodiments, the animal fibroblasts or fibroblast-like cells can include bovine fibroblasts or fibroblast-like cells.

[0008]

[0008] In one embodiment, disclosed herein is a method comprising the steps of: seeding isolated cells onto a scaffold, wherein the isolated cells may comprise an exogenous molecule; wherein the exogenous molecule may cause anchorage-independent growth or at least partially anchorage-independent growth based on a direct or indirect stimulus. In one embodiment, the cells may comprise immortalized cells. In one embodiment, the immortalized cells may comprise immortalized fibroblasts or fibroblast-like cells. In one embodiment, the immortalized fibroblasts or fibroblast-like cells may comprise immortalized bovine fibroblasts or fibroblast-like cells. In one embodiment, the molecule may comprise RNA, DNA, or a protein. In one embodiment, the DNA encodes a protein.

[0009]

[0009] In one embodiment, disclosed herein is a method comprising the steps of: seeding isolated cells onto a scaffold, wherein the cells may comprise an at least partially reversible exogenous molecular switch, an exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch, or both. In one embodiment, the isolated cells may comprise isolated immortalized cells.

[0010]

[0010] In one aspect, a method is disclosed herein comprising the following steps: transforming an isolated fibroblast into an isolated immortalized fibroblast or fibroblast-like cell; introducing into the isolated immortalized fibroblast or fibroblast-like cell an at least partially reversible exogenous molecular switch, an exogenous polynucleotide encoding an at least partially reversible exogenous molecular switch, or both, wherein each or both of the at least partially reversible exogenous molecular switch, the exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch causes the immortalized fibroblast or fibroblast-like cell to proliferate based on a direct or indirect stimulus at least partially fixation-independently or at least partially fixation-dependently; and causing the immortalized fibroblast or fibroblast-like cell to proliferate fixation-independently.

[0011]

[0011] Disclosed herein, in one aspect, is a modified cell comprising an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch or a combination thereof, wherein the at least partially reversible exogenous molecular switch causes the modified cell to grow at least partially fixation-independently or at least partially fixation-dependently based on a direct or indirect stimulus.

[0012]

[0012] Disclosed herein, in one embodiment, is a composition comprising: an isolated artificial dermal layer and a scaffold comprising immortalized fibroblasts or fibroblast-like cells cultured in vitro, wherein the immortalized fibroblasts or fibroblast-like cells can be at least partially in contact with the scaffold. In one embodiment, at least a portion of the cell layer of the isolated artificial dermal layer has been removed. In one embodiment, the scaffold can comprise a natural scaffold, a synthetic scaffold, or a combination thereof. In one embodiment, the scaffold can comprise a natural scaffold. In one embodiment, the scaffold can comprise a synthetic scaffold. In one embodiment, the scaffold can comprise an at least partially hollow structure. In some embodiments, the scaffold is made of collagen, cellulose, cotton, acetate, acrylic, latex, linen, nylon, rayon, velvet, modacrylic, saran, vinyon, wool, jute, hemp, bamboo, flax, alginate, fibronectin, poly-p-phenylene terephthalamide, polyethylene, polypropylene, carrageenan, agarose, fibrin, glass, silica, aramid, carbon, poly(tetrafluoroethylene), polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polyacrylonitrile, The artificial dermal layer may comprise chitosan, polyurethane, poly(urethane-urea), polyethylene phthalate, chitin, elastin, keratin, polyhydroxyalkanoate, dextran, pullulan, polyhyaluronic acid, poly(3-hydroxyalkanoate), poly(3-hydroxyoctanoate), poly(3-hydroxy fatty acid), poly(caprolactone), poly(para-dioxanone), laminin, zein, casein, gelatin, gluten, albumin, poly-L-lactic acid (PLA), polyglycolic acid (PGA), or any combination thereof. In some embodiments, the immortalized fibroblasts or fibroblast-like cells express CD10, CD73, CD44, CD90, type I collagen, type III collagen, prolyl-4-hydroxylase beta, or a combination thereof. In some embodiments, the artificial dermal layer may 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.

[0013]

[0013] In one embodiment, disclosed herein is a method comprising the steps of seeding transfected or transduced isolated cells onto a scaffold to form an artificial dermal layer. In one embodiment, the transfected or transduced isolated cells can comprise an exogenous polynucleotide. In one embodiment, the exogenous polynucleotide can encode: (i) a polypeptide that interacts with a tumor suppressor protein or a fragment thereof and alters the activity of the tumor suppressor protein or a fragment thereof, wherein the activity of the tumor suppressor protein or a fragment thereof can be measured by an in vitro assay, or (ii) a polynucleotide encoding a polypeptide that interacts with a tumor suppressor protein or a fragment thereof. In one embodiment, the transfected or transduced cells can be contacted with a culture medium. In one embodiment, the transfected or transduced cells, when contacted with a culture medium, can have: (i) increased collagen production compared to otherwise identical cells not contacted with a culture medium, (ii) at least partially increased differentiation compared to otherwise identical cells not contacted with a culture medium, or (iii) any combination of (i) and (ii). In some embodiments, the method can further include at least partially decellularizing the artificial dermal layer to form an at least partially decellularized dermal layer. In some embodiments, the method can further include tanning the at least partially decellularized artificial dermal layer to form synthetic leather. In some embodiments, the exogenous polynucleotide can encode an SV40 large T antigen (SV40-TAg) polypeptide, a telomerase (TERT) protein, a Bmi-1 protein, a cyclin D1 protein, a biologically active fragment of any of these, or any combination thereof. In some embodiments, the exogenous polynucleotide can include an SV40-TAg gene, a TERT gene, a BMI1 gene, a CCND1 gene, a transcript of any of these, an exon of any of these, or any combination thereof.In some embodiments, the exogenous polynucleotide may encode an SV40 large T antigen (SV40-TAg) protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode an hTERT protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode a Bmi-1 protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode a cyclin D1 protein, a biologically active fragment thereof, or any combination thereof. In some embodiments, the tumor suppressor protein or biologically active fragment thereof may include cyclin-dependent kinase 4, retinoblastoma, p53, a biologically active fragment of any of these, or any combination thereof. In some embodiments, after transfection or transduction, the cell growth cycle of the isolated cells may not be at least partially inhibited. In some embodiments, after transfection or transduction, the isolated cells may be allowed to grow for approximately 50 cell divisions. In some embodiments, after transfection or transduction, the isolated cells can be grown for about 70 cell divisions. In some embodiments, after transfection or transduction, the isolated cells can be grown for about 90 cell divisions. In some embodiments, after transfection or transduction, the isolated cells can be grown for about 100 cell divisions. In some embodiments, after transfection or transduction with an exogenous polynucleotide, the isolated cells can (a) proliferate, (b) avoid senescence, or (c) both, when the isolated cells are present in a growth medium. In some embodiments, prior to seeding, the transfected or transduced isolated cells can be expanded in a growth medium to form multiple transfected or transduced cells. In some embodiments, prior to seeding, the transfected or transduced isolated cells can be expanded in a container that at least partially inhibits cell adhesion. In some embodiments, the medium can comprise a growth medium, a tissue formation medium, or a combination thereof.In some embodiments, after seeding, the transfected or transduced isolated cells can be contacted with a tissue formation medium. In some embodiments, the growth medium can include growth factors, buffers, salts, sugars, amino acids, lipids, minerals, ECM proteins, or combinations thereof. In some embodiments, the salts can include inorganic salts. In some embodiments, the inorganic salts can include about 0.2 g / L calcium chloride, about 0.0001 g / L ferric nitrate·9H2O, about 0.09767 g / L magnesium sulfate (anhydrous), about 0.4 g / L potassium chloride, about 3.7 g / L sodium bicarbonate, about 6.4 g / L sodium chloride, about 0.109 g / L monobasic sodium phosphate (anhydrous), or any combination thereof. In some embodiments, the amino acids are about 0.084 g / L L-arginine·HCl, about 0.0626 g / L L-cystine·2HCl, about 0.03 g / L glycine, about 0.042 g / L L-histidine·HCl·H2O, about 0.105 g / L L-isoleucine, about 0.105 g / L L-leucine, about 1.46 g / L L-lysine·HCl, about 0.03 g / L L-methionine, about 0.06 ...0.105 g / L L-isoleucine, about 0.105 g / L L-leucine, about 1.46 g / L L-lysine·HCl, about 0.03 g / L L-methionine, about 0.066 g / L L-isoleucine, about 0.105 g / L L-leucine, about 0.066 g / L L-isoleucine, about 0.105 g / L L-leucine, about 0.066 g / L L-isoleucine, about 0.066 g / L L-isoleucine, about 0.066 g / L L-isoleucine, about 0.066 g / L L-isoleucine, about 0 The composition may contain about 0.042 g / L L-phenylalanine, about 0.042 g / L L-serine, about 0.095 g / L L-threonine, about 0.016 g / L L-tryptophan, about 0.12037 g / L L-tyrosine·2Na·2H2O, about 0.094 g / L L-valine, about 0.584 g / L L-glutamine, a stereoisomer of any of these, a salt of any of these, or any combination thereof. In some embodiments, the vitamins may include about 0.004 g / L choline chloride, about 0.004 g / L folic acid, about 0.0072 g / L myo-inositol, about 0.004 g / L niacinamide, about 0.004 g / L D-pantothenic acid (hemicalcium), about 0 g / L pyridoxal·HCl, about 0.004 g / L pyridoxine·HCl, about 0.0004 g / L riboflavin, about 0.004 g / L thiamine·HCl, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the sugar may include D-glucose, a stereoisomer thereof, a salt thereof, or any combination thereof.In some embodiments, the pH indicator may comprise about 0.0159 g / L phenol red·Na, about 0.11 g / L pyruvate·Na, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the growth medium may comprise amino acids, vitamins, inorganic salts, fetal bovine serum, antibiotics, antimycotics, or any combination thereof. In some embodiments, the tissue formation medium may comprise growth factors, buffers, salts, sugars, amino acids, lipids, minerals, ECM proteins, human platelet lysate, acid citrate dextrose, heparin, ascorbic acid, TGF-β1, normocin, serum, serum replacement, non-essential amino acids, antibiotics, antimycotics, or any combination thereof. In some embodiments, the tissue formation medium may further comprise about 0.1% to about 40% serum, serum replacement, or a combination thereof. In some embodiments, the amino acids may include glycine, alanine, L-arginine hydrochloride, L-asparagine-H2O, L-aspartic acid, L-cysteine ​​hydrochloride-H2O, L-cystine diHCl, L-glutamic acid, L-glutamine, L-histidine hydrochloride-H2O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate, L-valine, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the vitamins may include biotin, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the inorganic salt may include calcium chloride, copper sulfate, ferric nitrate, ferric sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, zinc sulfate, a stereoisomer of any of these, a salt of any of these, or any combination thereof.In some embodiments, the medium may further comprise D-glucose (dextrose), hypoxanthine Na, linoleic acid, lipoic acid, phenol red, putrescine 2HCl, sodium pyruvate, thymidine, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the serum replacement may be substantially free of animal-derived products, xeno-free, or a combination thereof. In some embodiments, the serum replacement may comprise growth factors, insulin, transferrin, cytokines, essential amino acids, non-essential amino acids, proteins, extracellular matrix proteins (ECM), laminin, fibronectin, vitronectin, cell adhesion peptides, RGD, extracellular matrix fragments, hormones, collagen, albumin, lipids, glycoproteins, protein fragments, or any combination thereof. In some embodiments, the serum may comprise fetal bovine serum (FBS), horse serum, fetal bovine serum, or any combination thereof. In some embodiments, the medium does not comprise TGF-beta. In some embodiments, after seeding, the transfected or transduced isolated cells may be contacted with a tissue formation medium. In some embodiments, the transfected or transduced isolated cells, when present in tissue formation medium, (a) at least partially increase collagen production, (b) have at least partially arrested cell proliferation, or (c) both, compared to otherwise comparable transfected or transduced isolated cells that have not been contacted with tissue formation medium. In some embodiments, the transfected or transduced isolated cells can at least partially differentiate when contacted with tissue formation medium. In some embodiments, after seeding, the transfected or transduced isolated cells can be contacted with a medium containing L-ascorbic acid 2-phosphate (AA2P), a salt thereof, a biologically active fragment thereof, or any combination thereof.In some embodiments, the transfected or transduced isolated cells, when present in a medium containing AA2P, a salt thereof, TGFBl, a biologically active fragment thereof, or a combination thereof, (a) have at least partially increased collagen production, (b) have at least partially arrested cell proliferation, or (c) both, compared to an otherwise comparable medium lacking AA2P, a salt thereof, TGFBl, a biologically active fragment thereof, or a combination thereof. In some embodiments, the transfected or transduced isolated cells can at least partially differentiate when contacted with the medium. In some embodiments, the transfected or transduced isolated cells can include isolated immortalized cells, isolated reprogrammed cells, isolated progenitor cells, isolated mesenchymal stem cells, or any combination thereof. The transfected or transduced isolated cells can be The transfected or transduced isolated cells may comprise isolated fibroblasts, isolated mesenchymal cells, isolated stem cell-derived cells, isolated umbilical cord stem cells, isolated amniotic tissue cells, isolated scar tissue cells, or any combination thereof. In some embodiments, the cells may exhibit markers of collagen production. In some embodiments, the cells may be isolated by flow cytometry. In some embodiments, the transfected or transduced isolated cells may be isolated from bovine, non-human mammals, reptiles, birds, sharks, kangaroos, fish, or eels. In some embodiments, the transfected or transduced isolated cells may be isolated from bovine, wherein the transfected or transduced isolated cells may comprise bovine fibroblasts. In some embodiments, the transfected or transduced isolated cells may be isolated from reptiles, wherein the transfected or transduced isolated cells may comprise turtle cells, snake cells, lizard cells, amphibian cells, crocodile cells, or alligator cells. In certain embodiments, the transfected or transduced isolated cell can be isolated from a non-human mammal, wherein the transfected or transduced isolated cell can comprise an antelope cell, a bear cell, a beaver cell, a bison cell, a boar cell, a camel cell, a caribou cell, a cat cell, a cow cell, a deer cell, a dog cell, an elephant cell, an elk cell, a fox cell, a giraffe cell, a goat cell, a rabbit cell, a horse cell, an ibex cell, a lion cell, a llama cell, a lynx cell, a mink cell, a moose cell, a bull cell, a peccary cell, a pig cell, a rabbit cell, a rhinoceros cell, a seal cell, a sheep cell, a squirrel cell, a tiger cell, a whale cell, a wolf cell, a yak cell, or a zebra cell. In some embodiments, the transfected or transduced isolated cell can be isolated from an avian species, where the transfected or transduced isolated cell can comprise a chicken cell, a duck cell, an emu cell, a goose cell, a ptarmigan cell, an ostrich cell, a pheasant cell, a pigeon cell, a quail cell, or a turkey cell. In some embodiments, the cell can comprise an animal cell.In some embodiments, the animal cells may include anserine cells, eagle cells, assinine cells, bovine cells, crab cells, dog cells, deer cells, crow cells, horse cells, elapine cells, elafin cells, cat cells, goat cells, lion cells, rabbit cells, wolf cells, murine cells, pavonine cells, fish cells, pig cells, rusine cells, snake cells, bear cells, fox cells, primate cells, sheep cells, bird cells, marsupial cells, reptile cells, lagomorph cells, or combinations thereof. In some embodiments, the cells may be derived from scar tissue, umbilical cord, or combinations thereof. In some embodiments, prior to seeding, the transfected or transduced cells may be selected for the presence of the exogenous polynucleotide. In some embodiments, the selection for the presence of the exogenous polynucleotide may include antibiotic selection. In some embodiments, the antibiotic may include puromycin. In some embodiments, the scaffold can comprise a porous material. In some embodiments, transfected or transduced isolated cells can be engrafted onto the scaffold. In some embodiments, the scaffold can comprise a synthetic material, a non-synthetic material, or a combination thereof. In some embodiments, the natural material can comprise silk, natural tissue adhesive, fibrin glue, collagen, basement membrane proteins, extracellular matrix, or a combination thereof.In some embodiments, the scaffold is made of polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polyamide 6,6 (PA6,6), polyamide 11 (PA11), polyvinylidene fluoride (PVDF), polyethylene furanoate (PEF), polyurethane (PU), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (P The materials may include PEGylated fibrin copolymers (P-fibrin), poly(glycerol sebacate) (PGS), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic acid) (PLGA), poly-D,L-lactic acid / polyethylene glycol / poly-D,L-lactic acid (PDLLA-PEG), hyaluronic acid (HA), carbon nanotubes, thermoplastic starch, lyocell / Tencel (cellulose), cotton, bast fibers, viscose bamboo, TiO2 nanofibers, cellulose materials, hydrogel materials, alginate, gelatin, nylon, polyester, silk, materials cross-linked with cell adhesion peptides, materials cross-linked with growth factors, or any combination thereof.

[0014]

[0014] Disclosed herein, in some embodiments, are modified cells comprising an exogenous molecule. In some embodiments, the exogenous molecule at least partially alters the activity of pRB or P53. In some embodiments, the modified cell may comprise an immortalized animal cell. In some embodiments, the animal cell may comprise a non-human animal cell. In some embodiments, the non-human animal cell may comprise a bovine cell. In some embodiments, the molecule may comprise RNA, DNA, a small molecule, a salt thereof, a polypeptide, a hormone, or a biologically active fragment thereof. In some embodiments, the molecule may comprise DNA, wherein the DNA may encode a polypeptide or a biologically active fragment thereof. In some embodiments, the molecule may comprise RNA, wherein the RNA may comprise mRNA, siRNA, or miRNA.

[0015]

[0015] In one embodiment, a method is disclosed herein that includes the following steps: contacting isolated transfected or transduced cells containing an exogenous polynucleotide with a medium containing about 2% to about 40% FBS, wherein the transfected or transduced cells, when present in the FBS-containing medium, (a) produce collagen, (b) have at least partially arrested cell growth, or (c) both, and wherein, after transfection or transduction, the activity of at least one of pRB or p53 can be at least partially altered in the isolated transfected or transduced cells. In one embodiment, the medium can contain about 20% FBS. In one embodiment, the medium can further contain L-ascorbic acid 2-phosphate (AA2P), a salt thereof, transforming growth factor beta 1 (TGFB1), a biologically active fragment thereof, or any combination thereof. In some embodiments, the exogenous polynucleotide may encode SV40 large T antigen, hTERT, Bmi-1, cyclin D1, a biologically active fragment of any of these, or any combination thereof. In some embodiments, the transfected or transduced isolated cell may be a fibroblast. In some embodiments, the transfected or transduced isolated cell may be isolated from a cow, a non-human mammal, a reptile, a bird, a shark, a kangaroo, a fish, or an eel. In some embodiments, the synthetic leather may be made into any article selected from the group consisting of bags, belts, watch straps, packaging, shoes, boots, footwear, gloves, clothing, vests, jackets, pants, hats, shirts, underwear, luggage, clutches, wallets, balls, backpacks, wallets, saddles, saddlery, chaps, whips, furniture, furniture accessories, upholstery, automobile car seats, automobile interiors, and any combination thereof.

[0016]

[0016] Disclosed herein, in an embodiment, is a composition comprising: (i) a transfected or transduced isolated cell comprising an exogenous polynucleotide, which can at least partially alter the activity of at least one of pRB or P53 in the transfected or transduced isolated cell after transfection or transduction, (ii) a scaffold, and (iii) a culture medium. In an embodiment, the transfected or transduced isolated cell can be at least partially contained on, in, or around the scaffold.

[0017]

[0017] In one embodiment, disclosed herein is an artificial dermal layer comprising: (i) transfected or transduced isolated cells comprising an exogenous polynucleotide and (ii) a scaffold. In one embodiment, after transfection or transduction, the activity of at least one of pRB or P53 can be at least partially altered in the transfected or transduced isolated cells. In one embodiment, the transfected or transduced isolated cells can be at least partially contained on, in, or around the scaffold. In one embodiment, at least a portion of the tissue can be at least partially decellularized. In one embodiment, at least a portion of the at least partially decellularized tissue can be tanned to form synthetic leather.

[0018]

[0018] Disclosed herein, in one aspect, is a composition comprising immortalized fibroblasts and a medium comprising an effective amount of: FBS, L-ascorbic acid 2-phosphate (AA2P) or a salt thereof, transforming growth factor beta 1 (TGFB1) or a biologically active fragment thereof, or any combination thereof, wherein the effective amount is an amount that induces reporter cells comprising a transfected or transduced polynucleotide to: The reporter cells may be present in the culture medium and be sufficient to increase (i) collagen production; (ii) collagen secretion; or (iii) both in the reporter cells and arrest cell growth, as determined by the following steps: transfecting or transducing the cells with a polynucleotide encoding SV40 large T antigen, a biologically active fragment thereof, hTERT, a biologically active fragment thereof, or any combination thereof; growing the cells in the culture medium and an otherwise comparable medium; comparing the growth rate of the cells grown in the culture medium to the otherwise comparable medium; and comparing the production of collagen produced in the culture medium to the otherwise comparable medium. Disclosed herein, in certain embodiments, is a composition comprising: (i) an isolated transfected or transduced cell containing an exogenous polynucleotide that, after transfection or transduction, can at least partially alter the activity of at least one of pRB and p53 in the transfected or transduced isolated cell, and (ii) the culture medium disclosed herein. Disclosed herein, in certain embodiments, is a composition comprising: (i) an isolated transfected or transduced cell comprising an exogenous polynucleotide that, after transfection or transduction, can at least partially alter the activity of at least one of pRB or P53 in the isolated transfected or transduced cell; and (ii) a culture medium as disclosed herein.

[0019]

[0019] Disclosed herein, in some embodiments, is a method comprising seeding immortalized bovine fibroblasts onto a scaffold to form an artificial dermal layer. In some embodiments, the method may further comprise at least partially decellularizing the artificial dermal layer to form an at least partially decellularized dermal layer. In some embodiments, the method may further comprise tanning the at least partially decellularized artificial dermal layer to form synthetic leather. In some embodiments, prior to seeding, the immortalized bovine fibroblasts can be expanded in culture to form a plurality of immortalized bovine fibroblasts. In some embodiments, the plurality of immortalized bovine fibroblasts can be propagated beyond the Hayflick limit. In some embodiments, the plurality of immortalized bovine fibroblasts can be propagated past about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. In some embodiments, after expansion, the plurality of immortalized bovine fibroblasts can be stored at a temperature below 0°C. In some embodiments, after storage, the plurality of immortalized bovine fibroblasts can be propagated in culture prior to seeding onto the scaffold. In some embodiments, culturing the immortalized bovine fibroblasts can include expanding the immortalized bovine fibroblasts. In some embodiments, after seeding the immortalized bovine fibroblasts onto the scaffold, the method can further include culturing the immortalized bovine fibroblasts on the scaffold to form an artificial dermal layer. In some embodiments, at least partially decellularizing can include contacting the artificial dermal layer with a salt solution. In some embodiments, contacting with the salt solution can include immersing the artificial dermal layer in the salt solution. In some embodiments, the salt can include sodium chloride. In some embodiments, the sodium chloride concentration can be about 30% to about 40%. In some embodiments, tanning can include crosslinking collagen in the artificial dermal layer. In some embodiments, tanning can include treating the isolated artificial dermal layer to produce leather. In some embodiments, tanning can produce synthetic leather similar to natural leather. In some embodiments, the tanning can include vegetable tanning, chrome tanning, aldehyde tanning, syntan tanning, bacterial dyeing, or any combination thereof.In some embodiments, vegetable tanning can include the use of tannins. In some embodiments, chrome tanning can include the use of chromium salts. In some embodiments, the chromium salts can include chromium sulfate. In some embodiments, the aldehyde can include a glutaraldehyde compound, an oxazolidine compound, or any combination thereof. In some embodiments, the syntan can include a synthetic tannin, an aromatic polymer, or any combination thereof. In some embodiments, tanning can be performed to convert the proteins in the artificial dermis layer into a stable material that will not decay, while allowing the material to maintain flexibility. In some embodiments, the pH of the cell layer or layered structure can be adjusted. In some embodiments, lowering the pH can include lowering to about 6, about 5, about 4, about 3, about 2, or about 1. In some embodiments, lowering the pH can include lowering to about 2.8 to 3.2. In some embodiments, after lowering the pH, the pH can then be raised. In some embodiments, raising the pH can include raising to about 3.8 to 4.2.

[0020]

[0020] Disclosed herein, in some embodiments, is a method comprising seeding immortalized bovine fibroblasts onto a scaffold to form an artificial dermal layer. In some embodiments, the method may further comprise tanning the artificial dermal layer to form synthetic leather. In some embodiments, prior to seeding, the immortalized bovine fibroblasts can be expanded in culture to form a plurality of immortalized bovine fibroblasts. In some embodiments, the plurality of immortalized bovine fibroblasts can be propagated beyond the Hayflick limit. In some embodiments, the plurality of immortalized bovine fibroblasts can be propagated past about 40 cell divisions, about 50 cell divisions, or about 60 cell divisions. In some embodiments, after expansion, the plurality of immortalized bovine fibroblasts can be stored at a temperature below 0°C. In some embodiments, after storage, the plurality of immortalized bovine fibroblasts can be propagated in culture prior to seeding onto the scaffold. In some embodiments, culturing the immortalized bovine fibroblasts can include expanding the immortalized bovine fibroblasts. In some embodiments, after seeding the immortalized bovine fibroblasts onto the scaffold, the method may further include culturing the immortalized bovine fibroblasts on the scaffold to form an artificial dermal layer. In some embodiments, the tanning may include cross-linking collagen in the artificial dermal layer. In some embodiments, the tanning may include treating the isolated artificial dermal layer to produce leather. In some embodiments, the tanning may produce synthetic leather similar to natural leather. In some embodiments, the tanning may include vegetable tanning, chrome tanning, aldehyde tanning, syntan tanning, bacterial dyeing, or any combination thereof. In some embodiments, the vegetable tanning may include using tannins. In some embodiments, the chrome tanning may include using chromium salts. In some embodiments, the chromium salts may include chromium sulfate. In some embodiments, the aldehyde may include a glutaraldehyde compound, an oxazolidine compound, or any combination thereof. In some embodiments, the syntan may comprise a synthetic tannin, an aromatic polymer, or any combination thereof.In some embodiments, tanning can be performed to convert the proteins in the artificial dermis layer into a stable material that will not decay, while allowing the material to maintain flexibility. In some embodiments, the pH of the cell layer or layered structure can be adjusted. In some embodiments, lowering the pH can include lowering to about 6, about 5, about 4, about 3, about 2, or about 1. In some embodiments, lowering the pH can include lowering to about 2.8 to 3.2. In some embodiments, after lowering the pH, the pH can then be raised. In some embodiments, raising the pH can include raising to about 3.8 to 4.2.

[0021]

[0021] Disclosed herein, in an embodiment, is a method comprising seeding cells onto a scaffold. In an embodiment, the cells can comprise an exogenous molecule. In an embodiment, the exogenous molecule can induce anchorage-independent growth or at least partially anchorage-independent growth based on a direct or indirect stimulus. In an embodiment, the cells can comprise immortalized cells. In an embodiment, the immortalized cells can comprise immortalized fibroblasts. In an embodiment, the immortalized fibroblasts can comprise immortalized bovine fibroblasts. In an embodiment, the molecule can comprise RNA, DNA, or protein.

[0022]

[0022] Disclosed herein, in an embodiment, is a method comprising seeding cells onto a scaffold. In an embodiment, the cells may comprise an at least partially reversible exogenous molecular switch, an exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch, or both. In an embodiment, the cells may be immortalized cells. In an embodiment, the method may further comprise growing the cells in one or more environments, e.g., a first environment, prior to seeding. In an embodiment, the cells may comprise an at least partially reversible exogenous molecular switch, an exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch, or both. In an embodiment, the cells may be a plurality of cells. In an embodiment, the plurality of cells may comprise a plurality of at least partially reversible exogenous molecular switches, exogenous polynucleotides encoding at least partially reversible exogenous molecular switches, or both. In an embodiment, the method may comprise a plurality of cells. In certain embodiments, the plurality of at least partially reversible exogenous molecular switches, the exogenous polynucleotides encoding the at least partially reversible exogenous molecular switches, or both, can be at least partially on during growth and at least partially off during seeding. In certain embodiments, the plurality of at least partially reversible exogenous molecular switches, the exogenous polynucleotides encoding the at least partially reversible exogenous molecular switches, or both, can be at least partially on during growth and at least partially off during seeding. In certain embodiments, the at least partially reversible exogenous molecular switches can individually be at least partially on during growth and at least partially off during seeding. In certain embodiments, the at least partially reversible exogenous molecular switches can individually be at least partially off during growth and at least partially on during seeding. In certain embodiments, the at least partially reversible exogenous molecular switches can be at least partially on during growth. In certain embodiments, the at least partially reversible exogenous molecular switches can be at least partially off during growth. In certain embodiments, the at least partially reversible exogenous molecular switches can be at least partially off during growth. In certain embodiments, the cell can be a eukaryotic cell.In some embodiments, the cells may be selected from the group consisting of primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, marsupial cells, reptilian cells, and lagomorph cells. In some embodiments, the first environment may be in suspension. In some embodiments, the at least partially reversible exogenous molecular switch may cause at least partially anchorage-independent or at least partially anchorage-dependent growth based on direct or indirect stimulation. In some embodiments, the at least partially anchorage-independent growth may be at least partially the result of increased expression of: integrin-linked kinase (ILK), cyclin D1, Cdk4, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), or a combination thereof. In some embodiments, the at least partially reversible exogenous molecular switch may be at least partially on during growth and at least partially off during seeding. In some embodiments, the at least partially reversible exogenous molecular switch may be at least partially off during growth and at least partially on during seeding. In some embodiments, the presence of a stimulus may cause an increase or decrease in expression of the exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch. In some embodiments, the absence of the stimulus may cause an increase or decrease in expression of the exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch. In some embodiments, growth may be in the presence of the stimulus. In some embodiments, growth may be in the absence of the stimulus. In some embodiments, the stimulus may be selected from the group consisting of a change in pH, light, temperature, current, microenvironment, the presence or absence of an ion, the level of an ion, the presence of one or more ion types, a mechanical stimulus, a change in culture medium composition, and any combination thereof. In some embodiments, the stimulus may include a change in temperature. In some embodiments, the at least partially reversible exogenous molecular switch may be reversible based on temperature. In some embodiments, the cells may be exposed to a temperature of about 28°C to about 34°C during growth.In some embodiments, the cells can be exposed to a temperature of about 37°C to about 41°C during or after seeding. In some embodiments, the scaffold can be at least partially natural or synthetic. In some embodiments, the scaffold can comprise at least one of the group consisting of silk, polylactide, polyglycolide, polyester, polycaprolactone, chitosan, hydrogel, and combinations thereof. In some embodiments, the cells can produce extracellular matrix proteins. In some embodiments, the extracellular matrix proteins can be selected from the group consisting of type I collagen, type III collagen, elastin, fibronectin, laminin, and combinations thereof. In some embodiments, the extracellular matrix proteins can comprise collage. In some embodiments, the method can further comprise producing at least a portion of a synthetic leather comprising the cells or a portion of a tissue developed therefrom. In some embodiments, the synthetic leather can comprise at least a portion of a tissue. In some embodiments, the tissue can comprise type I collagen. In some embodiments, the method can further comprise growing the cells in a first environment prior to seeding, and then adding, directly or indirectly, an at least partially reversible exogenous molecular switch to the growing cells. In some embodiments, the cells are seeded at a density of about 50,000 cells / cm. 2 ~about 1,000,000 cells / cm 2In some embodiments, seeding cells onto a scaffold can include seeding one side of the scaffold. In some embodiments, a second side of the scaffold can be seeded without inverting the scaffold. In some embodiments, seeding cells onto a scaffold can include seeding more than one side of the scaffold. In some embodiments, seeding can be sequential or simultaneous. In some embodiments, seeding can include seeding one side of the scaffold, then inverting the scaffold and seeding another side of the scaffold. In some embodiments, the cells or immortalized cells can be modified to have enhanced extracellular matrix production compared to an otherwise comparable wild-type cell that may not include the at least partially reversible exogenous molecular switch or a polynucleotide encoding the at least partially reversible exogenous molecular switch. In some embodiments, the extracellular matrix can include type I collagen, type III collagen, elastin, fibronectin, laminin, or a combination thereof. In some embodiments, the method may further comprise engineering the tissue to comprise the cells or immortalized cells. In some embodiments, the method may further comprise tanning the tissue. In some embodiments, the extracellular matrix may comprise type I collagen.

[0023]

[0023] Also disclosed herein are methods comprising transforming a cell into an immortalized cell. In some embodiments, the method may comprise introducing into the immortalized cell an exogenous polynucleotide encoding an at least partially reversible molecular switch, an at least partially reversible exogenous molecular switch, or both. In some embodiments, the at least partially reversible molecular switch, the exogenous polynucleotide encoding the at least partially reversible exogenous molecular switch, or both, may cause the immortalized cell to grow at least partially anchorage-independently or at least partially anchorage-dependently based on a direct or indirect stimulus. In some embodiments, the method may comprise anchorage-independently growing the immortalized cell. In some embodiments, the cell may be selected from the group consisting of primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, marsupial cells, reptile cells, and lagomorph cells. In some embodiments, the cell may be a bovine cell. In some embodiments, the cell may be a stem cell. In some embodiments, the stem cells may be selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells. In some embodiments, the cells may be selected from the group consisting of fibroblasts, animal adipose tissue-derived cells, umbilical cord-derived cells, keratinocytes, keratinocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, cuboidal cells, columnar cells, collagen-producing cells, and combinations thereof. In some embodiments, the cells may be fibroblasts or fibroblast-like cells. In some embodiments, the transformation may include increasing or decreasing the expression of an oncogene or a gene involved in the control of cell proliferation. In some embodiments, the cells may express a polypeptide or a biologically active fragment thereof encoded by: TERT, Bmi1, or any combination thereof. In some embodiments, the immortalized cells may express a polypeptide or a biologically active fragment thereof encoded by: TERT, CcnD1, Cdk4, or any combination thereof. In some embodiments, the method may further include exposing the immortalized cells to a stimulus.In some embodiments, the stimulus can be selected from the group consisting of a change in pH, light, temperature, current, microenvironment, the presence or absence of an ion, the level of an ion, the presence of one or more ion types, a change in mechanical stimulus, a change in medium composition, and any combination thereof. In some embodiments, the stimulus can include a change in temperature. In some embodiments, the temperature can be about 28°C to about 34°C. In some embodiments, the stimulus can cause the immortalized cells to grow at least partially anchorage-independently. In some embodiments, anchorage-independence can include growth in suspension. In some embodiments, at least partially anchorage-independent growth can be at least partially the result of an increase in expression of integrin-linked kinase (ILK), cyclin D1, Cdk4, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), or a combination thereof. In some embodiments, removal of the stimulus can cause the immortalized cells to grow at least partially anchorage-dependently. In some embodiments, the method can further include exposing the immortalized cells to a second stimulus. In some embodiments, the second stimulus may be selected from the group consisting of a change in pH, light, temperature, current, microenvironment, the presence or absence of an ion, the level of an ion, the presence of one or more ion types, a change in mechanical stimulus, a change in medium composition, and any combination thereof. In some embodiments, the second stimulus may include a change in temperature. In some embodiments, the temperature may be about 37°C to about 41°C. In some embodiments, the second stimulus may cause the immortalized cells to grow at least partially in an anchorage-dependent manner. In some embodiments, removal of the second stimulus may cause the immortalized cells to grow at least partially in an anchorage-independent manner. In some embodiments, the anchorage-dependence may include growth on a support. In some embodiments, the method may further include seeding the immortalized cells on a support. In some embodiments, the immortalized cells are grown at a density of about 50,000 cells / cm. 2 ~about 1,000,000 cells / cm 2In some embodiments, seeding the immortalized cells onto the support can include seeding one side of the support. In some embodiments, the second side of the support can be seeded without turning the support over. In some embodiments, seeding the immortalized cells onto the support can include seeding more than one side of the support. In some embodiments, seeding can be sequential or simultaneous. In some embodiments, seeding can include seeding one side of the support, then turning the support over and seeding another side of the support. In some embodiments, anchorage-dependent growth can be at least partially on, in, or around the support. In some embodiments, the support can include a scaffold. In some embodiments, the scaffold can be at least partially natural or synthetic. In some embodiments, the scaffold can include silk, polylactide, polyglycolide, polyester, polycaprolactone, chitosan, a hydrogel, or a combination thereof. In some embodiments, cells or immortalized cells can be engineered to have enhanced extracellular matrix production compared to otherwise comparable wild-type cells that may not contain an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch. In some embodiments, the extracellular matrix can include type I collagen, type III collagen, elastin, fibronectin, laminin, or a combination thereof. In some embodiments, the extracellular matrix protein can include collagen. In some embodiments, the method can further include engineering a tissue comprising the cells or immortalized cells disclosed herein. In some embodiments, the method can further include tanning a tissue comprising the immortalized cells disclosed herein. In some embodiments, the extracellular matrix can include type I collagen.

[0024]

[0024] Modified cells are also disclosed herein. In some embodiments, the modified cells can comprise an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch, or a combination thereof. In some embodiments, the modified cells can be immortalized bovine cells. In some embodiments, the at least partially reversible exogenous molecular switch can cause the modified cells to grow at least partially anchorage-independently or at least partially anchorage-dependently based on a direct or indirect stimulus. In some embodiments, the growth of the modified cells can be determined at least in part by a method selected from the group consisting of manual cell counting, automated cell counting, and indirect cell counting. In some embodiments, the modified cells can be selected from the group consisting of fibroblasts, animal adipose tissue-derived cells, umbilical cord-derived cells, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, cuboidal cells, columnar cells, collagen-producing cells, and combinations thereof. In some embodiments, the modified cells can be fibroblasts or fibroblast-like cells. In some embodiments, the modified cells can be derived from the group consisting of pluripotent stem cells, mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells. In some embodiments, the modified cells can be cells derived from a cell line comprising a plurality of cells. In some embodiments, the modified cells can express an exogenous oncogene or a gene involved in the control of cell proliferation. In some embodiments, the modified cells can express a polypeptide or biologically active fragment thereof encoded by: TERT, Bmil, or any combination thereof. In some embodiments, the modified cells can express a polypeptide or biologically active fragment thereof encoded by: TERT, CcnD1, Cdk4, or any combination thereof. In some embodiments, the modified cells can be modified to have enhanced extracellular matrix production compared to an otherwise comparable wild-type cell that does not comprise an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch.In some embodiments, the extracellular matrix may comprise type I collagen, type III collagen, elastin, fibronectin, laminin, or a combination thereof. In some embodiments, the extracellular matrix protein may comprise collagen. In some embodiments, the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch may be configured to at least partially increase or decrease expression in response to a stimulus. In some embodiments, increased expression of the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch may cause at least partial anchorage-dependent growth. In some embodiments, increased expression of the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch may cause at least partial anchorage-independent growth. In some embodiments, decreased expression of the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch may cause at least partial anchorage-dependent growth. In some embodiments, decreased expression of an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch can cause at least partial anchorage-independent growth. In some embodiments, at least partial anchorage-dependent growth can consume more, the same, or fewer nutrients or growth factors compared to anchorage-independent growth. In some embodiments, at least partial anchorage-independent growth can consume more, the same, or fewer nutrients or growth factors compared to at least partial anchorage-dependent growth. In some embodiments, at least partial anchorage-independent growth can be at least partially the result of increased expression of integrin-linked kinase (ILK), cyclin D1, Cdk4, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), or a combination thereof.In some embodiments, the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch can be a single switch or multiple switches. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch can encode a selectable marker. In some embodiments, the selectable marker can comprise a fluorescent protein. In some embodiments, the modified cell can comprise a recombinant selectable marker. In some embodiments, the selectable marker can be selected from the group consisting of an antibiotic resistance gene, a gene encoding a fluorescent protein, and a gene expressing an auxotrophic marker. In some embodiments, the modified cell can comprise a polynucleotide encoding the at least partially reversible exogenous molecular switch. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous switch can be located in the genome, extrachromosomal, or a combination thereof. In some embodiments, the stimulus can be an environmental stimulus. In some embodiments, the stimulus can be selected from the group consisting of a change in pH, light, temperature, current, microenvironment, the presence or absence of an ion, the level of an ion, a mechanical stimulus, the presence of one or more ion types, a change in culture medium composition, and any combination thereof. In some embodiments, the stimulus may include a change in temperature. In some embodiments, the temperature for at least partially anchorage-independent growth may be about 28°C to about 34°C. In some embodiments, the temperature for at least partially anchorage-dependent growth may be about 37°C to about 41°C. In some embodiments, the presence of the stimulus may cause anchorage-dependent growth. In some embodiments, the absence of the stimulus may cause anchorage-dependent growth. In some embodiments, the presence of the stimulus may cause anchorage-independent growth. In some embodiments, the absence of the stimulus may cause anchorage-independent growth. In some embodiments, the stimulus may be selected from the group consisting of the presence, absence, or change in level of an antibiotic, a protein, a chemical compound, a salt of any one of these, and any combination thereof.In some embodiments, the at least partially anchorage-independent growth can be at least partially the result of increased expression of: integrin-linked kinase (ILK), cyclin D1, Cdk4, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), or a combination thereof. In some embodiments, the engineered cells can be grown in a bioreactor. In some embodiments, the at least partially anchorage-independent growth can be at least partially in suspension. In some embodiments, the anchorage-dependent growth can be at least partially on, in, or around a scaffold. In some embodiments, the scaffold can be at least partially natural or synthetic. In some embodiments, the scaffold can comprise silk, polylactide, polyglycolide, polyester, polycaprolactone, chitosan, a hydrogel, or a combination thereof. In some embodiments, the engineered cells can comprise any one selected from the group consisting of a c-MycER system, a Tet-on system, a Tet-off system, and a combination thereof. In some embodiments, the modified cells may comprise any one selected from the group consisting of a Cre-LoxP system, TALENS, zinc finger, CRISPR system, or components thereof, and any combination thereof. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise DNA, RNA, or a combination thereof. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise DNA. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise cDNA. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise RNA. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise mRNA.In some embodiments, a polynucleotide encoding an at least partially reversible exogenous molecular switch can comprise an inducible promoter or operator, wherein the promoter or operator can be configured to repress or activate expression of a gene. In some embodiments, the promoter or operator can comprise any one selected from the group consisting of a tetracycline-regulated transcription unit, a dexamethasone-regulated transcription unit, a doxycycline-regulated transcription unit, a C-mycR transcription control unit, and any combination thereof. In some embodiments, a polynucleotide encoding an at least partially reversible exogenous molecular switch can be codon-optimized. In some embodiments, a polynucleotide encoding an at least partially reversible exogenous molecular switch can comprise an epigenetically modified base. In some embodiments, the epigenetically modified base can comprise a pyrimidine. In some embodiments, the pyrimidine can be cytosine or thymine. In some embodiments, the epigenetically modified base can comprise any one selected from the group consisting of a methylated base, a hydroxymethylated base, a formylated base, and a carboxylic acid-containing base. In some embodiments, the epigenetically modified base can comprise a hydroxymethylated base. In some embodiments, the hydroxymethylated base can include a 5-hydroxymethylated base. In some embodiments, the 5-hydroxymethylated base can include a 5-hydroxymethylcytosine. In some embodiments, the epigenetically modified base can include a methylated base. In some embodiments, the methylated base can include a 5-methylated base. In some embodiments, the 5-methylated base can include a 5-methylcytosine. In some embodiments, the isolated tissue can include the modified cells described herein. In some embodiments, the tissue can include a plurality of polyester fibers. In some embodiments, at least a portion of the tissue can be tanned.In some embodiments, at least a portion of the tissue can be tanned with a tanning agent including chromium, aluminum, zirconium, titanium, iron, sodium aluminum silicate, formaldehyde, glutaraldehyde, oxazolidine, isocyanate, carbodiimide, polycarbamoyl sulfate, tetrakis, hydroxyphosphonium sulfate, sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate, pyrogallol, catechol, syntan, or any combination thereof. In some embodiments, at least a portion of the tissue can further comprise an extracellular matrix. In some embodiments, the leather can comprise at least a portion of modified cells, their derivatives, their progeny, or isolated tissue. In some embodiments, the leather is selected from the group consisting of bags, belts, watch straps, packaging, shoes, boots, footwear, gloves, clothing, vests, jackets, pants, hats, shirts, underwear, luggage, clutches, wallets, balls, backpacks, wallets, saddles, saddlery, chaps, whips, furniture, furniture accessories, upholstery, automobile car seats, automobile interiors, and any combination thereof. In some embodiments, the modified cells can be in any one of the following forms: (a) a modified cell culture medium (e.g., a cell culture medium containing a cell type) and (b) a modified cell culture medium containing a cell type. In some embodiments, the modified cell culture medium can be in any one of the following forms: (a) a modified cell culture medium containing a cell type; (b) a modified cell culture medium containing a cell type; (c) a modified cell culture medium containing a cell type; (d) a modified cell culture medium containing a cell type; (e.g., a cell culture medium containing a cell type); (e.g., a cell culture medium containing a cell type); (f) a modified cell culture medium containing a cell type; (f) a modified cell culture medium containing a cell type; (f) a modified cell culture medium containing a cell type; (f) a modified cell culture medium containing a cell type; (f) a modified cell culture medium containing a cell type; (f) a modified cell culture medium containing a cell type; (f) a modified cell culture medium containing a cell type; In some embodiments, the at least partially reversible exogenous molecular switch or a polynucleotide encoding the at least partially reversible exogenous molecular switch can be introduced into the modified cells by transfection, electroporation, or transduction. In some embodiments, the at least partially reversible exogenous molecular switch or a polynucleotide encoding the at least partially reversible exogenous molecular switch can be introduced by any one selected from the group consisting of vectors, where the vector can be a virus, a virus-like particle, an adeno-associated virus vector, a liposome, a nanoparticle, a plasmid, a linear dsDNA, and combinations thereof. In some embodiments, the vector can include a plasmid. In some embodiments, the proliferation of the modified cells can be at least partially determined by a method selected from the group consisting of manual cell counting, automated cell counting, and indirect cell counting.In some embodiments, the growth of the modified cells can be determined by using a method selected from the group consisting of counting chambers, colony-forming unit counts, electrical resistance, flow cytometry, image analysis, stereoscopic cell counts, spectrophotometry, and impedance microbiology. In some embodiments, the method can include tanning the modified cells disclosed herein. In some embodiments, the tanning can include at least a portion of a tissue. In some embodiments, the tissue can include a layered structure. In some embodiments, the layered structure can include any one selected from the group consisting of a dermal layer, an epidermal layer, laminin, fibronectin, collagen, and combinations thereof. In some embodiments, the tissue can include any one selected from the group consisting of fibroblasts, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, and combinations thereof. In some embodiments, the method can include selecting or screening the modified cells. In some embodiments, synthetic leather can include the modified cells. In some embodiments, synthetic leather before tanning can include a portion of a tissue. In some embodiments, the tissue can be at least partially subjected to further processing. In some embodiments, the further processing may be selected from the group consisting of tanning, preserving, soaking, baiting, pickling, desalting, thinning, retanning, lubricating, crusting, wetting, sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, peeling, stuffing, whitening, 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 synthetic leather may comprise a biofabricated material. In some embodiments, the biofabricated material may comprise a zonal feature. In some embodiments, the culture vessel may comprise the modified cells disclosed herein.In some embodiments, the culture vessel may comprise any one selected from the group consisting of plastic, metal, glass, and combinations thereof. In some embodiments, the culture vessel may comprise an agent that causes the modified cells to adhere to at least a portion of the culture vessel. In some embodiments, the agent may comprise poly-L-lysine. In some embodiments, the manufacturing facility may comprise the modified cells. In some embodiments, the kit may comprise the modified cells disclosed herein. In some embodiments, the kit may further comprise a growth medium. In some embodiments, the kit may further comprise instructions for use.

[0025]

[0025] Modified cells are also disclosed herein. In some embodiments, the modified cells may comprise an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch, or a combination thereof. In some embodiments, the at least partially reversible exogenous molecular switch may cause the modified cells to grow at least partially fixation-independently or at least partially fixation-dependently based on a direct or indirect stimulus. In some embodiments, the growth of the modified cells may be determined at least in part by a method selected from the group consisting of manual cell counting, automated cell counting, and indirect cell counting. In some embodiments, the modified cells may be prokaryotic or eukaryotic cells. In some embodiments, the modified cells may be animal cells. In some embodiments, the modified cells may be isolated cells. In some embodiments, the modified cells may be non-human cells. In some embodiments, the modified cells may be human cells. In some embodiments, the modified cells may be selected from the group consisting of primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, and lagomorph cells. In some embodiments, the modified cells may be bovine cells. In some embodiments, the modified cells may be immortalized cells. In some embodiments, the modified cells may be selected from the group consisting of fibroblasts, animal adipose tissue-derived cells, umbilical cord-derived cells, keratinocytes, keratinocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, cuboidal cells, columnar cells, collagen-producing cells, and combinations thereof. In some embodiments, the modified cells may be fibroblasts or fibroblast-like cells. In some embodiments, the modified cells may be derived from the group consisting of pluripotent stem cells, mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells. In some embodiments, the modified cells may be derived from a cell line comprising multiple cells. In some embodiments, the modified cells may express an exogenous oncogene or a gene involved in the control of cell proliferation. In some embodiments, the modified cells can express a polypeptide or biologically active fragment thereof encoded by: TERT, Bmil, or any combination thereof.In some embodiments, the engineered cells can express a polypeptide or biologically active fragment thereof encoded by: TERT, CcnD1, Cdk4, or any combination thereof. In some embodiments, the engineered cells can be engineered to have enhanced extracellular matrix production compared to an otherwise comparable wild-type cell that does not comprise an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch. In some embodiments, the extracellular matrix can comprise type I collagen, type III collagen, elastin, fibronectin, laminin, or a combination thereof. In some embodiments, the extracellular matrix can comprise collagen. In some embodiments, the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch can be configured to at least partially increase or decrease expression in response to a stimulus. In some embodiments, increased expression of the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch can cause at least partial anchorage-dependent growth. In certain embodiments, increased expression of an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch can cause at least partial anchorage-independent growth. In certain embodiments, decreased expression of an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch can cause at least partial anchorage-dependent growth. In certain embodiments, decreased expression of an at least partially reversible exogenous molecular switch or a polynucleotide encoding an at least partially reversible exogenous molecular switch can cause at least partial anchorage-independent growth. In certain embodiments, at least partial anchorage-dependent growth can consume more, the same, or less nutrients or growth factors compared to anchorage-independent growth.In some embodiments, at least partially anchorage-independent growth may consume more, the same, or less nutrients or growth factors compared to at least partially anchorage-dependent growth. In some embodiments, the at least partially reversible exogenous molecular switch or the polynucleotide encoding the at least partially reversible exogenous molecular switch can be a single switch or multiple switches. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch can encode a selectable marker. In some embodiments, the selectable marker can comprise a fluorescent protein. In some embodiments, the modified cell can comprise a recombinant selectable marker. In some embodiments, the selectable marker can be selected from the group consisting of an antibiotic resistance gene, a gene encoding a fluorescent protein, and a gene expressing an auxotrophic marker. In some embodiments, the modified cell can comprise a polynucleotide encoding an at least partially reversible exogenous molecular switch. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch can be located in the genome, extrachromosomally, or a combination thereof. In some embodiments, the stimulus can be an environmental stimulus. In some embodiments, the stimulus may be selected from the group consisting of changes in pH, light, temperature, current, microenvironment, the presence or absence of ions, ion levels, mechanical stimuli, the presence of one or more ion types, changes in culture medium composition, and any combination thereof. In some embodiments, the stimulus may include a change in temperature. In some embodiments, the temperature for at least partially anchorage-independent growth may be about 28°C to about 34°C. In some embodiments, the temperature for at least partially anchorage-dependent growth may be about 37°C to about 41°C. In some embodiments, the presence of the stimulus may cause anchorage-dependent growth. In some embodiments, the absence of the stimulus may cause anchorage-dependent growth. In some embodiments, the presence of the stimulus may cause anchorage-independent growth. In some embodiments, the absence of the stimulus may cause anchorage-independent growth.In some embodiments, the stimulus may be selected from the group consisting of the presence, absence, or altered levels of an antibiotic, a protein, a chemical compound, a salt of any one of these, and any combination thereof. In some embodiments, the at least partially anchorage-independent growth may be at least partially the result of increased expression of the following: integrin-linked kinase (ILK), cyclin D1, Cdk4, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), or a combination thereof. In some embodiments, the modified cells may be grown in a bioreactor. In some embodiments, the at least partially anchorage-independent growth may be at least partially in suspension. In some embodiments, the anchorage-dependent growth may be at least partially on, in, or around a scaffold. In some embodiments, the scaffold may be at least partially natural or synthetic. In some embodiments, the scaffold may comprise silk, polylactide, polyglycolide, polyester, polycaprolactone, chitosan, a hydrogel, or a combination thereof. In some embodiments, the modified cells may comprise any one selected from the group consisting of a c-MycER system, a Tet-on system, a Tet-off system, and combinations thereof. In some embodiments, the modified cells may comprise any one selected from the group consisting of a Cre-LoxP system, TALENS, zinc finger, CRISPR system, or components thereof, and any combination thereof. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise DNA, RNA, or a combination thereof. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise DNA. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise cDNA. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise RNA. In some embodiments, the polynucleotide encoding the at least partially reversible exogenous molecular switch may comprise mRNA.In some embodiments, a polynucleotide encoding an at least partially reversible exogenous molecular switch can comprise an inducible promoter or operator, wherein the promoter or operator can be configured to repress or activate expression of a gene. In some embodiments, the promoter or operator can comprise any one selected from the group consisting of a tetracycline-regulated transcription unit, a dexamethasone-regulated transcription unit, a doxycycline-regulated transcription unit, a C-mycR transcription control unit, and any combination thereof. In some embodiments, a polynucleotide encoding an at least partially reversible exogenous molecular switch can be codon-optimized. In some embodiments, a polynucleotide encoding an at least partially reversible exogenous molecular switch can comprise an epigenetically modified base. In some embodiments, the epigenetically modified base can comprise a pyrimidine. In some embodiments, the pyrimidine can be cytosine or thymine. In some embodiments, the epigenetically modified base can comprise any one selected from the group consisting of a methylated base, a hydroxymethylated base, a formylated base, and a carboxylic acid-containing base. In some embodiments, the epigenetically modified base can comprise a hydroxymethylated base. In some embodiments, the hydroxymethylated base can include a 5-hydroxymethylated base. In some embodiments, the 5-hydroxymethylated base can include a 5-hydroxymethylcytosine. In some embodiments, the epigenetically modified base can include a methylated base. In some embodiments, the methylated base can include a 5-methylated base. In some embodiments, the 5-methylated base can include a 5-methylcytosine. In some embodiments, the isolated tissue can include the modified cells disclosed herein. In some embodiments, the tissue can include a plurality of polyester fibers. In some embodiments, at least a portion of the tissue can be tanned.In some embodiments, at least a portion of the tissue can be tanned with a tanning agent comprising: chromium, aluminum, zirconium, titanium, iron, sodium aluminum silicate, formaldehyde, glutaraldehyde, oxazolidine, isocyanate, carbodiimide, polycarbamoyl sulfate, tetrakishydroxyphosphonium sulfate, sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate, pyrogallol, catechol, syntan, or any combination thereof. In some embodiments, at least a portion of the tissue can further comprise an extracellular matrix. In some embodiments, leather can comprise at least a portion of the modified cells, their derivatives, their progeny, or isolated tissues disclosed herein. In some embodiments, the leather can be used to make bags, belts, watch straps, packaging, shoes, boots, footwear, gloves, clothing, vests, jackets, pants, hats, shirts, underwear, luggage, clutches, wallets, balls, or bags. The modified cells may be in the form of any one selected from the group consisting of packs, billfolds, saddles, saddlery, chaps, whips, furniture, furniture accessories, upholstery, automobile car seats, automobile interiors, and any combination thereof. In some embodiments, leather can comprise biofabricated materials. In some embodiments, the biofabricated materials can comprise zoned features. In some embodiments, the method can comprise contacting the modified cells disclosed herein with a stimulus. In some embodiments, the stimulus can comprise any one selected from the group consisting of a change in pH, light, temperature, current, microenvironment, the presence or absence of ions, mechanical stimuli, ion levels, the presence of one or more ion types, and any combination thereof. In some embodiments, the stimulus can comprise a change in temperature. In some embodiments, the modified cells can be grown at a temperature for at least partially anchorage-independent growth from about 28°C to about 34°C. In some embodiments, removal of the stimulus can attenuate at least partially anchorage-independent growth. In some embodiments, the modified cells can be grown at a temperature for at least partially anchorage-dependent growth from about 37°C to about 41°C. In some embodiments, removal of the stimulus can at least partially attenuate anchorage-dependent growth. In some embodiments, the at least partially reversible exogenous molecular switch or a polynucleotide encoding the at least partially reversible exogenous molecular switch can be introduced into the modified cells by transfection, electroporation, or transduction. In some embodiments, the at least partially reversible exogenous molecular switch or a polynucleotide encoding the at least partially reversible exogenous molecular switch can be introduced by any one selected from the group consisting of vectors, wherein the vector can be a virus, a virus-like particle, an adeno-associated virus vector, a liposome, a nanoparticle, a plasmid, a linear dsDNA, and combinations thereof. In some embodiments, the vector can comprise a plasmid.In some embodiments, the proliferation of modified cells can be determined at least in part by a method selected from the group consisting of manual cell counting, automated cell counting, and indirect cell counting. In some embodiments, the proliferation of modified cells can be determined by a method selected from the group consisting of counting chambers, colony-forming unit counting, electrical resistance, flow cytometry, image analysis, stereoscopic cell counting, spectrophotometry, and impedance microbiology. In some embodiments, the method can include tanning the modified cells disclosed herein. In some embodiments, the tanning can include at least a portion of a tissue. In some embodiments, the tissue can include a layered structure or a structure containing the modified cells described herein. In some embodiments, the layered structure can include any one selected from the group consisting of a dermal layer, an epidermal layer, laminin, fibronectin, collagen, and combinations thereof. In some embodiments, the tissue can include any one selected from the group consisting of fibroblasts, keratinocytes, keratinocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, and combinations thereof. In some embodiments, the method can include selecting or screening for the modified cells disclosed herein. In some embodiments, synthetic leather can include the modified cells disclosed herein. In some embodiments, synthetic leather can include a portion of tissue prior to tanning. In some embodiments, the tissue can be at least partially subjected to further processing. In some embodiments, the further processing can be selected from the group consisting of tanning, preserving, soaking, bating, pickling, desalting, thinning, retanning, lubricating, crusting, wetting, thumbing, shaving, re-pigmenting, neutralizing, dyeing, fat-liquoring, filling, peeling, stuffing, whitening, 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 synthetic leather can include biofabricated material.In some embodiments, the biofabricated material can include zone features. In some embodiments, the culture vessel can include the modified cells disclosed herein. In some embodiments, the culture vessel can include any one selected from the group consisting of plastic, metal, glass, and combinations thereof. In some embodiments, the culture vessel can include an agent that causes the modified cells to adhere to at least a portion of the culture vessel. In some embodiments, the agent can include poly-L-lysine. In some embodiments, the manufacturing facility can include the modified cells disclosed herein. In some embodiments, the kit can include the modified cells disclosed herein. In some embodiments, the kit can further include a growth medium. In some embodiments, the kit can further include instructions for use.

[0026] Incorporation by Reference

[0026] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0027] [Figure 1-1]Figure 1A shows population doubling time (hours) versus passage number. Unmodified bovine dermal fibroblasts (BDFs) can be expanded in vitro for approximately 40 population doublings (PDs), with a mean PDT of 35.5 hours. Primary BDFs vary their PDT over multiple passages, with a minimum PDT of 21.6 hours and a maximum PDT of 51.5 hours from passage 0 to passage 11. Figure 1B, in contrast, shows that VL-001 (SV40-TAg-transduced cells) cells can be expanded for at least 20 or more passages after puromycin selection, reaching a cumulative PD of 100 at passage 25. This number does not indicate senescence; rather, this is the time when the expansion experiment was stopped. The mean PDT of the VL-001 cell line (21.2 hours) is 40% shorter than that of unmodified primary BDFs. The VL-001 cell line also exhibited a more consistent PDT across successive passages, with a minimum PDT of 19.1 hours and a maximum PDT of 23.7 hours from passage 6 to passage 25, as shown in Figure 1A. [Figure 1-2] Figure 1C and Figure 1D show phase-contrast microscopy images of passage 9 unmodified bovine dermal fibroblasts (BDF) and passage 24 VL-001 (SV40-TAg transduced) cells. Although senescent cells have not been observed in the extensively passaged VL-001 cell line, senescent morphology can be identified in later-passaged unmodified primary BDF. [Figure 2]

[0028] Figure 2A shows a trichrome blue-stained cross-section of tissue derived from wild-type bovine dermal fibroblasts. Figure 2B shows a trichrome blue-stained cross-section of tissue derived from VL-001 (SV40-TAg-transduced) cells. Comparison of Figures 2A and 2B demonstrates that VL-001 cells can form tissue similar to wild-type dermal fibroblasts (BDF) on PET scaffolds. The images show tissue cross-sections stained with trichrome blue, highlighting collagen in the tissue. Wild-type bovine dermal fibroblasts and VL-001 (SV40-TAg-transduced cells) were seeded onto PET scaffolds at 500,000 cells / cm2. Wild-type bovine dermal fibroblasts were cultured in human platelet lysate (HPL)-based medium (DMEM + 10% HPL + ascorbic acid-2-phosphate (AA2P) + TGFB1 + ACD + Normocin) for 4 weeks. VL-001 was cultured in fetal bovine serum (FBS)-based medium (DMEM + 20% FBS + NEAA + antibiotic / antimycotic) for 2 weeks, after which AA2P was added to the FBS-based medium for another 2 weeks. [Figure 3]

[0029] Figure 3 shows a schematic diagram of the constructs used for cell transsection using SV40-T antigen (SV40-Tag) with a puromycin reporter (Puror) and the constructs used for green fluorescent protein (GFP) expression. [Figure 4]

[0030] Figure 4A shows -LV -Puro cells, Figure 4B shows -LV +Puro cells, Figure 4C shows SV40 TAg +Puro cells, and Figure 4D shows SV40 TAg +Puro cells. [Figure 5]

[0031] FIG. 5 shows the amount of collagen (ug / ml) in the culture medium for BDF and VL-001 cells grown in tissue formation medium and tissue formation medium+TGFb1+AA2P. [Figure 6]

[0032] Figure 6A shows a tissue scan image of BDF cells grown in HPL medium. Figure 6B shows a tissue scan image of BDF cells grown in HPL medium 10X at 10X magnification. Figure 6C shows a tissue scan and image of VL-001 cells grown in HPL medium. Figure 6D shows a tissue scan image of VL-001 cells grown in HPL medium at 10X magnification. Figure 6E shows a tissue scan image of VL-001 cells grown in 20% FBS medium. Figure 6F shows a tissue scan image of VL-001 cells grown in 20% FBS medium at 10X magnification. [Figure 7]

[0033] FIG. 7 shows the amount of collagen (ug / ml) in the culture medium for BDF and VL-001 cells grown in culture medium over the number of weeks in culture. [Figure 8]

[0034] FIG. 8 shows the amount of collagen (ug / g) normalized by wet weight for BDF and VL-001 cells. [Figure 9]

[0035] Figure 9A shows the VL-001 cell line after 0 weeks of growth in culture. Figure 9B shows VL-001 after 1 week of growth in culture. After 1 week of suspension culture, clear aggregate formation was observed. Figure 9C shows VL-001 after 2 weeks of growth in culture. Figure 9D shows VL-001 after 4 weeks of growth in culture. [Figure 10]

[0036] Figure 10 shows increased expression of the COL1 gene upon treatment with TGFB1 and AA2P. Expression levels of the COL1A1 and COL1A2 genes were upregulated in both primary bovine dermal fibroblasts (BDF) and an immortalized cell line (VL-001) upon exposure to transforming growth factor beta 1 (TGFB1) and ascorbic acid-2-phosphate (AA2P). [Figure 11]

[0037] Figures 11A and 11B each show an artificial skin layer grown from an immortalized bovine fibroblast cell line on a PLA scaffold. [Figure 12]

[0038] Figure 12 shows that tissue produced by VL-001 cells has similar total collagen protein levels as tissue derived from primary bovine dermal fibroblasts. Measurements of VL-001 S1 and VL-001 S2 were obtained from biopsies of the tissues shown in Figures 11A and 11B. [Figure 13]

[0039] Figure 13 shows picrosirius red (PSR) stained sections of tissue biopsies from an immortalized bovine fibroblast cell line on a PLA scaffold. The tissue biopsies were fixed in plastic resin, sectioned into 5 μm sections, and stained with picrosirius red (PSR). The staining shows that VL-001 cells can deposit collagen protein throughout the PLA scaffold. [Figure 14]

[0040] Figure 14 shows an image of VL-001 tissue in the crust phase (after drying to remove excess water). VL-001 cells were seeded onto bovine serum (BCS)-coated poly-L-lactic acid (PLA) three-dimensional nonwoven scaffolds and grown for 4 weeks in cell culture medium consisting of 5% hPL (human platelet lysate), heparin (2 mg / L), non-essential amino acids (1x concentration), ascorbic acid (82 μg / L), antibiotic-antimycotic (1x concentration; 100 units / mL penicillin, 100 μg / mL streptomycin, and 250 ng / mL Gibco amphotericin B). DETAILED DESCRIPTION OF THE INVENTION

[0028]

[0041] Some aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the features described herein. The features described herein can be implemented without one or more of the specific details or using other methods. The features described herein are not limited by the illustrated order of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events may be required to implement a method in accordance with features described herein.

[0029]

[0042] The terminology used herein may be for the purpose of describing particular cases only and is 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 dictates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0030]

[0043] 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 occur to a great extent or degree.

[0031]

[0044] As used herein, the term "fibroblast" may include connective tissue cells found in the skin and tendons of the body. Fibroblasts can be obtained from skin or tendon biopsies. Fibroblasts are also widely present in many tissues and organs other than skin and tendons. Fibroblasts may be a type of biological cell that synthesizes extracellular matrix and collagen. Fibroblasts may be a type of biological cell that produces the structural framework (stroma) for animal tissues and plays an important role in wound healing. Fibroblasts may be the most common cell of connective tissue in animals. Fibroblasts may comprise the majority of tissue layers grown in culture, which can be tanned into leather.

[0032]

[0045] As used herein, the term "fibroblast-like cells" can refer to fibroblasts that have been grown in culture, immortalized, or a combination thereof. Fibroblast-like cells can include cells that have differentiated to have a morphology, phenotype, or a combination thereof substantially similar to that of fibroblasts. Fibroblast-like cells can include genetic or phenotypic changes from fibroblasts while still expressing gene expression substantially similar to fibroblasts. Fibroblast-like cells can have similar characteristics to fibroblasts, such as the ability to synthesize collagen, extracellular matrix, the structural matrix (stroma) of animal tissues, or any combination thereof. Fibroblast-like cells can produce substantially similar levels of collagen compared to fibroblasts.

[0033]

[0046] As used herein, the term "pluripotent stem cell" can refer to any progenitor cell that has the potential to form any adult cell except the placenta.

[0047] As used herein, the term "embryonic stem cell" or "ES cell" or "ESC" can refer to a precursor cell that has the potential to form any adult cell.

[0034]

[0048] As used herein, the term "induced pluripotent stem cells" or "iPS cells" or "iPSCs" can refer to a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell (e.g., an adult somatic cell). Induced pluripotent stem cells can be identical to embryonic stem cells in their ability to form all adult cells, but they cannot be derived from an embryo.

[0035]

[0049] As used herein, the term "isolated" can refer to cells that have been removed from an animal or human body. Isolated cells can be grown in culture or in vitro. Isolated cells can be contacted with other isolated cells, a scaffold, a medium, or a combination thereof.

[0036]

[0050] As used herein, the terms "anchorage-dependent" and "anchorage-independent" growth can refer to cell growth in any medium, any condition, any device, or any combination thereof. Anchorage-independent growth can refer to cell growth while not attached to a support. Anchorage-dependent growth can refer to cell growth while at least partially attached to a support (e.g., a scaffold). Anchorage-independent growth can also be referred to as non-adherent growth or growth in suspension. In some embodiments, anchorage-independent growth and anchorage-dependent growth can include cells surrounded by medium. In some embodiments, anchorage-independent growth can include cells not in contact with another cell or a surface (e.g., a scaffold). In some embodiments, in anchorage-independent growth, the surface can be another cell. In some embodiments, anchorage-independent growth can be growth as a single cell in culture. In some embodiments, anchorage-dependent growth can be growth of cells in contact with multiple cells in culture (e.g., on a scaffold).

[0037]

[0051] As used herein, the terms "decellularize" or "decellularized" can refer to the removal of cells from a cell layer. The term "at least partially decellularized" can refer to the removal of at least some cells from a cell layer. "Decellularization" can refer to the process of removing cells to create a decellularized cell layer, and can be achieved by methods such as salt soaking or the use of detergents.

[0038]

[0052] As used herein, the term "synthetic leather" may mean that the skin equivalents described herein can serve as skin equivalents for any mammal or non-mammal. The disclosure can be practiced with humans and non-human mammals, such as non-human primates and bovine, ovine, porcine, equine, canine, and feline species, as well as rodents, such as mice, rats, and guinea pigs, members of the Lagomorpha family, including rabbits, fish, including sharks and stingrays, birds, including ostriches, and reptiles, including lizards, snakes, and crocodiles. In some embodiments, the synthetic leather can comprise artificial leather. In some embodiments, the synthetic leather can comprise cruelty-free leather, eco-friendly leather, plastic-free leather, or any combination thereof. In some embodiments, the synthetic leather can comprise a tanned artificial cell layer, a tanned at least partially decellularized cell layer, or a combination thereof. In some embodiments, the cell layer or at least partially decellularized cell layer can comprise a dermis layer, an epidermis layer, an at least partially decellularized dermis layer, an at least partially decellularized epidermis layer, or any combination thereof. In some embodiments, the mammalian synthetic leather that can be formed can depend on the source of cells used in the invention described herein, e.g., keratinocytes and fibroblasts; for example, if bovine keratinocytes and fibroblasts can be used to form a skin equivalent, bovine synthetic leather can be formed.

[0039]

[0053] The methods, compositions, kits, and systems disclosed herein can be directed to engineered cells for producing synthetic leather, artificial epidermal layers, artificial dermal layers, layered structures, products produced therefrom, and methods for producing the same. In some embodiments, the engineered cells can be animal cells, such as bovine cells. In some embodiments, the engineered cells can be immortalized. In some embodiments, the engineered cells can contain a molecular switch, where the molecular switch can be a polynucleotide. In some embodiments, the engineered cells can be included in synthetic leather. In some embodiments, the synthetic leather can include one or more layers. In some embodiments, one or more layers can contain cells, where the cells can be cultured in vitro. In some embodiments, the methods described herein can provide a high-throughput method for reliably, accurately, and reproducibly scaling up synthetic leather production to commercial levels. The advantages of the synthetic leather, artificial epidermis equivalent, artificial full-thickness skin equivalent, and the method for making the same disclosed herein include reproducibility, high throughput, and the production of customized tissue in an easily scalable manner with attractive appearance, texture, thickness, durability, or any combination thereof. In some embodiments, the full-thickness skin equivalent can include at least one dermis layer and at least one epidermis layer. In some embodiments, the full-thickness skin equivalent and the complete skin equivalent can be used interchangeably. In some embodiments, the skin layer can include the modified cells described herein.

[0040]

[0054] In some embodiments, the synthetic leather disclosed herein can include an artificial dermal layer containing fibroblasts, an artificial epidermal layer containing keratinocytes, or a combination thereof. In some embodiments, the dermal layer and epidermal layer can form a layered structure. In some embodiments, the synthetic leather can include one or more layered structures. In some embodiments, the synthetic leather can be tanned and further processed. In some embodiments, the cells forming the synthetic layer can include immortalized bovine fibroblasts. In some embodiments, the dermal layer can be placed on a scaffold, such as silk, to achieve the thickness and texture of natural leather. In some embodiments, the synthetic leather can include an artificial dermal layer containing the modified cells described herein.

[0041]

[0055] In some embodiments, a method for making synthetic leather can include forming a structure including an artificial dermis layer and tanning the structure. In some embodiments, the method can include further processing the artificial structure, for example, to achieve the thickness and texture of natural leather.

[0042]

[0056] In some embodiments, the synthetic leather can include a cell layer. In some embodiments, the synthetic leather can include multiple cell layers. In some embodiments, the synthetic leather can include an at least partially decellularized cell layer. In some embodiments, the cell layer can include a dermis layer, an epidermis layer, a tissue layer, a basement membrane, a basement membrane substitute, or any combination thereof. In some embodiments, the synthetic leather can further include subcutaneous tissue, scales, lamellae, dermal plates, or a combination thereof. In some embodiments, the synthetic layer can include a full-thickness skin equivalent. In some embodiments, the full-thickness skin equivalent can include any one or combination of the layers disclosed herein. In some embodiments, a portion of one or more cell layers in the synthetic leather can be removed. In some embodiments, removing at least a portion of one or more cell layers can include at least partial decellularization, shaving, or a combination thereof. In some embodiments, at least partial decellularization can include contacting the cell layer with a salt solution. In some embodiments, contacting with a salt solution can include immersion in a salt solution. In some embodiments, the salt solution can include sodium chloride, coarse salt crystals, a brine solution, or a combination thereof.In some embodiments, the brine solution is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102 5%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51 %,approximately 52%,approximately 53%,approximately 54%,approximately 55%,approximately 56%,approximately 57%,approximately 58%,approximately 59%,approximately 60%,approximately 61%,approximately 62%,approximately 63%,approximately 64%,approximately 65%,approximately 66%,approximately 67%,approximately 68%,approximately 69%,approximately 70%,approximately 71%,approximately 72%,approximately 73%,approximately 74%,approximately 75%,approximately 76%,approximately 77 The synthetic leather may comprise about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% salt. In some embodiments, the cell layer or at least partially decellularized cell layer can be tanned. In some embodiments, tanning can be performed after formation of one or more cell layers or layered structures. In some embodiments, the cell layer or layered structure can comprise modified cells. In some embodiments, tanning can be performed after at least partial decellularization of the cell layer. In some embodiments, the synthetic leather can be further processed. In some embodiments, the cell layer can comprise hair follicle cells, melanocytes, or a combination thereof.

[0043]

[0057] In some embodiments, the synthetic leather can include a dermal layer or at least a partially decellularized portion thereof. In some embodiments, the dermal layer can be an artificial dermal equivalent, for example, an artificial dermal layer formed in vitro.

[0044]

[0058] In some embodiments, the dermal layer can comprise connective tissue cells. In some embodiments, the dermal layer can comprise fibroblasts. In some embodiments, fibroblasts in the dermal layer can 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), cluster of differentiation 105 (CD105), type I collagen, type III collagen, prolyl-4-hydroxylase beta fibroblast, or a combination thereof. In some embodiments, the dermal layer can comprise other types of cells, such as immune cells, macrophages, adipocytes, or a combination thereof. In some embodiments, the dermal layer can comprise engineered cells. In some embodiments, the cell layer can comprise immortalized cells, bovine cells, fibroblasts, or any combination thereof. In some embodiments, the cell layer can comprise immortalized bovine fibroblasts.

[0045]

[0059] In some embodiments, the dermal layer can include matrix components in addition to cells. In some embodiments, the matrix components can include any one or more of collagen, elastin, extrafibrillar matrix, extracellular gel-like substances primarily composed of glycosaminoglycans, proteoglycans, glycoproteins, or any combination thereof. In some embodiments, the extracellular gel-like substances primarily composed of glycosaminoglycans can include hyaluronan.

[0046]

[0060] In some embodiments, the dermal layer can comprise a matrix support. In some embodiments, the matrix support can be a scaffold. In some embodiments, the matrix support can comprise a contracted collagen gel. In some embodiments, the pure collagen matrix can be a polyglycolic acid mesh or a collagen and glycosaminoglycan matrix coated with a silastic membrane (C-GAG), a biopolymer, or any combination thereof. In some embodiments, the biopolymer can comprise chitosan. In some embodiments, the matrix can be seeded with fibroblasts. In some embodiments, seeding with fibroblasts can generate an organotypic model. In some embodiments, the cell layer can comprise naturally occurring dermis, keratinocytes, or a combination thereof. In some embodiments, the naturally occurring dermis can be obtained from allogeneic cadaver skin. In some embodiments, the keratinocytes can form a sheet of keratinocytes. In some embodiments, the cell layer can comprise freeze-dried devitalized dermis from cadaver skin to support the keratinocyte sheet.

[0047]

[0061] In some embodiments, the thickness of a unit of leather can be reported in millimeters, ounces, or irons. In some embodiments, 1 ounce can be equal to 1 / 64 inch or 0.0156 inch or 0.396 millimeters. In some embodiments, 1 iron can be equal to 1 / 48 inch or 0.0208 inch or 0.53 millimeters.

[0048]

[0062] In some embodiments, the thickness of the dermal layer can be designed to suit the function or use of the synthetic leather. In some embodiments, the dermal layer can have a thickness of about 0.01 mm to about 50 mm. In some embodiments, the dermal layer can have a thickness of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the dermal layer can have a thickness of about 0.02 mm to 5 mm. For example, the dermal layer can have a thickness of about 0.1 mm to 0.5 mm. In some embodiments, the dermal layer can have a thickness of about 0.2 mm to 0.5 mm. In some embodiments, the thickness 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 embodiments, the thickness 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 thickness of at least about 50 mm.

[0049]

[0063] In some embodiments, the length of the dermal layer can be designed to suit the function or use of the synthetic leather. In some embodiments, the dermal layer can have a length of about 0.01 mm to about 50 mm. In some embodiments, the dermal layer can have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the dermal layer can have a length of about 0.02 mm to 5 mm. For example, the dermal layer can have a length of about 0.1 mm to 0.5 mm. In some embodiments, the dermal layer can have a length of about 0.2 mm to 0.5 mm. In some embodiments, the length 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 embodiments, the length 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 length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000 mm. In some embodiments, the dermal layer 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 dermal layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0050]

[0064] In some embodiments, the width of the dermal layer can be designed to suit the function or use of the synthetic leather. In some embodiments, the dermal layer can have a width of about 0.01 mm to about 50 mm. In some embodiments, the dermal layer can have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the dermal layer can have a width of about 0.02 mm to 5 mm. In some embodiments, the dermal layer can have a width of about 0.1 mm to 0.5 mm. In some embodiments, the dermal layer can have a width of about 0.2 mm to 0.5 mm. In some embodiments, 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 embodiments, 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, or 1000 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, 600, 700 cm. In some embodiments, the dermal layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0051]

[0065] In some embodiments, synthetic leather can include one or more dermal layers. In some embodiments, synthetic leather can 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. In some embodiments, when synthetic leather can include more than one dermal layer, a dermal layer can be disposed on top of another dermal layer. In some embodiments, synthetic leather can include two dermal layers, for example, a first dermal layer and a second dermal layer. In some embodiments, a first dermal layer can be disposed on top of a second dermal layer.

[0052]

[0066] In some embodiments, the dermal layer, or at least a partially decellularized portion thereof, can be layered, e.g., to have multiple sublayers. In some embodiments, the sublayers can have different compositions, e.g., different concentrations of fibers. In some embodiments, the sublayers of the dermal layer, or at least a partially decellularized portion thereof, can have different thicknesses, densities, or combinations thereof. In some embodiments, the dermal layer, or at least a partially decellularized portion thereof, can have a papillary dermis layer, a reticular dermis layer, at least a partially decellularized portion of either of these, or any combination thereof. In some embodiments, the papillary dermis layer, or at least a partially decellularized portion thereof, can comprise loose areolar connective tissue, loosely arranged fibers, at least a partially decellularized portion thereof, or any combination thereof. In some embodiments, the loosely arranged fibers can comprise collagen fibers. In some embodiments, the reticular dermis layer can comprise dense irregular connective tissue, including collagen fibers and dermal elastic fibers.

[0053]

[0067] In some embodiments, the dermal layer, or at least a partially decellularized portion thereof, can comprise a free collagen matrix or lattice, which can be contractile and homogeneous in all directions. In some embodiments, fibroblasts (e.g., immortalized bovine fibroblasts) and, if appropriate, other types of dermal cells can be distributed within a continuous collagen gel. In some embodiments, the dermal equivalent can comprise at least one matrix of type I collagen into which fibroblasts can be distributed. In some embodiments, the dermal equivalent can also contain other extracellular matrix components. In some embodiments, the extracellular matrix components can include collagen, such as collagen IV, laminin, entactin, fibronectin, proteoglycans, glycosaminoglycans, or hyaluronic acid. In some embodiments, the dermal layer can contain type IV collagen and laminin, entactin, or a combination thereof. In some embodiments, the concentrations of these various components can be adjusted. For example, in some embodiments, the concentration of laminin can be about 1% to about 15% of the final volume. In some embodiments, the concentration of collagen IV can be about 0.3% to about 4.5% of the final volume. In some embodiments, the concentration of entactin can be about 0.05% to about 1% of the final volume. In some embodiments, the collagen can be bovine, rat, or fish collagen, any other source of natural collagen, or collagen produced by genetic engineering that allows contraction in the presence of fibroblasts, or any combination thereof. In some embodiments, the collagen can be from a non-natural source. In some embodiments, the matrix can be a collagen gel that can be untensioned, resulting from both horizontal and vertical contraction, and that does not impose preferential organization of fibroblasts. In some embodiments, the matrix, also referred to as "free," cannot adhere to a support, and its volume can be modified without restriction to give it various thicknesses and diameters. In some embodiments, the thickness of the dermal equivalent can be at least 0.05 cm, and in some embodiments, approximately 0.05 to 2 cm.In some embodiments, the thickness can be increased without compromising the advantageous properties of the skin equivalent or synthetic leather. In some embodiments, the thickness can be from about 3 mm to about 20 cm or more. In some embodiments, the synthetic leather can include only the dermis layer.

[0054]

[0068] In some embodiments, the synthetic leather can include a skin layer (e.g., an artificial skin layer). In some embodiments, the skin layer can be an artificial skin equivalent, e.g., an artificial skin layer formed in vitro.

[0055]

[0069] In some embodiments, the epidermal layer can comprise one or more types of cells, including keratinocytes, melanocytes, Langerhans cells, Merkel cells, and inflammatory cells. In some embodiments, the epidermal layer can comprise keratinocytes. In some embodiments, the keratinocytes in the epidermal layer can comprise epidermal keratinocytes, basal keratinocytes, proliferative basal keratinocytes, differentiated suprabasal keratinocytes, or any combination thereof.

[0056]

[0070] In some embodiments, the epidermal layer may comprise modified cells. In some embodiments, the epidermal layer may comprise immortalized cells.

[0071] In some embodiments, the epidermal layer may comprise at least basal keratinocytes, e.g., keratinocytes that cannot differentiate. In some embodiments, the epidermal layer may further comprise partially differentiated keratinocytes as well as fully differentiated keratinocytes. In some embodiments, one or more epidermal layers in a synthetic leather may transition from undifferentiated basal keratinocytes to fully differentiated keratinocytes as they progress from the dermal-epidermal junction, where basal keratinocytes may be localized.

[0057]

[0072] In some embodiments, basal keratinocytes may express hemidesmosomes, which may serve to help secure the epidermal and dermal layers together. In some embodiments, basal keratinocytes may also serve to regenerate skin. In some embodiments, the epidermal layer in the synthetic leather herein may have basal keratinocytes that perform these functions. In some embodiments, synthetic leather containing such basal keratinocytes is capable of regeneration. In some embodiments, the distinction between basal keratinocytes and differentiated keratinocytes in one or more epidermal layers in synthetic leather may be that both E- and P-cadherin may be present in epidermal keratinocytes along the basement membrane zone (BMZ), while differentiated keratinocytes, which may be located away from the BMZ, may express only E-cadherin.

[0058]

[0073] In some embodiments, the basal keratinocytes of the epidermal layer can be aligned in a layer that directly contacts the dermal layer, which serves as a boundary between differentiated keratinocytes and fibroblasts. In alternative cases, gaps may exist between the basal keratinocytes and the dermal layer. Furthermore, gaps may exist between the basal keratinocytes and other basal keratinocytes, leaving gaps between the differentiated keratinocytes and the dermal layer. In these latter cases, where gaps may exist between the basal or differentiated keratinocytes and the dermal layer, the dermal and epidermal layers may not be in uniform contact with each other, but may be adjacent to each other. In some embodiments, the dermal and epidermal layers can be adjacent in that, while fluid may generally be present, other intervening materials, such as cells, collagen, matrix, or other support layers, are substantially absent between the dermal and epidermal layers.

[0059]

[0074] In some embodiments, 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), an isoform of tumor protein p63 (e.g., TAp63), laminin, beta 3 (LAMB3), and keratin 18 (KRT18).

[0060]

[0075] In some embodiments, the thickness of the skin layer can be designed to suit the function or use of the synthetic leather. In some embodiments, the skin layer can have a thickness of about 0.001 mm to about 10 mm. In certain embodiments, the epidermal layer can have a thickness of 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. In some embodiments, the epidermal layer can have a thickness of about 0.01 mm to about 2 mm. In some embodiments, the epidermal layer can have a thickness of about 0.1 mm to about 0.22 mm. In some embodiments, the thickness of the epidermal 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 embodiments, the thickness of the dermal 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 thickness values ​​described herein can be the thickness of the epidermal layer and basement membrane substitute.

[0061]

[0076] In some embodiments, the length of the skin layer can be designed to suit the function or use of the synthetic leather. In some embodiments, the skin layer can have a length of about 0.01 mm to about 50 mm. In some embodiments, the skin layer can have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the skin layer can have a length of about 0.02 mm to 5 mm. In some embodiments, the skin layer can have a length of about 0.1 mm to 0.5 mm. In some embodiments, the skin layer can have a length of about 0.2 mm to 0.5 mm. In some embodiments, 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 embodiments, the length 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 length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000 mm. In some embodiments, the epidermal layer 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 epidermal layer can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0062]

[0077] In some embodiments, the width of the skin layer can be designed to suit the function or use of the synthetic leather. In some embodiments, the skin layer can have a width of about 0.01 mm to about 50 mm. In some embodiments, the skin layer can have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the skin layer can have a width of about 0.02 mm to 5 mm. In some embodiments, the skin layer can have a width of about 0.1 mm to 0.5 mm. In some embodiments, the skin layer can have a width of about 0.2 mm to 0.5 mm. In some embodiments, 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 embodiments, 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, or 1000 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, 600, 700 cm. In some embodiments, the skin layer can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0063]

[0078] In some embodiments, synthetic leather can include one or more skin layers. In some embodiments, synthetic leather can 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. In some embodiments, when synthetic leather can include more than one skin layer, one skin layer can be disposed on another skin layer. In some embodiments, synthetic leather can include two skin layers, for example, a first skin layer and a second skin layer. In some embodiments, the first skin layer can be disposed on the second skin layer.

[0064]

[0079] In some embodiments, the epidermal layer can be stratified, e.g., having multiple sublayers. In some embodiments, the sublayers can have different cellular compositions, e.g., different types of keratinocytes. In some embodiments, the sublayers can comprise modified cells. In some embodiments, the sublayers of the epidermal layer can have different thicknesses and / or densities. In some embodiments, the epidermal layer can have one or more of a cornified layer (stratum corneum), a clear / translucent layer (stratum lucidum), a granular layer (stratum granulosum), a spinous layer (stratum spinosum), a basal / germinal layer (stratum basale / germinativum), or any combination thereof. In some embodiments, the epidermal layer can comprise a functional epidermal permeability barrier (e.g., an organized lipid bilayer in the stratum corneum). In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / stratum germinativum can have a thickness of about 0.0001 mm to about 5 mm. In some embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / stratum germinativum can 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 certain embodiments, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, or stratum basale / stratum germinativum can have a thickness of up to 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.

[0065]

[0080] In some embodiments, the epidermal layer may further comprise cells that produce pigment, e.g., melanin. In some embodiments, such pigment-producing cells may be melanocytes. In some embodiments, melanocytes in the epidermal layer may express one or more markers. In some embodiments, such markers may include, but are not limited to, SRY-box-containing gene 10 (Sox-10), microphthalmia-associated transcription factor (MITF-M), pre-melanosome protein (gp-100), dopachrome tautomerase (DCT), tyrosinase (TYR), and melan-A (MLANA). In some embodiments, the synthetic leather may not comprise an epidermal layer.

[0066]

[0081] In some embodiments, synthetic leather can include collagen and extracellular matrix components produced by cells in the dermal and / or epidermal layers disclosed herein. In some embodiments, synthetic leather can include at least a partially decellularized dermal and / or epidermal layer as disclosed herein. In some embodiments, synthetic leather does not include an epidermal layer. In some embodiments, synthetic leather can also include at least a portion of hair follicle cells, endothelial cells, smooth muscle cells, dermal papilla cells, immune system cells (e.g., lymphocytes, dendritic cells, mast cells, macrophages, or Langerhans cells), adipocytes, nerve cells, Schwann cells, and mixtures thereof. In some embodiments, synthetic leather can include at least a portion of engineered cells (e.g., cells containing molecular switches). Synthetic leather can include immortalized cells. In some embodiments, synthetic leather can include isolated cells. In some embodiments, synthetic leather can include cell lines.

[0067]

[0082] The synthetic leather can include at least a portion of a prokaryotic cell, a eukaryotic cell, or a combination thereof. In some embodiments, the synthetic leather can include at least a portion of a bacterial cell, such as E. coli. In some embodiments, the synthetic leather can include at least a portion of a eukaryotic cell (e.g., a bovine cell, a porcine cell, a human cell, or a budding yeast).

[0068]

[0083] In some embodiments, at least a portion of one or more cells in the synthetic leather may be genetically engineered cells. The term "genetically engineered" can refer to an artificial alteration to the nucleic acid content of a cell. Thus, genetically engineered cells can include cells containing alterations including the insertion, deletion, and / or substitution of one or more nucleotides in the genome of the cell, as well as the introduction of a self-replicating extrachromosomal nucleic acid inserted into the cell. Genetically engineered cells also include cells in which transcription of one or more genes has been altered, e.g., increased or decreased.

[0069]

[0084] In some embodiments, the cell may comprise a molecular switch. In some embodiments, the cell may not have a molecular switch. In some embodiments, the cell may be a eukaryotic cell (e.g., an animal cell) or a prokaryotic cell. In some embodiments, the cell may be a genetically engineered cell. In some embodiments, the cell may be an isolated cell. In some embodiments, the cell may be an immortalized cell. In some embodiments, the cell may comprise a human cell, a fibroblast, a stem cell, or any combination thereof. The cell may be an adipose tissue-derived cell (e.g., an adipocyte), a chondrocyte, an osteocyte, an osteoblast, a myofibroblast, a satellite cell, a myoblast, a muscle cell, a keratinocyte, a corneocyte, a melanocyte, a Langerhans cell, a basal cell, a smooth muscle cell, an umbilical cord cell, a pluripotent stem cell, a mesenchymal stem cell, an embryonic stem cell, or any combination thereof. In some embodiments, the cells can be selected from the group consisting of primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, marsupial cells, reptilian cells, and lagomorph cells. In some embodiments, the cells can be genetically engineered cells. The cells can comprise a cell line having a plurality of cells. In some embodiments, the cells can produce an extracellular matrix protein. In some embodiments, the cells can have enhanced extracellular matrix production compared to an otherwise comparable wild-type cell that does not contain the molecular switch. In some embodiments, the extracellular matrix protein can comprise collagen, type I collagen, type III collagen, elastin, fibronectin, laminin, or any combination thereof. In some embodiments, the engineered tissue can comprise the cells described herein.

[0070]

[0085] In some embodiments, the cell can comprise one or more molecular switches. In some embodiments, the cell may not have a molecular switch. In some embodiments, activation or inactivation of a switch can generate a cascade of molecular action within the engineered cell. In some embodiments, the cascade can comprise increased regulation of gene / protein expression. In some embodiments, the cascade can comprise decreased regulation of gene / protein expression. In some embodiments, the molecular switch can be a reversible molecular switch. In some embodiments, the molecular switch can be partially activated. In some embodiments, partial activation can result from a low level or concentration of a stimulus, such as an antibiotic, that drives expression of a gene. For example, partial activation can comprise decreased expression of a switch compared to full activation (e.g., a Tet-on system). In another example, partial activation can comprise increased expression of a switch compared to full activation (e.g., a Tet-off system). In some embodiments, the molecular switch can be active in a portion of the cell. In some embodiments, the portion of cells may comprise more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the activated cells. In some embodiments, the portion of cells may comprise less than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the activated cells. In some embodiments, the portion of cells may comprise about 5% to about 99%, 10% to about 30%, 20% to about 50%, 40% to about 70%, or about 50% to about 95% of the activated cells. In some embodiments, the switch may be a gene, multiple genes, a polynucleotide, or any combination thereof. In some embodiments, the polynucleotide may be DNA, RNA, or a combination thereof. In some embodiments, the molecular switch may comprise DNA, cDNA, RNA, siRNA, mRNA, miRNA, or any combination thereof. The molecular switch can increase or decrease gene expression after direct or indirect interaction with a stimulus. The molecular switch can be codon optimized.In some embodiments, the molecular switch can be integrated into the genome, extrachromosomal, or a combination of both. In some embodiments, the gene can be integrin-linked kinase (ILK), cyclin D1, CDK4, a gene in the p53-mediated apoptosis pathway, or any combination thereof. The gene can be derived from humans, mammals, birds, reptiles, amphibians, fish, invertebrates, viruses, bacteria, or any combination thereof. In some embodiments, the gene can be a fusion gene that can include one or more genes. In some embodiments, the gene can encode a temperature-sensitive switch, such as tsA58. In some embodiments, the gene can encode a Cre-LoxP recombinase system, a CRISPR system, an FLP-FRT system, a transcription activator-like effector nuclease (TALEN), a zinc finger, a methylation system, or any combination thereof. In some embodiments, the switch can be a tetracycline transcription unit, a dexamethasone-regulated transcription unit, a doxycycline-regulated transcription unit, a C-mycR-regulated transcription unit, an antibiotic-regulated transcription unit, a metabolite-regulated transcription unit, or any combination thereof. In some embodiments, the molecular switch can comprise a Tet-on, a Tet-off system, or any combination thereof. In some embodiments, the molecular switch can comprise a T-REx system. In some embodiments, in a Tet-on system, the system can be induced to express a gene when tetracycline, a derivative thereof, a salt thereof, or any combination thereof is introduced into the cellular environment. In some embodiments, in a Tet-off system, the system can be induced to repress a gene when tetracycline, a derivative thereof, a salt thereof, or any combination thereof is introduced into the cellular environment. In some embodiments, the molecular switch can be an estrogen receptor (ER) system. In some embodiments, the molecular switch can comprise an inducible system, for example, a mixture of an estrogen receptor system and a Tet-off system. In some embodiments, the switch can be a promoter, an operator, or a combination thereof. In some cases, the promoter or the operator can alter the expression of a gene.In some embodiments, the promoter, operator, or any combination thereof can be configured to activate or repress expression in the gene. In some embodiments, the switch can comprise a gene, a promoter, a polynucleotide sequence, or any combination thereof. In some embodiments, the molecular switch can cause at least partially anchorage-independent growth or at least partially anchorage-dependent growth based on a stimulus. In some embodiments, a change in expression of the molecular switch (e.g., an increase or decrease in expression) can cause at least partially anchorage-independent growth or at least partially anchorage-dependent growth. In some embodiments, the molecular switch can be at least partially on during growth. In some embodiments, the molecular switch can be at least partially off during growth.

[0071]

[0086] In some embodiments, the cells can comprise a selectable marker (e.g., a reporter construct). In some embodiments, the switch can comprise a selectable marker. In some embodiments, the selectable marker can comprise green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), EGFP, blue fluorescent protein, cyan fluorescent protein, orange fluorescent protein, a gene encoding any of these, or any combination thereof. In some embodiments, the selectable marker can comprise an antibiotic resistance gene (e.g., blasticidin, geneticin, mycophenolic acid, puromycin, zeocin, hygromycin b, a salt thereof, or any combination thereof), a fluorophore, a biosynthetic gene, an auxotrophic marker, or any combination thereof. In some embodiments, the selectable marker can be an antisense RNA. In some embodiments, the selectable marker can be used to select or screen for modified cells.

[0072]

[0087] In some embodiments, the switch can include an epigenetic modification. In some embodiments, the epigenetic modification can be passed on to daughter cells. In some embodiments, the epigenetic modification can be maintained for a single generation. In some embodiments, the epigenetic modification can be a nucleotide base, a sugar, or any combination thereof. In some embodiments, the epigenetic modification can include a pyrimidine, a purine, or any combination thereof. In some embodiments, the epigenetic modification can include a ribose, a deoxyribose, or any combination thereof. In some embodiments, the epigenetic modification can be on a cytosine, adenine, guanine, threonine, uracil, or any combination thereof. The epigenetically modified base can include a methylated base, a hydroxymethylated base, a formylated base, a carboxylic acid-containing base, or any combination thereof. In some embodiments, the epigenetically modified base can be a 5-methylated base, a 5-hydroxymethylated base, a 5-formylated base, a 5-carboxylated base, a 5-hydroxymethylcytosine, or any combination thereof.

[0073]

[0088] In some embodiments, the molecular switch can be introduced into cells by transfection, transformation, transduction, or injection. In some embodiments, transfection or transformation can include electroporation, bolistic particle delivery, sonoporation, or a combination thereof. In some embodiments, the polynucleotide encoding the molecular switch can be located genomically or extrachromosomally. In some embodiments, the molecular switch can be delivered to cells by a vector. In some embodiments, the vector can be a polynucleotide in the form of a plasmid. In some embodiments, the vector can be comprised of a liposome, a nanoparticle, or any combination thereof. The liposome can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, an archaeosome, a noisome, a novasome, a cryptosome, an emulsome, a vesosome, or any derivative thereof, or any combination thereof. Nanoparticles can include but are not limited to biopolymer nanoparticles, alginate nanoparticles, xanthan gum nanoparticles, cellulose nanoparticles, dendrimers, polymeric micelles, polyplexes, inorganic nanoparticles, nanocrystals, metal nanoparticles, quantum dots, protein nanoparticles, polysaccharide nanoparticles or any of their derivatives or any combination thereof.In some embodiments, vectors can be RNA virus vectors, which can include but are not limited to retrovirus, lentivirus, coronavirus, alphavirus, flavivirus, rhabdovirus, morbivirus, picornavirus, coxsackievirus or picornavirus or any part thereof, or any fragment thereof, or any combination thereof.In some embodiments, the vector may be a DNA viral vector, which may include, but may not be limited to, an adeno-associated viral (AAV) vector, an adenovirus, a hybrid adenovirus system, a hepadnavirus, a parvovirus, a papillomavirus, a polyomavirus, a herpesvirus, a poxvirus, a portion of any of these, a fragment of any of these, or any combination thereof.

[0074]

[0089] In some embodiments, the molecular switch can be controlled by a stimulus. In some embodiments, the stimulus can be a direct stimulus, an indirect stimulus, or any combination thereof. In some embodiments, the stimulus can be a gradient. A gradient stimulus can partially activate a molecular switch in a mixture of cells. In some embodiments, a gradient stimulus cannot activate a molecular switch in a fraction of cells of a cell culture. In some embodiments, a gradient stimulus can activate a molecular switch in a fraction of cells of a cell culture. In some embodiments, as the gradient increases or decreases, the molecular switch can be more or less activated. In some embodiments, cell growth, cell plating, or any combination thereof can be in the presence of the stimulus. In some embodiments, cell growth, cell plating, or any combination thereof can be in the absence of the stimulus. In some embodiments, the cells (e.g., immortalized cells having the switch) can be contacted with the stimulus. In some embodiments, the stimulus can cause the cells to grow at least partially anchorage-independently. In some embodiments, the stimulus can cause the cells to grow at least partially anchorage-dependently. In some embodiments, the absence of the stimulus can cause the cells to grow at least partially anchorage-independently. In some embodiments, the absence of a stimulus can cause cells to proliferate at least partially in an anchorage-dependent manner. Cells can be exposed to one stimulus or multiple stimuli (e.g., a second stimulus). The stimulus can be an environmental stimulus. In some embodiments, the environmental stimulus can include the presence, absence, or level of temperature, magnetic field, pH, light, electric current, microenvironment, ions, sound, air pressure, humidity, physical stimuli (e.g., mechanical stimuli), or any combination thereof. In some embodiments, the stimulus can be a change in culture medium. In some embodiments, the culture medium can include a culture medium. In some embodiments, the culture medium can include Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640, Eagle's Minimum Essential Medium, Ham's Nutrient Mixture, Iscove's Modified Dulbecco's Medium, or any combination thereof. In some embodiments, supplements can be added to the culture medium.In some embodiments, the supplements can include salts, buffering agents, phenol red, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, amino acids, carbohydrates, lipids, proteins, peptides, fatty acids, vitamins, elements, antibiotics, serum, cell culture media supplements, or any combination thereof. In some embodiments, the stimulus can be the presence, absence, or level of carbohydrates, lipids, nucleic acids, proteins, antibiotics, organic chemicals, inorganic chemicals, artificial chemicals, metabolites, or any combination thereof. In some embodiments, cells (e.g., modified cells described herein) may be exposed to temperatures of about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C during growth. In some embodiments, cells (e.g., modified cells described herein) may be exposed to a temperature of about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C during seeding. In some embodiments, the temperature difference between growth and seeding can be about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.In some cases, the temperature stimulus is from about 28°C to about 36°C, 28°C to about 37°C, 28°C to about 38°C, 28°C to about 39°C, 28°C to about 40°C, 28°C to about 41°C, 29°C to about 36°C, 29°C to about 37°C, 29°C to about 38°C, 29°C to about 39°C, 29°C to about 40°C, 29°C to about 41°C, to about 41°C, 30°C to about 36°C, 30°C to about 37°C, 30°C to about 38°C, 30°C to about 39°C, 30°C to about 40°C, 30°C to about 41°C, 31°C to about 36°C, 31°C to about 37°C, 31°C to about 38°C, 31°C to about 39°C, 31°C to about 40°C, 31°C to about 41°C , 32°C to about 36°C, 32°C to about 37°C, 32°C to about 38°C, 32°C to about 39°C, 32°C to about 40°C, 32°C to about 41°C, 33°C to about 36°C, 33°C to about 37°C, 33°C to about 38°C, 33°C to about 39°C, 33°C to about 40°C, 33°C to about 41°C, 34°C to about The temperature change can be to 36°C, 34°C to about 37°C, 34°C to about 38°C, 34°C to about 39°C, 34°C to about 40°C, 34°C to about 41°C, 35°C to about 36°C, 35°C to about 37°C, 35°C to about 38°C, 35°C to about 39°C, 35°C to about 40°C, or 35°C to about 41°C.In some cases, the temperature stimulus is from about 41°C to about 28°C, 41°C to about 29°C, 41°C to about 30°C, 41°C to about 31°C, 41°C to about 32°C, 41°C to about 33°C, 41°C to about 34°C, 41°C to about 35°C, 40°C to about 28°C, 40°C to about 29°C, 40°C to about 30°C, 40°C to about 35 ... to about 31°C, 40°C to about 32°C, 40°C to about 33°C, 40°C to about 34°C, 40°C to about 35°C, 39°C to about 28°C, 39°C to about 29°C, 39°C to about 30°C, 39°C to about 31°C, 39°C to about 32°C, 39°C to about 33°C, 39°C to about 34°C, 39°C to about 35°C , 38°C to about 28°C, 38°C to about 29°C, 38°C to about 30°C, 38°C to about 31°C, 38°C to about 32°C, 38°C to about 33°C, 38°C to about 34°C, 38°C to about 35°C, 37°C to about 28°C, 37°C to about 29°C, 37°C to about 30°C, 37°C to about 31°C, 37°C to about The temperature change can be to 32° C., 37° C. to about 33° C., 37° C. to about 34° C., 37° C. to about 35° C., 36° C. to about 28° C., 36° C. to about 29° C., 36° C. to about 30° C., 36° C. to about 31° C., 36° C. to about 32° C., 36° C. to about 33° C., 36° C. to about 34° C., or 36° C. to about 35° C. In certain embodiments, the molecular switch can be reversible upon a temperature stimulus.

[0075]

[0090] In some embodiments, the genetically engineered cells can contain genes for collagen production. In some embodiments, the collagen genes can be P4HA, P4HB, COL1A1, COL1A2, COL2A1, COL3A1, or any combination thereof. In some embodiments, the collagen genes can have altered promoters that can alter, e.g., increase or decrease, the expression of the collagen genes. In some embodiments, the collagen genes can be derived from humans. In some embodiments, the collagen genes can be derived from animals, mammals, birds, reptiles, amphibians, fish, invertebrates, or any combination thereof.

[0076]

[0091] In some embodiments, the synthetic leather can comprise immortalized cells, tissues derived therefrom, or any combination thereof. In some embodiments, the exogenous polynucleotide can encode (i) a polypeptide capable of interacting with a tumor suppressor protein or a fragment thereof and altering the activity of the tumor suppressor protein or a fragment thereof, (ii) a polynucleotide capable of encoding a polypeptide that interacts with a tumor suppressor protein or a fragment thereof, or (iii) a combination of (i) and (ii). In some embodiments, the activity of the tumor suppressor protein or a fragment thereof can be measured by an in vitro assay. In some embodiments, the immortalized cells can comprise a molecular switch. In some embodiments, the synthetic leather can be derived from non-immortalized cells, tissues derived therefrom, or any combination thereof. The immortalized cells can comprise fibroblasts, adipose tissue-derived cells, umbilical cord cells, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, cuboidal cells, columnar cells, collagen-producing cells, or any combination thereof. In some embodiments, the immortalized cells may comprise or be derived from pluripotent stem cells, induced pluripotent stem cells, mesenchymal stem cells, or embryonic stem cells. In some embodiments, the immortalized cells may have characteristics similar to any cell type described herein. In some embodiments, the immortalized cells may have a mutation. In some embodiments, the immortalized cells may comprise an exogenous gene. In some embodiments, an exogenous gene or genes may cause immortalization. In some embodiments, the exogenous gene is hTERT, TERT, Bmil, CcnDl, a mutant of Cdk4, Cdk4, TAg (SV40 large T), SV40, c-myc, H-ras, Ela, c-mMycER. TAM, E6, E7, HER-2, SRC, EGFR, Abl, AtkO2, Am11, Axl, Bcl, Dbl, EGFR, ERBB, Ets-1, Fms, Fos, Fps, Gli, Gsp, Her2, Hox11, Hst, Il-3, Int-2, Jun, Kit, KS3, K-SAM, Lbc, Lck, L-myc, Lyl-1, Lyt-10, Mas, MDM-2, Mll, Mos, Myb, Neu, N-myc, Ost, Pax-5, Pim-1, PRAD-1, Ras-K, Ras-N, Ret, Ros, Ski, Sis, Set, Src, Tal1, Tan1, Tiam1, Tsc2, Trk, or any combination thereof. In some embodiments, the immortalized cell can have an exogenous gene, which can be a fusion gene that can include one or more genes. In some embodiments, the immortalized cells can have a protein product or a biologically active fragment thereof capable of inducing immortalization. In some embodiments, the immortalized cells can remove the exogenous gene after cell division (e.g., by a Cre-LoxP system or a CRISPR system). In some embodiments, the exogenous gene can be derived from a human. In some embodiments, the exogenous gene can be derived from a mammal, a bird, a reptile, an amphibian, a fish, an invertebrate, a virus, a bacterium, or any combination thereof. In some embodiments, the immortalized cells can have random mutations or multiple mutations. In some embodiments, the mutations can be generated by UV mutagenesis, chemical mutagenesis, or any combination thereof. In certain cases, the immortalized cells can have targeted mutations, for example, targeted mutations can be generated by a CRISPR system. In some embodiments, the mutations can be in cell cycle genes, oncogenes, metabolic genes, or any combination thereof. In some embodiments, the immortalized cells can have mutations in genes, promoter regions, intragenic regions, intergenic regions, or any combination thereof. In some embodiments, the genes can include oncogenes, cell cycle genes, or combinations thereof, hi some embodiments, the immortalized cells can have increased or decreased expression of oncogenes or genes involved in the control of cell proliferation.In some embodiments, the immortalized cells can have a molecular switch. In some embodiments, the immortalized cells can be conditionally immortalized cells. In some embodiments, the conditionally immortalized cells can exhibit an immortalized cell phenotype under a specific environmental stimulus, or a differentiated cell phenotype under a different environmental stimulus. In some embodiments, the immortalized cells can undergo about 30 cell divisions, about 40 cell divisions, about 50 cell divisions, about 60 cell divisions, about 70 cell divisions, about 80 cell divisions, about 90 cell divisions, about 100 cell divisions, about 150 cell divisions, about 200 cell divisions, about 250 cell divisions, about 300 cell divisions, about 350 cell divisions, about 400 cell divisions, about 450 cell divisions, about 500 cell divisions, The cells can be propagated past about 550 cell divisions, about 600 cell divisions, about 650 cell divisions, about 700 cell divisions, about 750 cell divisions, about 800 cell divisions, about 850 cell divisions, about 900 cell divisions, about 950 cell divisions, about 1000 cell divisions, about 5,000 cell divisions, about 10,000 cell divisions, about 50,000 cell divisions, or about 100,000 cell divisions.

[0077]

[0092] In some embodiments, the methods described herein can include growing immortalized cells in a first environment (wherein the immortalized cells can include a reversible exogenous molecular switch that can be at least partially activated or partially silenced by the presence or absence of a stimulus); and seeding the immortalized cells onto a scaffold. In some embodiments, the environment can include a bioreactor, an incubator, a container, a scaffold, growth conditions (e.g., growth medium, temperature, aeration, in suspension), or any combination thereof.

[0078]

[0093] In some embodiments, the cells described herein can be included in a kit. In some embodiments, the kit can include growth medium, instructions for use, packaging, or any combination thereof.

[0079]

[0094] In some embodiments, synthetic leather can have at least one component of natural skin, such as melanocytes, hair follicles, sweat glands, and nerve endings. In some cases, synthetic leather can be distinguished from normal natural skin by its lack of at least one of these components. In some embodiments, synthetic leather can contain all of these components, including at least one cell that exhibits an abnormal phenotype or has an altered genotype.

[0080]

[0095] In some embodiments, additional components can be added to the synthetic leather. Such additional components can include myoepithelial cells, duct cells, secretory cells, alveolar cells, Langerhans cells, Merkel cells, adhesion cells, mammary gland cells, or any mixture thereof. In some embodiments, the synthetic leather can include one or more of the following: nerve cells, connective tissue (including bone, cartilage, osteogenic cells, and cells that differentiate into chondrocytes, as well as lymphatic tissue), epithelial cells (including endothelial cells that line cavities and blood vessels or channels, exocrine epithelial cells, epithelial absorptive cells, keratinizing epithelial cells, and extracellular matrix-secreting cells), and undifferentiated cells (e.g., embryonic cells, stem cells, and other progenitor cells).

[0081]

[0096] In some embodiments, the synthetic leather can include hair follicles. The hair follicles can include one or more structures, including a papilla, matrix, root sheath, bulge, infundibulum, arrector pili muscle, sebaceous gland, apocrine sweat gland, or any combination thereof. The hair follicles can include one or more hair follicle cells, including dermal papilla cells, outer root sheath cells, or any combination thereof. In some embodiments, the hair follicles can be in the epidermal layer. In some embodiments, the hair follicles can be in the dermal layer. In some embodiments, the hair follicle cells can be differentiated from progenitor cells, such as 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%, or 100% of the hair follicle cells can be differentiated from induced pluripotent stem cells.

[0082]

[0097] In some embodiments, the synthetic leather may lack hair, blood vessels, sebaceous glands, hair follicles, oil glands, nerves, or any combination thereof.

[0098] In some embodiments, synthetic leather can include hair. In some embodiments, synthetic leather can include hair in one or more layered structures. In some embodiments, synthetic leather can include fur. In some embodiments, the hair (e.g., fur) can be natural, synthetic, or a combination thereof. In some embodiments, the hair (e.g., fur) can be grown from cells in the synthetic leather or added to the synthetic leather from an exogenous source. In some embodiments, synthetic leather can be free of any hair.

[0083]

[0099] In some embodiments, at least a portion of one or more cells in the synthetic leather can be differentiated from precursor cells, such as stem cells. In some embodiments, the synthetic leather can be produced from modified cells or tissue containing the modified cells disclosed herein. In some embodiments, the fibroblasts in the synthetic leather can be differentiated from stem cells. In some embodiments, the keratinocytes in the synthetic leather can be differentiated from stem cells. In some embodiments, the melanocytes in the synthetic leather can be differentiated from stem cells.

[0084]

[0100] In some embodiments, stem cells can include embryonic stem cells (ESCs), adult stem cells, somatic stem cells, tissue-specific stem cells, mesenchymal stem cells, induced pluripotent stem cells (iPSCs), or any combination thereof. In some embodiments, stem cells can be totipotent, pluripotent, or multipotent. In some embodiments, stem cells can include adult stem cells, umbilical cord blood stem cells, or a combination thereof. Embryonic stem cells can be derived from fertilized embryos that may be less than one week old. Induced pluripotent stem cells can be obtained by inducing the expression of one or more of Oct3, Oct4, Sox2, Klf4, TERT, Bmil, CcnD1, Cdk4, SV40 large T antigen, c-Myc, or fragments of any of these genes in any somatic cell. In some embodiments, somatic cells can include adult somatic cells. In some embodiments, somatic cells can include fibroblasts. In some embodiments, the exogenous vector can include or encode a gene that induces pluripotency. In some embodiments, the exogenous vector can include a plasmid. In some embodiments, induced pluripotent stem cells can be obtained by an active protein product or a biologically active fragment thereof. In some embodiments, one or more other genes can also be induced to reprogram somatic cells into induced pluripotent stem cells. In some embodiments, the genes for inducing pluripotency can include NANOG, UTF1, LIN28, SALL4, NR5A2, TBX3, ESSRB, DPPA4, SV40LT, REM2, MDM2, and cyclin D1. In some embodiments, the genes can be derived from humans. In some embodiments, the genes can be derived from mammals, birds, reptiles, amphibians, fish, invertebrates, or any combination thereof.

[0085]

[0101] In some embodiments, various delivery methods can be used to regulate gene expression to reprogram somatic cells into iPSCs. In some embodiments, exemplary delivery methods can include naked DNA delivery, adenoviral vectors, electrical delivery, chemical delivery, mechanical delivery, polymer-based systems, microinjection, retroviral vectors (e.g., MMLV-derived retroviruses), lentiviral vectors (e.g., excisable lentiviruses), or any combination thereof. In some embodiments, the somatic cells can comprise adult somatic cells. In some embodiments, the somatic cells can be transfected with a vector for delivery of a gene that induces pluripotency. In some embodiments, the vector can comprise a viral vector. In some embodiments, the vector can comprise a retroviral vector. In some embodiments, the gene that induces pluripotency can include Oct3, Oct4, Sox2, Klf4, TERT, Bmil, CcnD1, Cdk4, SV40 large T antigen, c-Myc, any fragment thereof, or any combination thereof. In some embodiments, Sendai virus can be used as a delivery system. In some embodiments, the somatic cells can comprise adult somatic cells. In some embodiments, somatic cells can be transfected with an extrachromosomal vector. In some embodiments, the extrachromosomal vector can comprise a plasmid. In some embodiments, the extrachromosomal vector can deliver Oct3, Oct4, Sox2, Klf4, TERT, Bmil, CcnDl, Cdk4, SV40 large T antigen, c-Myc, any fragment thereof, or any combination thereof.

[0086]

[0102] In some embodiments, methods and compositions comprising cells are disclosed herein. In some embodiments, the cells can comprise cells derived from an animal. In some embodiments, the cells can be derived from a mammal, a bird, a reptile, an amphibian, a fish, an invertebrate, or any combination thereof. In some embodiments, the cells can be obtained from a biopsy. In some embodiments, the cells can be immortalized. In some embodiments, the synthetic leather can comprise modified cells derived from any cell type described herein and can include a molecular switch.

[0087]

[0103] In some embodiments, the synthetic leather can include cells derived from an animal. In some embodiments, the synthetic leather can include cells derived from a mammal. In some embodiments, the cells derived from a mammal can include mammalian cells. In some embodiments, the mammal can include a non-human mammal. In some embodiments, the non-human mammal can be an antelope, bear, beaver, bison, wild boar, camel, caribou, cat, cow, deer, dog, elephant, elk, fox, giraffe, goat, rabbit, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, ox, peccary, pig, rabbit, rhinoceros, seal, sheep, lamb, squirrel, tiger, whale, wolf, yak, or zebra. In some embodiments, the animal can be a primate, cow, sheep, pig, horse, dog, cat, rodent, lagomorph, fish, bird, or reptile. In some embodiments, the animal can include a reptile. In some embodiments, the reptile may comprise a crocodile, alligator, or snake. In some embodiments, 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 attracted the attention of a community through notoriety or public fame from previous activities. "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.

[0088]

[0104] In some embodiments, the synthetic leather can include cells from other species. In some embodiments, the cells can be derived from birds. In some embodiments, birds can include chickens, ducks, emus, geese, grouse, ostriches, pheasants, pigeons, quails, or turkeys. In some embodiments, the cells can be derived from reptiles, such as turtles, snakes, lizards, amphibians, crocodiles, or alligators. In some embodiments, the cells can be derived from amphibians. In some embodiments, amphibians can include frogs, toads, salamanders, or newts. In some embodiments, the cells can be derived from fish. In some embodiments, fish can include anchovy, bass, catfish, carp, cod, eel, flounder, pufferfish, grouper, haddock, halibut, herring, mackerel, mahi-mahi, manta ray, marlin, orange roughy, perch, pike, cod, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, or Alaska pollock.

[0089]

[0105] In some embodiments, the cells in the synthetic leather can be derived from the same species. In some embodiments, all of the cells in the synthetic leather can be bovine cells. In some embodiments, the synthetic leather can contain cells derived from multiple species. In some embodiments, the synthetic leather can contain bovine cells and alligator cells. In some embodiments, the synthetic leather can contain cells derived from at least 2, 3, 4, 5, 6, 7, 8, or 10 species.

[0090]

[0106] In some embodiments, the precursors of the cells in the synthetic leather can also be derived from the sources described herein. In some embodiments, the modified cells can include any cell in the molecular switch, somatic cells, primary cells used in the synthetic cells, dermal layer cells, epidermal layer cells, or synthetic cells, and their precursors can be derived from the sources described herein. In some embodiments, somatic cells can be reprogrammed into iPSCs.

[0091]

[0107] In some embodiments, the synthetic leather can include one or more layered structures. In some embodiments, the layered structure can be formed by placing a first type of layer on top of a second type of layer. In some embodiments, the first type of layer and the second type of layer can be the same or different. In some embodiments, the layered structure can be formed by placing an epidermal layer on a dermal layer. In some embodiments, the layered structure can be formed by placing an epidermal layer on a dermal layer with a basement membrane substitute therebetween. In some embodiments, the layered structure can include multiple layers of dermis.

[0092]

[0108] In some embodiments, the layered structure can comprise two or more layers. In some embodiments, the layered structure can comprise 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 embodiments, the layered structure can comprise 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. In some embodiments, the layered structure can comprise 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.

[0093]

[0109] In some embodiments, the layered structure can comprise one or more types of cells described herein. In some embodiments, the layered structure can comprise cells in the dermis layer, such as fibroblasts, cells in the epidermis layer, or any combination thereof. In some embodiments, the cells in the epidermis layer can comprise keratinocytes. In some embodiments, the layered structure can comprise modified cells having a switch. In some embodiments, the layered structure can comprise immortalized cells having an exogenous molecular switch.

[0094]

[0110] In some embodiments, the layered structure can have a thickness of about 0.001 mm to about 100 mm. In some embodiments, the layered structure can have a thickness of about 0.005 mm to about 50 mm, about 0.005 to about 10 mm, 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 embodiments, the thickness 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, 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, or 100 mm. In some embodiments, the thickness of the layered structure can be at most 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.

[0095]

[0111] In some embodiments, the length of the layered structure can be designed to suit the function or use of the synthetic leather. In some embodiments, the layered structure can have a length of about 0.01 mm to about 50 mm. In some embodiments, the layered structure can have a length of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. In some embodiments, the layered structure can have a length of about 0.02 mm to 5 mm. In some embodiments, the layered structure can have a length of about 0.1 mm to 0.5 mm. In some embodiments, the layered structure can have a length of about 0.2 mm to 0.5 mm. In some embodiments, the length 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 embodiments, the length 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 length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, or 1000 mm. In some embodiments, the layered structure 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 layered structure can have a length of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0096]

[0112] In some embodiments, the width of the layered structure can be designed to suit the function or use of the synthetic leather. The layered structure can have a width of about 0.01 mm to about 50 mm. For example, the layered structure can have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. For example, the layered structure can have a width of about 0.02 mm to 5 mm. For example, the layered structure can have a width of about 0.1 mm to 0.5 mm. For example, the layered structure can have a width of about 0.2 mm to 0.5 mm. In some embodiments, 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 embodiments, 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, or 1000 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, 600, 700 cm. In some embodiments, the layered structure can have a width of at least about 50, 60, 70, 80, 90, 100, 200, 300, 400 m.

[0097]

[0113] The layered structure can 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. In some embodiments, the ratio of fibroblasts to keratinocytes can be about 20:1 to about 3:1, about 20:1 to about 4:1, about 20:1 to about 5:1, about 20:1 to about 10:1, or about 20:1 to about 15:1. In some embodiments, the layered structure can comprise any of the cells disclosed herein. In some embodiments, the layered structure can comprise any of the modified cells disclosed herein.

[0098]

[0114] The layered structure can 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. In certain embodiments, the ratio of fibroblasts to melanocytes can be about 20:1 to about 3:1, about 20:1 to about 4:1, about 20:1 to about 5:1, about 20:1 to about 10:1, or about 20:1 to about 15:1.

[0099]

[0115] The layered structure can 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. In certain embodiments, the ratio of keratinocytes to melanocytes can be about 20:1 to about 3:1, about 20:1 to about 4:1, about 20:1 to about 5:1, about 20:1 to about 10:1, or about 20:1 to about 15:1.

[0100]

[0116] One type of cell in the layered structure can 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. One type of cell in the layered structure can 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 can comprise 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.

[0101]

[0117] In some embodiments, synthetic leather is disclosed herein. In some embodiments, methods for forming synthetic leather are disclosed herein. In some embodiments, the synthetic leather can include at least a portion of a layer of cells, an at least partially decellularized layer of cells, one or more layered structures, one or more at least partially decellularized layered structures, or any combination thereof. In some embodiments, the synthetic leather can be formed by one or more layered structures, dermis, or epidermis. In some embodiments, the synthetic leather can 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.

[0102]

[0118] In some embodiments, synthetic leather can be of various thicknesses. In some embodiments, synthetic leather can have a thickness similar to that of natural leather. In some embodiments, 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 mm to about 10 mm, about 0.01 mm to about 10 mm, about 0.1 mm 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 embodiments, the thickness 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, 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, or 100 mm. In some embodiments, the thickness of the synthetic leather can be at most 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 thickness of the synthetic leather can be about 1.2 mm.

[0103]

[0119] The synthetic leather may have a length of about 0.01 mm to about 50 mm. 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 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm 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 embodiments, 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 embodiments, 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 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, 700 mm, or 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.

[0104]

[0120] Synthetic leather can have a width of about 0.01 mm to about 50 mm. For example, synthetic leather can have a width of about 0.01 mm to about 10 mm, about 0.01 mm to about 8 mm, about 0.01 mm to about 5 mm, about 0.02 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.1 mm to about 5 mm, about 0.1 mm to about 2 mm, about 0.1 mm to about 1 mm, about 0.1 mm to about 0.8 mm, or about 0.1 mm to about 0.5 mm. For example, synthetic leather can have a width of about 0.02 mm to 5 mm. For example, synthetic leather can have a width of about 0.1 mm to 0.5 mm. For example, synthetic leather can have a width of about 0.2 mm to 0.5 mm. In some embodiments, 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 embodiments, 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 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, 700 mm, or 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.

[0105]

[0121] In some embodiments, synthetic leather can include biofabricated materials. In some embodiments, leather can include biofabricated materials. In some embodiments, the biofabricated materials can include cells, modified cells, or tissues disclosed herein. In some embodiments, the biofabricated materials can include modified cells described herein. In some embodiments, the biofabricated materials can include zonal characteristics. Zonal characteristics can include one or more zones in the biofabricated material. In some embodiments, a zone can have one or more properties that can differ from one or more adjacent zones. In some embodiments, properties that can differ from zone to zone include color, breathability, stretch, tear strength, softness, stiffness, abrasion resistance, heat transfer to enable heating or cooling, electromagnetic, luminescence, reflectivity, antibacterial, antifungal, antibacterial, strength, fragrance, and combinations thereof.

[0106]

[0122] The synthetic leather can further include a basement membrane substitute. The basement membrane substitute can be between two cell layers, for example, between the dermis and the epidermis. From a structural and / or biochemical standpoint, the basement membrane substitute can be a dermal-epidermal junction similar to that present in vivo. From a biochemical standpoint, the basement membrane substitute can include components of the basement membrane, lamina densa, lamina lucidum, and subbasal zone, such as collagen IV, collagen VII, laminin 5, entactin, fibronectin, or any combination thereof.

[0107]

[0123] The basement membrane substitute in the synthetic leather can be urinary basement membrane (UBM), liver basement membrane (LBM), amniotic membrane, 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 some 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 in Bye et al., Journal of Biomaterials and Tissue Engineering, Vol. 4, 1-7, 2014.

[0108]

[0124] In some embodiments, cells, cell layers, layered structures, or synthetic leather can be seeded onto the scaffold. In some embodiments, the scaffold can include a support. In some embodiments, the scaffold can provide a particular hardness (e.g., resistance to tearing), elasticity, or both. In some embodiments, the synthetic leather can include part or all of the scaffold. In some embodiments, the synthetic leather may not include the scaffold. In some embodiments, after assisting in the formation of layers in the synthetic leather, the scaffold can be removed from the final synthetic leather product. In some embodiments, the scaffold included in the synthetic leather can degrade after a period of time. In some embodiments, the scaffold can be degradable, biodegradable, bioabsorbable, resorbable, or any combination thereof.

[0109]

[0125] In some embodiments, the scaffold can be made from natural materials, synthetic materials, or any combination thereof. In some embodiments, the scaffold can include a support. In some embodiments, the support can include a support for cell growth. In some embodiments, the scaffold can be formed using a net made of a bioabsorbable synthetic polymer. In some embodiments, the scaffold can be formed by attaching a nylon net to a silicone film. In some embodiments, the scaffold can include a two-layer structure of a collagen sponge and a silicone sheet. In some embodiments, the scaffold can be formed using an atelocollagen sponge. In some embodiments, the scaffold can be fabricated into a sheet. In some embodiments, the scaffold can be formed by matching collagen sponges with different pore sizes. In some embodiments, the acellular dermal matrix (ADM) can be formed using fibrin glue, allogeneic skin, or a combination thereof that has been at least partially decellularized.

[0110]

[0126] In some embodiments, the scaffold can comprise natural materials such as collagen (e.g., collagen matrix), natural adhesives (e.g., fibrin adhesives, cold adhesives, animal adhesives, blood albumin adhesives, casein adhesives, or plant adhesives such as starch and dextrin adhesives). In some embodiments, the scaffold can comprise polylactide, polyglycolide, polycaprolactone, hydrogel, or any combination thereof. In some embodiments, the scaffold can comprise silk. In some embodiments, the scaffold can be made from silk. In some embodiments, the scaffold can comprise silk fibroin, cellulose, cotton, acetate, acrylic, latex fiber, linen, nylon, rayon, velvet, modacrylic, olefin polyester, saran, vinyon, wool, jute, hemp, bamboo, flax, or combinations thereof. In some embodiments, the scaffold can comprise fibers. In some embodiments, the fibers may be silk, cotton, wool, linen, cellulose, particularly extracted from wood, plants, or algae, polyamide, modified cellulose, poly-p-phenylene terephthalamide, acrylic fibers, such as polymethyl methacrylate or poly-2-hydroxyethyl methacrylate acrylic fibers, polyolefin fibers, such as polyethylene or polypropylene fibers, glass, silica, aramid, carbon, such as a form of graphite, poly(tetrafluoroethylene), insoluble collagen, polyester, polyvinyl chloride, polyvinylidene chloride, polyvinyl alcohol, polyacrylonitrile, chitosan, polyurethane, poly(urethane-urea), polyethylene phthalate, and blends of polymers, such as blends of the above polymers, for example, fibers formed from polyamide / polyester fibers, or any combination thereof. In some embodiments, the modified cellulose may include rayon, viscose, acetate, rayon acetate, or any combination thereof.

[0111]

[0127] In some embodiments, the scaffold can include a polymer. In some embodiments, the polymer can include a biopolymer. In some embodiments, the biopolymer can include, but is not limited to, chitin, chitosan, elastin, collagen, keratin, or polyhydroxyalkanoates. In some embodiments, the polymer can be biodegradable, biostable, or a combination thereof. In some embodiments, the polymer in the scaffold can be a natural polymer. In some embodiments, exemplary natural polymers can include polysaccharides such as alginate, cellulose, dextran, pullulan, polyhyaluronic acid, chitin, poly(3-hydroxyalkanoates), poly(3-hydroxyoctanoates), poly(3-hydroxy fatty acids), or any combination thereof. In some embodiments, the polymer is polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polyamide 6,6 (PA6,6), polyamide 11 (PA11), polyvinylidene fluoride (PVDF), polyethylene furanoate (PEF), polyurethane (PU), polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polylactic acid (PLA), polycaprolactone (PCL). , polybutylene succinate (PBS), poly(glycolic) acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyvinyl alcohol (PVOH), alginate, copolymer PEGylated fibrin (P-fibrin), polyglycerol sebacate (PGS), poly-L-lactic acid (PLLA), poly-lactic-co-glycolic acid (PLGA), poly-D,L-lactic acid / polyethylene glycol / poly-D,L-lactic acid (PDLLA-PEG), hyaluronic acid (HA), or any combination thereof. In some embodiments, the scaffold can also comprise a chemical derivative of a natural polymer. In some embodiments, the chemical derivative can include substitution and / or addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, another chemical modification, or any combination thereof.In some embodiments, the natural polymer can be selected from proteins such as collagen, zein, casein, gelatin, gluten, and serum albumin. In some embodiments, 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 (e.g., poly-D,L-lactic-co-glycolic acid (PLGA)), and hyaluronic acid.

[0112]

[0128] In some embodiments, the scaffold can be bioabsorbable. In some embodiments, a bioabsorbable scaffold can be a non-cytotoxic structure or material that can house or support living cells and hold them in a desired configuration for a period of time. In some embodiments, the term "bioresorbable" can refer to any material that the body can break down into non-toxic byproducts that can be excreted from or metabolized within the body. In certain embodiments, exemplary bioabsorbable materials for the scaffold can 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(acrylate esters), poly(methacrylate esters), poly(vinyl alcohol), hyaluronic acid, chondroitin sulfate, heparin, dermatan sulfate, versican, copolymers, blends of polymers, mixtures of polymers, oligomers containing bioabsorbable linkages, or any combination thereof.

[0113]

[0129] In some embodiments, the scaffold can be a mesh. In some embodiments, the mesh can be a network of materials (e.g., threads, strings, strands, fibers, or any combination thereof) that can be woven or otherwise connected. In some embodiments, the mesh can comprise materials that can be artificial, biological, or any combination thereof. In some embodiments, the mesh can have pores that can be regular or irregular in size, regular or irregular in shape, regular or irregular in pattern, or any combination thereof. In some embodiments, the mesh can be two-dimensional or three-dimensional. In some embodiments, the mesh can have pores that are about 10 nm to 10 cm in diameter, spacing, or any combination thereof. In some embodiments, the pore size can generally be about 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 15 mm, 2 cm, 2.5 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. The mesh can have a diameter of about 20 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 12 mm, 15 mm, 2 cm, 2.5 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. In some embodiments, the strands (e.g., fibers, yarns, webs, etc.) or materials forming the mesh can have a diameter of about 50 nm to about 10 mm. In some embodiments, the mesh may not be a scaffold.

[0114]

[0130] In some embodiments, the scaffold can be a support structure for cell growth, hi some embodiments, the scaffold can be permeable to fluids and nutrients so that cell culture medium can contact the surface of the cell layer.

[0115]

[0131] In some embodiments, the scaffold can include a three-dimensional structure. In some embodiments, the scaffold can be porous. In some embodiments, cells can be seeded within the scaffold. In some embodiments, the scaffold can have a variety of thicknesses. In some embodiments, the scaffold can have a thickness suitable for forming a cell layer. In some embodiments, the scaffold can 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. In some embodiments, the scaffold can have a thickness of about 0.5 mm to 1 mm. In some embodiments, the scaffold can 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 embodiments, the scaffold can 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 can have the length and / or width of a cell layer to be disposed and / or grown on the scaffold. In some embodiments, the scaffold can have the length and / or width of a cell layer described herein. In some embodiments, the scaffold can include pore sizes less than 1 nanometer. In some embodiments, the scaffold can include pore sizes greater than 1 nanometer. In some embodiments, the scaffold can include pore sizes between 10 μm and 900 μm.

[0116]

[0132] In some embodiments, a cell layer may not be formed on the scaffold. In some embodiments, a dermal layer may not be formed on the scaffold (e.g., collagen matrix). In some embodiments, the synthetic leather does not include a scaffold.

[0117]

[0133] In some embodiments, the synthetic leather can include one or more pigments. In some embodiments, one or more layers of the synthetic leather can be colored. In some embodiments, the pigments in the synthetic leather can be natural pigments produced in the cells that form the synthetic leather. In some embodiments, the pigments can include melanins, including eumelanins (e.g., brown eumelanins and black eumelanins), pheomelanins, neuromelanins, or any combination thereof. In some embodiments, the pigments in the synthetic leather can be exogenous pigments, such as leather pigment dyes.

[0118]

[0134] In some embodiments, the synthetic leather can include collagen. In some embodiments, collagen can refer to any member of a family of at least 28 different collagen types. In some embodiments, collagen can be characterized by a repeating triplet of amino acids (-(Gly-XY)n-), such that approximately one-third of the amino acid residues in collagen can be glycine. In some embodiments, X can be proline and Y can be hydroxyproline. In some embodiments, the collagen structure can have twisted triplet units of peptide chains of different lengths. In some embodiments, the synthetic leather can include collagen from more than one species. In some embodiments, the synthetic leather can include collagen from different animals. In some embodiments, different animals can produce collagen with different amino acid compositions, which can result in different properties (and differences in the resulting leather). In some embodiments, collagen fiber monomers can be produced from alpha chains approximately 1050 amino acids long, such that the triple helix takes the form of a rod approximately 300 nm long with a diameter of approximately 1.5 nm.

[0119]

[0135] In some embodiments, the synthetic leather can include one or more types of collagen. In some embodiments, the engineered cells described herein can secrete collagen. In some embodiments, the collagen included in the synthetic leather can include fibrillar collagen, non-fibrillar collagen, or a combination thereof. In some embodiments, the fibrillar collagen can include type I, type II, type III, type V, type XI collagen, or any combination thereof. In some embodiments, the non-fibrillar collagen can include fibril-associated collagen with interrupted triple helices, short-chain collagen, basement membrane collagen, multiplexin (multiple triple-helical domains with interruptions), MACIT collagen (membrane-associated collagen with interrupted triple helices), or any combination thereof. In some embodiments, the fibril-associated collagen with interrupted triple helices can include type IX, type XII, type XIV, type XVI, type XIX collagen, or any combination thereof. In some embodiments, the short-chain collagen can include type VIII, type X collagen, or any combination thereof. In some embodiments, the basement membrane collagen can include type IV collagen. In some embodiments, the multiplexin can comprise collagen type XV, type XVIII, or a combination thereof, hi some embodiments, the MACIT collagen can comprise collagen type XIII, type XVII, or a combination thereof.

[0120]

[0136] In some embodiments, collagen may be included in one or more portions of the synthetic leather. In some embodiments, one or more dermal layers, one or more epidermal layers, or a combination thereof may contain the collagen disclosed herein. In some embodiments, one or more layered structures in the synthetic leather may contain the collagen disclosed herein. In some embodiments, if a portion of the synthetic leather is removed during the process, collagen may also be included in the removed product.

[0121]

[0137] In some embodiments, the collagen in the synthetic leather can be derived from one or more sources. In some embodiments, the collagen can be produced by collagen-producing cells in the synthetic leather. In some embodiments, the collagen can be added separately to the leather. In some embodiments, the synthetic leather can include collagen produced by collagen-producing cells and collagen that is added separately.

[0122]

[0138] In some embodiments, at least a portion of the collagen in the synthetic leather may be produced by collagen-producing cells. In some embodiments, the method for forming the synthetic leather may include using collagen-producing cells. In some embodiments, the synthetic leather may include collagen-producing cells. In some embodiments, the collagen-producing cells may include modified cells, immortalized cells, epithelial cells, fibroblasts, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, smooth muscle cells, or combinations thereof. In some embodiments, the epithelial cells may include squamous cells, cuboidal cells, columnar cells, basal cells, or any combination thereof. In some embodiments, the fibroblasts may include dermal fibroblasts. In some embodiments, the keratinocytes may include epidermal keratinocytes, basal keratinocytes, proliferative basal keratinocytes, differentiated suprabasal keratinocytes, or any combination thereof. In some embodiments, the collagen in the synthetic leather may be produced by one or more types of collagen-producing cells.

[0123]

[0139] In some embodiments, collagen production can be induced in cells by adding ascorbic acid, an ascorbic acid analog, a salt thereof, or any combination thereof at a concentration of about 1 mM (millimolar) to about 5 M. In some embodiments, collagen fibrillation can be induced by adding a salt or combination of salts, which can include, for example, Na3PO4, K3PO4, KCl, and NaCl. In some embodiments, the salt concentration of each salt can be about 10 mM to about 5 M.

[0124]

[0140] In some embodiments, cells can be genetically engineered to contain a collagen gene or multiple collagen genes. In some embodiments, the genetically engineered cells can include epithelial cells, fibroblasts, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, smooth muscle cells, immortalized cells, cells with molecular switches, or combinations thereof. In some embodiments, epithelial cells can include squamous epithelial cells, cuboidal cells, columnar cells, basal cells, or combinations thereof. In some embodiments, fibroblasts can include dermal fibroblasts. In some embodiments, keratinocytes can include epithelial keratinocytes, basal keratinocytes, proliferative basal keratinocytes, differentiated suprabasal keratinocytes, or combinations thereof. In some embodiments, collagen genes can include P4HA, P4HB, COL1A1, COL1A2, COL2A1, COL3A1, or any combination thereof. In some embodiments, collagen genes can have an altered promoter that can alter (e.g., increase or decrease) collagen gene expression. In some embodiments, a collagen gene can be placed downstream of the engineered expression system. The collagen gene can be from a human, mammal, bird, reptile, amphibian, fish, invertebrate, or any combination thereof.

[0125]

[0141] In some embodiments, the synthetic leather can further include one or more additives. Such additives can enhance commercial appeal (e.g., appearance, color, or odor). In some embodiments, exemplary additives can include minerals, fibers, fatty acids, amino acids, proteins, or any combination thereof. In some embodiments, the additive can be an odorant, pigment, dye, resin, polymer, or any combination thereof.

[0126]

[0142] In some embodiments, the additive can include a matrix protein, a proteoglycan, an antioxidant, a perfluorocarbon, a hormone, a growth factor, or any combination thereof. In some embodiments, the growth factor can be a protein, a polypeptide, or a complex of polypeptides, including a cytokine (e.g., which can be produced by a cell and affect itself and / or various other adjacent or distant cells). In some embodiments, the growth factor can affect the growth and / or differentiation of specific types of cells, either developmentally or in response to a number of physiological or environmental stimuli. In some embodiments, the growth factor can include a hormone. In some embodiments, the growth factor can include insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), PDGF-AA, PDGF-AB, hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-alpha), transforming growth factor beta (TGF-β), TGFpi, TGFP3, epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, or any combination thereof. In some embodiments, 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.

[0127]

[0143] In some embodiments, the additives can include preservatives, such as antimicrobial preservatives, such as calcium propionate, sodium nitrate, sodium nitrite, sulfites (e.g., sulfur dioxide, sodium bisulfate, potassium bisulfite, etc.), disodium ethylenediaminetetraacetic acid (EDTA), and antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

[0128]

[0144] In some embodiments, the synthetic leather may include extracellular matrix or connective tissue, hi some embodiments, the synthetic leather may further include collagen, keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, or any combination thereof.

[0129]

[0145] In some embodiments, the synthetic leather can be patterned. For example, the synthetic leather can be patterned with an antelope, bear, beaver, bison, wild boar, camel, caribou, cat, cow, deer, dog, elephant, elk, fox, giraffe, goat, rabbit, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, ox, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, zebra, turtle, snake, crocodile, alligator, dinosaur, frog, toad, salamander, newt, chicken, The skin may be patterned according to the skin pattern of an animal selected from duck, emu, 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, cod, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, pollock, and combinations thereof. In some embodiments, 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, loch ness monster, sasquatch, thunderbird, yeti, chupacabra, and combinations thereof.

[0130]

[0146] In some embodiments, synthetic leather can be fabricated to resemble traditional animal skin, hide, or leather products and design parameters (e.g., cell type, additives, size, shape). In some embodiments, synthetic leather can include a cell layer characterized by a composition that can be substantially similar to traditional animal skin, hide, 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% to 35% protein, and about 1% to 25% fat. In some embodiments, keratinocytes in the cell layer can be aligned. In some embodiments, keratinocytes can be aligned by application of an electric field. For example, keratinocytes can be aligned by application of a mechanical stimulus, such as cyclic stretching and relaxation of a substratum. In some embodiments, aligned (e.g., electrically oriented and mechanically oriented) keratinocytes can have substantially the same orientation relative to each other, as can be found in many animal skin tissues.

[0131]

[0147] In some embodiments, the synthetic leather herein can be at least a portion of a leather article. For example, the synthetic leather can be used as a substitute for natural leather in a leather article. Exemplary leather articles include watch straps, belts, suspenders, packaging, shoes, boots, footwear, gloves, clothing (e.g., tops, bottoms, and outerwear), luggage, bags (e.g., handbags with or without shoulder straps), clutch bags, wallets, purses, billfolds, key pouches, credit card cases, pencil cases, backpacks, cases, wallets, saddles, saddlery, whips, travel goods (e.g., trunks, suitcases, travel bags, beauty cases, or toiletries), rucksacks, portfolios, briefcases, briefcases, attaché cases, pet goods (e.g., leather leashes or collars), hunting and fishing goods (e.g., gun cases, knife cases, or holsters for hard firearms), stationary articles (e.g., letter paper, book covers, camera cases, eyeglass cases, cigarette cases, cigar cases, jewelry cases, or cell phone holsters), or sporting goods (e.g., balls, such as basketballs, soccer balls, or footballs). For example, the leather article can be a watch wrap. For example, the leather article can be a belt. For example, the leather article can be a bag.

[0132]

[0148] In some embodiments, synthetic leather or a portion thereof can also be used as a skin graft, such as an allograft or xenograft for transplantation into a subject. For example, synthetic leather, the dermis layer, the epidermis layer, and / or layered structure can be a source of a skin graft for allograft or xenograft. In some embodiments, synthetic leather, the dermis layer, the epidermis layer, and / or layered structure can be produced using genetically modified cells to reduce immune rejection in the recipient of the graft.

[0133]

[0149] Disclosed herein, in some embodiments, is a method for developing engineered cells containing a molecular switch. In some embodiments, producing synthetic leather can include forming an artificial dermal layer, forming an artificial epidermal layer, forming a tissue, or a combination thereof. In some embodiments, the artificial dermal layer, artificial epidermal layer, tissue, or a combination thereof can be at least partially decellularized. In some embodiments, the at least partial decellularization can include contact with a salt solution. In some embodiments, the method can further include tanning at least a portion of the artificial dermal layer, artificial epidermal layer, artificial tissue, or any combination thereof. In some embodiments, the cells in the synthetic leather, e.g., the cells in the tissue, can include immortalized cells having the molecular switch. In some cases, the method can include placing a first cell layer (e.g., an epidermal layer) on a second cell layer (e.g., a dermal layer), thereby forming a layered structure, and tanning at least a portion of the layered structure. In some embodiments, the method can further include removing at least a portion of the cell layer (e.g., the epidermal layer). In some embodiments, at least a portion of the cell layer can be removed prior to tanning. In some embodiments, the methods herein can further comprise generating the molecular switch and the immortalized cell.

[0134]

[0150] In some embodiments, a method for producing a molecular switch is disclosed herein. In some embodiments, the molecular switch can be formed by genetically engineering a cell to contain exogenous DNA that can be programmed to induce a phenotype after exposure to a stimulus. In some embodiments, the cell can be an animal cell, such as a bovine cell. In some embodiments, the cell can be a fibroblast or a stem cell. In some embodiments, the cell can be an immortalized cell. In some embodiments, the molecular switch can be introduced into a cell by transfection, transduction, electroporation, or any combination thereof. In some embodiments, the exogenous DNA can be introduced into a host cell by electroporation or by a viral vector. In some embodiments, the molecular switch or a polynucleotide encoding the molecular switch can be introduced by a vector, where the vector can be a virus, a virus-like particle, an adeno-associated virus vector, a liposome, a nanoparticle, a plasmid, a linear dsDNA, and combinations thereof. In some embodiments, the molecular switch can be in the genome, or the molecular switch can be extrachromosomal, for example, a plasmid.

[0135]

[0151] In some embodiments, forming a molecular switch can include adding a gene, promoter, polynucleotide sequence, or any combination thereof to a cell to drive a desired response to a stimulus. In some embodiments, the response can be a growth phenotype modification (e.g., immortalization), a reporter signal (e.g., expression of a fluorophore), a change in expression, a transcriptional change, or any combination thereof. In some embodiments, the response can be an increase or decrease in gene expression. In some embodiments, the stimulus can be an environmental stimulus (e.g., temperature). In some embodiments, the stimulus can be an antibiotic (e.g., tetracycline). In some embodiments, the molecular switch can be configured to grow cells in an anchorage-independent, anchorage-dependent, or any combination thereof. In some embodiments, a stimulus can be applied to induce a change from anchorage-independent growth to anchorage-dependent growth, or a stimulus can be applied to induce a change from anchorage-dependent growth to anchorage-independent growth. In some embodiments, the stimulus can induce the removal or addition of a gene using a Cre-Lox system, a CRISPR system, or any combination thereof. In some embodiments, anchorage-independent growth can be the result of altered expression (e.g., increased or decreased) of integrin-linked kinase (ILK), cyclin D1, Cdk4, ST6, N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), or a combination thereof. In some embodiments, anchorage-dependent growth can be the result of altered expression of genes in the P53-mediated apoptosis pathway. In some embodiments, anchorage-independent growth can be achieved by modulation of integrin signaling, cell cycle, apoptosis pathways (e.g., cloning cell cycle genes into molecular switches), or any combination thereof.

[0136]

[0152] In some embodiments, immortalized cells can comprise any cell type. In some embodiments, immortalized cells can be made from fibroblasts, adipose tissue-derived cells, umbilical cord cells, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, cuboidal cells, columnar cells, collagen-producing cells, or any combination thereof. In some embodiments, immortalized cells can be made from any cell type. In some embodiments, immortalized cells can be made from primate cells, bovine cells, ovine cells, porcine cells, equine cells, canine cells, feline cells, rodent cells, avian cells, marsupial cells, reptiles, lagomorph cells, or any combination thereof. In some embodiments, immortalized cells can be made from pluripotent stem cells, induced pluripotent stem cells, mesenchymal stem cells, or embryonic stem cells. In some embodiments, immortalized cells can have characteristics similar to any of the cell types described herein. In some embodiments, immortalized cells can have mutations. In some embodiments, immortalized cells can be generated by transfection, transduction, electroporation, or any combination thereof, of exogenous gene(s). In some embodiments, a vector can carry the exogenous gene. In some embodiments, the vector can be a virus, a virus-like particle, an adeno-associated virus vector, a liposome, a nanoparticle, a plasmid, a linear dsDNA, or a combination thereof. In some embodiments, the vector can comprise a plasmid. In some embodiments, the exogenous gene can be hTERT, TERT, Bmil, CcnDl, a mutant of Cdk4, Cdk4, TAg (SV40 large T), SV40, c-myc, H-ras, Ela, c-mMycER. TAM, E6, E7, HER-2, SRC, EGFR, Abl, Atk02, Am11, Axl, Bcl, Dbl, EGFR, ERBB, Ets-1, Fms, Fos, Fps, Gli, Gsp, Her2, Hox11, Hst, Il-3, Int-2, Jun, Kit, KS3, K-SAM, Lbc, Lck, L-myc, Lyl-1, Lyt-10, Mas, MDM-2, Mll, Mos, Myb, Neu, N-Myc, Ost, Pax-5, Pim-1, PRAD-1, Ras-K, Ras-N, Ret, Ros, Ski, Sis, Set, Src, Tal1, Tan1, Tiam1, Tsc2, Trk, or a combination thereof. In some embodiments, immortalization can be from an exogenous gene, which can be a fusion gene that can include one or more genes. The exogenous gene may be integrated into a chromosome or may be extrachromosomal. In some embodiments, immortalization may be derived from a protein product or a biologically active fragment thereof that can induce immortalization. In some embodiments, the immortalized cell can remove the exogenous gene after cell division (e.g., by a Cre-LoxP system or a CRISPR system). In some embodiments, the exogenous gene may be derived from a human. In some embodiments, the exogenous gene may be derived from a mammal, a bird, a reptile, an amphibian, a fish, an invertebrate, a virus, a bacterium, or any combination thereof. In some embodiments, immortalization may be derived from random mutation or multiple mutations. In some embodiments, the mutation may be generated by UV mutagenesis, chemical mutagenesis, or any combination thereof. In some cases, immortalization may be derived from targeted mutation, for example, the targeted mutation may be generated by a CRISPR system. In some embodiments, the mutation may be in a cell cycle gene, an oncogene, a metabolic gene, or any combination thereof. In some embodiments, immortalization can result from mutations in oncogenes, cell cycle genes, genes, promoter regions, intragenic regions, intergenic regions, or any combination thereof. In some embodiments, immortalized cells can have increased or decreased expression of oncogenes or genes involved in the control of cell proliferation. In some embodiments, immortalized cells can contain proteins that act via competitive inhibition.In some embodiments, competitive inhibition may involve modifying the activity of a tumor suppressor gene, a cell cycle gene, or a combination thereof. In some embodiments, the Hayflick limit or Hayflick number refers to the finite number of cell doublings that primary cells can undergo. In some embodiments, immortalized cells can be propagated beyond the Hayflick limit. In some embodiments, immortalized cells can be propagated past about 40 cell divisions, about 50 cell divisions, about 60 cell divisions, about 70 cell divisions, about 80 cell divisions, about 90 cell divisions, about 100 cell divisions, about 200 cell divisions, about 300 cell divisions, about 400 cell divisions, about 500 cell divisions, about 600 cell divisions, about 700 cell divisions, about 800 cell divisions, about 900 cell divisions, about 1000 cell divisions, about 10,000 cell divisions, or about 100,000 cell divisions. In some embodiments, immortalized cell lines can be propagated sufficiently to produce at least 1 million square feet of synthetic leather per year. In some embodiments, the immortalized cells can have a molecular switch. In some embodiments, the immortalized cells can be conditionally immortalized cells. Conditionally immortalized cells can exhibit an immortalized cell phenotype under certain environmental stimuli or a differentiated cell phenotype under different environmental stimuli.

[0137]

[0153] A cell layer can be formed by preparing a plurality of multicellular bodies containing one or more types of cells and arranging such multicellular bodies to form a cell layer. For example, a cell layer can be formed by arranging a plurality of multicellular bodies adjacent to each other, where the plurality of multicellular bodies can be fused to form a planar layer. In some embodiments, cells can be grown in three dimensions. In some embodiments, cells can be grown in suspension.

[0138]

[0154] In some embodiments, forming a cell layer can include using a scaffold. In some embodiments, the cell layer can be formed by placing a plurality of multicellular bodies on a scaffold. In some embodiments, the forming step can include placing or placing the multicellular bodies on a support substrate that allows the multicellular bodies to fuse together to form a layer (e.g., a substantially planar layer). In some embodiments, the multicellular bodies or layers can be placed horizontally and / or vertically adjacent to each other. In some embodiments, forming a cell layer may not require a scaffold.

[0139]

[0155] In some embodiments, the cell layer can be formed by embedding cells in a medium or gel. In some embodiments, the dermal layer can be formed using fibroblasts embedded in collagen I or fibrin gel. In some embodiments, other types of medium can also be used. In some embodiments, the medium can encourage fibroblasts to secrete a sufficient amount of extracellular matrix to enable long-term maintenance of the epidermis without the need for a collagen gel.

[0140]

[0156] In some embodiments, there may be various methods for producing multicellular bodies having the characteristics described herein, e.g., comprising one or more modified cells disclosed herein. In some embodiments, multicellular bodies can be produced from a cell paste containing a plurality of cells, e.g., having a desired cell density and viscosity. In some embodiments, the multicellular bodies can comprise modified cells. In further cases, the cell paste can be molded into a desired shape and allowed to mature (e.g., incubate) to form the multicellular body. In some embodiments, elongated multicellular bodies can be produced by molding a cell paste containing a plurality of cells into an elongated shape (e.g., a cylinder). In further cases, the cell paste can be incubated in a controlled environment to allow the cells to adhere and / or aggregate with one another to form the elongated multicellular body. In some embodiments, multicellular bodies can be produced by molding a cell paste containing a plurality of living cells in a device that holds the cell paste in a three-dimensional shape. In some embodiments, the cell paste can be incubated in a controlled environment while it is held in a three-dimensional shape for a sufficient time to produce a body with sufficient cohesion to support itself on a flat surface, as described herein.

[0141]

[0157] In some embodiments, a cell paste can be provided by (A) mixing cells or cell aggregates (of one or more cell types) and cell culture medium (e.g., in a predetermined ratio) to result in a cell suspension, and (B) compacting the cell suspension to produce a cell paste with a desired cell density and viscosity. Compaction can be achieved in a number of ways, for example, by concentrating a particular cell suspension obtained from cell culture to achieve the desired cell concentration (density), viscosity, and consistency required for the cell paste. In some embodiments, a relatively dilute cell suspension from cell culture can be centrifuged for a determined time to achieve a cell concentration in the pellet that allows molding in a mold. Tangential flow filtration ("TFF") can be another suitable method for concentrating or compacting cells. In some embodiments, a compound can be combined with the cell suspension to impart the required extrusion properties. Suitable compounds include collagen, hydrogels, Matrigel, nanofibers, self-assembled nanofibers, gelatin, and fibrinogen. One or more ECM components (or derivatives of ECM components) can also be included by resuspending the cell pellet in one or more physiologically acceptable buffers containing the ECM components (or derivatives of the ECM components) and centrifuging the resulting cell suspension again to form a cell paste.

[0142]

[0158] In some embodiments, various methods can be used to shape the cell paste. For example, in certain embodiments, the cell paste can be manually shaped or pressed (e.g., after concentration / compression) to achieve a desired shape. As a further example, the cell paste can be introduced (e.g., aspirated) into a preformed device, such as a micropipette (e.g., a capillary pipette), which shapes the cell paste to fit the interior surface of the device. In some embodiments, 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 shape. In some embodiments, the cell paste can be shaped by depositing it into a preformed mold, such as a plastic mold, a metal mold, or a gel mold. In some embodiments, centrifugal casting or continuous casting can be used to shape the cell paste.

[0143]

[0159] In some embodiments, the cell paste can be further matured. In some embodiments, the cell paste can be incubated at about 37° C. for a period of time (which may be cell type dependent) to promote adhesion and / or coherence. Alternatively, or in addition, the cell paste can be maintained in the presence of cell culture medium containing factors and / or ions to promote adhesion and / or coherence.

[0144]

[0160] The multicellular bodies can be placed on a support substrate to generate 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 by an automated machine such as a biofabricator.

[0145]

[0161] The support substrate can be permeable to fluids, gases, and nutrients, allowing cell culture medium to contact all surfaces of the multicellular bodies and / or layers during placement and subsequent fusion. In certain embodiments, the support substrate can be made from natural biomaterials such as collagen, fibronectin, laminin, and other extracellular matrices. In certain embodiments, the support substrate can be made from synthetic biomaterials such as hydroxyapatite, alginate, agarose, polyglycolic acid, polylactic acid, and copolymers thereof. In certain embodiments, the support substrate can be solid, semi-solid, or a combination of solid and semi-solid support elements. In certain embodiments, the support substrate can be planar to facilitate the fabrication of planar layers. In certain embodiments, the support substrate can be raised or elevated above a non-permeable surface, such as a portion of the cell culture environment (e.g., a Petri dish, cell culture flask, etc.) or a bioreactor. A permeable, elevated support substrate can contribute to preventing premature cell death, enhance cell proliferation, and promote the fusion of multicellular bodies to form a layer.

[0146]

[0162] Once the assembly of the layers is complete, tissue culture medium can be poured on top of the construct. In some embodiments, the tissue culture medium can enter the spaces between the multicellular bodies and support the cells in the multicellular bodies. In some embodiments, the multicellular bodies in the three-dimensional construct can be fused to one another to generate a layer (e.g., substantially planar) for use in forming synthetic leather. By "fuse," "fused," or "fusion" it can mean that the cells of adjacent multicellular bodies adhere and / or cohere to one another, either directly through interactions between cell surface proteins or indirectly through interactions between the cells and ECM components or derivatives of ECM components. In some embodiments, the fused layer can be completely fused, and the multicellular bodies can become substantially continuous. Alternatively, the fused layer can be substantially fused or partially fused, and the cells of the multicellular bodies become adhered and / or cohere to the degree necessary to allow for complete movement and manipulation of the layer.

[0147]

[0163] 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 embodiments, the multicellular bodies fuse to form a layer in an environment having conditions suitable for promoting the growth of the cell types contained in the multicellular bodies. In some embodiments, the fusion occurs over a period of about 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes and increments therein. In other cases, the fusion occurs over a period of 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, the fusion is carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 14 days and increments therein. In further cases, the fusion is carried out for about 2 hours to about 24 hours. Factors related to the fusion time can include the cell type, cell type ratio, culture conditions, and the presence of additives such as growth factors.

[0148]

[0164] Once fusion of the layers is complete, the layers and the support substrate can be separated. In some embodiments, the layers and the support substrate can be separated when fusion of the layers can be substantially or partially complete, but the cells of the layers can still adhere, cohere, or any combination thereof to the extent necessary to allow the layers to be moved, manipulated, and stacked without disrupting the layers. The layers and the support substrate can be separated by standard procedures for melting, dissolving, or decomposing the support substrate. In some embodiments, the support substrate can be dissolved by, for example, a temperature change, light, or other stimulus that does not adversely affect the layers. In some cases, the support substrate can be made of a flexible material and peeled from the layers. The separated layers can be transferred to a bioreactor for further maturation. In some embodiments, the separated layers mature and further fuse after incorporation into an artificial animal skin, hide, or leather product.

[0149]

[0165] Alternatively, the layer and the support substrate may not be separated, and the support substrate decomposes or biodegrades prior to packaging, freezing, sale, or consumption of the assembled artificial animal skin, hide, or leather product.

[0150]

[0166] The cell layer can be formed over a period of time. In some embodiments, the cell layer, e.g., the epidermal layer or 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, or 300 days. In some embodiments, the dermal layer can be formed within about 1 to 15 days, e.g., 5 to 10 days or 10 to 12 days. In some embodiments, the dermal layer can be formed within about 5 to 25 days, e.g., 14 to 15 days.

[0151]

[0167] The present disclosure provides methods for producing synthetic leather with improved barrier function. In some embodiments, the methods include providing a medium containing keratinocytes and ascorbic acid and linoleic acid; and culturing the keratinocytes under conditions such that synthetic leather with improved barrier function can be formed. In some embodiments, the culture conditions include culturing at about 50-95% humidity, e.g., about 75% humidity. In some embodiments, the ascorbic acid can be provided at a concentration of about 10-100 micrograms / ml. In yet further cases, the linoleic acid can be provided at a concentration of about 5-80 micromolar. The present disclosure is not limited to synthetic leather formed from a particular source of keratinocytes. 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 some embodiments, synthetic leather can be formed by engineered cells containing a molecular switch. In some embodiments, the modified cells may be immortalized cells (e.g., immortalized bovine fibroblasts). In yet further embodiments, the keratinocytes express exogenous wild-type or mutant Krüppel-like factor (GKLF). In yet further embodiments, the keratinocytes may be derived from two different sources. In other embodiments, the synthetic leather has a surface capacitance of about 40 to about 240 pF. In some preferred embodiments, the skin equivalent has a surface capacitance of about 80 to about 120 pF. In other preferred embodiments, the content of ceramides 5, 6, and 7 in the skin equivalent may be about 20 to about 50% of the total ceramide content. In yet other preferred embodiments, the content of ceramide 2 in the skin equivalent may be about 10 to about 40% of the total ceramide content. In still further embodiments, the present disclosure provides a skin equivalent produced by the method described immediately above.

[0152]

[0168] Multiple cell layers can be arranged to form a layered structure, thus producing the synthetic leather described herein. In some embodiments, the dermal and epidermal layers can be formed separately and assembled by placing the epidermal layer on top of the dermal layer (e.g., when both the epidermal and dermal layers can be fully formed). In some embodiments, the epidermal layer can be grown on top of the dermal layer. In some cases, a basement membrane or basement membrane substitute can be placed between the dermal and epidermal layers. For example, the cell layers can be manually placed in contact with each other, or can be deposited in place by an automated computer-assisted machine, such as a biofabricator, according to a computer script.

[0153]

[0169] 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 histological analysis (e.g., at least 3-5 days). Using the methods provided herein, there can be a low risk of improperly formed layered structures or full-thickness skin equivalents.

[0154]

[0170] Multiple cell layers can be assembled in various ways. In some embodiments, an epidermal layer and a dermal layer (with or without a basement membrane substitute) can be placed on a scaffold (e.g., silk) to achieve the thickness and tensile strength of, for example, natural leather. In some embodiments, an epidermal layer and multiple dermal layers (with or without a basement membrane substitute) can be assembled without the use of a scaffold. Such an assembly can achieve a thickness and tensile strength similar to natural leather. In some embodiments, an epidermal layer and multiple dermal layers (with or without a basement membrane substitute) can be placed on a scaffold (e.g., silk) to achieve a thickness and tensile strength similar to natural leather. In some embodiments, the leather has a tensile strength of at least about 10, 20, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, or 300 kgf / cm.2 In some embodiments, the leather has a tensile strength of about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, or 300 kgf / cm 2 In certain embodiments, the tensile strength test can be performed according to American Society for Testing and Materials (ASTM) standards.

[0155]

[0171] Multiple cell layers can be assembled to form synthetic leather (e.g., a full-thickness skin equivalent). The synthetic leather can include a top, a middle, and a bottom. The top can include an epidermal layer. For example, the top can be a single epidermal layer. The middle section can include a basement membrane substitute. In some embodiments, the middle section does not have a basement membrane substitute. For example, the middle section can have a layer of negligible thickness. The bottom can have one or more dermal layers. In some embodiments, the bottom has a single dermal layer disposed on a scaffold (e.g., silk). In some embodiments, the bottom has multiple dermal layers (e.g., up to five layers) without any scaffold. In some embodiments, the bottom has multiple dermal layers stacked on top of each other and disposed on a scaffold (e.g., silk).

[0156]

[0172] The adhesiveness between the epidermal layer and the dermal layer can be strong enough to resist splitting of the layers. In some embodiments, the cell layers can be assembled by gluing onto the scaffold. Natural or synthetic adhesives can be used for assembly. The natural adhesive can 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, plant glue (e.g., starch, dextrin glue, Canada balsam, pine resin-based glue, cocconia, gum arabic, postage stamp gum, latex, library paste, methylcellulose, mucus, resorcinol resin, or urea-formaldehyde resin), or any combination thereof. Synthetic adhesives can be acrylonitrile, cyanoacrylate (e.g., n-butyl-2-cyanoacrylate adhesive), acrylic, resorcinol adhesive, epoxy resin, epoxy putty, ethylene-vinyl acetate, phenol-formaldehyde resin, polyamide, polyester resin, polyethylene, polypropylene, polysulfide, polyurethane, polyvinyl acetate (including white glue (e.g., Elmer's glue) and yellow carpenter's glue (aliphatic resin)), polyvinyl alcohol, polyvinyl chloride (PVC), polyvinyl chloride emulsion (PVCE), polyvinylpyrrolidone rubber cement, silicone, and styrene-acrylic copolymer. For example, assembly can be performed using fibrin adhesive. For example, assembly can be performed using n-butyl-2-cyanoacrylate adhesive.

[0157]

[0173] In some embodiments, cell layers (e.g., substantially planar layers) can be stacked to form synthetic leather. The cell layers can have an orientation determined by the arrangement, pattern, or orientation of the multicellular bodies. In some embodiments, each layer can be stacked in a specific orientation relative to the supporting substrate and / or one or more other layers. For example, one or more layers can be stacked in an orientation that includes a rotation relative to the supporting substrate and / or underlying layer, where the rotation can be between 0.1 and 180 degrees, e.g., 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 layers can be oriented substantially the same.

[0158]

[0174] Once the layer stacking is complete, the layers in the three-dimensional construct can be fused together to produce synthetic leather. In some embodiments, the layers are fused in a cell culture environment (e.g., a Petri dish, a cell culture flask, a bioreactor, etc.). In some embodiments, the fusion occurs over a period of about 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 minutes, and increments therein. In other cases, the fusion occurs over a period of 1 to 48 hours, e.g., 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 over a period of about 2 hours to about 24 hours.

[0159]

[0175] In some embodiments, cells or cell layers can be cultured under various cell culture conditions. In some embodiments, cells and cell layers can be incubated in a medium, a bioreactor, an incubator, or any combination thereof. In some embodiments, the medium can include a culture medium. In some embodiments, cells or cell layers can be cultured in vitro. For example, the dermal layer and / or epidermal layer can be cultured in vitro. Alternatively, cells or cell layers can be cultured in vivo. For example, the dermal layer and / or epidermal layer can be cultured in vivo. In some embodiments, the medium can include a culture medium disclosed herein. In some embodiments, the culture medium can include a growth medium, a tissue formation medium, or a combination thereof, as disclosed herein. In some embodiments, cells, cell layers, portions thereof, or any combination thereof can be cultured in the medium. In some embodiments, the medium can include a supplement. In some embodiments, the supplement can be a natural supplement, a synthetic supplement, or a combination thereof. In some embodiments, the supplement can be an additive. In some embodiments, one or more of the supplements can induce the production and assembly of extracellular matrix from the engineered cells, thus enhancing the natural appearance of the synthetic leather. In some embodiments, the supplements can include ECM components such as collagen, fibrin, growth factors, small molecules, polymers, sulfate, carrageenan, or any combination thereof. In some embodiments, the small molecules can include ascorbic acid. In some embodiments, the polymers can include dextran. In some embodiments, the cells can be grown in a cell culture medium. In some embodiments, the cell culture medium can include a growth medium, a tissue formation medium, or a combination thereof. In some embodiments, the culture medium can include Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640, Eagle's Minimum Essential Medium, Ham's Nutrient Mixture, Iscove's Modified Medium, Dulbecco's Medium, or any combination thereof.In some embodiments, supplements such as salts, buffering agents, phenol red, HEPES, amino acids, carbohydrates, lipids, proteins, peptides, fatty acids, vitamins, elements, media supplements, antibiotics, serum, serum replacement, or any combination thereof can be added to the culture medium. In some embodiments, the medium can contain about 0%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% serum, serum replacement, or a combination thereof. In certain embodiments, the medium may contain about 0.1% to about 10%, about 10% to about 20%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% serum, serum substitute, or a combination thereof.

[0160]

[0176] In some embodiments, the medium can comprise a growth medium, a tissue formation medium, or a combination thereof. In some embodiments, after seeding, the transfected or transduced isolated cells can be contacted with the tissue formation medium. In some embodiments, the growth medium can comprise growth factors, buffers, salts, sugars, amino acids, lipids, minerals, ECM proteins, or a combination thereof. In some embodiments, the salt can comprise an inorganic salt. In some embodiments, the inorganic salt can comprise calcium chloride, ferric nitrate, magnesium sulfate (anhydrous), potassium chloride, sodium bicarbonate, sodium chloride, basic sodium phosphate (anhydrous), or any combination thereof. In some embodiments, the amino acids can include L-arginine·HCl, L-cystine·2HCl, glycine, L-histidine·HCl·H2O, L-isoleucine, L-leucine, L-lysine·HCl, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine·2Na·2H2O, L-valine, L-glutamine, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the vitamins can include choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxal·HCl, pyridoxine·HCl, riboflavin, thiamine·HCl, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the sugars can include D-glucose, a stereoisomer thereof, a salt thereof, or any combination thereof. In some embodiments, the pH indicator can include phenol red·Na, pyruvate·Na, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the growth medium can include amino acids, vitamins, inorganic salts, fetal bovine serum, antibiotics, antifungals, or any combination thereof.In some embodiments, the tissue formation medium can include growth factors, buffers, salts, sugars, amino acids, lipids, minerals, ECM proteins, human platelet lysate, acid citrate dextrose, heparin, ascorbic acid, TGF-β1, normocin, serum, serum replacement, non-essential amino acids, antibiotics, antifungals, or any combination thereof. In some embodiments, the tissue formation medium can further include serum, serum replacement, or a combination thereof. In some embodiments, the amino acids can include glycine, alanine, L-arginine hydrochloride, L-asparagine-H2O, L-aspartic acid, L-cysteine ​​hydrochloride-H2O, L-cystine diHCl, L-glutamic acid, L-glutamine, L-histidine hydrochloride-H2O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate, L-valine, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the vitamins can include biotin, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the inorganic salt can include calcium chloride, copper sulfate, ferric nitrate, ferric sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, zinc sulfate, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the medium can further include D-glucose (dextrose), hypoxanthine Na, linoleic acid, lipoic acid, phenol red, putrescine 2HCl, sodium pyruvate, thymidine, any stereoisomer thereof, any salt thereof, or any combination thereof. In some embodiments, the serum replacement can be substantially free of animal-derived products, can be xeno-free, or a combination thereof.In some embodiments, the serum replacement can include growth factors, insulin, transferrin, cytokines, essential amino acids, non-essential amino acids, proteins, extracellular matrix proteins (ECM), laminin, fibronectin, vitronectin, cell adhesion peptides, RGD, extracellular matrix fragments, hormones, collagen, albumin, lipids, glycoproteins, protein fragments, or any combination thereof. In some embodiments, the serum can include fetal bovine serum (FBS), horse serum, fetal bovine serum, or any combination thereof. In some embodiments, the medium does not contain TGF-beta. In some embodiments, after seeding, the transfected or transduced isolated cells can be contacted with a tissue formation medium. In some embodiments, contacting the cells with a tissue formation medium can at least partially differentiate or form the cells into another cell or tissue type. In some embodiments, the transfected or transduced isolated cells, when present in tissue formation medium, (a) at least partially increase collagen production, (b) have at least partially arrested cell proliferation, or (c) both, compared to otherwise comparable transfected or transduced isolated cells not contacted with tissue formation medium. In some embodiments, the transfected or transduced isolated cells can be at least partially differentiated when contacted with tissue formation medium. In some embodiments, after seeding, the transfected or transduced isolated cells can be contacted with a medium containing L-ascorbic acid 2-phosphate (AA2P), a salt thereof, a biologically active fragment thereof, or any combination thereof. In certain embodiments, the transfected or transduced isolated cells, when present in a medium containing AA2P, a salt thereof, TGFBl, a biologically active fragment thereof, or a combination thereof, (a) at least partially increase collagen production, (b) have at least partially arrested cell proliferation, or (c) both, compared to an otherwise comparable medium lacking AA2P, a salt thereof, TGFBl, a biologically active fragment thereof, or a combination thereof.In some embodiments, the transfected or transduced isolated cells can be at least partially differentiated upon contact with culture medium.

[0161]

[0177] In some embodiments, the stimuli can include the presence, absence, or levels of carbohydrates, lipids, nucleic acids, proteins, antibiotics, organic chemicals, inorganic chemicals, artificial chemicals, metabolites, or any combination thereof. In some embodiments, cells, cell layers, or combinations thereof can be cultured with one or more stimuli. In some embodiments, the stimuli can be applied before growth, during growth, after growth, or any combination thereof.

[0162]

[0178] In some embodiments, single cell propagation can be performed in a shaker, spinner flask, or any combination thereof. In some embodiments, tissue growth can include growth with or without shaking. In some embodiments, tissue growth can include initial growth with high-speed shaking followed by growth with little or no shaking. In some embodiments, tissue growth can include initial growth with little or no shaking followed by growth with high-speed shaking. In some embodiments, the little or no shaking can be at most 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 5, 3, 2, 1, or 0 revolutions per minute (RPM). In some embodiments, the little or no shaking can be for more than about 6 hours, 12 hours, 18 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the little or no shaking can be for less than about 6 hours, 12 hours, 18 hours, 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the little or no shaking can be for more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 passages. In some embodiments, the little or no shaking can be for less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 passages. In some embodiments, the high speed shaking can be at least 60, 80, 100, 120, 140, 160, 180, 200, 220, or 240 RPM. In some embodiments, the high speed shaking can be 180-240, 200-240, 160-220, 60-160, 60-140, 60-120, 60-100, 60-90, 60-80, 80-160, 80-140, 80-120, 80-100, or 80-90 RPM. In some embodiments, the high speed shaking can be for more than about 2, 3, 4, 5, 7, 10, 15, or 20 passages. In some embodiments, the high speed shaking can be for less than about 2, 3, 4, 5, 7, 10, 15, or 20 passages.In some embodiments, shaking can be performed at an initial speed and then increased to a higher speed, in some embodiments, shaking can be performed at an initial speed and then decreased to a lower speed, in some embodiments, shaking can be at a constant speed.

[0163]

[0179] In some embodiments, the proliferation of the modified cells can be determined by manual cell counting, automated cell counting, indirect cell counting, or any combination thereof. In some embodiments, cell counting can include counting chambers, colony-forming unit counting, electrical resistance, flow cytometry, image analysis, stereocytometry, spectrophotometry, impedance microbiology, automated cell counting, microscopy, Coulter counters, automated imaging cell counters, droplet digital polymerase chain reaction (PCR), quantitative PCR, metabolic measurements, or any combination thereof.

[0164]

[0180] In some embodiments, anchorage-independent cells are capable of anchorage-independent growth for at least 1, 2, 3, 4, 5, 7, 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 34, 35, 37, or 40 cell divisions. Anchorage-independent cells may be capable of anchorage-dependent growth for at least 1, 2, 3, 4, 5, 7, 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 34, 35, 37, or 40 cell divisions. Cell division may also be referred to as passaging herein. In some embodiments, anchorage-independent cells may be capable of anchorage-independent growth indefinitely. In some embodiments, anchorage-dependent cells may be capable of anchorage-independent growth indefinitely. In some embodiments, anchorage-independent cells may be capable of anchorage-independent growth for at least 1 passage, 5 passages, or at least 35 passages. In some embodiments, the anchorage-dependent cells may be capable of anchorage-dependent growth for at least 1 passage, 5 passages, or at least 35 passages. In some embodiments, the cells may be programmed to switch from anchorage-independent growth to anchorage-dependent growth after a set amount of divisions. In some embodiments, the cells may be programmed to switch from anchorage-dependent growth to anchorage-independent growth after a set amount of divisions. In some embodiments, the stimulus can control the programmed switch. In some embodiments, the stimulus may not control the programmed switch. For example, cells in anchorage-independent growth can undergo 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 or 50 rounds of division before switching to anchorage-dependent growth.In some embodiments, cells in anchorage-dependent growth can switch to anchorage-independent growth after 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, or 50 rounds of division. In some embodiments, anchorage-independent cells can be at least partially incapable of adhesive growth. In some embodiments, anchorage-dependent cells can be at least partially incapable of growth in suspension. In some embodiments, at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the anchorage-independent cells may be incapable of adherent growth, hi some embodiments, at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the anchorage-dependent cells may be incapable of growth in suspension.

[0165]

[0181] In some embodiments, the cells can be seeded onto the scaffold. In some embodiments, the cells may not be seeded onto the scaffold. In some embodiments, the cells are seeded at a density of about 100 cells / cm. 2 ~about 10,000,000 cells / cm 2 , 1,000 cells / cm 2 ~about 1,000,000 cells / cm 2 , 100,000 cells / cm 2 ~about 10,000,000 cells / cm 2 , 10,000 cells / cm 2 ~about 100,000 cells / cm 2 , 50,000 cells / cm 2 ~about 200,000 cells / cm 2 , 100,000 cells / cm 2 ~about 1,000,000 cells / cm 2 , 100,000 cells / cm 2 ~about 500,000 cells / cm2 or approximately 100,000 cells / cm 2 ~about 2,500,000 cells / cm 2 In certain embodiments, the cells may be seeded at a density of about 10, 100, 10,000, 50,000, 75,000, 90,000, 100,000, 125,000, 150,000, 175,000, 200,000, 1,000,000, or 10,000,000 cells / cm. 2 In some embodiments, the cells are seeded at about 10, 100, 10,000, 50,000, 75,000, 90,000, 100,000, 125,000, 150,000, 175,000, 200,000, 1,000,000, or 10,000,000 cells / cm. 2 In some embodiments, cells can be seeded at a density less than 1000 kJ / cm. In some embodiments, cells can be seeded on one side of a scaffold. In some embodiments, a scaffold can be a substrate. In some embodiments, cells can be seeded on more than one side of a scaffold, for example, on the top and bottom of a scaffold. A scaffold can be seeded sequentially and simultaneously. In some embodiments, a scaffold can be seeded on more than one side of a scaffold without flipping the scaffold. For example, a second side of a scaffold can be seeded without flipping the scaffold. In some embodiments, a scaffold can be seeded on one side, then the scaffold can be flipped over and seeded on another side of the scaffold. In some embodiments, the seeded cells can comprise a biofabricated material. In some embodiments, the biofabricated material can comprise the cells, modified cells, or tissues disclosed herein.

[0166]

[0182] In some embodiments, the cells and cell layers described herein can be cultured under anchorage-independent conditions, anchorage-dependent conditions, or any combination thereof. In some embodiments, anchorage-independent growth, anchorage-dependent growth, or both can occur in a vessel, container, or a combination thereof. In some embodiments, anchorage-independent or anchorage-dependent growth can be measured on cells that may not have received a stimulus, cells that may not contain a switch, or any combination thereof. In some embodiments, anchorage-dependent growth or anchorage-independent growth can be determined by manual cell counting, automated cell counting, indirect cell counting, or any combination thereof. In some embodiments, at least partially anchorage-dependent growth can consume more, the same, or fewer nutrients compared to at least partially anchorage-independent growth. In some embodiments, the vessel or container can be glass, metal, plastic, or any combination thereof. In some embodiments, the vessel or container can contain a coating that allows cells to adhere to at least a portion of the vessel or container. In some embodiments, the coating can be poly-L-lysine, poly-D-lysine, laminin, collagen, bovine fibronectin, gelatin, extracellular matrix, laminin, entactin, HSPG, bovine gelatin, or any combination thereof. In some embodiments, the container or vessel can hold a total volume of at least about 0.5 mL, 1 mL, 5 mL, 25 mL, 100 mL, 250 mL, 500 mL, 1000 mL, 2 L, 5 L, 10 L, 100 L, 1000 L, 5000 L, or about 10,000 L. In some embodiments, in anchorage-independent growth conditions, the cells grow at a volume of about 1 e 1 cells / mL, 1e 2 cells / mL, 1e 3 cells / mL, 1e 4 cells / mL, 1e 5 cells / mL, 1e 6 cells / mL, 1e 7 cells / mL, 1e 8 cells / mL, 1e 9 cells / mL or 1e 10In some embodiments, cells in anchorage-independent growth can be grown to less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% confluence. In some embodiments, cells in anchorage-independent growth can be grown to greater than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% confluence. In some embodiments, cells in anchorage-independent growth can be grown in suspension. In anchorage-dependent growth conditions, cells can be grown to about 1e 1 cells / mL, 1e 2 cells / mL, 1e 3 cells / mL, 1e 4 cells / mL, 1e 5 cells / mL, 1e 6 cells / mL, 1e 7 cells / mL, 1e 8 cells / mL, 1e 9 cells / mL or 1e 10 In some embodiments, anchorage-independent growing cells can be expanded to approximately 1 e 5 cells / mL, 1e 6 cells / mL, 1e 7 cells / mL, 1e 8 cells / mL, 1e 9 cells / mL or 1e 10 When the cells reach a density of about 1 e cells / mL, they can switch to anchorage-dependent growth. In some embodiments, cells undergoing anchorage-dependent growth can grow at a density of about 1 e 5 cells / mL, 1e 6 cells / mL, 1e 7 cells / mL, 1e 8 cells / mL, 1e 9 cells / mL or 1e 10 When cells reach a density of approximately 50,000 cells / cm, they can switch to anchorage-dependent growth. Under anchorage-dependent growth conditions, cells grow at a density of approximately 50,000 cells / cm. 2 ~about 9,000,000 cells / cm 2 , 200,000 cells / cm 2 ~about 4,500,000 cells / cm2 , 250,000 cells / cm 2 ~about 4,000,000 cells / cm 2 , 500,000 cells / cm 2 ~about 2,000,000 cells / cm 2 or 1,000,000 cells / cm 2 ~about 6,000,000 cells / cm 2 In some embodiments, cells undergoing anchorage-dependent growth can be grown to a confluence of greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, cells undergoing anchorage-dependent growth can be grown to a confluence of less than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Cells in anchorage-dependent growth conditions can be grown at least partially on, in, or around a scaffold. In some embodiments, cells undergoing anchorage-dependent growth can be grown at least partially without a scaffold.

[0167]

[0183] In some embodiments, the cells and structures disclosed herein can be cultured at a specific air humidity. For example, a cell layer (e.g., modified cells, dermal layer, or epidermal 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 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%.

[0168]

[0184] In some embodiments, cells and tissues (e.g., immortalized cells having a switch) can be grown in a bioreactor or device that supports a biologically active environment. The device or bioreactor can include cell stacks, roller bottles, shakers, flasks, stirred tank suspension bioreactors, high cell density fixed-bed perfusion bioreactors, incubators, or any combination thereof. In some embodiments, the bioreactor can be a scalable modular system from cell culture production and can include 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, or more than 50 vessels. In some embodiments, the bioreactor can be a scalable system, a stackable system, a modular system, or any combination thereof. Furthermore, in some embodiments, the bioreactor can be configured to allow for easy and rapid scale-up. Additionally, the bioreactor can include a motion control system configured to rock or tilt the container, which can create dynamic flow within the chamber, which can increase the viability of the cell culture within the container. In some embodiments, the bioreactor can include a container in which cells can be grown, a temperature control system, a gas inlet / outlet system configured to control the gas concentration within the container, an agitator configured to mix the growth medium within the container, and any number of inlet and outlet ports for fluid transport. In some embodiments, the cell culture can be suspended in the growth medium within the bioreactor container. In some embodiments, the bioreactor contains a computer system for inputting commands. In some embodiments, a user can program settings that can dictate the rate of agitation, desired pH, temperature, and / or dissolved oxygen level. In some embodiments, the system can adjust these parameters based on the parameters entered by the user.In some embodiments, a user or the system can manually or automatically siphon / exchange medium or other components, for example, using a pump operably connected to a port in the container via tubing or piping. In some embodiments, medium can continuously flow into and out of the container of the system. In some embodiments, the system can utilize static motion (i.e., the bioreactor typically remains stationary during growth). In some embodiments, the systems described herein can include a motion control system configured to rock, rotate, or tilt the container, which can create dynamic flow within the chamber, which can increase the viability of the cell culture within the container.

[0169]

[0185] In some embodiments, the methods herein can include tanning at least a portion of the synthetic leather, e.g., at least a portion of the tissue. In some embodiments, the tissue can include fibroblasts, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, adipocytes, smooth muscle cells, epithelial cells, modified cells, immortalized cells, or any combination thereof. Any of the cells described herein can be immortalized cells. In some embodiments, the tissue can include a layered structure. In some embodiments, the tissue can include a dermis layer. In some embodiments, the tissue can include an epidermis layer. Tanning can make the synthetic leather resemble natural leather, which can be a durable and flexible material created by tanning animal hides and skins, often bovine hides. Tanning, as used herein, can refer to the process of treating animal skin to produce leather. Tanning can be carried out 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), syntanning (using synthetic tannins, aromatic polymers), bacterial staining, etc. In some embodiments, the tanning can be metal-free. In some embodiments, the tanning can be an environmentally friendly process.

[0170]

[0186] In some embodiments, tanning can be performed to convert proteins in hides / skin into a stable material that will not decay, while allowing the material to remain flexible. Chromium can be used as a tanning material. In some cases, the tanning can include chromium, aluminum, zirconium, titanium, iron, sodium aluminum silicate, formaldehyde, glutaraldehyde, oxazolidine, isocyanate, carbodiimide, polycarbamoyl sulfate, tetrakishydroxyphosphonium sulfate, sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate, pyrogallol, catechol, syntan, or any combination thereof. In some embodiments, tanning can be performed on modified cells in a scaffold. In some embodiments, the tissue that can be tanned can include a fiber or multiple fibers (e.g., polyester fiber, synthetic fiber, natural fiber). 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. In some embodiments, the pH described herein can be at least 1. In some embodiments, the pH described herein can be 14 or less.

[0171]

[0187] In some embodiments, tanning can be performed on a cell layer, such as a dermal layer, an epidermal layer, modified cells, an immortalized cell layer, laminin, fibronectin, collagen, or any combination thereof. In some embodiments, tanning can be performed on a tissue (e.g., tissue derived from modified cells). Tanning can also be performed on a layered structure, such as a layered structure comprising at least a dermal layer. In some cases, tanning can be performed on synthetic leather. For example, tanning can be performed after forming a cell layer, such as a dermal layer or an epidermal layer. For example, tanning can be performed after forming a layered structure. Tanning can also be performed on cells containing a switch. For example, tanning can be performed on a layer of immortalized cells containing a molecular switch for anchorage-independent and anchorage-dependent growth.

[0172]

[0188] In some embodiments, tanning can be performed on dehydrated cells or cell layers. The moisture content of the synthetic leather after dehydration can be about 5%, 10%, 15%, 20%, 30%, 35%, 40%, 50%, 60%, 70%, or 80% or less. In some embodiments, dehydration can include decellularizing the cells to prevent shrinkage. In some embodiments, decellularization can be performed using ethanol, propyl alcohol, isopropyl alcohol, butanol, methane, ethane, methyl acetate, ethyl acetate, butyl acetate, ethylene carbonate, ethereal hydrochloride, dichloromethane, chloroethylene, DMSO, acetone, diethyl ether, an organic solvent, or any combination thereof.

[0173]

[0189] In some embodiments, tanning can be performed by modifying extracellular matrix (ECM) materials. Tanning can be performed by modifying collagen in the ECM. Tanning can be performed using a tanning agent, such as chromium(III) sulfate ([Cr(H2O)6]2(SO4)3). Chromium(III) sulfate dissolves to form the hexaaquachromium(III) cation, [Cr(H2O)6] 3+At higher pH levels, chromium(III) compounds can be produced, which at higher pH levels can be active in tanning by crosslinking collagen subunits through a process called oleation. Some ligands include sulfate anions, collagen carboxyl groups, amine groups from amino acid side chains, and masking agents. Masking agents can be carboxylic acids, such as acetic acid, used to suppress the formation of polychromium(III) chains. Masking agents can allow tanners to further increase the pH to increase collagen reactivity without inhibiting the penetration of the chromium(III) complex. Tanning can increase the spacing between protein chains in collagen (e.g., from 10 to 17 Å), consistent with crosslinking by polychromium species of the type resulting from oleation and oxolation. Chromium can be crosslinked to collagen. Chromium-tanned leather can contain approximately 4% to 5% chromium. This efficiency can be characterized by the leather's increased hydrothermal stability and its resistance to shrinkage in heated water. Other tanning agents can be used to tan lamellar bodies and modify collagen.

[0174]

[0190] In some embodiments, tanning can be carried out using other minerals. In some embodiments, tanning can be carried out using agents based on alum, zirconium, titanium, iron salts, or combinations thereof. Tanning can be carried out at least partially free of chromium.

[0175]

[0191] In some embodiments, tanning can be performed by vegetable tanning. In some embodiments, vegetable tanning can include soaking the hide in a plant-based extract. In some embodiments, the plant-based extract can include extracts of chestnut bark, oak bark, hemlock bark, mangrove bark, acacia bark, oak bark, cuera alba bark, mimosa bark, olive leaf, oak bark, or any combination thereof. In some embodiments, tanning can be performed by wet white tanning (e.g., tanning with aldehydes, polycarbamoyl sulfate, or any combination thereof). In some embodiments, tanning can be performed using a mineral-free tanning system including Granofin® Easy F90 and ProSpread™. In certain embodiments, retanning can be performed using Densotan® A, Catalix® 150, Lipoderm® N, Lipoderm® LA, Basyntan® NL, Relugan® Soft Ap, Tanicor® VX, Basyntan® SW, Granofin® TAP, Melioderm® HF Brown, formic acid, Terogtan, Lipsol MSG, Catalix® 150, Brun dye, or any combination thereof.

[0176]

[0192] In some embodiments, the modified cells, immortalized cells, cell layers, layered structures, and synthetic leathers produced herein can be further processed after tanning. In some embodiments, the methods provided herein further include one or more leather processing steps (e.g., those used in traditional leather formation). Examples of processing steps include preserving, soaking, liming, unhairing, lining, splitting, deliming, reliming, baiting, degreasing, freezing, bleaching, coloring, pickling, desalting, tanning, retanning (e.g., if color may be lost during processing), thinning, retanning, lubricating, crusting, wetting, thumbing, shaving, re-pigmenting, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixing, setting, drying, conditioning, milling (e.g., dry milling), staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, and tumbling. In some embodiments, the lubricant can be a fat, biological oil, mineral oil, synthetic oil, cod oil, sulfonated oil, polymer, resin, organofunctional siloxane, or any combination thereof. In some embodiments, the lubricant can comprise a surfactant, anionic surfactant, cationic surfactant, cationic polymeric surfactant, anionic polymeric surfactant, amphiphilic polymer, fatty acid, modified fatty acid, nonionic hydrophilic polymer, nonionic hydrophobic polymer, polyacrylic acid, polymethacrylic, acrylic, natural rubber, synthetic rubber, resin, amphiphilic anionic polymer, amphiphilic anionic copolymer, amphiphilic cationic polymer, amphiphilic cationic copolymer, or combinations thereof. In some embodiments, the lubricant emulsion or suspension can be in water, alcohol, ketone, and other solvents.

[0177]

[0193] In some embodiments, synthetic leather can be shaped by controlling the number, size, and arrangement of the multicellular bodies and / or layers used to construct, for example, animal skin, hide, or leather. In other cases, the animal skin, hide, or leather can be shaped by, for example, cutting, pressing, molding, or stamping. The shape of the synthetic leather can be made to resemble traditional animal skin, hide, or leather products.

[0178]

[0194] In some embodiments, the methods herein can include removing a portion of the synthetic leather produced herein. In some embodiments, the methods can include removing at least a portion of the epidermis layer to form a removed product. For example, the removing can be shaving.

[0179]

[0195] In some embodiments, the methods herein can include coloring synthetic leather. In some embodiments, coloring can be achieved by introducing pigment-producing cells (e.g., melanocytes) into the synthetic leather. In some embodiments, the synthetic leather can include functional live melanocytes. The melanocytes can have a location similar to that in human skin. In some embodiments, melanin can be constitutively produced by melanocytes. In some embodiments, melanin can be transferred to keratinocytes. In some embodiments, melanocytes can be 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).

[0180]

[0196] In some embodiments, cells in different layers of synthetic leather, such as keratinocytes, melanocytes, or fibroblasts, can be derived from, e.g., differentiated from, precursor cells, such as stem cells. In other cases, primary cells or cultured cells derived from primary cells can be used to form cell layers for making synthetic leather. In some embodiments, modified cells containing molecular switches can be used to form cell layers for making synthetic leather. In some embodiments, cells can be differentiated by contacting them with a tissue formation medium.

[0181]

[0197] In some embodiments, 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 the synthetic leather, artificial epidermal equivalents, artificial full-thickness skin equivalents, and methods for making the same disclosed herein include reproducible, high-throughput, and easily scalable production of customized tissues with attractive appearance, texture, thickness, and durability. In some embodiments, the methods described herein can result in an increased yield of one or more of the epidermal layer, dermal layer, layered structure, or synthetic leather. In some embodiments, the increased yield 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 cost of producing synthetic leather, artificial epidermal layers, artificial dermal layers, layered structures, and products made therefrom. In some embodiments, the methods disclosed herein can produce synthetic leather, artificial epidermal layers, artificial dermal layers, layered structures, and products made therefrom with uniform thickness. In some embodiments, the synthetic leather, artificial epidermal layers, artificial dermal layers, layered structures, and products made therefrom can have substantially uniform thickness, length, and / or width. In some embodiments, cells in any one or more of the epidermal layer, dermal layer, and layered structure can be uniformly distributed. In some embodiments, cells in any one or more of the epidermal layer, dermal layer, and layered structure can be non-uniformly distributed. [Example]

[0182] Example 1. Development of immortalized bovine fibroblast cells.

[0198] In some embodiments, immortalized bovine fibroblast cell lines can provide a consistent, sustainable source of cells to support the demands of industrial-scale leather production. In some embodiments, most primary cells can have a finite number of cell doublings, known as the Hayflick number, which correlates with the shortening of chromosomes after each doubling. In some embodiments, once this number is reached, the cells tend to undergo death. In some embodiments, non-immortalized or primary cell lines can have the capacity to produce enough cells for at least 1 million square feet of leather per year. In some embodiments, more hides may need to be produced to meet market demand. In some embodiments, increased production can be achieved by immortalizing primary cells. In some embodiments, immortalizing primary cells can allow the cells to replicate indefinitely. In some embodiments, cell lines can be further engineered to improve yield and reduce costs. In some embodiments, cells can be grown in 2D. In some embodiments, 2D growth can include cells adhering to and growing on tissue culture plastic. In some embodiments, the cells can be modified to grow in a 3D environment, such as a suspension culture, to increase production efficiency. In some embodiments, the cells can be engineered to grow or differentiate without relying on expensive components such as growth factors and cell attachment factors.

[0183]

[0199] In some embodiments, the TERT and Bmil genes can be inserted into a plasmid vector to generate immortalized bovine fibroblasts. In some embodiments, the plasmid can be transfected into a fibroblast cell line. In some embodiments, the plasmid can contain gfp, so successful transfection can be determined using a fluorescent selectable marker. In some embodiments, selected fibroblast cell lines carrying the plasmid can be purified and expanded. In some embodiments, the fibroblast cell line can exhibit an immortalized phenotype, remain diploid, secrete normal extracellular matrix, and have controlled growth. In some embodiments, the immortalized cell line can be frozen for future use.

[0184]

[0200] Alternatively, immortalization can be achieved by expressing exogenous genes such as SV40 large T antigen (SV40-TAg), telomerase (TERT), BMI1, and CCND1, or by interfering with the function of cell cycle regulators such as CDK4, retinoblastoma protein (RB), and p53. SV40-TAg can be a nonstructural protein derived from Simian Virus 40 (SV40). Ectopic expression of SV40-TAg can result in cell transformation by blocking the function of RB and p53. RB and p53 can act as negative cell cycle regulators. RB can control DNA replication by inhibiting cell cycle progression from G1 to S phase. SV40-TAg can block the function of the RB protein, resulting in cell cycle progression. p53 can be involved in controlling growth cycle progression from G1 to S phase, apoptosis, or senescence. The interaction between SV40-TAg and p53 can interfere with p53 function, resulting in cell cycle progression and cell survival.

[0185]

[0201] In some embodiments, ectopic expression of SV40-TAg can immortalize primary cells from various species and cell types. In some embodiments, ectopic expression of SV40-TAg can immortalize primary cells from human embryonic fibroblasts, hamster dermal fibroblasts, bovine fetal liver cells, bovine peritoneal macrophages, bovine dental papilla cells, bovine embryonic lung cells, or any combination thereof. Disclosed herein are methods that include using SV40-TAg to immortalize other bovine primary cells, such as skin fibroblasts, preadipocytes, amniotic fluid cells, umbilical cord stem cells, and any combination thereof.

[0186]

[0202] In some embodiments, collagen may comprise the most abundant protein in bovine dermal tissue. In some embodiments, the dermis can be divided into papillary and reticular regions. In some embodiments, the papillary region can produce higher-quality leather, called grain leather. In some embodiments, the tissue can consist of densely packed collagen fibers (primarily types I and III) mixed with elastic fibers (elastin) and extrafibrillar fibers such as fibronectin embedded in a polysaccharide gel. In some embodiments, the tanning process can include a series of process steps to chemically crosslink the collagen while removing the polysaccharide gel or other tissue components. In some embodiments, the crosslinked collagen can then be further processed and dyed to create a leather-like texture. In some cases, expression of SV40-TAg can have an effect on collagen production. In some embodiments, transduction of SV40-TAg in the human lung fibroblast cell line WI38 can result in decreased collagen production. In some embodiments, transduction of SV40-TAg can have a limited effect on type I and type III collagen production in human ligament cells. In some embodiments, the role of SV40-mediated transformation on collagen production may be cell line dependent. In some embodiments, it may be important to demonstrate that cell lines generated by using SV40-TAg may be capable of producing collagen that leads to leather production.

[0187] Example 2. Stable bovine dermal fibroblast (BDF) cell line generation

[0203] In one embodiment, to generate stable bovine dermal fibroblast (BDF) cell lines, primary BDFs were transduced at passage 5 (P5) using a lentiviral vector to stably express the SV40-TAg-T2A-Puror fragment (Puror = puromycin resistance gene) under the CMV promoter. Green fluorescent protein (GFP) was expressed under another CMV promoter for visualization (Figure 3). After transduction, cells were cultured in 10% fetal bovine serum (FBS) medium (Table 1) for 3 days to allow transgene expression. To select cells carrying the SV40-TAg-T2A-Puror sequence, 0.5 μg / ml puromycin was added to the cell culture for 2 days, and then the puromycin concentration was increased to 0.75 μg / ml for an additional 7 days. BDF cells transduced with the SV40-TAg-T2A-Puror fragment survive under puromycin selection and are able to express GFP (Figures 4A, 4B, 4C, and 4D). The SV40-TAg-T2A-Puror-transduced cells that survived puromycin selection were subsequently designated VL-001 cell line and were grown at 6000 cells / cm. 2 The cells were repeatedly passaged at 100°C for 1 hour to test their proliferation potential.

[0188]

[0204]

[0189] [Table 1]

[0190]

[0205] Unmodified BDFs can be expanded in vitro for approximately 40 population doublings (PDs), with a mean PDT of 35.5 h. Primary BDFs were found to vary their PDT over multiple passages, with a minimum PDT of 21.6 h and a maximum PDT of 51.5 h from passage 0 to passage 11 (Figure 2A). In contrast, immortalized cell line (VL-001) cells were expanded for at least 20 passages after puromycin selection, reaching a cumulative PD of 100 at passage 25 (Figure 1B). This number does not indicate senescence; rather, this is the time when the expansion experiment was stopped. The mean PDT of the VL-001 cell line (21.2 h) was 40% shorter than that of unmodified primary BDFs. The VL-001 cell line also showed a more consistent PDT across successive passages. The VL-001 cell line has a minimum PDT of 19.1 hours and a maximum PDT of 23.7 hours from passage 6 to passage 25 (Figure 1A). Furthermore, no senescent cells were observed in the extensively passaged VL-001 cell line, whereas senescent morphology can be identified in later-passaged unmodified primary BDFs (Figure 1C - white arrows indicating senescent cells).

[0191] Collagen production in 2d culture

[0206] Having demonstrated that the cells could proliferate for more PDs than unmodified fibroblasts, further experiments focused on whether the immortalized VL-001 fibroblast cell line could produce collagen. It was demonstrated that VL-001 cells, when grown in 2D on tissue culture plastic, were able to secrete similar levels of collagen compared to unmodified primary BDNF (Figure 5). Supplementation of human platelet lysate (HPL)-based medium with TGFb1 and ascorbic acid 2-phosphate (AA2P) significantly increased collagen production in both VL-001 and unmodified BDNF, suggesting that VL-001 possesses a similar collagen production mechanism to unmodified BDNF (Figure 5).

[0192] Tissue generation and collagen production on 3D scaffolds

[0207] Experiments were conducted to determine whether the immortalized VL-001 fibroblast cell line could generate 3D tissue on porous polyester nonwoven scaffolds similar to unmodified primary BDF. In some cases, unmodified BDF could form consistent tissue on porous scaffolds in 4 weeks when cultured in HPL-based medium (Table 1). However, the VL-001 cell line was unable to do so; instead, the cells formed aggregates and did not spread on the polyester material when cultured in the same HPL-based medium (Figures 6A, 6B, 6C, 6D, 6E, and 6F). Following this observation, a second experiment was conducted to determine whether a similar media formulation (high-glucose Dulbecco's Modified Eagle's Medium - DMEM) used for 2D cell culture of VL-001 containing 10% fetal bovine serum could be used to form tissue. Therefore, we supported VL-001 tissue formation on polyester scaffolds using DMEM with 20% FBS (higher serum enriched for growth factors and cell attachment proteins) and 2X non-essential amino acids. By changing the culture medium from HPL medium to 20% FBS medium (Table 1), we found that VL-001 cells could form 3D tissue on the scaffolds. VL-001 cells, like unmodified BDF, were able to grow between support fibers (Figures 6A, 6B, 6C, 6D, 6E, and 6F). Figure 6A shows a tissue scan of BDF cells grown in HPL medium. Figure 6B shows a 10x magnification tissue scan of BDF cells grown in HPL medium. Figure 6C shows a tissue scan and image of VL-001 cells grown in HPL medium. Figure 6D shows a 10x magnification tissue scan of VL-001 cells grown in HPL medium. Figure 6E shows a tissue scan image of VL-001 cells grown in 20% FBS medium. Figure 6F shows a 10x magnification tissue scan image of VL-001 cells grown in 20% FBS medium. Furthermore, VL-001 cells cultured in 20% FBS medium can produce free collagen in the culture medium (Figure 7) and deposit collagen fibrils in tissues, similar to unmodified BDF cultured in HPL-based medium (Figure 8).

[0193] Suspension culture The VL-001 cell line rapidly proliferated under 2D culture conditions. Here, we demonstrated that the VL-001 cell line can grow in 3D suspension culture as aggregates. Cell aggregation still relied on cell / cell or cell / protein contact. VL-001 cells were seeded on ultra-low attachment plates at 1000 cells / cm2. After one week of suspension culture, obvious aggregate formation was observed. The number and size of the aggregates increased over three weeks (Figures 9A, 9B, 9C, and 9D).

[0194] Example 3. Development of cells with molecular switches for anchorage-independent and anchorage-dependent growth

[0209] To develop the molecular switch, integrin-linked kinase (Ilk) is inserted into a plasmid vector under the control of a temperature-sensitive promoter. The temperature-sensitive promoter allows expression of Ilk when cells are grown at 33°C and inhibits expression when cells are grown at 37°C to 39°C. The plasmid is transfected into bovine dermal fibroblasts. When Ilk is expressed, the bovine dermal fibroblasts exhibit an anchorage-independent growth phenotype. To support cell growth, the cells are grown under anchorage-independent growth conditions at a temperature of 33°C. Once the cells reach the desired density (e.g., 5e 7 Once the cell count reaches 100 cells / mL, the temperature is changed to 39°C to attenuate Ilk expression in the dermal fibroblasts. The temperature shift shifts the cells to anchorage-dependent growth, allowing them to adhere to the scaffold and begin to form tissue. The temperature is then lowered to 37°C to support normal cell growth while still reducing the expression of the transfected Ilk. The resulting tissue is used to create synthetic leather.

[0195] Example 4. Development of cells with a molecular switch for immortalization

[0210] To generate the molecular switch, the hTERT and SV40 genes are inserted into a plasmid vector under the control of the Cre-LoxP system. The Cre-LoxP system allows for the excision of the hTERT and SV40 genes when cells are grown under conditions in which the Cre protein is expressed. The plasmid is transfected into bovine dermal fibroblasts. Under non-inducing conditions, hTERT and SV40 are expressed, and the bovine dermal fibroblasts exhibit an immortalized cell phenotype. Under non-inducing conditions, the cells are grown under anchorage-independent growth conditions. Once the cells reach the desired density (e.g., 5e 7 Once the cell density reaches 1000 cells / mL, an inducer is added to express the Cre recombinase protein, which excises the hTERT and SV40 genes from the dermal fibroblasts. Excision of hTERT and SV40 allows the cells to adhere to the scaffold and begin to form tissue. The tissue produced is used to generate synthetic leather.

[0196] Example 5. Development of immortalized cells with molecular switches

[0211] To generate immortalized bovine fibroblasts, mutant versions of the TERT, CCND1, and CDK4 genes are inserted into a plasmid vector and transfected into bovine fibroblasts. The plasmid contains the neo gene (an antibiotic selectable marker) to determine successful transfection. Cells are exposed to geneticin and survivors are selected. Selected fibroblast cell lines carrying the plasmid are purified and expanded. The fibroblast cell lines exhibit an immortalized phenotype, remaining diploid, secreting normal extracellular matrix, and exhibiting controlled growth. Second, to create a molecular switch for growth of immortalized cells in suspension, the cyclin D1 gene is cloned with a Tet-inducible promoter. The construct is contained in an integrating viral vector and transfected into immortalized cells, where it is integrated into the chromosome. The immortalized cells containing the molecular switch are grown in culture medium containing tetracycline, which drives expression of the cyclin D1 gene and thus induces anchorage-independent growth. When the cells reach approximately 5 e 7After reaching a concentration of 1000 cells / mL, the cells are harvested and the medium is replaced with medium without tetracycline. In the absence of inducer, cyclin D1 expression is reduced and anchorage-dependent growth on the scaffold occurs. The cells begin to form the tissue used to produce leather.

[0197] Example 6. Preparation of epidermal layers from immortalized bovine fibroblasts carrying a molecular switch

[0212] Immortalized bovine fibroblasts carrying the molecular switch were cultured in 0.07 mM Ca medium supplemented with human keratinocyte growth supplement. 2+ A suspension of immortalized bovine fibroblasts (2.21 x 10 cells) is plated 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. 5 / cm 2 Inserts) are seeded.

[0198]

[0213] On day 3 (D3) after seeding, the medium is switched to CnT-02-3D (CELLnTEC) or CnT-3D Barrier (CELLnTEC). On day 4, the immortalized bovine fibroblasts are air-exposed by supplying CnT-02-3D or CnT-3D Barrier to the bottom of the insert. From day 4 onwards, the immortalized bovine fibroblasts are fed CnT-02-3D or CnT-3D Barrier daily until harvest. The immortalized bovine fibroblasts are grown in a humid (100% RH) or dry incubator (50% RH) 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 pan.

[0199]

[0214] To control for possible changes in osmolality, the medium is refreshed daily. No significant changes in osmolality, as measured by a Micro Osmometer (Precision Systems), are detected using this protocol. 12-well inserts are used for transepithelial electrical resistance (TEER) measurements, light microscopy, and electron microscopy, while 6-well inserts are used for transepidermal water loss (TEWL) measurements and immunoblotting.

[0200] Example 7. Cultivation of epidermal layers

[0215] Immortalized bovine fibroblasts carrying the molecular switch were inoculated at 2.0–2.5 × 10 cells per well onto 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 .

[0201]

[0216] On day 3 (D3) after seeding, the medium was switched to CnT-02-3D (CELLnTEC) or CnT-3D Barrier (CELLnTEC). On day 4, cells were air-exposed by feeding the bottom of the insert with CnT-02-3D or CnT-3D Barrier. From day 4 onwards, the epidermal layer was fed with CnT-02-3D or CnT-3D Barrier daily and harvested on day 14.

[0202] Example 8. Preparation of support substrate

[0217] To prepare a 2% agarose solution, dissolve 2 g of Ultrapure low melting point (LMP) agarose in 100 mL of ultrapure water / buffer solution (1:1, v / v). The buffer solution can optionally be PBS (Dulbecco's phosphate-buffered saline 1x) or HBSS (Hank's balanced salt solution 1x). The agarose solution can be placed in a beaker containing warm water (above 80°C) and kept on a hot plate until the agarose is completely dissolved. As long as the temperature is above 36°C, the agarose solution will remain liquid. Below 36°C, a phase transition occurs, the viscosity increases, and eventually the agarose forms a gel.

[0203]

[0218] To prepare the agarose support substrate, 10 mL of liquid 2% agarose (temperature >40°C) is deposited into a 10 cm diameter Petri dish and spread evenly to form a uniform layer. The agarose is allowed to form a gel at 4°C in a refrigerator.

[0204] Example 9. Fabrication of synthetic leather containing immortalized bovine fibroblasts, keratinocytes, and melanocytes carrying a molecular switch

[0219] The protocol may be outlined as follows: a) contacting a solution of immortalized bovine fibroblasts carrying the molecular switch with collagen, followed by incubation for a time sufficient for the immortalized bovine fibroblas...

Claims

[Claim 1] The invention described herein.

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