WO2014201406A1 - Engineered leather and methods of manufacture thereof - Google Patents

Abstract

Engineered animal skin, hide, and leather comprising a plurality of layers of collagen formed by cultured collagen-producing cells. Layers may be formed by cultured collagen-producing cells that are grown to confluence to form a sheet of collagen, the sheets are treated to prevent contractions, e.g., by decellularizing them with ethanol, and then the sheets are stacked and adhered together by seeding the region between the them with additional collagen-forming cells. Additional filler material may be added between the layers, such as reconstituted collagen or collagen pulp. Multiple layers may be stacked and adhered in this manner. Finally, the sheets may be treated to form leather, by modifying the collagen using a tanning (or a modified tanning) process.

Description

ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF

CROSS REFERENCE TO RELATED APPLICATION

[0001] This patent application claims priority to U.S. Provisional patent application no. 61/834,867 filed on 6/13/2013, and titled "ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF".

[0002] This patent application may be related to U.S. Patent Application No. 13/853,001, filed March 28, 2013 and titled "ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF," which is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0003] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BACKGROUND

[0004] Leather is used in a vast variety of applications, including furniture upholstery, clothing, shoes, luggage, handbag and accessories, and automotive applications. Currently, skins of animals are used as raw materials for natural leather. However, skins from livestock pose environmental concerns because raising livestock requires enormous amounts of feed, pastureland, water, and fossil fuel. Livestock also produces significant pollution for the air and waterways.

[0005] In addition, use of animal skins to produce leather is objectionable to socially conscious individuals. The global leather industry slaughters more than a billion animals per year. Most of the leather comes from countries with no animal welfare laws or have laws that go largely or completely unenforced. Leather produced without killing animals would have tremendous fashion novelty and appeal.

[0006] Although synthetic leather was developed to address some of these concerns, it lacks the quality, durability, and prestige of natural leather. Thus far, scientifically sound and industrially feasible processes have not been developed to produce natural leather. Accordingly, there is a need for a solution to demands for alternative to leather produced from live animals.

[0007] Natural leather is typically a durable and flexible material created by the tanning of animal rawhide and skin, often cattle hide. Tanning is generally understood to be the process of treating the skins of animals to produce leather. Tanning may be performed in any number of well-understood ways, including vegetable tanning (e.g., using tannin), chrome tanning (chromium salts including chromium sulfate), aldehyde tanning (using glutaraldehyde or oxazolidine compounds), syntans (synthetic tannins, using aromatic polymers), and the like.

[0008] Natural leather is typically prepared in three main parts: preparatory stages, tanning, and crusting. Surface coating may also be included. The preparatory stages prepare the hide/skin for tanning, and unwanted raw skin components are removed. The preparatory stages may include: preservation, soaking (rehydrating), liming, de-hairing, de-fleshing (removing subcutaneous material), splitting, re-liming, deliming (to remove de-hairing and liming chemicals), bating (protein proteolysis), degreasing, frizzing, bleaching, pickling (changing pH), de-pickling, etc.

[0009] Tanning is performed to convert proteins in the hide/skin into a stable material that will not putrefy, while allowing the material to remain flexible. Chromium is the most commonly used tanning material. The pH of the skin/hide may be adjusted (e.g., lowered, e.g. to pH 2.8-3.2) to enhance the tanning; following tanning the pH may be raised ("basification" to a slightly higher level, e.g., pH 3.8-4.2).

[00010] Crusting refers to the post-tanning treatment that may include coloring (dying), thinning, drying or hydrating, and the like. Examples of crusting techniques include: wetting (rehydrating), sammying (drying), splitting (into thinner layers), shaving, neutralization (adjusting pH to more neutral level), retanning, dyeing, fatliquoring, filling, stuffing, stripping, whitening, fixation of unbound chemicals, setting, conditioning, softening, buffing, etc.

[00011] In practice, the process of converting animal skin into leather may include sequential steps such as: unhairing/dehairing, liming, deliming and bateing, pickling, tanning,

neutralizing/Dyeing and Fat liquoring, drying and finishing. The dehairing process may chemically remove the hair (e.g., using an alkali solution), while the liming step (e.g., using an alkali and sulfide solution) may further complete the hair removal process and swell ("open up") the collagen. During tanning, the skin structure may be stabilized in the "open" form by replacing some of the collagen with complex ions of chromium. Depending on the compounds used, the color and texture of the leather may change. Tanned leather may be much more flexible than an untreated hide, and also more durable.

[00012] Skin, or animal hide, is formed primarily of collagen, a fibrous protein. Collagen is a generic term for a family of at least 28 distinct collagen types; animal skin is typically type 1 collagen (so the term collagen is typically assumed to be type 1 collagen), although other types of collagen may be used in forming leather. Collagens are characterized by a repeating triplet of amino acids, -(Gly-X-Y)„-, so that approximately one-third of the amino acid residues are in collagen are glycine. X is often proline and Y is often hydroxyproline. Thus, the structure of collagen may consist of twined triple units of peptide chains of differing lengths. Different animals may produce different amino acid compositions of the collagen, which may result in different properties (and differences in the resulting leather). Collagen fiber monomers may be produced from alpha-chains of about 1050 amino acids long, so that the triple helix takes the form of a rod of about 300 nm long, with a diameter of 1.5 nm. In the production of extracellular matrix by fibroblast skin cells, triple helix monomers may be synthesized and the monomers may self-assemble into a fibrous form. These triple helices may be held together by salt links, hydrogen bonding, hydrophobic bonding, and covalent bonding. Triple helices can be bound together in bundles called fibrils, and fibril bundles come together to create fibers. Fibers typically divide and join with each other throughout a layer of skin. Variations of the crosslinking or linking may provide strength to the material. Fibers may have a range of diameters. In addition to type I collagen, skin (hides) may include other types of collagen as well, including type III collagen (reticulin), type IV collagen, and type VII collagen.

[00013] Previous attempts to make engineered leathers have proven unsuccessful or impractical. For example, EP1589098 describes a method of growing fibroblasts seeded onto three-dimensional bioactive scaffolds. The scaffolds may be made from collagen waste material from a tanning process ("split"), microparticles of pure collagen, particle of collagen waste material, or synthetic scaffolds (e.g., made of polymers such as HYAFF). The addition of the scaffold material complicates and increases the expense of their proposed process, and also affects the properties of an leather produced this way.

[00014] Described herein are engineered leathers that replicate much of the structures and properties of natural leathers, but may be processed in a much simpler manner, and may address many of the problems of natural and previously-described engineered leathers including those identified above. SUMMARY OF THE DISCLOSURE

[00015] Disclosed herein are engineered animal skin, hide, and leather, and methods of producing the same. In certain embodiments, disclosed herein is an engineered animal skin, hide, or leather comprising a plurality of layers of extracellular matrix, ECM, (e.g., collagen) formed from cultured cells.

[00016] For example, the cultured cells (with and without ECM) may be animal cells cultured in vitro. In certain embodiments, disclosed herein is an engineered animal skin, hide, or leather formed of a plurality of layers of animal cells comprising one or more types of collagen-releasing cells. Any collagen-releasing cell may be used, including skin cells. In certain embodiments, the collagen-releasing cells provided herein are non-human cells. It should be understood that although skin cells are described and illustrated herein, any collagen-producing cell (e.g., cell that can produce or be induced to produce collagen ECM) may be used with any of the methods described herein to produce the engineered leather described. Collagen may refer to collagen or to ECM generally, including ECM with collagen of one or more types. In particular, collagen-releasing may be smooth muscle cells (SMCs). Thus, collagen ECM producing cells may include muscle cells (including smooth muscle cells) and the like.

[00017] For example, described herein are methods of making a engineered leather, which may also be referred to as cultured leather, or synthetic leather. The methods may include: culturing a first group of collagen-releasing cells to form a plurality of sheets of collagen; treating the plurality of sheets to prevent contractions thereby forming a plurality of non-contractile sheets; placing a second group of collagen-releasing cells on a first sheet from the plurality of non-contractile sheets; placing a second sheet from the plurality of non-contractile sheets on top of the first sheet to form a first stack; and culturing the first stack until the first sheet and second sheet are adherent.

[00018] In some variations, the method further comprises processing the first stack to modify the collagen. The method may include processing the first stack by tanning to modify the collagen.

[00019] In some variations, placing the second sheet comprises rolling the second sheet onto a mandrel and unrolling the second sheet onto the first sheet.

[00020] In some variations, culturing the first group of collagen-releasing cells comprises culturing the cells to confluence. In some variations, the first collagen-releasing cells comprise smooth muscle cells. The first and second groups of collagen-releasing cells may comprise the same type of cells.

[00021] In some variations, treating comprises de-cellularizing the plurality of sheets to kill the collagen-releasing cells. Treating may comprise treating the plurality of sheets with ethanol to kill the collagen-releasing cells.

[00022] In some variations, culturing comprises growing the first group of collagen-releasing cells in culture without a scaffold.

[00023] In some variations, placing the second group of collagen-releasing cells comprises seeding the second group of collagen-releasing cells on the first sheet before placing the second sheet on the first sheet.

[00024] The method may also include adding a filler material between the first and second sheets before placing the second sheet on the first sheet.

[00025] In some variations, placing the second group of collagen-releasing cells comprises adding a filler material comprising the second group of collagen-releasing cells between the first and second sheets before placing the second sheet on the first sheet.

[00026] In some variations, the method further comprises adding a collagen filler material between the first and second sheets before placing the second sheet on the first sheet.

[00027] The method may further comprise adding a filler material comprising a reconstituted collagen between the first and second sheets before placing the second sheet on the first sheet.

[00028] In some variations, the method includes adding a filler material comprising a collagen pulp between the first and second sheets before placing the second sheet on the first sheet.

[00029] In some variations, the method includes increasing the height of the first stack by placing additional collagen-releasing cells on the first stack and then placing an additional sheet from the plurality of non-contractile sheets or an additional stack comprising adherent sheets from the plurality of non-contractile sheets onto the first stack, and culturing the first stack and additional sheet or additional stack until the first stack and additional sheet or additional stack are adherent.

[00030] The method may include sequentially increasing the height of first stack by repeating the steps of placing additional collagen-releasing cells on the first stack, placing an additional sheet from the plurality of non-contractile sheets or an additional stack comprising adherent sheets from the plurality of non-contractile sheets onto the first stack, and culturing the first stack and additional sheet or additional stack until the first stack and additional sheet or additional stack are adherent.

[00031] The method may include comprising placing additional collagen-releasing cells on the second sheet and placing an additional sheet from the plurality of non-contractile sheets onto the second sheet to increase the height of the first stack.

[00032] Also described herein are methods of making a synthetic leather, the method comprising: culturing collagen-releasing cells to confluency to form a plurality of sheets of collagen; decellularizing the plurality of sheets to prevent contractions; seeding collagen-releasing cells on a first sheet from the plurality of decellularized sheets; placing a second sheet from the plurality of decellularized sheets on top of the first sheet to form a stack; and culturing the stack until the first sheet and second sheet are adherent.

[00033] Also described herein are methods of making a synthetic leather, the method comprising: growing collagen-releasing cells in culture without a scaffold to form a plurality of sheets of collagen; decellularizing the plurality of sheets; forming a stack by placing a second sheet from the plurality of decellularized sheets on top of a first sheet from the plurality of decellularized sheets with collagen-releasing cells between the first and second sheets; and culturing the stack until the first sheet and second sheet are adherent.

[00034] The engineered leather described herein, e.g., using a processes such as those descried herein, may be grossly similar (if not identical) to natural leathers. However, these engineered leathers may include numerous differences rising from the method of formation, using cultured cells. For example. The sheets of extracellular matrix formed and stacked (and completely or partially adhered and/or fused) as described herein may be formed of the cultured skin cells so that each layer is substantially homogenous within the layer. Unlike natural leathers, the engineered leathers described herein may be completely free of muscle (e.g., papillary muscle), hair and hair follicles, blood vessels, and the like, as the material forming the leather is grown from cultured cells. During the formation process, the engineered leather may be formed to precise dimensions, including thickness, and without the need to prepare the material as is necessary with natural hides, including liming, de-hairing, splitting, fleshing, etc.

[00035] The collagen-producing cells may comprise epithelial cells, fibroblasts, keratinocytes, comeocytes, melanocytes, Langerhans cells, basal cells, or a combination thereof. The epithelial cells may comprise squamous cells, cuboidal cells, columnar cells, basal cells, or a combination thereof. The fibroblasts may be dermal fibroblasts. The keratinocytes may be epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or a combination thereof. The engineered leather of claim 1 , further comprising an extra-cellular matrix or connective tissue.

[00036] In some variations, the engineered leather further comprises one or more components selected from the group consisting of keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, and a combination thereof.

[00037] The animal cells may be derived from mammals selected from the group consisting of antelope, bear, beaver, bison, boar, camel, caribou, cat, cattle, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, oxen, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, and zebra, and a combination thereof. The animal cells may be derived from reptiles selected from the group consisting of turtle, snake, crocodile, and alligator, or combinations thereof. The animal cells may be derived from birds selected from the group consisting of chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, and turkey, or combinations thereof. The animal cells may be derived from fish selected from the group consisting of anchovy, bass, catfish, carp, cod, eel, flounder, fugu, grouper, haddock, halibut, herring, mackerel, mahi mahi, manta ray, marlin, orange roughy, perch, pike, pollock, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, and walleye, or combinations thereof.

[00038] In general, the engineered leather may be formed without the need for a structural scaffold.

[00039] At least one of the layers of the engineered leather may comprise a ratio of animal fibroblasts to animal keratinocytes between about 20: 1 to about 3: 1. The engineered leather layers may be substantially free of non-differentiated keratinocytes, fibroblasts, or epithelial cells.

[00040] In general, the engineered leather may be patterned. For example, the engineered leather may be patterned after a skin pattern of an animal selected from antelope, bear, beaver, bison, boar, camel, caribou, cat, cattle, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, oxen, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, zebra, turtle, snake, crocodile, alligator, dinosaur, frog, toad, salamander, newt, chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, turkey, anchovy, bass, catfish, carp, cod, eel, flounder, fugu, grouper, haddock, halibut, herring, mackerel, mahi mahi, manta ray, marlin, orange roughy, perch, pike, pollock, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, walleye, and a combination thereof. The pattern may be a skin pattern of a fantasy animal selected from 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 a combination thereof.

[00041] Any appropriate number of layers may be used, and may be selected based on the desired thickness. As the sheets are formed and layered atop one another, in some variations, the cells in one or the layers may be killed or allowed to die. For example cells that are in a sheet that is already layered in the body may be allowed to die (e.g., for lack of nutrients) or more preferably, it may be specifically treated to kill/remove the cells to prevent contraction. Additional cells may be seeded between the layers to adhere them. For example, an engineered leather may include a plurality of layers, e.g., comprising about 2 to about 50 layers, 2 to about 40 layers, 2 to about 30 layers, etc.

[00042] Any of the engineered leather described herein may be colored, e.g., comprising one or more colorants or pigments, or patterned.

[00043] Also described herein are methods of producing engineered leather. In general, these methods allow the production of engineered leather to any desired thickness from cultured collagen-producing (e.g., skin) cells, eliminating the need for some of the more resource-intensive and polluting steps associated with traditional leather making, including, e.g., de-hairing, soaking, fleshing, liming/deliming, splitting, and bleaching.

[00044] For example in some variations the method includes: (1) culturing one or more types of skin cells in vitro without a scaffold to form a plurality of collagen sheets from one more types of collagen-releasing cells; (2) treating the cells to prevent contraction; (3) combining non-contractile layers with additional collagen-releasing cells and/or filler; (4) allowing the layers to adhere, then repeating the process of steps (2) (and in some variations) and (3) as necessary to form a body having a desired volume; and processing the body by tanning. [00045] In any of the method of forming the engineered leather described herein the method may include one or more processing steps in addition to the tanning step. The tanning step may be performed in any appropriate manner, such as chrome tanning (using chromium salt). The method may further include processing the layered body using one or more additional processing steps. Additional processing steps may include: preserving, soaking, bating, pickling, depickling, thinning, retanning, lubricating, crusting, wetting, sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixating, setting, drying, conditioning, milling, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, and tumbling.

[00046] The method of forming engineered leather may use any appropriate cell(s), including skin and muscle (e.g., smooth muscle) cells. For example, the skin cells may comprise epithelial cells, fibroblasts, keratinocytes, corneocytes, melanocytes, Langerhans cells, basal cells, or a combination thereof. The epithelial cells may comprise squamous cells, cuboidal cells, columnar cells, basal cells, or a combination thereof. The fibroblasts may be dermal fibroblasts. The keratinocytes may be epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or a combination thereof.

[00047] In some variations, the step of forming the plurality of sheets comprises forming a plurality of sheets of the one or more types of skin cells and extracellular matrix material including collagen and one or more components selected from the group consisting of: keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, and a combination thereof.

[00048] The collagen-producing cells may be derived from mammals selected from the group consisting of antelope, bear, beaver, bison, boar, camel, caribou, cat, cattle, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, oxen, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, and zebra, and a combination thereof. The animal skin cells may be derived from reptiles selected from the group consisting of turtle, snake, crocodile, and alligator, or combinations thereof. The animal skin cells are derived from birds selected from the group consisting of chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, and turkey, or combinations thereof. The animal skin cells are derived from fish selected from the group consisting of anchovy, bass, catfish, carp, cod, eel, flounder, fugu, grouper, haddock, halibut, herring, mackerel, mahi mahi, manta ray, marlin, orange roughy, perch, pike, pollock, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, and walleye, or combinations thereof.

[00049] As mentioned, the engineered leather described herein may be patterned. For example, the method may include aligning the skin cells to form a pattern. [00050] Similarly, any appropriate number of sheets/layers may be selected, which may determine the thickness of the engineered lather. For example, the engineered leather may comprise about 2 to about 50 layers, about 2 to about 40 layers, about 2 to about 30 layers, etc.

[00051] Any of the methods described herein may also include coloring or dying the engineered leather. For example, the method may include dying the layered body using one or more colorants or pigments.

BRIEF DESCRIPTION OF THE DRAWINGS

[00052] FIG. 1 shows an overview of the method of making engineered leather as discussed herein. In this example, to grow leather, cells are taken from an animal through a simple biopsy, and cells are then isolated and multiplied in a cell culture medium. The cells are then allowed (and/or induced) to produce collagen, as they would naturally after the cells are spread out so that the collagen forms sheets. The sheets are then treated to prevent contraction, and the thin non-contracting (e.g., decellularized) sheets are stacked on top of one another after seeding with additional collagen-forming cells, like filo pastry, to form thicker sheets which are allowed to adhere by the action of the collagen-releasing cells. Finally, leather is formed from this multilayered structure through a shortened tanning process to modify the collagen.

[00053] FIG. 2A-C illustrate one example of forming a stack of sheets of collagen formed as described to create an engineered leather.

[00054] FIG. 3 illustrates the forming of a filler material of collagen pulp.

[00055] FIG. 4 illustrates one variation of a method of forming an engineered leather using non-contracting sheets of collagen and filler material.

[00056] FIG. 5 illustrates another variation of a method of forming an engineered leather using non-contracting sheets of collagen and filler material. Decellularized collagen sheets may be prepared and covered with live collagen-secreting cells (e.g., fibroblasts). Pelleted collagen (shown as two discs of 4.7cm diameter) is placed on a decellularized sheet. Subsequently, the construct is covered with another decellularized collagen sheet. Cells on the inner surface of the decellularized sheets may connect the sheets (along the white areas) and cells may attach more strongly to the decellularized sheets than to the pelleted collagen discs. The construct may then be fold on itself, resulting in a layered structure. There are two decellularized layers between the pelleted collagen discs in this example.

[00057] FIGS. 6A and 6B illustrate another variation of a method of forming an engineered leather using non-contracting sheets of collagen and filler material.

[00058] FIGS. 7A and 7B illustrate the operation of a mandrel device for forming engineered leather. [00059] FIG. 8 is a photograph of a sample of engineered leather formed by a method such as that shown in FIG. 5.

DETAILED DESCRIPTION

[00060] Tissue engineering technology offers new opportunities to produce animal skin, hide, or leather that are not associated with the environmental degradation of raising livestock. Tissue engineering has been defined as an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Langer R, Vacanti JP, Tissue Engineering, Science 260(51 10):920-926 (May 1993).

[00061] Tissue engineered products made using traditional materials and methods are limited in size due to the short distances gases and nutrients can diffuse to nourish interior cells. Also, existing techniques fail to provide adequate speed and throughput for mass production of engineered products. As a result, existing tissue engineering methods result in unappealing thin sheets and pastes on a commercially infeasible scale.

[00062] Thus, an objective of the animal skin, hide, or leather, and methods of making the same disclosed herein is to provide commercially viable and appealing animal skin, hide, or leather. Another objective is to provide high-throughput methods that reliably, accurately, and reproducibly scale up to commercial levels. Advantages of the animal skin, hide, or leather, and methods of making the same disclosed herein include, but are not limited to, production of customized tissues in a reproducible, high throughput and easily scalable fashion while keeping precise control of pattern formation, particularly in cases of multiple cell types, which may result in engineered animal skin, hide, or leather with appealing appearance, texture, thickness, and durability.

[00063] Disclosed herein are engineered animal hide and leather, and methods of producing the same. In certain embodiments, disclosed herein is engineered animal skin, hide, or leather comprising a plurality of layers of animal cells comprising one or more types of skin cells, wherein said animal cells are cultured in vitro. In certain embodiments, each layer of animal cells provided herein is biofabricated. The sheets/layer of animal cells provided herein are typically grown without a structural scaffold.

[00064] In general, the methods described herein may include first forming sheets of collagen by growing (culturing) collagen-releasing cells and allowing (or stimulating) them to secrete collagen. In culture the cells may be grown to confluence on a substrate such as the bottom of a cell culture dish, flask, roller, chamber (e.g., rotating chamber) or the like. Cells may be derived from tissue extracts/explants, immortalized cell lines, or manipulated (transgenic) cell lines, or any variation thereof. In some variations, the cell may be grown to complete confluence (e.g., 100% confluence), in which cells are inhibited from further growth but may continue to produce or be stimulated to produce and release collagen. In some variations, the cells may not be grown to complete confluence, (e.g., approximately 99% confluence, 95 % confluence, 90% confluence, 85% confluence, 80% confluence, etc.). Cells may be cultured until greater than 80% confluence, greater than 85% confluence, greater than 90% confluence, greater than 95% confluence and/or just under full (100%) confluence.

[00065] The sheets of collagen may be treated to prevent contractions. Cultured cells, including collagen-releasing cells, may begin contracting during culture. The cultured cells forming the multiple sheets of collagen may be treated while growing, and/or towards the end of the growth. Typically, the sheets of collagen are treated before contraction begins, or before contraction above a threshold occurs. In general, it is desirable to prevent contractions because the contracting cells in a sheet of collagen may cause the sheet to deform, which may be undesirable when forming an engineered leather.

[00066] In some variations, treatment of the sheets of collagen or treatment of the cells forming the sheets of collagen) may include killing the cells. Thus the sheets of collagen including the cultured cells may be treated by decellularlizing (e.g., removing or killing) the collagen-releasing cells. In some variations, this may include treating the collagen sheets with ethanol. After treatment, the resulting sheets of collagen may be referred to as "non-contractile" sheets or "decellularized" sheets (for sheets treated to kill and/or remove the collagen-releasing cells).

[00067] After treating the sheets to form non-contractile sheets, the sheets may be washed, (e.g., rinsed, or the like) to remove the material used to treat the cells. Other additional treatments may also be performed. Thereafter, an engineered leather of a desired thickness may be formed using the non-contractile sheets. For example, the sheets may be stacked either sequentially or simultaneously, and adhered together. Adhesion may be achieved by again seeding the sheets (e.g., one surface of the sheet) so that collagen-releasing cells can communicate with one or both sides of the non-contractile sheets. This second group of cells may be the same cells used to grow the collagen sheets, or they may be different types of cells (though also collagen-releasing), or different distributions of cell types where multiple types of cells are used to grow the collagen sheets.

[00068] The sheets along with the second group of collagen-releasing cells may then be allowed to grow (e.g., cultured) until sufficient adhesion is achieved between the two sheets. Sufficient adhesion may be determined by time in culture (e.g., about: 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc.), or it may be empirically determined. For example the "stack" of sheets (two sheets being adhered together by the seeded second group of collagen-releasing cells) may be allowed to grow for more than about 4 days, more than about 5 days, more than about 6 days, more than about 7 days, more than about 8 days; and/or less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about

5 days, etc., expressly including between about 2-9 days, between about 4-7 days, about 5 days, etc.

[00069] In some variations a filler material may be inserted between the sheets before culturing them to allow them to adhere. Any appropriate filler material, but particularly collagenous filler material may be used. The filler material may be useful to increase the thickness of the stack forming the engineered leather, and/or reducing the number of collagen layers used to form an engineered leather. Example of filler materials include reconstituted collagen, and/or collagen pulp (e.g., mechanically sheered collagen). The reconstituted collagen may be depolymerized (e.g., collagen monomers or small polymers). Filler material may be added to a desired thickness. In some variations the filler material may be seeded with cells from the second group of collagen-releasing cells, in addition or alternatively to the collagen-releasing cells seeded onto one or both layers to be stacked onto each other around the filler material.

[00070] When adding additional sheets of collagen to a stack (e.g., non-contractile sheets of collagen) forming an artificial leather, additional sheets may be added either sequentially or in parallel, or a combination of both, in which a small number (e.g. less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, 2, etc.) of sheets may be stacked atop a first of non-contractile sheet of collagen and the sheets may be allowed to adhere by culturing the second group of collagen-releasing cells placed between the two or more sheets (with or without filler material). In this manner, a "stack" of collagen sheets that have been adhered may be formed. There after the dimensions (e.g., thickness) of the stack can be increased by combining two or more stacks and allowing them to adhere by culturing one (or more) atop the other with or without filler material after seeding them with some of the second group of collagen-releasing cells.

Cells

[00071] Many cell types may be used for the collagen-releasing cells used to produce the sheets/layers of collagen, and thus the engineered skin, hide, and leather products described herein. The collagen-releasing cells may be homogenous (e.g., of the type of cell) or they may be a mixture of collagen-releasing cells and/or cells that release other ECM materials, and/or cells that do not release collagen. In some embodiments, the engineered animal skin, hide, and leather products are designed to resemble traditional animal skin, hide, and leather products and the cell types are chosen to approximate those found in traditional animal skin, hide, and leather products. In further embodiments, the engineered animal skin, hide, and leather products, and sheet/layers include animal epidermis, basement membrane, dermis, hypodermis, scale, scute, osteoderm, or a combination thereof. In some embodiments, animal epidermis provided herein comprises stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, stratum germinativum, stratum basale, or a combination thereof. In some embodiments, animal dermis provided herein comprises stratum papillare, stratum reticulare, or a combination thereof. In some embodiments, animal scale provided herein comprises placoid scale, cosmoid scale, ganoid scale, elasmoid scale, cycloid scale, ctenoid scale, crenate scale, spinoid scale, or a combination thereof.

[00072] In certain embodiments, animal cells provided herein comprise epithelial cells, fibroblasts, keratinocytes, comeocytes, melanocytes, Langerhans cells, basal cells, or a combination thereof. In some embodiments, epithelial cells provided herein comprise squamous cells, cuboidal cells, columnar cells, basal cells, or a combination thereof. In some embodiments, fibroblasts provided herein are dermal fibroblasts. In some embodiments, keratinocytes provided herein are epithelial keratinocytes, basal keratinocytes, proliferating basal keratinocytes, differentiated suprabasal keratinocytes, or a combination thereof.

[00073] In certain embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is between about 20: 1 to about 3: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is between about 20: 1 to about 4: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is between about 20: 1 to about 5 : 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is between about 20: 1 to about 10: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is between about 20: 1 to about 15: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 25: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 24: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 23: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 22: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 21 : 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 20: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 19: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 18: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 17: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 16: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 15: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 14: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 13: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 12: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 1 1 : 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 10: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 9: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 8: 1.

In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 7: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 6: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 5: 1. In some embodiments, the ratio of animal fibroblasts to animal keratmocytes provided herein is about 4: 1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 3 : 1. In some embodiments, the ratio of animal fibroblasts to animal keratinocytes provided herein is about 2: 1.

[00074] In certain embodiments, animal cells provided herein are substantially free of non-differentiated keratinocytes, fibroblasts, or epithelial cells.

[00075] In other embodiments, the engineered animal skin, hide, or leather products include neural cells, connective tissue (including bone, cartilage, cells differentiating into bone forming cells and chondrocytes, and lymph tissues), epithelial cells (including endothelial cells that form linings in cavities and vessels or channels, exocrine secretory epithelial cells, epithelial absorptive cells, keratinizing epithelial cells, and extracellular matrix secretion cells), and undifferentiated cells (such as embryonic cells, stem cells, and other precursor cells), among others.

[00076] In certain embodiments, engineered animal skin, hide, or leather further comprises an extracellular matrix or connective tissue. In certain embodiments, engineered animal skin, hide, or leather further comprises one or more components selected from the group consisting of collagen, keratin, elastin, gelatin, proteoglycan, dermatan sulfate proteoglycan, glycosoaminoglycan, fibronectin, laminin, dermatopontin, lipid, fatty acid, carbohydrate, and a combination thereof.

[00077] In some embodiments, the cells are obtained from commercial sources. In certain embodiments, the cells are derived from, by way of non-limiting examples, mammals, birds, reptiles, fish, crustaceans, moliusks, cephalopods, insects, non-arthropod invertebrates, and combinations thereof. In some embodiments, the animal cells human cells. In certain

embodiments, the animal cells provided herein are non-human cells. In some embodiments, suitable cells are derived from mammals such as antelope, bear, beaver, bison, boar, camel, caribou, cat, cattle, deer, dog, elephant, elk, fox, giraffe, goat, hare, horse, ibex, kangaroo, lion, llama, lynx, mink, moose, oxen, peccary, pig, rabbit, seal, sheep, squirrel, tiger, whale, wolf, yak, and zebra, or combinations thereof. In some embodiments, suitable cells are derived from birds such as chicken, duck, emu, goose, grouse, ostrich, pheasant, pigeon, quail, and turkey, or combinations thereof.

[00078] In some embodiments, suitable cells are derived from reptiles such as turtle, snake, crocodile, and alligator, or combinations thereof.

[00079] In some embodiments, suitable cells are derived from fish such as anchovy, bass, catfish, carp, cod, eel, flounder, fugu, grouper, haddock, halibut, herring, mackerel, mahi mahi, manta ray, marlin, orange roughy, perch, pike, salmon, sardine, shark, snapper, sole, stingray, swordfish, tilapia, trout, tuna, and walleye, or combinations thereof.

[00080] In some embodiments, suitable cells are derived from amphibians such as frog, toad, salamander, newt, or combinations thereof.

[00081] In some embodiments, suitable cells are derived from crustaceans such as crab, crayfish, lobster, prawn, and shrimp, or combinations thereof.

[00082] In some embodiments, suitable cells are derived from mollusks such as abalone, clam, conch, mussel, oyster, scallop, and snail, or combinations thereof.

[00083] In some embodiments, suitable cells are derived from cephalopods such as cuttlefish, octopus, and squid, or combinations thereof.

[00084] In some embodiments, suitable cells are derived from insects such as ants, bees, beetles, butterflies, cockroaches, crickets, damselflies, dragonflies, earwigs, fleas, flies, grasshoppers, mantids, mayflies, moths, silverfish, termites, wasps, or combinations thereof.

[00085] In some embodiments, suitable cells are derived from non-arthropod invertebrates (e.g., worms) such as flatworms, tapeworms, flukes, threadworms, roundworms, hookworms, segmented worms (e.g., earthworms, bristle worms, etc.), or combinations thereof.

Additives

[00086] In some embodiments, the engineered animal skin, hide, or leather products, include one or more additives. In further embodiments, one or more additives are selected from: minerals, fiber, fatty acids, and amino acids. In some embodiments, the engineered animal skin, hide, or leather products, sheet/layers include one or more additives to enhance the commercial appeal (e.g., appearance, color, odor, etc.). In further embodiments, the engineered skin, hide, and leather products, sheet/layers include one or more colorants, and/or one or more odorants.

[00087] In some embodiments, the engineered animal skin, hide, or leather products, engineered sheet/layers include one or more of: matrix proteins, proteoglycans, antioxidants, perfluorocarbons, and growth factors. The term "growth factor," as used herein, refers to a protein, a polypeptide, or a complex of polypeptides, including cytokines, that are produced by a cell and which can affect itself and/or a variety of other neighboring or distant cells. Typically growth factors affect the growth and/or differentiation of specific types of cells, either developmentally or in response to a multitude of physiological or environmental stimuli. Some, but not all, growth factors are hormones. Exemplary growth factors are insulin, insulin-like growth factor (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), fibroblast growth factors (FGFs), including basic FGF (bFGF), platelet-derived growth factors (PDGFs), including PDGF-AA and PDGF-AB, hepatocyte growth factor (HGF), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-P), including TGFpi and TGFP3, epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), IL-8, and the like.

[00088] In some embodiments, the engineered animal skin, hide, or leather products, engineered sheet/layers include one or more preservatives known to the art. In some embodiments, the preservatives are antimicrobial preservatives including, by way of non-limiting examples, calcium propionate, sodium nitrate, sodium nitrite, sulfites (e.g., sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.) and disodium ethylenediammetetraacetic acid (EDTA). In some embodiments, the preservatives are antioxidant preservatives including, by way of non-limiting examples, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

Engineered animal skin, hide, and leather

[00089] Disclosed herein, in some embodiments, is engineered animal skin, hide, or leather products. In some embodiments, the engineered animal skin, hide, or leather products are further processed by any known methods in the art. Examples of known methods of processing include processing by preserving, soaking, liming, unhairing, fleshing, splitting, deliming, reliming, bating, degreasing, frizzing, bleaching, pickling, depickling, tanning, thinning, retanning, lubricating, crusting, wetting, sammying, shaving, rechroming, neutralizing, dyeing, fatliquoring, filling, stripping, stuffing, whitening, fixating, setting, drying, conditioning, milling, staking, buffing, finishing, oiling, brushing, padding, impregnating, spraying, roller coating, curtain coating, polishing, plating, embossing, ironing, glazing, and tumbling. It is significant that the methods described herein do not require any of the pre-processing steps that are necessary when using natural animal hide, including de-hairing (unhairing), liming, fleshing, splitting deliming, reliming, etc. The layered bodies formed as described herein may be formed of any appropriate length, and the collagen (and other ECM molecules) formed by the cultured cells, resulting in a layered body that does not include structures such as hair follicles, blood vessels, muscle (e.g., arrector pili muscle), etc.

[00090] In general, engineered leather described herein may be tanned (or processed by a similar process) to modify the extracellular matrix material. As discussed above, one of the principle components of the ECM is collagen (and particularly Type I collagen). Tanning may modify the collagen. For example, one tanning agent, chromium(III) sulfate

([Cr(H20)6]2(S04)3), has long been regarded as the most efficient and effective tanning agent. Chromium(III) sulfate dissolves to give the hexaaquachromium(III) cation, [Cr(H20)6]3+, which at higher pH undergoes processes called olation to give polychromium(III) compounds that are active in tanning, being the cross-linking of the collagen subunits. Some ligands include the sulfate anion, the collagen's carboxyl groups, amine groups from the side chains of the amino acids, as well as masking agents. Masking agents are carboxylic acids, such as acetic acid, used to suppress formation of polychromium(III) chains. Masking agents allow the tanner to further increase the pH to increase collagen's reactivity without inhibiting the penetration of the chromium(III) complexes. Collagen's high content of hydroxyproline allows for significant cross-linking by hydrogen bonding within the helical structure. Ionized carboxyl groups

(RC02-) are formed by hydrolysis of the collagen by the action of hydroxide. This conversion may occur during the liming process, before introduction of the tanning agent (chromium salts). The ionized carboxyl groups may coordinate as ligands to the chromium(III) centers of the oxo-hydroxide clusters. Tanning may increase the spacing between protein chains in collagen (e.g., from 10 to 17 A), consistent with cross-linking by polychromium species, of the sort arising from olation and oxolation. The chromium may be cross-linked to the collagen. Chromium's ability to form such stable bridged bonds explains why it is considered one of the most efficient tanning compounds. Chromium-tanned leather can contain between 4 and 5% of chromium. This efficiency is characterized by its increased hydrothermal stability of the leather, and its resistance to shrinkage in heated water. Other tanning agents may be used to tan the layered body and modify the collagen.

[00091] In some embodiments, the engineered animal skin, hide, or leather products are substantially-free of pathogenic microorganisms. In further embodiments, controlled and substantially sterile methods of cell preparation, cell culture, sheet/layer preparation, and engineered animal skin, hide, or leather preparation result in a product substantially-free of pathogenic microorganisms. In further embodiments, an additional advantage of such a product is increased utility and safety.

[00092] In some embodiments, the engineered animal skin, hide, or leather products are shaped. In further embodiments, the animal skin, hide, or leather is shaped by, for example, controlling the number, size, and arrangement of the non-contractile sheets/layers used to construct the animal skin, hide, or leather. In other embodiments, the animal skin, hide, or leather is shaped by, for example, cutting, pressing, molding, or stamping. In some embodiments, the shape of the animal skin, hide, or leather product is selected to resemble a traditional animal skin, hide, or leather product. Examples

[00093] The following illustrative examples are representative of embodiments of the methods of forming bodies that can be tanned to form engineered leather. The examples described herein and are not meant to be limiting.

Example 1

[00094] Example 1 illustrates a sample of artificial leather made by forming collagen sheets (from collagen-releasing cells), decellularizing them to prevent contractions, and adhering them by seeding the decellularized sheets with a second set of collagen-releasing cells, then incubating them to adhere the sheets. FIG. 2 further illustrates this example. This sample material was formed entirely from the interconnected fibrous collagen secreted by the cells. In this example, the cells were initially cultured in rectangular culture (e.g., Petri) dishes. Once sufficient amount of collagen has been secreted (and before contraction), the sample was "decellularized", in this example with alcohol (i.e. it is fixed with absolute ethanol for 10 minutes, which kills the cells but does not remove them; here decellularization thus means a process which results in the absence of live cells). This prevents the contraction of the collagen sheet. The intact collagen sheet may then be removed. Sheets can then piled on top of each other with live cells between them to make sure that neighboring sheets naturally interconnect; cells may attach to the sheets, reorganize and probably interconnect the collagen meshes in the neighboring sheets. As shown in FIG. 2B, the sheets may be sprinkled with live cells and the sheets layered atop each other (stacked or piled). The living cells may adhere to the native collagen and attach neighboring sheets to one another. By secreting collagen the cells may interconnect the existing collagen meshes within the sheets, resulting in a globally interconnected 3D network. This results in a "natural" layered structure, as illustrated in FIG. 2C. In this example, the layers (9 layers) were prepared using fibroblasts as the first and second set of collagen-releasing cells. The layers were combined at approximately the same time and seeded with the second set of collagen-releasing cells, then incubated/cultured for 6 more days. Alternatively, the sheets may be combined in a pairwise fashion. For example, pairs of decellularized sheets maybe layered (after placing collagen-releasing cells between them) and cultured to adhere. These two sheets may then be cultured until the sheets are sufficiently adherent (e.g., in one example, approximately six days). These smaller stacks may then be combined (e.g., after again decellularlizing in some variations), seeded with cells, and again cultured to adhere. Additional sheets or groups (stacks) of sheets may be added in this manner to increase the volume, e.g., height, of the stack. In some variations, multiple sheets may be combined (e.g., 3, 4, 5, 6, 7, 8, 9, etc. The limit may be the diffusion limit of the culture medium).

The sample may then be processed (tanned) to modify the collagen to produce leather. Example 2

[00095] As discussed above, in some variations, collagen pulp may be used to in the manufacture of engineered leather. For example, the collagen pulp may be used as a filler material, as illustrated below. In one variation, an engineered leather was formed using just collagen pulp as illustrated in FIG. 3 (107, 109), made entirely from pelleted collagen.

[00096] In an initial step 101, collagen-producing cells may be grown to secrete collagen (e.g., collagen sheets). As in any of the variations, the collagen-producing cells may be grown in culture medium and in a culture dish (for example a roller bottle, as shown). Thus, the cells secrete collage to form a sheet along the wall of the roller bottle. These collagen sheets may be used to make the collagen pulp as described; alternatively, in some variations, the collagen pulp may be formed from other collagen sources (include animal hide/skin waste material, etc.).

[00097] Collagen pulp may be obtained by blending/grinding the collagen produced by collagen-producing cells such as dermal fibroblasts and preparing a pulp. For example, the collage may be removed from a roller bottle 101 (e.g., with trypsin and vigorous shaking) and blended 103. As shown in FIG. 3, the process resembles the production of paper. Relatively thick samples can be formed in a relatively convenient way (i.e. pouring the pulp into a dish of desired shape and size. However, the interconnected fibrous collagen mesh is destroyed by the process; long fibers are probably chopped up 103.

[00098] The collagen pulp 105 may then be shaped/formed into a layer. For example, as shown in FIG. 3, the layer may be formed using a filter 107 (e.g., a bottle top filter). "Pour pulp" represents a process in which the collagen recovered from the roller bottle is blended (e.g., while in a pickling solution), centrifuged into a pellet, re-suspended to the desired volume and poured out (e.g., into a petri dish) to dry. After the sheet is dry, it is removed and usually washed with water. In one exemplary test material, the sample was completely dried until it became hard and slightly curled up. It was then placed in waterjust long enough so that it could be made flat and left slightly moist so it could be treated.

[00099] In some variations, as mentioned, the collagen pulp may be used as a filler material, as illustrated in Example 3.

Example 3

[000100] In one example, a hybrid structure, combining the collagen sheets formed as in Example 1 , with the collagen pulp as formed in example 2. In FIG. 4, and example of an engineered leather having 9 decellularized layers, formed as in example 1, are combined 401 and then a layer of pelleted collagen (collagen pulp) followed by 1 additional decellularlized collagen sheet, and then another pelleted layer, followed by 6 decellularized layers is formed. This forms a combined sandwiched construct 403 with alternating layers of collagen sheets 401 and pelted collagen pulp 405. The sheets/layers may be allowed to adhere by seeding with

collagen-releasing cells and culturing until they are adherent, as discussed above. In this example, the 9 and 6 layered constructs were prepared prior to the assembly of overall sample.

The sample may then be processed (tanned) to modify the collagen to produce leather. The sheets and pulp (as well as the sandwiched structure 403) shown are not to scale.

[000101] FIG. 5 illustrates another example of an engineered leather formed by combining decellularized sheets 501 of collagen produced as discussed above, with collagen pulp 503. In this example, the sample material was manipulated to fold 505 over on itself an thereby double its thickness. The filler material (collagen pulp 503) adds thickness to the final engineered leather 507. FIG. 8 illustrates a tanned sample 801 prepared as illustrated in FIG. 5.

[000102] In this example, the disc's diameter is 4.3 cm. Chromium tanning solution (we used Waytan 175 from Elementis), was diluted half and half with water. The sample was placed in the solution for 1 hours at 32-38°C. Magnesium oxide was then added and the temperature is raised to 46-49°C. The final pH is around 3-4. The sample was then rinsed in water. The moist sample is placed in the dye solution for 2 hours and then fatliquored with Wasco Truetan soft tan oil.

Example 4

[000103] Another filler material that may be used is reconstituted collagen. FIG. 6 illustrates two different samples prepared using reconstituted collagen as a filler material between collagen sheets grown from cultured collagen-releasing cells. For example, three sheets of

fibroblast-secreted collagen were grown to confluence, then fixed with alcohol and washed before applying new live cells. The fixation step is performed to stop detachment and the gradual shrinkage of these sheets while in culture due to contraction. The decellularized (fixed) collagen sheets (one each) 601 are then seeded with live fibroblasts. For example, 3 mg/ml collagen solution was prepared containing 1 million cells/ml and poured over the fixed sheets until an approximately 1 mm thickness was achieved. These three composite sheets were incubated for two days under regular culture conditions (although longer incubation could be used).

[000104] On the third day the top of the constructs were reseeded with live cells, and cells were allowed to attach (e.g., waiting approximately 15 minutes) to attach to the collagen substrate, then the three sheets were piled on the top of each other. The stack was then incubated (e.g., for >3 days in culture) until the layers had adhered sufficiently so as not to separate during a tanning process. This multilayered fixed sheet/reconstituted collagen construct was detached from the dish and tanned for 1 hr, starting at 32°C and reaching 39°C at the end of the tanning process, then a small amount of magnesium oxide was added and the temperature was gradually raised to 49°C, in approximately 40 minutes. The constructs were washed in DI water until the bluish color due to the chromium disappeared.

[000105] In this example shown in FIG. 6A, one of the samples (SV 603) has only two fixed sheet/reconstituted collagen layers. The tanning of this sample was done in the original Petri dish to avoid excessive manipulation of the sheets, and since the dish is probably coated with collagen, the sheet in contact with the dish was attaching very strongly to the dish, possibly due to the chromium treatment. The second sample 605 (SVI) shown in FIG. 6B has three composite layers. In this example, a collagen gel 609 is the filler material. The reconstituted collagen gel (e.g., commercially available collagen made in 3 mg/ml solution) may be combined with cells such as 1 mill/ml fibroblasts and allowed to gel, as mentioned above. In some variations, another filler material that may be used includes pelleted collagen (e.g., pelleted from collagen pulp as described above).

[000106] In another test, a sample was prepared consisting of decellularized layers of SMC and blended collagen. In this example, 5 SMC decellularized sheets of collagen were prepared. They were interconnected via live cells as discussed above (allowed to incubate). Three sheets of collagen were prepared in this manner and between pairs of these sheets, reconstituted collagen was used as a filler layer as discussed above, for a total of 2 layers of filler material. The sample was incubated to permit complete adhesion and then tanned. In this example the sample size was 10 x 7 cm². Larger samples have also been prepared.

[000107] For example, samples of size 24x24 cm² have been prepared by initially growing the collagen-releasing cells in 24x24 cm culture plates. In this example, three SMC decellularized layers are interconnected with SMC live cells.

[000108] The larger the sheets, however, the more difficult it may be to transfer then for stacking (forming the stack). Smaller samples are typically manually transferred, e.g., manually lifting individual sheets from the culture dish and transferring them to be placed on the other sheets. In some variations, the sheets may be placed using a rolling technique, e.g., rolling onto a mandrel.

[000109] In general a mandrel is a roller that may include an attachment for grasping or securing one end or edge of the collagen sheet so that it can be rolled on the rest of the mandrel. The roller may be any appropriate diameter; the sheet may be rolled up onto itself around the mandrel and later rolled off. Thus, the attachment may be disengaged to release the sheet of collagen.

[000110] FIGS. 7 A and 7B illustrate one variation of a mandrel, in which the mandrel is made of polyacrylic tubing. The tubing has a slit/groove on its lateral surface, parallel with the longitudinal axis. A thin steel wire fits into this groove, secured at both ends by rubber o-rings as shown in

FIG. 7A. When mounted, the wire sits in the groove, flush with the surface of the mandrel. One end of the sheet is detached, pulled over the mandrel and secured into the slit using the wire, then the sheet is rolled onto the mandrel, and can then be transferred to another dish.

[000111] In some variations, the mandrel may be used to stack multiple collagen sheets

(decellularlized collagen sheets) that have been reseeded directly onto the mandrel. In this example, a first decellularlized collagen sheet that has been rinsed and reseeded with

collagen-releasing cells is rolled onto the mandrel as discussed above. Thereafter, the wire (beneath layers of the collagen sheet wrapped on the mandrel) can be pulled out from under the sheet, as shown in FIG. 7B, and another sheet can be secured to the mandrel over the already rolled sheet, then this process is repeated so that sheets may be sequentially rolled onto the mandrel. Multiple sheets can be rolled onto each other, then the entire construct transferred the into a small roller bottle. Beside securing the wire to the mandrel, the o-rings at both ends of the mandrel may ensure that the surface of the sheets are not rubbed/dragged to the wall of the bottle, which can then be incubated to allow them to adhere.

[000112] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[000113] As mentioned above, terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

[000114] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[000115] Although the terms "first" and "second" may be used herein to describe various features/elements, these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[000116] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or "approximately," even if the term does not expressly appear. The phrase "about" or

"approximately" may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1 % of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[000117] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[000118] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term "invention" merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

CLAIMS What is claimed is:

1. A method of making a synthetic leather, the method comprising:

culturing a first group of collagen-releasing cells to form a plurality of sheets of

collagen;

treating the plurality of sheets to prevent contractions thereby forming a plurality of non-contractile sheets;

placing a second group of collagen-releasing cells on a first sheet from the plurality of non-contractile sheets;

placing a second sheet from the plurality of non-contractile sheets on top of the first sheet to form a first stack; and

culturing the first stack until the first sheet and second sheet are adherent.

2. The method of claim 1 , further comprising processing the first stack to modify the

collagen.

3. The method of claim 1 , further comprising processing the first stack by tanning to modify the collagen.

4. The method of claim 1 , wherein placing the second sheet comprises rolling the second sheet onto a mandrel and unrolling the second sheet onto the first sheet.

5. The method of claim 1 , wherein culturing the first group of collagen-releasing cells

comprises culturing the cells to confluence.

6. The method of claim 1, where the first collagen-releasing cells comprise smooth muscle cells.

7. The method of claim 1, wherein the first and second groups of collagen-releasing cells comprise the same type of cells.

8. The method of claim 1 , wherein treating comprises de-cellularizing the plurality of sheets to kill the collagen-releasing cells.

9. The method of claim 1, wherein treating comprises treating the plurality of sheets with ethanol to kill the collagen-releasing cells.

10. The method of claim 1, wherein culturing comprises growing the first group of collagen-releasing cells in culture without a scaffold.

1 1. The method of claim 1, wherein placing the second group of collagen-releasing cells comprises seeding the second group of collagen-releasing cells on the first sheet before placing the second sheet on the first sheet.

12. The method of claim 1, further comprising adding a filler material between the first and second sheets before placing the second sheet on the first sheet.

13. The method of claim 1, wherein placing the second group of collagen-releasing cells comprises adding a filler material comprising the second group of collagen-releasing cells between the first and second sheets before placing the second sheet on the first sheet.

14. The method of claim 1, further comprising adding a collagen filler material between the first and second sheets before placing the second sheet on the first sheet.

15. The method of claim 1, further comprising adding a filler material comprising a

reconstituted collagen between the first and second sheets before placing the second sheet on the first sheet.

16. The method of claim 1, further comprising adding a filler material comprising a collagen pulp between the first and second sheets before placing the second sheet on the first sheet.

17. The method of claim 1, further comprising increasing the height of the first stack by

placing additional collagen-releasing cells on the first stack and then placing an additional sheet from the plurality of non-contractile sheets or an additional stack comprising adherent sheets from the plurality of non-contractile sheets onto the first stack, and culturing the first stack and additional sheet or additional stack until the first stack and additional sheet or additional stack are adherent.

18. The method of claim 1, further comprising sequentially increasing the height of first stack by repeating the steps of placing additional collagen-releasing cells on the first stack, placing an additional sheet from the plurality of non-contractile sheets or an additional stack comprising adherent sheets from the plurality of non-contractile sheets onto the first stack, and culturing the first stack and additional sheet or additional stack until the first stack and additional sheet or additional stack are adherent.

19. The method of claim 1, further comprising placing additional collagen-releasing cells on the second sheet and placing an additional sheet from the plurality of non-contractile sheets onto the second sheet to increase the height of the first stack.

20. A method of making a synthetic leather, the method comprising:

culturing collagen-releasing cells to confluency to form a plurality of sheets of

collagen;

decellularizing the plurality of sheets to prevent contractions;

seeding collagen-releasing cells on a first sheet from the plurality of decellularized sheets;

placing a second sheet from the plurality of decellularized sheets on top of the first sheet to form a stack; and

culturing the stack until the first sheet and second sheet are adherent.

21. A method of making a synthetic leather, the method comprising:

growing collagen-releasing cells in culture without a scaffold to form a plurality of sheets of collagen;

decellularizing the plurality of sheets;

forming a stack by placing a second sheet from the plurality of decellularized sheets on top of a first sheet from the plurality of decellularized sheets with

collagen-releasing cells between the first and second sheets; and

culturing the stack until the first sheet and second sheet are adherent.