Thin and ultra-thin leather and production methods thereof

Abstract

The present invention relates to synthetic leather products obtained from cell culture In particular, the invention provides thin and ultra-thin cultivated leather and methods for producing thin and ultra-thin cultivated leather.

Description

[0001] Thin and ultra-thin leather and production methods thereof

[0002] The present invention relates to thin and ultra-thin cultivated leather and methods for producing thin and ultra-thin cultivated leather.

[0003] BACKGROUND

[0004] Natural animal leather is a product obtained from the chemical treatment of animal hides and skins to prevent decomposition and putrefaction, and increase the thermal, chemical and microbiological stability of the fresh hide or skin. Natural animal leather has been used for centuries as a versatile material to produce a variety of long -lasting items, including clothing, footwear, containers, furniture, tools, and decorative, sports, and specialist technical equipment. However, due to its provenance, the maximum and minimum dimensions of natural animal leather broadly depend on the dimensions of the animal skin it was derived from.

[0005] Consequently, the thickness of natural animal leather is limited by the natural range of the thickness of animal hide or skin and the ability to split it without compromising the overall integrity of the material. In practice, this means that leather produced from animal hides or skins is limited to a minimum thickness of about 0.4-0.6 mm, after which point it loses its structural integrity and durability. Thinner leather represents a concept material with great potential and of particular interest to applications where high-resistance, low-bulk properties are required. Previously, there have been no successful efforts to create extremely thin leathers, as this is impractical / impossible by using natural animal skins. Accordingly, previous efforts to create cultivated leather have been focused on creating thicker leather equivalents.

[0006] The aim of the invention is to address some of the problems in the prior art, and provide new and improved methods, compositions, and cellular composites for use in producing thin (0.2-0.4 mm thick) and even ultra-thin (<0.2 mm thick) cultivated leather.

[0007] SUMMARY OF INVENTION

[0008] In one aspect, the invention provides a method for producing thin cultivated leather, the method comprising (a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) repeating step (a) at least once to independently produce further cell layers; (c) contacting the primary cell layer with the further cell layers to produce a cellular composite; (d) culturing the cellular composite in serum-free medium; and (e) tanning the cellular composite to produce thin cultivated leather; wherein the cellular composite is between about 0.2 mm and 0.8 mm thick and wherein the thin cultivated leather is at most 0.4 mm thick.

[0009] Suitably, in the methods of the invention, the method may comprise a further step (f), wherein the thin cultivated leather produced in step (e) is be split to produce at least two independent tanned tissue sheets.In another aspect, the invention provides a method for producing ultra-thin cultivated leather, the method comprising (a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) optionally repeating step (a) at least once to independently produce further cell layers and contacting the primary cell layer with the further cell layers to produce a cellular composite; (c) culturing the primary cell layer or the cellular composite in serum-free medium; and (d) tanning the primary cell layer or the cellular composite to produce ultra-thin cultivated leather; wherein the primary cell layer is between about 0.005 mm and 0.2 mm thick or the cellular composite is between about 0.01 mm and 0.4 mm thick and the ultra-thin cultivated leather is less than 0.2 mm thick.

[0010] Suitably, in the methods of the invention, the primary cell layer and / or the cellular composite and / or the cultivated leather may not comprise a scaffold.

[0011] Suitably, in the methods of the invention, the fibroblasts may be dermal fibroblasts. Suitably, in the methods of the invention, the composition may further comprise adipocytes and / or adipose progenitor cells. Suitably, in the methods of the invention, the adipose progenitor cells may be pre-adipocytes, optionally wherein the pre-adipocytes may be hypodermal pre-adipocytes.

[0012] Suitably, in the methods of the invention, the serum-free medium may comprise one or more macromolecular crowding (MMC) agents. Suitably, in the methods of the invention, the MMC may be selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0013] Suitably, in the methods of the invention, the culturing step to produce a primary cell layer may be for at least about 7 days, preferably at least about 14 days. Suitably, in the methods of the invention, the culturing of the cellular composite may be for at least about 7 days.

[0014] Suitably, in the methods of the invention, the tanning may be selected from chrome tanning, vegetable tanning, or chrome-free tanning.

[0015] In another aspect, the invention provides a thin cultivated leather obtained by any method of the invention, wherein the thin cultivated leather has an average thickness of between 0.2 and 0.4 mm, wherein the onset temperature of the leather is more than 80°C.

[0016] In another aspect, the invention provides an ultra-thin cultivated leather obtained by any method of the invention, wherein the ultra-thin cultivated leather has an average thickness of less than 0.2 mm, wherein the onset temperature of the leather is more than 80°C.

[0017] Suitably, in the cultivated leather of the invention, the cultivated leather may not comprise a scaffold. Suitably, in the cultivated leather of the invention, the onset temperature of the leather may be more than 85°C, more than 90°C, more than 95°C, more than 100°C, or more than 105°C.Suitably, in the cultivated leather of the invention, the leather may have a tensile strength of >10 N / mm2. In another aspect, the invention provides a thin cultivated leather wherein the thin cultivated leather has an average thickness of between 0.2 and 0.4 mm, wherein the onset temperature of the leather is more than 80°C.

[0018] In another aspect, the invention provides an ultra-thin cultivated leather wherein the ultra-thin cultivated leather has an average thickness of less than 0.2 mm, wherein the onset temperature of the leather is more than 80°C.

[0019] Suitably, in the cultivated leather of the invention, the cultivated leather may not comprise a scaffold. Suitably, in the cultivated leather of the invention, the leather may have an average thickness of 0.109 mm. Suitably, in the cultivated leather of the invention, the leather may have an average thickness of less than 0.051 mm. Suitably, in the cultivated leather of the invention, the leather may have an average thickness of 0.045 mm.

[0020] Suitably, in the cultivated leather of the invention, the onset temperature of the leather may be more than 85°C, more than 90°C, more than 95°C, more than 100°C, or more than 105°C.

[0021] Suitably, in the cultivated leather of the invention, the leather may have a tensile strength of >10 N / mm2. In another aspect, the invention provides a cellular composite wherein the cellular composite has an average thickness of at most 0.8 mm, wherein the onset temperature of the cellular composite is more than 69°C when pickled and / or more than 74°C when raw.

[0022] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0023] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0024] Various aspects of the invention are described in further detail below.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0027] Figure 1 shows a schematic outlining the process for producing thin and ultra-thin cultivated leather. The schematic describes the various steps of the process to create scaffold-free animal tissues (celllayers), assemble such cell layers into cellular composites, and the taming of scaffold-free animal tissues (cell layers) or cellular composites into thin and ultra-thin cultivated leather.

[0028] Figure 2 shows cell proliferation during cell layer production in the process for producing cultivated leather. E. Derm dermal cells were cultured for 7 or 14 days in mono- (M) or co-culture with CPF4-A hypodermal cells (C) with serum-free proliferation medium (SPM; light grey bars) or serum -containing medium (w / FBS; dark grey bars). Total cell number, expressed as a percentage of the corresponding day control (mono-culture w / FBS at day 7 or 14), corresponded to average ± S.D. of three independent experiments (n =3; 4-. p = 0.05).

[0029] Figure 3 shows tissue deposition during cell layer production in the process for producing cultivated leather. E. Derm dermal cells cultured for 14 days in mono- (M) or co-culture with CPF4-A hypodermal cells (C) with serum-free proliferation medium (SPM) were subsequently maintained for an additional 7 or 14 days with serum-free tissue production medium (STPM) or serum-containing medium (w / FBS; dark grey bars). Total tissue deposition, expressed as percentage of collagen mass of the corresponding day control (mono-culture w / FBS at day 7 or 14), corresponded to average ± S.D. of three independent experiments (n =3;

[0030]

[0031] and 4-. p = 0.05 and 0.01, respectively).

[0032] Figure 4 shows tissue assembly into scaffold-free cellular composites in the process for producing cultivated leather. Scaffold-free cellular composites were made from 50 (A) and 25 layers of scaffold-free cell layers (B) assembled into a single stack. After recovery from a liquid environment, these robust scaffold-free cellular composites retained their shape, size, and integrity even after drying and extensive handling and stretching (C). Scale bars = 2 cm (A-C).

[0033] Figure 5 shows the viability and integrity of scaffold-free cell layers and scaffold-free composites thereof in the process for producing cultivated leather. A scaffold-free cell layer (1 -layer) and the 50-and 25 -layer cellular composites made from their assembly were analysed for cell viability using the Calcein AM / Propidium iodide live / dead cell staining assay (top panels), and for integrity after pickling under intense agitation (bottom panels). Outcomes of integrity tests are indicated below bright-field photographs. Results are representative of three independent experiments (n = 3). Scale bars = 100 pm (top panels), 2 mm (bottom panels).

[0034] Figure 6 shows the free amino acid content of scaffold-free cellular composites in the process for producing cultivated leather. Total free amino acid contents, expressed as pg of amino acid per g of dry tissue, corresponded to the average ± S.D. of three 25- and 50-layer scaffold-free cellular composites (light and dark grey bars, respectively), along with that from porcine dermis samples (black bars), here used as control for natural animal tissue.

[0035] Figure 7 shows the hydrolysed amino acid content of scaffold-free cellular composites in the process for producing cultivated leather. Total hydrolysed amino acid contents, expressed as mg of amino acidper g of dry tissue, corresponded to the average ± S.D. of three 25- and 50-layer scaffold-free cellular composites (light and dark grey bars, respectively), along with that from porcine dermis samples (black bars), here used as control for natural animal tissue.

[0036] Figure 8 shows the ultrastructural analysis of scaffold-free cellular composites in the process for producing cultivated leather. The panels show low-energy scanning electron microscopy (SEM) imaging of 25- (A) and 50-layer scaffold-free cellular composites (B) and of porcine dermis (C). They also show high-energy SEM imaging of 25- (D) and 50-layer scaffold-free cellular composites (E) and of porcine dermis (F). Scale bars = 0.2 mm (A-C), 10 pm (D-F).

[0037] Figure 9 shows the compositional analysis of thin and ultra-thin cultivated leather. Scaffold-free cellular composites made from 50-layers of scaffold-free cell layers assembled into a single stack, are shown before (A) and after (B) vegetable tanning. Single cell layers are also suitable to be tanned, retaining their shape and structural integrity (C) while acquiring greater mechanical stability (inset). (D) Measurement of thickness of thin (i) and ultra-thin scaffold-free cultivated leather (ii) made from 50-layer cellular constructs and from single cell layers, respectively. Picrosirius Red staining (E) and Sudan IV staining (F) were used to characterise the composition of cultivated leather made from scaffold-free cellular composites with 50 (i) and 25 layers of tissue (ii), and compared with unstained controls (iii). Scale bars = 2 cm (A-B), 1 mm (C), 100 pm (D).

[0038] Figure 10 shows the structural analysis of thin and ultra-thin cultivated leather. The panels show low-energy scanning electron microscopy (SEM) imaging of leather made from a 100-layer scaffold-free cellular construct (A), and from porcine dermis (B). They also show high-energy SEM of cultivated leather made from 100- (C) and 50-layer (D) scaffold-free cellular composites, along with that of an untanned scaffold-free cellular construct (E) and of tanned porcine dermis (F). Scale bars = 0.1 mm (A), 0.5 mm (B), 1 pm (C-F).

[0039] Figure 11 shows the tensile analysis of thin and ultra-thin cultivated leather. Tensile testing specimens were cut into dogbone shapes (A) and thickness measured at the shoulder-gauge (arrows) and gauge regions (arrowhead). (B) Ultra-thin cultivated leather specimens produced from 25- (i) and 12-layer (ii) scaffold-free cellular composites showing an average thickness of 0.109 mm and 0.045 mm, respectively, were successfully tested along with a sample of natural animal leather (iii) which was 0.765 mm-thick. The data also shows the stress-strain profile (C) and tensile test results (D) of ultrathin cultivated leather of different thickness (i, ii) and of natural animal leather (iii).

[0040] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.DETAILED DESCRIPTION

[0041] The present invention is based on the inventors’ surprising finding that cell layers can be produced from animal cells cultured in serum-free medium, and can be assembled into a cellular composite, which can be tanned to produce cultivated leather, thin cultivated leather and ultra-thin cultivated leather.

[0042] The inventors have surprisingly identified that the use of serum -free media (comprising macromolecular crowding agents) prevents contraction (shrinkage) of cell layers and cellular composites thereof during the production of cultivated leather. Unwanted contraction is typically seen in the methods of producing cultivated leather in the art. This could be in part attributable to lot variations from serum, a well-known feature affecting consistency in cell performance and tissue culture production. The presence of serum in media is also expected to increase variation in yield by promoting spontaneous cell contraction that leads to cell and tissue loss during culture. The method of this invention circumvents the requirement for mechanical restraints during the production process. Additionally, the absence of spontaneous cell contraction allows for the production of larger pieces of leather. Moreover, the use of serum -free media (comprising macromolecular crowding agents) during cell layer production and during the culturing of cellular composites thereof results in increased extracellular matrix component (ECM) production and deposition, in turn resulting in high concentrations of ECM (such as collagen) within cell layers and between cell layers within cellular composites. The inventors have surprisingly shown that even a single cell layer is intact after pickling (the first step of leather production), which the inventors attribute (without wishing to be bound by theory) to the increased ECM (such as collagen) deposition during the cell layer production. Without wishing to be bound by theory, the inventors believe it is the increased ECM (such as collagen) deposition between cell layers within the cellular composites that results in cellular composites that retain structure during tanning.

[0043] The inventors have surprisingly identified that when fibroblasts are co-cultured with pre-adipocytes, there is an increase in both cell proliferation and ECM production during cell layer production in comparison to fibroblast monoculture. This observation is seen regardless of whether the media is serum -containing or serum -free.

[0044] The inventors have also shown that the cultivated leather, including thin and ultra-thin cultivated leather, produced by the method of the invention possesses physical properties comparable to or enhanced over that of natural animal leather. Surprisingly, these properties are achieved without the requirement of any type of supporting scaffold, filler, or glue to achieve said properties at any stage during the production process or in the cultivated leather itself. This is surprising given that cell layers and composites thereof without scaffolds would be assumed to be too fragile to be subjected to the tanning process. Without wishing to be bound by theory, the inventors believe this may be due to the high concentration of ECM within the cell layers and between the cell layers within the cellular composite. Surprisingly, the cultivated leather of the present invention outperforms natural leather invarious physical properties (such as thermal stability and tensile strength) when considering that these properties may correlate linearly with thickness. Accordingly, the present inventors have provided a method capable of producing cultivated leather with thicknesses below the limits of what is possible for natural leather, but with comparable or improved properties.

[0045] Moreover, the cultivated leather of the invention has organoleptic properties similar to that of leather obtained from natural skin.

[0046] Additionally, the thickness of the cultivated leather can be precisely controlled by selecting the number of cell layers to be assembled into the cellular composite for subsequent tanning to produce thin or ultrathin cultivated leather. This allows for unlimited possibilities of cultivated leather products produced by the method of the present invention. The cultivated leather, including thin and ultra-thin cultivated leather, produced by the method of the present invention represent materials of particular interest to applications where thermal stability, high tensile strength, and low-bulk properties are required.

[0047] Surprisingly, the inventors identified that the cellular composites (and therefore the cultivated leather products produced therefrom) have significantly higher concentrations of elastin than porcine dermis. Without wishing to be bound by theory, the inventors believe that the higher concentrations of elastin may account for the higher onset temperatures of cellular composites (and the cultivated leather thereof) because of the higher thermostability of elastin than collagen.

[0048] Additionally, the reduced concentration of several ECM proteins, especially sGAGs and HA, in the cellular composites of the present invention in comparison to natural animal skin allows the simplification of the tanning process because sGAGs and HA need to be removed during the tanning process anyway.

[0049] Furthermore, the inventors observed that the cellular composites of the invention have an enhanced lipid content in comparison to natural animal skin. Without wishing to be bound by theory, the inventors believe that this is due to the enhanced cell density, and this enhanced cell density may promote more even cell distribution within the cellular composite and in turn facilitate the fatliquoring step during tanning.

[0050] Previous efforts to produce cultivated leather have been focused on producing cultivated leather equivalent to the thickness observed in leather produced from natural animal skins. This may be because producing leather of < 0.2 mm thickness from natural animal skins is impossible. Accordingly, there has been no effort to produce thin or ultra-thin cultivated leather.

[0051] Thin cultivated leather and ultra-thin cultivated leather may find use in leather products where thin and / or ultra-thin leather is desirable. Non-limiting examples of where thin and / or ultra-thin leather may be desirable include sportswear (for example, football boots) sports equipment, vehicle seats, nautical and aeronautical furnishings, technical and scientific tools and instruments, furniture and upholstery,bookbinding, accessories (for example, phone cases, key rings), and fashion (for example, handbags, gloves, jackets).

[0052] Thin and ultra-thin cultivated leather

[0053] In one aspect, the present invention provides a method for producing cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather. The aim of this method is to produce a cultivated leather product, a thin cultivated leather product and / or an ultra-thin cultivated leather product.

[0054] In one aspect, the present invention provides a method for producing thin cultivated leather, the method comprising:

[0055] (a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) repeating step (a) at least once to independently produce further cell layers;

[0056] (c) contacting the primary cell layer with the further cell layers to produce a cellular composite; (d) culturing the cellular composite in serum-free medium; and

[0057] (e) tanning the cellular composite to produce thin cultivated leather; wherein the cellular composite is between about 0.2 mm and 0.8 mm thick and wherein the thin cultivated leather is at most 0.4 mm thick.

[0058] In another aspect, the present invention provides a method for producing thin cultivated leather, the method comprising:

[0059] (a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) repeating step (a) at least once to independently produce further cell layers;

[0060] (c) contacting the primary cell layer with the further cell layers to produce a cellular composite; (d) culturing the cellular composite in serum-free medium; and

[0061] (e) tanning the cellular composite to produce thin cultivated leather; wherein the thin cultivated leather is at most 0.4 mm thick.

[0062] In another aspect, the present invention provides a method for producing ultra-thin cultivated leather, the method comprising:

[0063] (a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) optionally repeating step (a) at least once to independently produce further cell layers and contacting the primary cell layer with the further cell layers to produce a cellular composite;(c) culturing the primary cell layer or the cellular composite in serum-free medium; and (d) tanning the primary cell layer or the cellular composite to produce ultra-thin cultivated leather; wherein the primary cell layer is between about 0.005 mm and 0.2 mm thick or the cellular composite is between about 0.01 mm and 0.4 mm thick and the ultra-thin cultivated leather is less than 0.2 mm thick.

[0064] In another aspect, the present invention provides a method for producing ultra-thin cultivated leather, the method comprising:

[0065] (a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) optionally repeating step (a) at least once to independently produce further cell layers and contacting the primary cell layer with the further cell layers to produce a cellular composite;

[0066] (c) culturing the primary cell layer or the cellular composite in serum-free medium; and (d) tanning the primary cell layer or the cellular composite to produce ultra-thin cultivated leather; wherein the ultra-thin cultivated leather is less than 0.2 mm thick.

[0067] In another aspect, the present invention provides a method for producing cultivated leather, the method comprising:

[0068] a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) repeating step (a) at least once to independently produce further cell layers;

[0069] (c) contacting the primary cell layer with the further cell layers to produce a cellular composite; (d) culturing the cellular composite in serum-free medium; and

[0070] (e) tanning the cellular composite to produce cultivated leather.

[0071] Suitably, the method of producing cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather is an in vitro method.

[0072] As used herein, the term “cultivated leather” refers to leather produced in vitro from cells of animal origin. In the art, cultivated leather may also be known as cultured leather, slaughter-free leather, in vitro leather, lab-grown leather, cell-based leather, cellular leather, clean leather, artificial leather, and / or synthetic leather. The term “animal origin” refers to cells originally derived from but not directly obtained from an animal. A cell that is originally derived from but not directly obtained from an animal is, for example, a cell that has arisen as a result of a cell directly obtained from an animal undergoing cell division in cell culture. In another example, a cell that is originally derived from but not directly obtained from an animal is a cell that has been directly obtained from an animal and has undergonedifferentiation in cell culture. Accordingly, in the context of the present disclosure, animal -like cells are cells that have been produced in vitro by culturing a cell that itself was indirectly obtained from an animal, or a cell that was directly obtained from an animal. A cell that has been directly obtained from an animal may be physically removed from an animal (for example by a biopsy) or from animal tissues (for example from an animal carcass). Cells that are indirectly obtained from an animal may be from an established cell line or cell strain.

[0073] The thickness of the cultivated leather of the invention is not restricted. Suitably, the cultivated leather of the invention may be at least a similar thickness to, more thick or less thick than leather obtained from a natural animal skin. Leather produced from natural animal skin is typically between 0.6 and 4.4 mm thick, depending on the animal species, part of the animal body, and type of leather (full grain, top grain, nubuck leather etc.). As outlined in the Examples herein, an example of leather produced from natural animal skin has a thickness of 0.765 mm.

[0074] Suitably, the cultivated leather may be ultra-thin cultivated leather. In the context of the present invention, the term “ultra-thin cultivated leather” refers to cultivated leather less than 0.2 mm thick. Suitably, the ultra-thin cultivated leather may be less than 0.2 mm thick, less than 0.18 mm thick, less than 0.16 mm thick, less than 0.14 mm thick, less than 0.12 mm thick, less than 0.10 mm thick, less than 0.08 mm thick, less than 0.06 mm thick, less than 0.04 mm thick, less than 0.02 mm thick, or less than 0.01 mm thick. Suitably, the ultra-thin cultivated leather may be less than 0.2 mm thick.

[0075] Suitably, the cultivated leather may be thin cultivated leather. In the context of the present invention, the term “thin cultivated leather” refers to cultivated leather at most about 0.4 mm thick.

[0076] Suitably, the thin cultivated leather may be at most 0.2 mm thick, at most 0.22 mm thick, at most 0.24 mm thick, at most 0.26 mm thick, at most 0.28 mm thick, at most 0.3 mm thick, at most 0.32 mm thick, at most 0.34 mm thick, at most 0.36 mm thick, at most 0.38 mm thick, or at most 0.4 mm thick. Suitably, the thin cultivated leather may be at most 0.4 mm thick.

[0077] The skilled person would appreciate that thin cultivated leather, in the context of the present invention, could be referred to as being between 0.2 and 0.4 mm thick.

[0078] Suitably the thin cultivated leather may be between 0.22 and 0.4 mm thick, between 0.24 and 0.4 mm thick, between 0.26 and 0.4 mm thick, between 0.28 and 0.4 mm thick, between 0.3 and 0.4 mm thick, between 0.32 and 0.4 mm thick, between 0.34 and 0.4 mm thick, between 0.38 and 0.4 mm thick. Suitably, the thin cultivated leather may be between 0.2 and 0.22 mm thick, between 0.2 and 0.24 mm thick, between 0.2 and 0.26 mm thick, between 0.2 and 0.28 mm thick, between 0.2 and 0.3 mm thick, between 0.2 and 0.32 mm thick, between 0.2 and 0.34 mm thick, between 0.2 and 0.36 mm thick, between 0.2 and 0.38 mm thick, between 0.2 and 0.4 mm thick. Suitably, the thin leather may be between 0.2 and 0.4 mm thick.Suitably, the cultivated leather has an average thickness of 0.109 mm. Suitably, the cultivated leather has an average thickness of less than 0.051 mm. Suitably, the cultivated leather has an average thickness of 0.045 mm.

[0079] As used herein, the term “average thickness” means that across a leather or cultivated leather sample, the stated value is the average of a sample of thickness measurements taken.

[0080] Suitably, in each and every step comprising the method of producing cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, an exogenous scaffold is not utilised. Accordingly, the resulting cultivated leather, thin cultivated leather and ultra-thin cultivated leather may not comprise a scaffold.

[0081] Suitably, the cultivated leather, thin cultivated leather and ultra-thin cultivated leather may not comprise a scaffold. As used herein, the term “scaffold” refers to an exogenous scaffold. In other words, the term “scaffold” refers to supporting materials that are not derived from the cells themselves. Exogenous scaffolds are typically made of polymeric biomaterials and provide the structural support for cell attachment and subsequent tissue development. Non-limiting examples of exogenous scaffolds include natural exogenous materials derived from plants (e.g., cellulose and its derivatives), fungus (e.g., chitin and its derivatives), animals (e.g., ECM, such as decellularised ECM, keratin and its derivatives), or algae (e.g., alginate and its derivatives), and those produced from synthetic polymers. Non-limiting synthetic polymers include PLA (polylactic acid), PGA (polyglycolide), PLGA (poly(lactic-co-glycolic acid)), PCL (Poly caprolactone), poly (propylene fumarate), and GFRP (glass fiber reinforced plastic). Suitably, the cultivated leather, thin cultivated leather and ultra-thin cultivated leather may not comprise an exogenous scaffold.

[0082] In the context of the present disclosure, the ECM is not classified as a scaffold, as it is natively produced by the cells of the cultivated leather. Accordingly, with the ECM present in the cultivated leather described herein, it may be said the cultivated leather is scaffold-free, or free of an exogenous scaffold. Suitably, cultivated leather, thin cultivated leather and ultra-thin cultivated leather may have organoleptic properties at least similar to leather obtained from an animal. Organoleptic properties are the properties of a product or substance that act on the senses. Organoleptic properties of natural leather may be selected from the fullness, softness, grain tightness, grain smoothness, colour uniformity, and smell of the leather.

[0083] As exemplified in the Examples section herein, the inventors surprisingly identified that the cultivated leather of the present invention has physical properties (for example, thermal stability and tensile strength) similar to and / or enhanced over that of natural leather when leather thickness is taken into consideration.Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may have physical properties at least similar to or enhanced over leather obtained from an animal. As used herein, the term “similar” refers to having a resemblance in appearance, character, or quantity, without being identical. As used herein, the term “enhanced” refers to an intensified, increased, or improved quality, value, or extent of. Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may have similar tensile strength, tear strength, tensile stress, displacement, elongation, thermal stability, density / mass, water resistance, waterproofhess, fire resistance, colourfastness, and / or endurance to flexing and rubbing to leather obtained from an animal. Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may have enhanced strength, tear strength, tensile stress (tensile strength), displacement, elongation and / or thermal stability over leather obtained from an animal. Suitably, these similar or enhanced physical properties may take into consideration the thickness of the cultivated leather. For example, cultivated leather with a thickness of 0.1 mm may have a lower tensile stress than a 0.7 mm leather (produced from natural animal skin); however, when taking into consideration thickness (0.1 mm versus 0.7 mm), the cultivated leather may have a similar or enhanced tensile stress over the leather produced from natural animal skin. Suitably, these similar or enhanced physical properties may be present regardless of the thickness of the cultivated leather. Methods of measuring these physical properties will be known by the person skilled in the art; however, examples include tensile testing (using instruments for measuring tensile strength include Shimadzu Autograph AGS-X tensile tester) and thermal stability measurements using differential scanning calorimetry (DSC), as outlined in the examples section herein. DSC methods provide the onset temperatures, peak temperatures, and endset temperatures (°C) of the material being tested.

[0084] As used herein, the term “onset temperature” in the context of DSC, relates to the thermal stability of a material, such as a leather or cellular composite, and the “onset temperature” of leather and related materials is usually considered the shrinkage temperature of the material i.e. the heat shrinkage temperature, at which the material starts to melt. As used herein, the term “peak temperature” in the context of DSC refers to the measure of the temperature at which the melting of the material reaches its maximum rate. As used herein, the term “endset temperature” in the context of DSC refers to the temperature at which the melting of the material comprising a leather or cellular composite is considered complete.

[0085] Suitably, the onset temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 75°C, more than about 80°C, more than about 85°C, more than about 90°C, more than about 95 °C, more than about 100°C, or more than about 105 °C. Suitably, the onset temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 80°C (when vegetable tanned). Suitably, the onset temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 100°C (when chromium tanned).Suitably, the onset temperature of the thin cultivated leather may be more than about 75 °C, more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, or more than about 105 °C. Suitably, the onset temperature of the thin cultivated leather may be more than about 80°C (when vegetable tanned). Suitably, the onset temperature of the thin cultivated leather may be more than about 100°C (when chromium tanned).

[0086] Suitably, the onset temperature of the ultra-thin cultivated leather may be more than about 75 °C, more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, or more than about 105°C. Suitably, the onset temperature of the ultra-thin cultivated leather may be more than about 80°C (when vegetable tanned). Suitably, the onset temperature of the ultra-thin cultivated leather may be more than about 100°C (when chromium tanned).

[0087] Suitably, the peak temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95 °C, more than about 100°C, more than about 105 °C, may be more than about 110°C, or more than about 115°C. Suitably, the peak temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 80°C (when vegetable tanned). Suitably, the peak temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 105 °C (when chromium tanned).

[0088] Suitably, the peak temperature of the cultivated leather may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, more than about 105°C, may be more than about 110°C, or more than about 115°C. Suitably, the peak temperature of the cultivated leather may be more than about 80°C (when vegetable tanned). Suitably, the peak temperature of the cultivated leather may be more than about 105 °C (when chromium tanned).

[0089] Suitably, the peak temperature of the thin cultivated leather, may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, more than about 105 °C, may be more than about 110°C, or more than about 115°C. Suitably, the peak temperature of the thin cultivated leather, may be more than about 80°C (when vegetable tanned). Suitably, the peak temperature of the thin cultivated leather may be more than about 105 °C (when chromium tanned). Suitably, the peak temperature of the ultra-thin cultivated leather, may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, more than about 105 °C, may be more than about 110°C, or more than about 115°C. Suitably, the peak temperature of the ultra-thin cultivated leather, may be more than about 80°C (when vegetable tanned). Suitably, the peak temperature of the ultra-thin cultivated leather, may be more than about 105 °C (when chromium tanned).

[0090] Suitably, the endset temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 80°C, more than about 85°C, more than about 90°C, morethan about 95 °C, more than about 100°C, more than about 105 °C, may be more than about 110°C, or more than about 115 °C. Suitably, the endset temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 85°C (when vegetable tanned). Suitably, the endset temperature of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may be more than about 105 °C (when chromium tanned).

[0091] Suitably, the endset temperature of the cultivated leather may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, more than about 105°C, may be more than about 110°C, or more than about 115°C. Suitably, the endset temperature of the cultivated leather may be more than about 85 °C (when vegetable tanned). Suitably, the endset temperature of the cultivated leather may be more than about 105 °C (when chromium tanned).

[0092] Suitably, the endset temperature of the thin cultivated leather, may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, more than about 105 °C, may be more than about 110°C, or more than about 115 °C. Suitably, the endset temperature of the thin cultivated leather, may be more than about 85°C (when vegetable tanned). Suitably, the endset temperature of the thin cultivated leather may be more than about 105 °C (when chromium tanned). Suitably, the endset temperature of the ultra-thin cultivated leather, may be more than about 80°C, more than about 85°C, more than about 90°C, more than about 95°C, more than about 100°C, more than about 105 °C, may be more than about 110°C, or more than about 115 °C. Suitably, the endset temperature of the ultra-thin cultivated leather, may be more than about 85°C (when vegetable tanned). Suitably, the endset temperature of the ultra-thin cultivated leather, may be more than about 105 °C (when chromium tanned).

[0093] As outlined in the examples herein, the present inventors have shown that the cultivated leather can withstand significant amounts of force and meet the minimum tensile strength requirements for natural animal leather applications such as making watch straps. The maximum force, tensile strength, displacement, and elongation of cultivated leather may be measured from stress-strain curves generated by testing the material (such as cultivated leather) under applied force using tensile testers such as the Shimadzu Autograph AGS-X tensile tester.

[0094] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may have a maximum force of at least about 1 N, at least about 2 N, at least about 3 N, at least about 4 N, at least about 5 N, at least about 6 N, at least about 7 N, at least about 8 N, at least about 9 N, at least about 10 N, at least about 11 N, at least about 12 N, at least about 13 N, at least about 14 N, at least about 15 N, at least about 16 N, or at least about 20 N.

[0095] As used herein, the terms “maximum force” and “max force” are interchangeable and in the context of cultivated leather refer to the maximum applied tensile force (N) the cultivated leather can withstand prior to failure (breakage) of the cultivated leather. As used herein, the term “tensile force” refers to aforce (force = mass of an object * acceleration) acting on a material typically acting to elongate the material, such as a pulling force or a stretching force. Therefore, the maximum force is the maximum tensile force the cultivated leather can withstand whilst being stretched or pulled before breaking. Typically, maximum force is measured in units of force, such as Newtons (N).

[0096] Suitably, the cultivated leather may have a maximum force of at least about 1 N, at least about 5 N, at least about IO N, at least about 15 N, at least about 20 N, at least about 30 N, at least about 40 N, at least about 50 N, at least about 60 N, at least about 70 N, at least about 80 N, at least about 90 N, at least about 100 N, at least about 150 N, at least about 200 N, at least about 300 N, at least 400, or at least about 500 N.

[0097] Suitably, the thin cultivated leather may have a maximum force of at least about 1 N, at least about 5 N, at least about 10 N, at least about 15 N, at least about 20 N, at least about 30 N, at least about 40 N, at least about 50 N, at least about 60 N, at least about 70 N, at least about 80 N, at least about 90 N, at least about 100 N, at least about 150 N, at least about 200 N, at least about 300 N, at least about 400 N, or at least about 500 N.

[0098] Suitably, the ultra-thin cultivated leather may have a maximum force of at least about 1 N, at least about 5 N, at least about 10 N, at least about 15 N, at least about 20 N, at least about 30 N, at least about 40 N, at least about 50 N, at least about 60 N, at least about 70 N, at least about 80 N, at least about 90 N, at least about 100 N, at least about 150 N, at least about 200 N, at least about 300 N, at least about 400N, or at least about 500 N. Suitably, the ultra-thin cultivated leather may have a maximum force of at least about 5 N.

[0099] Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may have a maximum force of at most about 1 N, at most about 5N, at most about 10 N, at most about 15 N, at most about 20 N, at most about 30 N, at most about 40 N, at most about 50 N, at most about 60 N, at most about 70 N, at most about 80 N, at most about 90 N, at most about 100 N, at most about 150 N, at most about 200 N, at most about 300 N, at most about 400 N, or at most about 500 N.

[0100] Suitably, the cultivated leather may have a maximum force of at most about 1 N, at most about 5N, at most about 10 N, at most about 15 N, at most about 20 N, at most about 30 N, at most about 40 N, at most about 50 N, at most about 60 N, at most about 70 N, at most about 80 N, at most about 90 N, at most about 100 N, at most about 150 N, at most about 200 N, at most about 300 N, at most about 400 N, or at most about 500 N.

[0101] Suitably, the thin cultivated leather may have a maximum force of at most about 1 N, at most about 5N, at most about 10 N, at most about 15 N, at most about 20 N, at most about 30 N, at most about 40 N, at most about 50 N, at most about 60 N, at most about 70 N, at most about 80 N, at most about 90 N, at most about 100 N, at most about 150 N, at most about 200 N, at most about 300 N, at most about 400 N, or at most about 500 N.Suitably, the ultra-thin cultivated leather, may have a maximum force of at most about 1 N, at most about 5N, at most about IO N, at most about 15 N, at most about 20 N, at most about 30 N, at most about 40 N, at most about 50 N, at most about 60 N, at most about 70 N, at most about 80 N, at most about 90 N, at most about 100 N, at most about 150 N, at most about 200 N, at most about 300 N, at most about 400 N, or at most about 500 N. Suitably, the ultra-thin cultivated leather, may have a maximum force of at most about 16 N.

[0102] Suitably, the ultra-thin cultivated leather may have a maximum force of between about 4 N and 16 N. Suitably, the ultra-thin cultivated leather may have a maximum force of between about 4.6 N and 15.8 N. Suitably, the ultra-thin cultivated leather may have a maximum force of about 4.6 N. Suitably, the ultra-thin cultivated leather may have a maximum force of about 15.8 N.

[0103] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may have a maximum displacement of at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, at least about 10 mm, at least about 20 mm, at least about 50 mm, or at least about 100 mm.

[0104] As used herein, the term “displacement” in the context of cultivated leather refers to the maximum strain (or stretch) of the cultivated leather under applied tensile force prior to failure (breakage) of the cultivated leather. Typically, displacement is in units of length, such as mm. Accordingly, if a sample is 10 mm long and is elongated 2 mm prior to breakage, the displacement will be 2 mm.

[0105] Suitably, the cultivated leather may have a displacement of at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, at least about 10 mm, at least about 20 mm, at least about 50 mm, or at least about 100 mm.

[0106] Suitably, the thin cultivated leather may have a displacement of at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, at least about 10 mm, at least about 20 mm, at least about 50 mm, or at least about 100 mm.

[0107] Suitably, the ultra-thin cultivated leather may have a displacement of at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, at least about 10 mm, at least about 20 mm, at least about 50 mm, or at least about 100 mm. Suitably, the ultra-thin cultivated leather may have a displacement of at least about 4 mm.

[0108] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may have a maximum displacement of at most about 2 mm, at most about 3 mm, at most about 4 mm, at most about5 mm, at most about 6 mm, at most about 7 mm, at most about 8 mm, at most about 9 mm, at most about 10 mm, at most about 20 mm, at most about 50 mm, or at most about 100 mm.

[0109] Suitably, the cultivated leather, may have a displacement of at most about 2 mm, at most about 3 mm, at most about 4 mm, at most about 5 mm, at most about 6 mm, at most about 7 mm, at most about 8 mm, at most about 9 mm, at most about 10 mm, at most about 20 mm, at most about 50 mm, or at most about 100 mm.

[0110] Suitably, thin cultivated leather may have a displacement of at most about 2 mm, at most about 3 mm, at most about 4 mm, at most about 5 mm, at most about 6 mm, at most about 7 mm, at most about 8 mm, at most about 9 mm, at most about 10 mm, at most about 20 mm, at most about 50 mm, or at most about 100 mm.

[0111] Suitably, the ultra-thin cultivated leather may have a displacement of at most about 2 mm, at most about 3 mm, at most about 4 mm, at most about 5 mm, at most about 6 mm, at most about 7 mm, at most about 8 mm, at most about 9 mm, at most about 10 mm, at most about 20 mm, at most about 50 mm, or at most about 100 mm. Suitably, the ultra-thin cultivated leather may have a maximum displacement of at most about 9 mm.

[0112] Suitably, the ultra-thin cultivated leather may have a displacement of between about 4 mm and 9 mm. Suitably, the ultra-thin cultivated leather may have a maximum displacement of between about 4.5 mm and 8.5 mm. Suitably, the ultra-thin cultivated leather may have a maximum displacement of about 4.5 mm. Suitably, the ultra-thin cultivated leather may have a maximum displacement of about 8.5 mm. Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may have a tensile strength of >1 N / mm2, >2 N / mm2, >3 N / mm2, >4 N / mm2, >5 N / mm2, >6 N / mm2, >7 N / mm2, >8 N / mm2, >9 N / mm2, >10 N / mm2, >11 N / mm2, >12 N / mm2, >13 N / mm2, >14 N / mm2, >15 N / mm2, >20 N / mm2, >30 N / mm2, >40 N / mm2, >50 N / mm2, >60 N / mm2, >70 N / mm2, >80 N / mm2, >90 N / mm2, or >100 N / mm2. Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather, may have a tensile strength of >10 N / mm2.

[0113] As used herein, the terms “tensile stress”, “tensile strength”, and “peak stress” are interchangeable and in the context of cultivated leather refers to the tensile force the cultivated leather can take across a specific area before it either permanently stretches or fails (breaks). More specifically, tensile strength = Force / Area, and in turn is the force exerted per unit area in the stretch of the material. Typically, tensile strength is measured in force / area, such as N / mm2.

[0114] Suitably, the cultivated leather may have a tensile strength of >1 N / mm2, >2 N / mm2, >3 N / mm2, >4 N / mm2, >5 N / mm2, >6 N / mm2, >7 N / mm2, >8 N / mm2, >9 N / mm2, >10 N / mm2, >11 N / mm2, >12 N / mm2, >13 N / mm2, >14 N / mm2, >15 N / mm2, >20 N / mm2, >30 N / mm2, >40 N / mm2, >50 N / mm2, >60 N / mm2,>70 N / mm2, >80 N / mm2, >90 N / mm2, or >100 N / mm2. Suitably, the cultivated leather may have a tensile strength of >10 N / mm2.

[0115] Suitably, the thin cultivated leather may have a tensile strength of >1 N / mm2, >2 N / mm2, >3 N / mm2, >4 N / mm2, >5 N / mm2, >6 N / mm2, >7 N / mm2, >8 N / mm2, >9 N / mm2, >10 N / mm2, >11 N / mm2, >12 N / mm2, >13 N / mm2, >14 N / mm2, >15 N / mm2, >20 N / mm2, >30 N / mm2, >40 N / mm2, >50 N / mm2, >60 N / mm2, >70 N / mm2, >80 N / mm2, >90 N / mm2, or >100 N / mm2. Suitably, the thin cultivated leather may have a tensile strength of >10 N / mm2.

[0116] Suitably, the ultra-thin cultivated leather may have a tensile strength of >1 N / mm2, >2 N / mm2, >3 N / mm2, >4 N / mm2, >5 N / mm2, >6 N / mm2, >7 N / mm2, >8 N / mm2, >9 N / mm2, >10 N / mm2, >11 N / mm2, >12 N / mm2, >13 N / mm2, >14 N / mm2, >15 N / mm2, >20 N / mm2, >30 N / mm2, >40 N / mm2, >50 N / mm2, >60 N / mm2, >70 N / mm2, >80 N / mm2, >90 N / mm2, or >100 N / mm2. Suitably, the ultra-thin cultivated leather may have a tensile strength of >10 N / mm2.

[0117] Suitably, the ultra-thin cultivated leather may have a tensile strength of between 9 N / mm2and 14 N / mm2. Suitably, the ultra-thin cultivated leather may have a tensile strength of between 9.2 N / mm2and 13.2 N / mm2. Suitably, the ultra-thin cultivated leather may have a tensile strength of 9.2 N / mm2. Suitably, the ultra-thin cultivated leather may have a tensile strength of 13.2 N / mm2.

[0118] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may have an elongation of at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, or at least about 150%. As used herein, the term “elongation” in the context of the cultivated leather of the invention refers to the increase in length of the cultivated leather under applied tensile force prior to failure (breakage) of the cultivated leather as a proportion of cultivated leather pre-testing. Typically, elongation is measured as a percentage (%). Accordingly, if a sample is 10 mm long and is elongated 2 mm prior to breakage, the elongation will be 20%.

[0119] Suitably, the cultivated leather may have an elongation of at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, or at least about 150%.

[0120] Suitably, the ultra-thin cultivated leather may have an elongation of at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, or at least about 150%.

[0121] Suitably, the ultra-thin cultivated leather may have an elongation of at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, or at least about 150%. Suitably, the ultra-thin cultivated leather may have an elongation of at least about 8%.

[0122] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may have an elongation of at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 80%, at most about 90%, at most about 100%, or at most about 150%.

[0123] Suitably, the cultivated leather may have an elongation of at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 80%, at most about 90%, at most about 100%, or at most about 150%.

[0124] Suitably, the thin cultivated leather may have an elongation of at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 80%, at most about 90%, at most about 100%, or at most about 150%.

[0125] Suitably, the ultra-thin cultivated leather may have an elongation of at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 80%, at most about 90%, at most about 100%, or at most about 150%. Suitably, the ultra-thin cultivated leather may have an elongation of at most about 16%.

[0126] Suitably, the ultra-thin cultivated leather may have an elongation of between about 8 and about 16%. Suitably, the ultra-thin cultivated leather may have an elongation ofbetween about 8.4 and about 15.8%. Suitably, the ultra-thin cultivated leather may have an elongation of about 8.4%. Suitably, the ultra-thin cultivated leather may have an elongation of about 15.8%.Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may have at least similar compositions to leather obtained from an animal. Suitably, the cultivated leather, thin cultivated leather, and ultra-thin cultivated leather, may comprise ECM proteins as observed in leather produced from natural animal skins.

[0127] Suitably, the cultivated leather may comprise ECM proteins as observed in leather produced from natural animal skins. Suitably, the thin cultivated leather may comprise ECM proteins as observed in leather produced from natural animal skins. Suitably, the ultra-thin cultivated leather may comprise ECM proteins as observed in leather produced from natural animal skins.

[0128] Suitably, the ECM proteins may be cell-specific ECM components. Suitably, the ECM proteins may comprise one or more of: soluble and insoluble collagen, hyaluronic acid, non-sulphated and sulphated glucosaminoglycans (GAGs and sGAGs, respectively), elastin, fibronectin, decorin, and biglycan. Suitably, the ECM proteins may cross-link. Suitably, the cross-linking of ECM proteins may form multi-stratified, structured cell layers.

[0129] As outlined herein, the inventors surprisingly identified that the cellular composites (and therefore the cultivated leather products produced therefrom) have significantly higher concentrations of elastin than porcine dermis. Without wishing to be bound by theory, the inventors believe that because elastin is more thermostable than collagen, higher concentrations might account for the higher onset temperatures observed for cellular composites and cultivated leather produced therefrom.

[0130] Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather may have a reduced, similar, or enhanced concentration of ECM proteins in comparison to leather produced from natural animal skins.

[0131] Suitably, the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather may have an enhanced concentration of total elastin in comparison to leather produced from natural animal skin. Suitably, the cultivated leather may have enhanced levels of total elastin in comparison to leather produced from natural animal skin. Suitably, the thin cultivated leather may have enhanced levels of total elastin in comparison to leather produced from natural animal skin. Suitably, the ultra-thin cultivated leather may have enhanced levels of total elastin in comparison to leather produced from natural animal skin.

[0132] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather may have a total elastin concentration of at least about 50 mg / g dry tissue, at least about 60 mg / g dry tissue, at least about 70 mg / g dry tissue, at least about 80 mg / g dry tissue, at least about 90 mg / g dry tissue, at least about 100 mg / g dry tissue, at least about 110 mg / g dry tissue, at least about 120 mg / g dry tissue, at least about 130 mg / g dry tissue, at least about 140 mg / g dry tissue, or at least about 150 mg / g dry tissue.Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather may have a total elastin concentration of at least about 120 mg / g dry tissue.

[0133] Suitably, the cultivated leather may have a total elastin concentration of at least about 50 mg / g dry tissue, at least about 60 mg / g dry tissue, at least about 70 mg / g dry tissue, at least about 80 mg / g dry tissue, at least about 90 mg / g dry tissue, at least about 100 mg / g dry tissue, at least about 110 mg / g dry tissue, at least about 120 mg / g dry tissue, at least about 130 mg / g dry tissue, at least about 140 mg / g dry tissue, or at least about 150 mg / g dry tissue. Suitably, the cultivated leather may have a total elastin concentration of at least about 120 mg / g dry tissue.

[0134] Suitably, the thin cultivated leather may have a total elastin concentration of at least about 50 mg / g dry tissue, at least about 60 mg / g dry tissue, at least about 70 mg / g dry tissue, at least about 80 mg / g dry tissue, at least about 90 mg / g dry tissue, at least about 100 mg / g dry tissue, at least about 110 mg / g dry tissue, at least about 120 mg / g dry tissue, at least about 130 mg / g dry tissue, at least about 140 mg / g dry tissue, or at least about 150 mg / g dry tissue. Suitably, the thin cultivated leather may have a total elastin concentration of at least about 120 mg / g dry tissue.

[0135] Suitably, the ultra-thin cultivated leather may have a total elastin concentration of at least about 50 mg / g dry tissue, at least about 60 mg / g dry tissue, at least about 70 mg / g dry tissue, at least about 80 mg / g dry tissue, at least about 90 mg / g dry tissue, at least about 100 mg / g dry tissue, at least about 110 mg / g dry tissue, at least about 120 mg / g dry tissue, at least about 130 mg / g dry tissue, at least about 140 mg / g dry tissue, or at least about 150 mg / g dry tissue. Suitably, the ultra-thin cultivated leather may have a total elastin concentration of at least about 120 mg / g dry tissue.

[0136] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather may have total elastin concentrations of at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold of that of leather produced from natural animal skin. Suitably, the cultivated leather, thin cultivated leather, and / or ultrathin cultivated leather may have total elastin concentrations of at least about 4-fold of that of leather produced from natural animal skin. Suitably, the natural animal skin is porcine dermis.

[0137] Suitably, the cultivated leather may have total elastin concentrations of at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5 -fold of that of leather produced from natural animal skin. Suitably, the cultivated leather may have total elastin concentrations of at least about 4-fold of that of leather produced from natural animal skin. Suitably, the natural animal skin is porcine dermis.

[0138] Suitably, the thin cultivated leather may have total elastin concentrations of at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5 -fold of that of leather produced from natural animal skin. Suitably, the thin cultivatedleather may have total elastin concentrations of at least about 4-fold of that of leather produced from natural animal skin. Suitably, the natural animal skin is porcine dermis.

[0139] Suitably, ultra-thin cultivated leather may have total elastin concentrations of at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5 -fold, at least about 4-fold, at least about 4.5-fold, or at least about 5 -fold of that of leather produced from natural animal skin. Suitably, the ultrathin cultivated leather may have total elastin concentrations of at least about 4-fold of that of leather produced from natural animal skin. Suitably, the natural animal skin is porcine dermis.

[0140] Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather may have a similar concentration of sGAGs to leather produced from natural animal skin. Suitably, the cultivated leather may have a similar concentration of sGAGs to leather produced from natural animal skin. Suitably, the thin cultivated leather may have a similar concentration of sGAGs to leather produced from natural animal skin. Suitably, the ultra-thin cultivated leather may have a similar concentration of sGAGs to leather produced from natural animal skin. Suitably, the natural animal skin is porcine dermis. Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather may have a total sGAGs concentration of at least about 1 mg / g dry tissue, at least about 1.5 mg / g dry tissue, at least about 2 mg / g dry tissue, at least about 2.5 mg / g dry tissue, at least about 3 mg / g dry tissue, or at least about 3.5 mg / g dry tissue. Suitably, the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather may have a total sGAGs concentration of at least about 2.5 mg / g dry tissue.

[0141] Suitably, the cultivated leather may have a total sGAGs concentration of at least about 1 mg / g dry tissue, at least about 1.5 mg / g dry tissue, at least about 2 mg / g dry tissue, at least about 2.5 mg / g dry tissue, at least about 3 mg / g dry tissue, or at least about 3.5 mg / g dry tissue. Suitably, the cultivated leather may have a total sGAGs concentration of at least about 2.5 mg / g dry tissue.

[0142] Suitably, the thin cultivated leather may have a total sGAGs concentration of at least about 1 mg / g dry tissue, at least about 1.5 mg / g dry tissue, at least about 2 mg / g dry tissue, at least about 2.5 mg / g dry tissue, at least about 3 mg / g dry tissue, or at least about 3.5 mg / g dry tissue. Suitably, the thin cultivated leather may have a total sGAGs concentration of at least about 2.5 mg / g dry tissue.

[0143] Suitably, the ultra-thin cultivated leather may have a total sGAGs concentration of at least about 1 mg / g dry tissue, at least about 1.5 mg / g dry tissue, at least about 2 mg / g dry tissue, at least about 2.5 mg / g dry tissue, at least about 3 mg / g dry tissue, or at least about 3.5 mg / g dry tissue. Suitably, the ultra-thin cultivated leather may have a total sGAGs concentration of at least about 2.5 mg / g dry tissue.

[0144] Suitably, the ECM proteins may comprise fibrillar collagens. Suitably, the collagen fibrils may be deposited in very dense layers, arranged in loose bundles.Suitably, the collagen comprising the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, may be cross-linked. Suitably, the collagen comprising the cultivated leather may be cross-linked. Suitably, the collagen comprising the thin cultivated leather may be cross-linked. Suitably, the collagen comprising the ultra-thin cultivated leather may be cross-linked.

[0145] The cultivated leather may be heterogeneous in structure. The heterogeneity of the structure may be obtained from the types of cells used in the method of cultivating leather in vitro.

[0146] Culturing

[0147] In the aspects of the present invention, the method for producing cultivated leather, thin cultivated leather and / or ultra-thin leather, comprises a step of culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer. Suitably, the method for producing cultivated leather, thin cultivated leather, and / or ultra-thin leather, may comprise a step repeating the step of culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer at least once to independently produce further cell layers.

[0148] As used herein, the term “culturing” refers to keeping cells in an artificial environment under conditions favouring growth, differentiation, continued viability, and / or ECM production and deposition. Culturing may be on a substrate or in suspension. As used herein, the term “culturing” can also refer to keeping a cell layer, animal tissue or cellular composite in an artificial environment under conditions favouring growth, differentiation, continued viability, and / or ECM production and deposition. Culturing may be on a substrate or in suspension.

[0149] As used herein, the terms "cell culture medium", “culture medium”, and "medium" (plural "media" in each case) refer to a nutritive solution for cultivating live cells and may be used interchangeably. Suitable substrates for culturing the cells may comprise naturally occurring and / or synthetic substrates. Non-limiting examples of naturally occurring substrates include ECM proteins, such as collagen, fibronectin, laminin, and hyaluronic acid. Non-limiting examples of synthetic substrates for cell culture include poly-L-lysine, glass, metal, polyester, and polystyrene. Suitably, the composition comprising one or more cells may be seeded on a substrate, wherein the substrate is polystyrene.

[0150] Suitably, during each and every stage of culturing, the culturing may occur at 37°C and 5% CO2 in a humidified environment. Suitably, during each and every stage of culturing, the cell media may be exchanged every 3-4 days of culture.

[0151] Suitably, a composition comprising one or more cells may be cultured (or incubated) in a serum-free medium at 37°C and 5% CO2 in a humidified environment to produce a primary cell layer. Suitably, during the step of culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, the cell media may be exchanged every 3-4 days of culture.Suitably, a composition comprising one or more cells, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells, may be cultured for 14 days in serum-free medium at 37°C and 5% CO2 in a humidified environment to produce a primary cell layer, wherein the cell media is exchanged every 3-4 days. Suitably, a composition comprising one or more cells, wherein the one or more cells comprise fibroblasts, may be cultured for 14 days in serum-free medium at 37°C and 5% CO2 in a humidified environment to produce a primary cell layer, wherein the cell media is exchanged every 3-4 days.

[0152] Suitably, the composition comprising one or more cells may be seeded on a substrate at a cell density of at least about IxlO3cells / cm2to at least about IxlO7cells / cm2. Suitably, the composition comprising one or more cells may be seeded on a substrate at a cell density of at least about IxlO3cells / cm2, IxlO4cells / cm2, IxlO5cells / cm2, IxlO6cells / cm2, or IxlO7cells / cm2. Suitably, the composition comprising one or more cells may be seeded on a substrate at a cell density of at least about IxlO4cells / cm2. Suitably, the composition comprising one or more cells, wherein the one or more cells comprise fibroblasts, may be seeded on a substrate at a cell density of at least about IxlO4cells / cm2.

[0153] As outlined in the examples herein, the present inventors have surprisingly found that when a composition comprising fibroblasts and / or pre-adipocytes is cultured this results in both an increase of cell number and an increase in ECM (such as collagen) production.

[0154] This finding gives rise to the example of culturing a composition comprising fibroblasts during primary cell layer production and independent further cell layer production. In the case where a composition comprising of one type of cells, such as fibroblasts, is cultured, this is referred to as “mono-culture” herein.

[0155] As used herein, the term “fibroblast” refers to a cell that has differentiated and become specialised in synthesising ECM, such as collagen. Typically, fibroblasts may be characterised by the native environment in which they reside. For example, fibroblasts may be characterised into skin fibroblasts, lung fibroblasts, skeletal muscle fibroblasts, and heart fibroblasts.

[0156] Suitably, the fibroblast is a skin fibroblast. Non-limiting examples of skin fibroblasts include papillary dermal fibroblasts, reticular dermal fibroblasts, hypodermal fibroblasts, dermal sheath fibroblasts, or dermal papilla fibroblasts. Papillary dermal fibroblast markers may include CD26 / Dpp4+, Dlklneg, Scal / Ly6aneg, Blimp 1+, EphB2+, Lrigl+, and / or Trpsl+. Reticular dermal fibroblasts markers may include CD26 / Dpp4negDlkl+Scal / Ly6aneg. Hypodermal fibroblasts markers may include CD26neg, Dlklneg, and / or Scal / Ly6a . Dermal sheath fibroblasts markers may include aSMA+, Itga8+, Itga5+, Col l lai . Acan+, CD200+, MyhlO+, Mlck+, and / or Myl9+. Dermal papilla fibroblasts markers may include Sox2+, Lefl+, Crabpl+, Rspo3+, Corin4, Vcrsican . and / or Alkaline phosphatase4. Pan-fibroblast markers may include Pdgfira , Dpt4, Colla24, Col3al4, Twist24, and / or Vimentin4.As used herein, the term “fibroblast progenitor cell” refers to a cell having the ability to differentiate into a mature fibroblast. Non-limiting examples of fibroblast progenitor cells include a common fibroblast progenitor cell, a reticular fibroblast progenitor cell, a hypodermal fibroblast progenitor cell, and a papillary dermal fibroblast progenitor cell. Common fibroblast progenitor cell markers may include Pdgfrof, DLK+, and / or LRIG1+. Papillary dermal fibroblast progenitor cell markers may include Pdgfrof, DLK . BLIMP1+and / or LRIG1+. Reticular fibroblast progenitor cell markers may include Pdgfrof, DLK+, BLIMP 1 . Hypodermal fibroblast progenitor cell markers may include Pdgfra . DLK+, BLIMP 1 .

[0157] Suitably, fibroblasts and / or fibroblast progenitor cells may be either naturally occurring in the adult body or the embryo of an animal or generated artificially by bioengineering. Suitably, the animal may be extant or extinct. An artificially generated fibroblast and / or fibroblast progenitor cell may be described as an artificial fibroblast and / or fibroblast progenitor cell. An artificial fibroblast and / or fibroblast progenitor cell may be engineered to produce more ECM, deposit more ECM, have increased proliferation, be immortalised, be adapted to reduced nutrient media (minimal media), express molecular machines or bionanites, express molecular tags and / or other DNA-coded data, have resistance to shear or oxidative stress, or have resistance to microbial or phage infection. An artificial fibroblast and / or fibroblast progenitor cell may have the ability to cross-link ECM components, such as collagen, or to express cytokines, hormones, enzymes, vitamins, dyes, and / or growth factors from the same or different species.

[0158] Suitably, the fibroblast and / or fibroblast progenitor cell may be isolated from an adult animal and / or animal embryo.

[0159] Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts and / or fibroblast progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts and / or fibroblast progenitor cells.

[0160] Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts. Suitably, the composition may comprise, consist of, or substantially consist of fibroblast progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblast progenitor cells.

[0161] Suitably, the fibroblast may be a dermal fibroblast. Dermal fibroblasts are found within the dermis layer of skin and are responsible for generating connective tissue which organises the stratified squamous epithelial cells of the epidermis into a unified tissue. Suitably, the dermal fibroblast may be an equine dermal fibroblast. Suitably, the dermal fibroblast may be E. Derm dermal cells. E. Derm dermal cellsare a fibroblast of skin and are derived from the dermis of Equus caballus (Horse). E. Derm dermal cells are also known as E.Derm, E. Derm (NBL-6), NBL-6, Eq.Derm, and ECID. Suitably, the fibroblast may be a horse fibroblast.

[0162] As the person skilled in the art would appreciate, other animal dermal fibroblasts may find utility in this invention. Non-limiting examples include avian dermal fibroblasts (e.g., chicken, ostrich, dodo), mammalian dermal fibroblasts (e.g., mouse, cow, pig, human), reptilian dermal fibroblasts (e.g., snake, crocodile), fish dermal fibroblasts, amphibian dermal fibroblasts, and mollusc dermal fibroblasts. Suitably, the fibroblast may be chicken dermal fibroblast. Suitably, the chicken dermal fibroblast may be UMNSAH / DF-1.

[0163] UMNSAH / DF-1 (ATCC) is a spontaneously immortalised chicken (Gallus gallus) cell line derived from a 10-day old embryo in East Lansing Line (ELL-0) eggs. UMNSAH / DF-1 are dermal fibroblasts. As outlined in the examples herein, the present inventors have surprisingly found that when a composition comprising fibroblasts is co-cultured with pre-adipocytes, an increase in cell proliferation, and collagen production and deposition is observed. As used herein, the term “co-cultured” refers to culturing (i.e. keeping the cells in an artificial environment under conditions favouring growth, differentiation, and / or continued viability) two or more different cell types.

[0164] Suitably, the composition may further comprise adipocytes and / or adipose progenitor cells. Suitably, the composition may further comprise adipose progenitor cells. Suitably, the adipose progenitor cells are pre-adipocytes. Suitably, the composition may further comprise adipocytes.

[0165] As used herein, the term “adipocyte” refers to a cell that has differentiated and become specialised in the synthesis and / or storage of fat. In vivo, adipocytes are derived from mesenchymal stem cells which give rise to adipocytes through adipogenesis. Suitably, the adipocyte may be selected from the group consisting of a white adipocyte, beige adipocyte, and brown adipocyte. An adipocyte marker includes adiponectin (ADIPOQ), peroxisome proliferator-activated receptor y (PPARy), fatty acid-binding protein 4 (FABP4), cytoplasmic glycerol-3 -phosphate dehydrogenase (GPDH), Uncoupling Protein 1 (UCP1), and / or leptin (LEP), among others, and depending on adipocyte type. Suitably, the adipocyte may be a hypodermal adipocyte.

[0166] The term “adipose progenitor cell” refers to a cell having the ability to differentiate into a mature adipocyte. Suitably the adipose progenitor cell may be selected from the group consisting of a preadipocyte, an adipose stem cell and a mesenchymal stem cell. Suitably, the adipose progenitor cell (such as a pre-adipocyte cell) may be positive for CD34, CD90 and CD271 and negative for CD31, CD45, CD104b, CD105, and CD146.

[0167] Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts and adipocytes. Suitably, the composition may comprise, consist of, or substantially consist of fibroblastsand adipocytes. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts and adipocytes.

[0168] Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts and adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts and adipose progenitor cells

[0169] Suitably, the composition may comprise, consist of, or substantially consist of fibroblast progenitor cells and adipocytes. Suitably, the composition may comprise, consist of, or substantially consist of fibroblast progenitor cells and adipocytes. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblast progenitor cells and adipocytes.

[0170] Suitably, the composition may comprise, consist of, or substantially consist of fibroblast progenitor cells and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblast progenitor cells and adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblast progenitor cells and adipose progenitor cells.

[0171] Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, fibroblast progenitor cells, and adipocytes. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, fibroblast progenitor cells, and adipocytes. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts, fibroblast progenitor cells, and adipocytes

[0172] Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, fibroblast progenitor cells, and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, fibroblast progenitor cells, and adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts, fibroblast progenitor cells, and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, adipocytes and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, adipocytes and adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts, adipocytes and adipose progenitor cells

[0173] Suitably, the composition may comprise, consist of, or substantially consist of fibroblast progenitor cells, adipocytes and adipose progenitor cells. Suitably, the composition may comprise, consist of, orsubstantially consist of fibroblast progenitor cells, adipocytes and adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblast progenitor cells, adipocytes and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, fibroblast progenitor cells, adipocytes and adipose progenitor cells. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts, fibroblast progenitor cells, adipocytes and adipose progenitor cells. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts, fibroblast progenitor cells, adipocytes and adipose progenitor cells.

[0174] Suitably, the adipose progenitor cell is a pre-adipocyte. Suitably, the pre-adipocyte may be a porcine pre-adipocyte cell. Suitably, the pre-adipocyte may be a hypodermal pre-adipocyte. Suitably, the pre-adipocyte may be from the porcine hypodermis. Suitably, the pre-adipocyte may be a porcine hypodermal pre-adipocyte. Suitably, the porcine hypodermal pre-adipocyte may be a CPF4-A cell. Suitably, the composition may comprise, consist of, or substantially consist of fibroblasts and preadipocytes. By substantially consist of it is meant that at least about 90%, at least about 95%, or at least about 99% of the cells in the composition are fibroblasts and pre-adipocytes.

[0175] Suitably, the composition may comprise dermal fibroblasts and hypodermal pre-adipocytes at a percent ratio.

[0176] Suitably, the composition may comprise dermal fibroblasts and hypodermal pre-adipocytes at a 99:1 percent ratio, at a 98:2 percent ratio, at a 97:3 percent ratio, at a 96:4 percent ratio, at a 95:5 percent ratio, or at a 90:10 percent ratio. Suitably, the composition may comprise dermal fibroblasts and hypodermal pre-adipocytes at a 95:5 percent ratio. Suitably, the composition comprising dermal fibroblasts and hypodermal pre-adipocytes may be seeded on a substrate at a cell density of at least about IxlO3cells / cm2to at least about 1x107 cells / cm2. Suitably, the composition comprising one or more cells may be seeded on a substrate at a cell density of at least about IxlO3cells / cm2, IxlO4cells / cm2, IxlO5cells / cm2, or IxlO6cells / cm2, or IxlO7cells / cm2. Suitably, the composition comprising dermal fibroblasts and hypodermal pre-adipocytes may be seeded on a substrate at a cell density of at least about IxlO4cells / cm2.

[0177] As the skilled person would appreciate, culturing a composition of one or more cells, wherein the one or more cells are, for example, fibroblast progenitor cells, may result in a composition further comprising fibroblasts if at least a proportion of fibroblast progenitor cells differentiate into fibroblasts during culture. Likewise, culturing a composition of one or more cells, wherein the one or more cells are, for example, fibroblast cells and adipose progenitor cells (such as pre-adipocytes), may result in a composition further comprising adipocytes if at least a proportion of adipose progenitor cells (such as pre-adipocytes) differentiate into adipocytes during culture.The person skilled in the art would appreciate that the fibroblasts, fibroblast progenitor cells, adipocytes, and / or adipose progenitor cells may be from the same animal origin or distinct animal origin. Suitably, the fibroblasts, fibroblast progenitor cells, adipocytes, and / or adipose progenitor cells are from the same animal origin. Suitably, the fibroblasts, fibroblast progenitor cells, adipocytes, and / or adipose progenitor cells are from a distinct animal origin as outlined above. Suitably, the fibroblast and / or fibroblast progenitor cell may be from Equine origin. Suitably, the adipocyte and / or adipose progenitor cells may be from Porcine origin. Suitably, the adipose progenitor cells may be pre-adipocyte cells, wherein the pre-adipocyte cells are from Porcine origin.

[0178] Various natural animal tissue samples are discussed in the Examples as comparators for cultivated leather. These include mammalian dermis, porcine dermis, horse skin, reticular dermis, and papillary dermis. Mammalian dermis may be derived from an appropriate mammal. Reticular dermis is the thick bottom layer of the dermis. The reticular dermis has blood vessels and connective tissue that supports the skin. The papillary dermis is the thin top layer of the dermis. The papillary dermis has connective tissue and blood vessels that give nutrients to the epidermis (the outer layer of the skin). The Examples show that in porcine dermis the collagen fibres are arranged in long, highly-compacted, regionally-aligned, and considerably irregular bundles.

[0179] Producing primary cell layer and further cell layers

[0180] The compositions of the invention outlined above may be cultured to produce at least one cell layer. Suitably, the at least one cell layer comprises fibroblasts and / or fibroblast progenitor cells and may further comprise adipocytes and / or adipose progenitor cells.

[0181] As used herein, the term “cell layer” refers to a population of cells at least one cell in thickness. The cell layer may be irregular in structure or a globule. In the context of the present disclosure, where reference is made to an “animal tissue”, the animal tissue is a cell layer.

[0182] Suitably, the cell layer may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more cells in thickness. Suitably, the cell layer may be a monolayer, wherein the monolayer is 1 cell thick. Suitably, the cell layer may be at least 2 cells in thickness.

[0183] Suitably, the cell layer may be at least about 2 to at least about 4, at least about 2 to at least about 5, at least about 2 to at least about 6, at least about 2 to at least about 7, at least about 2 to at least about 8, at least about 2 to at least about 9, at least about 2 to at least about 10, at least about 2 to at least about 12, at least about 2 to at least about 15, at least about 2 to at least about 20, , at least about 2 to at least about 30, at least about 2 to at least about 40, at least about 2 to at least about 50, at least about 2 to at least about 60, at least about 2 to at least about 70, at least about 2 to at least about 80, at least about 2 to at least about 90, at least about 2 to at least about 100, at least about 2 to at least about 150, at least about 2 to at least about 200, or at least about 2 to at least about 250 cells in thickness.Suitably, the cell layer may be about 3 to about 5 cells in thickness. Suitably, the cell layer may be 3 to 5 cells in thickness.

[0184] Suitably, the cell layer may be at least about 0.005 mm thick, at least about 0.01 mm thick, at least about 0.02 mm thick, at least about 0.04 mm thick, at least about 0.06 mm thick, at least about 0.08 mm thick, at least about 0.1 mm thick, at least about 0.15 mm thick, or at least about 0.2 mm thick.

[0185] Suitably, the cell layer may be between about 0.005 mm and about 0.2 mm thick, between about 0.01 mm and about 0.2 mm thick, between about 0.02 mm and about 0.2 mm thick, between about 0.04 mm and about 0.2 mm thick, between about 0.06 mm and about 0.2 mm thick, between about 0.08 mm and about 0.2 mm thick, between about 0.1 mm and about 0.2 mm thick, or between about 0.15 mm and about 0.2 mm thick.

[0186] Suitably, the cell layer may be between about 0.005 mm and about 0.2 mm thick, between about 0.005 mm and about 0.15 mm thick, between about 0.005 mm and about 0.1 mm thick, between about 0.005 mm and about 0.1 mm thick, between about 0.005 mm and about 0.08 mm thick, between about 0.005 mm and about 0.06 mm thick, between about 0.005 mm and about 0.04 mm thick, between about 0.005 mm and about 0.02 mm thick, or between about 0.005 mm and about 0.01 mm thick.

[0187] Suitably, the cell layer may be between about 0.005 mm and about 0.2 mm thick.

[0188] The initial cell layer is referred to as the “primary cell layer” herein i.e. this is the first cell layer produced. Cell layers which are not the primary cell layer are referred to as “further cell layers”. Further cell layers have been produced independently of the primary cell layer. In other words, the further cell layers are produced separately by culturing a composition comprising one or more cells.

[0189] Accordingly, the primary cell layer may be between about 0.005 mm and about 0.2 mm thick.

[0190] Suitably, the step of culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer is repeated at least once to produce at least one further cell layer. Accordingly, the further cell layers may be between about 0.005 mm and about 0.2 mm thick. This step may be repeated to produce at least 2, at least 12, at least 25, at least 50, at least 100, at least 200, at least 500, or at least 1000 cell layers in total. Suitably, this step may be repeated to produce at most 12, at most 25, at most 50, at most 100, at most 200, at most 500, or at most 1000 cell layers in total. As appreciated by the person skilled in the art, production of a further cell layer is not restricted to the same composition used for the primary cell layer. For example, the primary cell layer could be produced from the culture of a composition comprising fibroblasts and at least one of the further cell layers could be produced from the culture of a composition comprising fibroblasts and pre-adipocytes.

[0191] Suitably, the step of culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer may not utilise an exogenous scaffold. Suitably, the step repeating the stepof culturing a composition comprising one or more cells in serum-free medium to independently produce further cell layers may not utilise an exogenous scaffold.

[0192] Suitably, in the method of producing cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather, each step may not utilise an exogenous scaffold.

[0193] In turn, the resulting cell layers (primary cell layers and further cell layers) may not comprise a scaffold. Suitably, the cell layers (primary cell layers and further cell layers) may not comprise a scaffold. As used herein, the term “scaffold” refers to an exogenous scaffold. Suitably, the cell layers (primary cell layers and further cell layers) may not comprise an exogenous scaffold.

[0194] In the context of the present disclosure, the ECM is not classified as a scaffold, as it is natively produced by the cells of the cell layers. Accordingly, with the ECM present in the cell layers (primary cell layers and further cell layers) described herein, it may be said the cell layers are scaffold-free, or free of an exogenous scaffold.

[0195] Suitably, the cell layers produced in serum-free medium may have a higher wet weight than cell layers produced in serum -containing medium. Suitably, the cell layers produced in serum -free medium may have a lower range of variation in wet weight than cell layers produced in serum -containing medium. Suitably, the cell layers produced in serum-free medium may have a higher production yield compared to cell layers produced in serum-containing medium. Suitably, the cell layers produced in serum-free medium may have a lower range of variation in production yield than cell layers produced in serumcontaining medium.

[0196] Suitably, the cell layers produced in serum-free medium may have a lower cell layer area shrinkage than cell layers produced in serum -containing medium.

[0197] Suitably, the cell layers produced in serum -free medium may have at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% lower cell layer area shrinkage than cell layers produced in serum -containing medium. Suitably, the cell layers produced in serum-free medium may have at least 5.5% lower cell layer area shrinkage than cell layers produced in serum-containing medium. Suitably, the cell layers produced in serum-free medium may have at least 14% lower cell layer area shrinkage than cell layers produced in serumcontaining medium. Suitably, the cell layers produced in serum-free medium may have at least 18% lower cell layer area shrinkage than cell layers produced in serum -containing medium.

[0198] Suitably, the cell layers produced in serum -free medium may have a wet weight of at least about 1.5 mg / cm2, at least about 1.8 mg / cm2, at least about 2.0 mg / cm2, at least about 2.1 mg / cm2, at least about 2.5 mg / cm2, at least about 2.7 mg / cm2, at least about 3.0 mg / cm2, at least about 4.0 mg / cm2, at leastabout 5.0 mg / cm2, at least about 7.0 mg / cm2, at least about 10.0 mg / cm2, at least about 20.0 mg / cm2, or at least about 30.0 mg / mm2. Suitably, the cell layers produced in serum-free medium may have a wet weight of at least about 2.1 mg / cm2(fibroblast monoculture). Suitably, the cell layers produced in serum -free medium may have a wet weight of at least about 2.7 mg / cm2(fibroblast and pre-adipocyte co-culture).

[0199] Suitably, the cell layers produced in serum-free medium from monoculture of fibroblasts and / or coculture of fibroblasts and pre-adipocytes may have a production yield of at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 140%, at least about 145%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, or at least about 500% of cell layers produced by fibroblast monocultures in serum -containing medium.

[0200] Suitably, the cell layers produced in serum-free medium from monoculture of fibroblasts may have a production yield of at least about 115% of cell layers produced by fibroblast monocultures in serumcontaining medium.

[0201] Suitably, the cell layers produced in serum-free medium from co-culture of fibroblasts and pre-adipocytes may have a production yield of at least about 150% of cell layers produced by fibroblast monocultures in serum -containing medium.

[0202] Serum-free medium

[0203] As outlined in the examples herein, the present inventors have surprisingly found that when a composition comprising fibroblasts is cultured in serum-free medium, or co-cultured with pre-adipocytes in serum-free medium this results in both an increase of cell number and an increase in ECM (such as collagen) production.

[0204] Suitably, the culturing may be in a serum -free medium.

[0205] As shown herein, the inventors show that the culturing of compositions to produce primary cell layers and / or further cell layers in the absence of serum (serum-free medium) prevent the shrinkage of cell layers.

[0206] Cell culture media wherein there is no detectable serum are referred to herein as “serum-free” cell culture media. Typically, for media that contains serum, the serum is added as a supplement at the start of, or during cell culture. The term “serum -free” cell culture medium therefore includes cell culture media which have not been supplemented with serum. The term “serum-free” is very well known in the art.

[0207] As would be clear to a person of skill in the art, “serum -free” media may comprise a number of additives and supplements, provided that it does not contain detectable levels of serum.Without wishing to be bound by theory, the inventors believe that it is due to the medium being serum-free that the cell layers (and cellular composites produced therefrom) produced by the method of the invention are resistant to contraction / shrinkage (i.e., the cell layers are non-contractile). Or in other words, the cell layers and cellular composites of the present invention do not exhibit shrinkage.

[0208] Suitably, the primary cell layers and / or further cell layers are non-contractile. As used herein, the term “non-contractile” refers to the absence of cell layer contraction. Methods of producing cultivated leather typically exhibit shrinkage during various steps of their methods and require mechanical restraints. Mechanical restraints can be used to clamp the edges of the cell layers or cellular composites and stretch them or prevent them from contracting or shrinking. The method of the present invention does not require mechanical restraints at any stage.

[0209] Suitably, in the step of culturing the composition comprising one or more cells to produce a cell layer, the composition may comprise a macromolecular crowding (MMC) agent. Therefore, in the context of the method for cultivating leather in vitro, it can be said that the step of culturing the composition comprising one or more cells to produce a primary and / or further cell layer is performed in the presence of an MMC agent.

[0210] Macromolecular crowding (MMC) is a biophysical phenomenon based on the principles of excluded-volume effect. It involves the addition of macromolecules to culture media. Following the principles of excluded volume effect (two molecules cannot occupy the same space at the same time), MMC significantly increases rates and kinetics of biochemical reactions and biological processes. According to the excluded volume effect theory, the volume of a solution that is excluded to a particular molecule is dependent on the sum of nonspecific hindrances (governed by size and shape) and electrostatic repulsions (governed by electrical charge) between the background molecules. Crowding is a result of the reduction of the available solvent volume by a macromolecule, which can be mobile or fixed. Crowding hinders solute diffusion, thereby increasing the effective solute concentration. This, in turn, increases the chemical potential of the solute. Crowding can therefore shift reaction equilibria and change the rates of chemical reactions. Crowding has therefore been used extensively to study polymer looping dynamic properties, DNA structure, condensation, replication, and stability, for example. Macromolecular crowding influences many critical processes including cell adhesion, migration, proliferation as well as extracellular matrix formation and remodelling.

[0211] These effects have been shown herein to positively affect the cell proliferation and ECM (such as collagen) production and deposition by fibroblasts alone or co-cultured with pre-adipocyte and / or adipocyte cells during the steps of culturing compositions to produce primary cell layers and further cell layers.Several MMC agents are known. In the context of the serum-free medium for culturing compositions described herein, the MMC agent may be selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0212] Suitably, the MMC agent may be selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0213] Suitably, the step of culturing compositions may be performed in serum-free medium comprising a MMC agent. Suitably, the MMC agent may be selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0214] Suitable, in the methods of the present discourse, the step of culturing compositions may be performed in serum-free media comprising an MMC agent selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

[0215] Polyethylene glycol (PEG) is a commonly used macromolecular crowder having effects on in vitro experiments, such as influencing ligand affinity and rate of enzymatic reaction and promoting extracellular matrix deposition when used with serum. Polyethylene glycol is prepared by polymerization of ethylene oxide and is commercially available over a wide range of molecular weights, from 300 Da to 10,000 kDa (Alister et al., Angewandte Chemie International Edition Volume 48, Issue 7 p. 1248-1252). For example, Polyethylene Glycol 8 kDa (PEG8) is a non-toxic polyether with a hydrophilic head allowing dilution in aqueous solutions. Macromolecular crowding induced by PEG8 can modulate reactions by increasing substrate binding at high concentrations as well as increasing proliferation bacterial strains. The molecular structure of PEG8 is well known, see for example Sigma-Aldrich, Cas Number 25322-68-3, linear formula: H(OCH2CH2)nOH and Hyun-Jun Jang et al., Toxicol Res. 2015 Jun; 31(2): 105-136.

[0216] An alternative PEG that may be used herein is PEG35. The molecular structure of PEG35 is also well known, see for example Hyun-Jun Jang et al., Toxicol Res. 2015 Jun; 31(2): 105-136.

[0217] Polyvinylpyrrolidone 40 kDa (PVP40) is a water-soluble polymer with a variety of uses including in beverage stabilization and medical uses where it is used as plasma volume expander. PVP40 in combination with serum has also been shown to be an effective macromolecular crowder with treatment increasing both collagen type I and proliferation of human dermal fibroblasts. The molecular structure of PVP40 is well known, see for example Sigma Aldrich, Cas Number 9003-39-8, linear formula (C6H9NO)n. and Kariduraganavar et al., Natural and Synthetic Biomedical polymers; chapter 1, 2014, pages 1 to 31.

[0218] Polyvinylpyrrolidone 360 kDa (referred to herein as PVP or PVP360) used as a macromolecular crowder together with serum has been shown to increase human dermal fibroblast cell proliferation and extracellular matrix deposition as indicated by increases in collagen type I production. The molecularstructure of PVP360 is also well known. See for example Kariduraganavar et al., Natural and Synthetic Biomedical polymers; chapter 1, 2014, pages 1 to 31.

[0219] Carrageenans (also known as carrageenins) are a family of natural linear sulphated polysaccharides that are extracted from red edible seaweeds. The most well-known and still most important red seaweed used for manufacturing the hydrophilic colloids to produce carrageenan is Chondrus crispus (Irish moss) which is a dark red parsley-like plant that grows attached to the rocks. Carrageenans are widely used in the food industry, for their gelling, thickening, and stabilizing properties. Their main application is in dairy and meat products, due to their strong binding to food proteins.

[0220] All carrageenans are high-molecular-weight polysaccharides and mainly made up of alternating 3-linked b-D-galac-topyranose (G-units) and 4-linked a-D-galactopyranose (D-units) or 4-linked 3,6-anhydro-a-D-galactopyranose (DA-units), forming the disaccharide repeating unit of carrageenans. There are three main commercial classes of carrageenan: Kappa carrageenan, Iota carrageenan and Lambda carrageenan. The molecular structures of different types of carrageenan are well known, see for example Hilliou, Adv Food Nutr Res. 2014;72: 17-43.

[0221] Suitably, the carrageenan may be lambda carrageenan. Carrageenans encompass a family of sulphated galactans originally extracted from red seaweed, where they have been found to play key structural functions. Traditionally, carrageenans are produced and used as crude extracts comprising different combinations of three molecular species defined by their sulphation and the presence or absence of anhygalactose. The lambda-carrageenan contains about 35% ester sulfate and no anhygalactose, making it highly soluble in water and unable to form gels. In contrast, the iota-carrageenan and kappa-carrageenan contain less ester sulfate and about 30-35% of 3,6-anhydrogalactose, making them insoluble in cold water and forming thermo-reversible gels in hot aqueous solutions. The different physicochemical and biological properties of lambda-carrageenan demonstrates that this molecular species is altogether distinct from the iota and kappa species, as well as from crude carrageenan extracts. Carrageenan has been proposed to be a promising macromolecular crowder (MMC) for tissue engineering due to its ability to increase extracellular matrix production. Treatment of adipose-derived stem cells with carrageenan and serum has been shown to enhance extracellular matrix deposition of collagen type I, II and V, to increase cell proliferation as well as increasing osteogenesis, chondrogenesis and decreasing adipogenesis.

[0222] Dextran sulphate (also known as DxS) is a biocompatible polyanionic polymer. It is a highly branched polysaccharide with 1,6 and 1,4 glycosidic linkages, with approximately 2.3 sulphate groups per glucosyl unit. Dextran sulphate is understood to facilitate deposition of extracellular matrix proteins, such as collagen.

[0223] Ficoll® 70 (also known as Poly(sucrose-co-epichlorhydrin)) is used as a macromolecular crowding agent in studies of cell volume signaling and protein refolding. It may be used in tissue engineering andmacromolecular conformation research for the development, evaluation and use of macromolecular crowding (MMC) systems and configurations. The molecular structure of Ficoll® 70 is well known, see for example Sigma Aldrich, Cas Number 72146-89-5, and CN102690364A.

[0224] Ficoll® 400 (also known as Polysucrose 400) is a non-ionic synthetic polymer of sucrose used for cell separation and organ isolation. The molecular structure of Ficoll® 400 is well known, see for example Sigma Aldrich, Cas Number 26873-85-8, and CN102690364A and https : / / pubchem.ncbi .nlm .nih.gov / compound / Ficoll-400.

[0225] PSS (also known as poly(sodium 4-styrenesulfonate)) is a sulfonated anionic polymer capable of enhancing collagen deposition. The molecular structure of PSS is well known, see for example Sigma Aldrich, Cas Number 25704-18-1.

[0226] The MMC agents described herein may be used as a single supplement (wherein only one MMC agent is added to the serum-free medium), or they may be used in combination. Suitable combinations may be identified by a person of skill in the art. For example, a combination of at least two MMC agents may be used. Alternatively, a combination of at least three or at least four MMC agents may be used. The MMC agents may be used at any appropriate concentration within the serum-free medium described herein.

[0227] Suitable final concentrations of MMC agents that are used in combination may be determined based on the disclosure provided herein, using routine methods known in the art.

[0228] Suitably, the culturing step to produce a primary cell layer and / or further cell layer may be in serum-free medium comprising DMEM / F12 basal medium supplemented with L-alanyl-L-glutamine dipeptide, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, Proliferum-M™ growth factor cocktail, and London-Mix™ macromolecular crowding solution. As the person skilled in the art would appreciate, other MMC agents or solutions may be used in place of London-Mix™ macromolecular crowding solution, and other growth factor cocktails can be used in place of Proliferum-M™ growth factor cocktail. Suitably, the culturing step to produce a primary cell layer and / or further cell layer may be in serum -free medium comprising DMEM / F12 basal medium supplemented with L-alanyl-L-glutamine dipeptide, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, a growth factor cocktail, and a macromolecular crowding solution.

[0229] Suitably, the culturing step to produce a primary cell layer and / or further cell layer may be in serum-free medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 5% (v / v) Proliferum-M™ growth factor cocktail, and 4% (v / v) London-Mix™ macromolecular crowding solution. In the Examples, this serum-free medium is referred to as serum-free proliferation medium (SPM). Suitably, the culturing step to produce a primary cell layer and / or further cell layer may be in serum-free medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution. In the Examples, this serum -free medium is referred to as serum -free tissue production medium (STPM).

[0230] Suitably, the culturing step to produce a primary cell layer may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, for at least about 7 days, for at least about 8 days, for at least about 9 days, for at least about 10 days, for at least about 11 days, for at least about 12 days, for at least about 13 days, for at least about 14 days, for at least about 21 days, or for at least about 28 days. Suitably, the culturing step to produce a primary cell layer may be for at least about 7 days. Suitably, the culturing step to produce a primary cell layer may be for at least about 14 days.

[0231] Suitably, the culturing step to produce a further cell layer may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, for at least about 7 days, for at least about 8 days, for at least about 9 days, for at least about 10 days, for at least about 11 days, for at least about 12 days, for at least about 13 days, for at least about 14 days, for at least about 21 days, or for at least about 28 days. Suitably, the culturing step to produce a further cell layer may be for at least about 7 days. Suitably, the culturing step to produce a further cell layer may be for at least about 14 days.

[0232] Suitably, the culturing step to produce a primary cell layer may comprise a first period and a second period. Suitably, the first period and second period of the culturing step to produce a primary cell layer may run concurrently. In other words, the second period will follow the first period.

[0233] Suitably, the first period of the culturing step to produce a primary cell layer may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, or for at least about 7 days. Suitably, the first period of the culturing step to produce a primary cell layer may be for at least about 7 days. Suitably, the first period of the culturing step to produce a primary cell layer may be for at least about 14 days.

[0234] Suitably, the second period of the culturing step to produce a primary cell layer may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, or for at least about 7 days. Suitably, the second period of the culturing step to produce a primary cell layer may be for at least about 7 days. Suitably, the second period of the culturing step to produce a primary cell layer may be for at least about 14 days.

[0235] Suitably, the culturing step to produce a further cell layer may comprise a first period and / or a second period. Suitably, the first period and second period of the culturing step to produce a further cell layer may run concurrently. In other words, the second period will follow the first period.Suitably, the first period of the culturing step to produce a further cell layer may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, or for at least about 7 days. Suitably, the first period of the culturing step to produce a further cell layer may be for at least about 7 days. Suitably, the first period of the culturing step to produce a further cell layer may be for at least about 14 days.

[0236] Suitably, the second period of the culturing step to produce a further cell layer may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, or for at least about 7 days. Suitably, the second period of the culturing step to produce a further cell layer may be for at least about 7 days. Suitably, the second period of the culturing step to produce a further cell layer may be for at least about 14 days.

[0237] Suitably, the first period of the culturing step to produce a primary cell layer and / or further cell layer may be in serum -free medium comprising DMEM / F12 basal medium supplemented with L-alanyl-L-glutamine dipeptide, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, Proliferum-M™ growth factor cocktail, and London-Mix™ macromolecular crowding solution. As the person skilled in the art would appreciate, other MMC agents or solutions may be used in place of London-Mix™ macromolecular crowding solution, and other growth factor cocktails can be used in place of Proliferum-M™ growth factor cocktail.

[0238] Suitably, the first period of the culturing step to produce a primary cell layer and / or further cell layer may be in serum -free medium comprising DMEM / L12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 5% (v / v) Proliferum-M™ growth factor cocktail, and 4% (v / v) London-Mix™ macromolecular crowding solution. This serum-free medium may also be referred to herein as serum-free proliferation medium (SPM).

[0239] The second period of the culturing step to produce a primary cell layer and / or further cell layer may be in serum-free medium with higher concentrations of compounds required for ECM production. This is in view of enhancing ECM production and deposition. Lor example, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate may be utilised at a higher concentration. Additionally, MMC agents may be used at a higher concentration.

[0240] Suitably, the second period of the culturing step to produce a primary cell layer and / or further cell layer may be in serum -free medium comprising DMEM / F12 basal medium supplemented with L-alanyl-L-glutamine dipeptide, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, Proliferum-M™ growth factor cocktail, and London-Mix™ macromolecular crowding solution. As the person skilled in the art would appreciate, other MMC agents or solutions may be used in place of London-Mix™ macromolecular crowding solution, and other growth factor cocktails can be used in place of Proliferum-M™ growth factor cocktail.Suitably, the second period of the culturing step to produce a primary cell layer and / or further cell layer may be in serum -free medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution. This serum-free medium may also be referred to herein as serum-free tissue production medium (STPM).

[0241] Suitably, the culture medium utilised in the methods of the present disclosure may comprise a further component selected from the group consisting of L-glutamine, L-alanyl-L-glutamine dipeptide, a natural and / or synthetic peptide growth factor (such as insulin, an insulin growth factor, a fibroblast growth factor, a transforming growth factor, a hepatocyte growth factor, a platelet-derived growth factors, and / or an epithelial growth factor), a neuregulin, a morphogenic protein, a vitamin (for example ascorbic acid, all-trans retinoic acid, thiamine, calciferol, tocopherol, riboflavin, niacin, biotin, pantothenic acid, folic acid, cobalamin, pyridoxine, and / or phytonadione), a carrier molecule (for example albumin, fetuin, and / or transferrin), an amino alcohol (such as ethanolamine, carbinoxamine, or chlorphenoxamine), a trace metal (such as selenium, zinc, aluminium, manganese, molybdenum, and / or iron) and a lipid (such as cholesterol, linoleic, stearic, oleic, palmitic, and / or uric acid), or analogues thereof, or activators or inhibitors of their molecular pathways.

[0242] Producing the cellular composite

[0243] Suitably, the method for producing cultivated leather, thin cultivated leather and / or ultra-thin leather may comprise a step of contacting the primary cell layer with the further cell layers to produce a cellular composite. Suitably, the method for producing cultivated leather may comprise a step of contacting the primary cell layer with the further cell layers to produce a cellular composite. Suitably, the method for producing thin cultivated leather may comprise a step of contacting the primary cell layer with the further cell layers to produce a cellular composite. Suitably, the method for producing ultra-thin cultivated leather may comprise a step of contacting the primary cell layer with the further cell layers to produce a cellular composite.

[0244] Suitably, the cellular composite of the present invention may comprise a primary cell layer and at least one further cell layer. As outlined elsewhere herein, the primary cell layer and further cell layers comprise fibroblasts, pre-adipocytes, and / or adipocytes. Accordingly, the cellular composite of the present invention may comprise fibroblasts, pre-adipocytes, and / or adipocytes.

[0245] Accordingly, the method of the present invention may provide a step for producing a cellular composite for tanning. This step may be referred to as “tissue assembly” herein. The aim of this step is to produce a cellular composite.

[0246] As used herein, the term “cellular composite” refers to an isolated artificial cell structure. The cellular composite may also be referred to as “cellular construct” herein. The cellular composite provides an exvivo organ structure that closely represents in vivo organ structure. In the present case, the organ is the skin, and more specifically the dermis layer of the skin.

[0247] As used herein, the term “contacting” refers to bringing the primary cell layer sufficiently close in proximity to at least one further cell layer to allow interaction. Suitably, contacting may mean physically contacting some or all of the primary cell layer with some or all of at least one further cell layer. Therefore, the step of contacting the primary cell layer with the further cell layers to produce a cellular composite results in a layered structure, comprising a primary cell layer and at least one further cell layer.

[0248] Suitably, the cellular composites may be produced (assembled) by the consecutive stacking of cell layers. Suitably, the cell layers may be stacked or assembled and kept in place by a glass plate presser. Suitably, the cellular composite may comprise at least 2 cell layers, at least 3 cell layers, at least 4 cell layers, at least 5 cell layers, at least 6 cell layers, at least 7 cell layers, at least 8 cell layers, at least 9 cell layers, at least 10 cell layers, at least 12 cell layers, at least 20 cell layers, at least 25 cell layers, at least 30 cell layers, at least 35 cell layers, at least 40 cell layers, at least 45 cell layers, at least 50 cell layers, at least 75 cell layers, at least 100 cell layers, at least 150 cell layers, at least 200 cell layers, or at least 250 cell layers.

[0249] Suitably, the cellular composite may comprise at most 2 cell layers, at most 3 cell layers, at most 4 cell layers, at most 5 cell layers, at most 6 cell layers, at most 7 cell layers, at most 8 cell layers, at most 9 cell layers, at most 10 cell layers, at most 12 cell layers, at most 20 cell layers, at most 25 cell layers, at most 30 cell layers, at most 35 cell layers, at most 40 cell layers, at most 45 cell layers, at most 50 cell layers, at most 75 cell layers, at most 100 cell layers, at most 150 cell layers, at most 200 cell layers, or at most 250 cell layers.

[0250] Suitably, the cellular composite may comprise at least 12 cell layers. Suitably, the cellular composite may comprise at most 12 cell layers. Suitably, the cellular composite may comprise 12 cell layers. Suitably, a cellular composite comprising 12 cell layers may be used to produce ultra-thin cultivated leather.

[0251] Suitably, the cellular composite may comprise at least 25 cell layers. Suitably, the cellular composite may comprise at most 25 cell layers. Suitably, the cellular composite may comprise 25 cell layers. Suitably, a cellular composite comprising 25 cell layers may be used to produce ultra-thin cultivated leather.

[0252] Suitably, the cellular composite may comprise at least 50 cell layers. Suitably, the cellular composite may comprise at most 50 cell layers. Suitably, the cellular composite may comprise 50 cell layers. Suitably, a cellular composite comprising 50 cell layers may be used to produce thin cultivated leather.Suitably, the cellular composite may comprise at least 100 cell layers. Suitably, the cellular composite may comprise at most 100 cell layers. Suitably, the cellular composite may comprise 100 cell layers. Suitably, a cellular composite comprising 100 cell layers may be used to produce thin cultivated leather.

[0253] Culturing the cellular composite

[0254] Suitably, the method for producing cultivated leather, thin cultivated leather and / or ultra-thin leather may comprise a step of culturing the cellular composite. The step of culturing the cellular composite is to promote the cellular composite to produce additional ECM, depositing both within and between each cell layer to create a biological glue capable of converting the layered cell layers into a single fused cellular composite.

[0255] Suitably, the method for producing cultivated leather may comprise a step of culturing the cellular composite. Suitably, the method for producing thin cultivated leather may comprise a step of culturing the cellular composite. Suitably, the method for producing ultra-thin cultivated leather may comprise a step of culturing the cellular composite.

[0256] In the aspects of the present invention, the step of culturing the cellular composite is in serum-free medium. Suitable serum-free medium is mentioned elsewhere herein. Suitably, the serum-free medium may comprise one or more macromolecular crowding (MMC) agents. Suitable MMC agents are mentioned elsewhere herein.

[0257] Suitably, the cellular composite may be cultured in serum-free medium for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, or at least about 28 days. Suitably, the cellular composite may be cultured in serum-free medium for at least about 7 days.

[0258] Suitably, the serum-free medium for culturing the cellular composite of the present invention may comprise DMEM / F12 basal medium supplemented with L-alanyl-L-glutamine dipeptide, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, Proliferum-M™ growth factor cocktail, and London-Mix™ macromolecular crowding solution. As the person skilled in the art would appreciate, other MMC agents or solutions may be used in place of London-Mix™ macromolecular crowding solution, and other growth factor cocktails can be used in place of Proliferum-M™ growth factor cocktail. Suitably, the serum-free medium for culturing the cellular composite of the present invention may comprise DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution. As the person skilled in the art would appreciate, other MMC agents or solutions may be used in place of London-Mix™macromolecular crowding solution, and other growth factor cocktails can be used in place of Proliferum-M™ growth factor cocktail. This serum-free medium is herein referred to as STPM.

[0259] Suitably, the culturing of the cellular composite may comprise a first period and a second period. Suitably, the first period and second period of culturing of the cellular composite may run concurrently. In other words, the second period will follow the first period.

[0260] Suitably, the first period of culturing the cellular composite may be for at least about 1 day, for at least about 2 days, for at least about 3 days, for at least about 4 days, for at least about 5 days, for at least about 6 days, for at least about 7 days, for at least about 8 days, for at least about 9 days, for at least about 10 days, for at least about 11 days, for at least about 12 days, for at least about 13 days, for at least about 14 days, for at least about 21 days, or for at least about 28 days. Suitably, the first period of culturing the cellular composite may be for at least about 7 days.

[0261] Suitably, the first period of culturing the cellular composite may be in serum-free medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution; i.e. STPM.

[0262] Suitably, the second period of culturing the cellular composite may comprise riboflavin / UV treatment. Riboflavin / UV treatment comprises adding medium including riboflavin to cover the cellular composite and incubating the cellular composite in this medium. This incubation may take place in the dark, may be at 37°C and / or may be for 30 minutes. The Riboflavin / UV treatment then comprises exposing the cellular composite to UVA light. This exposure may be for 150 seconds. The cellular composite may then be removed from the medium and washed to remove any media residues.

[0263] Suitably, the second period of culturing the cellular composite may comprise rounds of UV treatment in serum -free medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution (STPM) supplemented with 0.1% (w / v) riboflavin. As the person skilled in the art would appreciate, other MMC agents or solutions may be used in place of London-Mix™ macromolecular crowding solution, and other growth factor cocktails can be used in place of Proliferum-M™ growth factor cocktail. This serum -free medium may herein be referred to as serum -free tissue assembly medium (STAM).

[0264] Suitably, the second period of culturing the cellular composite may comprise at least 1 round of riboflavin / UV treatment, wherein 1 round is for one side of the cellular composite. Suitably, the second period of culturing the cellular composite may comprise at least 1 round, at least 2 rounds, at least 3 rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least9 rounds, at least 10 rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds, at least 15 rounds, or at least 16 rounds of riboflavin / UV treatment.

[0265] As used herein, the term “UV treatment” refers to the exposure of an object, such as a cellular composite, with ultraviolet light. Suitably, the wavelength of UV radiation used in UV treatment may lie in the range of 100-400 nm. UV radiation may be subdivided into UVA (315-400 nm), UVB (280- 315 nm), and UVC (100-280 nm). Suitably, the wavelength of UV radiation used in UV treatment may be UVA (315-400 nm), UVB (280-315 nm), and / or UVC (100-280 nm)

[0266] Suitably, the second period of culturing the cellular composite may comprise a different treatment to enhance cross-linking of ECM proteins between cell layers and within cell layers of the cellular composite. Non-limiting examples of treatments applicable to the second period of culturing the cellular composite include at least 1 round of genipin treatment, at least 1 round of paraformaldehyde treatment, and / or at least 1 round of glutaraldehyde treatment. Suitably, the second period of culturing the cellular composite may comprise at least 1 round of genipin treatment, at least 1 round of paraformaldehyde treatment, and / or at least 1 round of glutaraldehyde treatment.

[0267] As used herein, the term “genipin treatment” refers to exposing an object, such as a cellular composite, to genipin. Genipin is a chemical compound found in Genipa americcinci fruit extract. It is an aglycone derived from an iridoid glycoside called geniposide which is also present in fruit of Gardenia jasminoides. Genipin is an excellent natural cross-linker for proteins (such as collagen and / or elastin), gelatin, and chitosan cross-linking.

[0268] As used herein, the term “paraformaldehyde treatment” refers to exposing an object, such as a cellular composite, to paraformaldehyde (PFA). PFA is the polymeric form of formaldehyde. Once PFA is depolymerized, the resulting formaldehyde can be utilised to cross-link proteins, such as collagen and / or elastin.

[0269] As used herein, the term “glutaraldehyde treatment” refers to exposing an object, such as a cellular composite, to glutaraldehyde. Glutaraldehyde is an organic compound with the formula (CFF CHO^. The molecule consists of a five-carbon chain doubly terminated with formyl (CHO) groups. Glutaraldehyde (and its hydrates) can crosslink proteins, such as collagen and / or elastin, through condensation.

[0270] Suitably, the cellular composite may not comprise a scaffold. Suitably, the cellular composite may not comprise an exogenous scaffold. In this context, it is meant that the cellular composite does not comprise an artificially provided scaffold, such as a biocompatible scaffold. The cellular composite may, however, comprise a natively produced scaffold by the cells of the cellular composite. Such a scaffold may be for example ECM produced by the cells of the cellular composite.Suitably, the invention provides a cellular composite wherein the cellular composite has an average thickness of at most 0.8 mm, wherein the onset temperature of the cellular composite is more than 69°C when pickled and / or more than 74°C when raw.

[0271] Suitably, the cellular composite may have an average thickness of at most about 0.1 mm, at most about 0.2 mm, at most about 0.3 mm, at most about 0.4 mm, at most about 0.5 mm, at most about 0.6 mm, at most about 0.7 mm, or at most about 0.8 mm. Suitably, the cellular composite may have an average thickness of at most about 0.8 mm. Suitably, the cellular composite may have an average thickness of at most about 0.19 mm. Suitably, the cellular composite may have an average thickness of at most about 0.36 mm. Suitably, the cellular composite may have an average thickness of about 0.19 mm. Suitably, the cellular composite may have an average thickness of about 0.36 mm.

[0272] Suitably, the average thickness of a cell layer comprised within a cellular composite may be 0.0063 ± 0.0004 mm and 0.0059 ± 0.0002 mm for thinner (0.19 mm) and thicker (0.36 mm) cellular composites, respectively. Suitably the average thickness of a cell layer comprised within a cellular composite may be at most about 0.001 mm, at most about 0.002 mm, atmost about 0.003 mm, at most about 0.004 mm, at most about 0.005 mm, at most about 0.006 mm, at most about 0.007 mm, at most about 0.008 mm, at most about 0.010 mm, atmost about 0.020 mm, atmost about 0.05 mm, at most about 0.1 mm, at most about 0.15 mm, or at most about 0.2 mm. Suitably, the cell layers comprised within a cellular composite of the present invention are between 0.005 mm and 0.2 mm thick.

[0273] As the person skilled in the art would appreciate, the thickness of the cell layers and the number of cell layers used to produce a cellular composite will directly impact the thickness of the resulting cellular composite. Suitably, the thickness of the cell layers and / or number of cell layers can be altered to produce cellular composites of varying thickness.

[0274] Accordingly, the methods described here establish the close direct correlation between the number of cell layers in a cellular composite and the average thickness of the resulting cellular composite, which consequently defines the thickness of the final cultivated leather product. Therefore, this demonstrates the advantage of the method of producing thin and ultra-thin leather using a rational, bottom-up approach, where the number of scaffold-free cell layers and / or their thickness can be exploited to tune the thickness of the final cultivated leather product.

[0275] The thermal stability (such as onset temperature) of the cellular composite may depend on whether the cellular composite has been pickled or not. Where the cellular composite has not been pickled, it may be referred to as “raw” herein. “Pickling” uses a low pH acidic salt solution to stabilize animal tissue prior to the tanning process and stop deterioration.

[0276] Suitably, the onset temperature of the cellular composite when pickled is more than about 60°C, more than about 65 °C, more than about 69°C, more than about 70°C, more than about 75 °C, more than about 80°C, more than about 85°C, or more than about 90°C. Suitably, the onset temperature of the cellularcomposite when pickled is more than about 69°C. Onset temperature may be measured by DSC as described elsewhere herein.

[0277] Suitably, the peak temperature of the cellular composite when pickled is more than about 70°C, more than about 71°C, more than about 72°C, more than about 73°C, more than about 74°C, more than about 75°C, more than about 76°C, more than about 78°C, more than about 80°C, more than about 85°C, or more than about 90°C. Suitably, the peak temperature of the cellular composite when pickled is more than about 71°C. Peak temperature may be measured by DSC as described elsewhere herein.

[0278] Suitably, the endset temperature of the cellular composite when pickled is more than about 70°C, more than about 71°C, more than about 72°C, more than about 73°C, more than about 74°C, more than about 75°C, more than about 76°C, more than about 78°C, more than about 80°C, more than about 85°C, or more than about 90°C. Suitably, the endset temperature of the cellular composite when pickled is more than about 73°C. Endset temperature may be measured by DSC as described elsewhere herein.

[0279] Suitably, the onset temperature of the cellular composite when raw is more than about than 70°C, more than about than 72°C, more than about than 74°C, more than about than 76°C, more than about than 78°C, more than about than 80°C, more than about than 82°C, more than about than 84°C, more than about than 86°C, more than about than 88°C, or more than about than 90°C. Suitably, the onset temperature of the cellular composite when raw is more than about 74°C.

[0280] Suitably, the peak temperature of the cellular composite when raw is more than about 70°C, more than about 71°C, more than about 72°C, more than about 73°C, more than about 74°C, more than about 75°C, more than about 76°C, more than about 78°C, more than about 80°C, more than about 85°C, or more than about 90°C. Suitably, the peak temperature of the cellular composite when raw is more than about 76°C.

[0281] Suitably, the endset temperature of the cellular composite when raw is more than about 70°C, more than about 71°C, more than about 72°C, more than about 73°C, more than about 74°C, more than about 75°C, more than about 76°C, more than about 77°C, more than about 78°C, more than about 79°C, more than about 80°C, more than about 85°C, or more than about 90°C. Suitably, the endset temperature of the cellular composite when raw is more than about 78°C.

[0282] In the context of the present disclosure, the ECM is not classified as a scaffold, as it is natively produced by the cells of the cell layers. Accordingly, with the ECM present in the cellular composite described herein, it may be said the cellular composite is scaffold-free, or free of an exogenous scaffold.

[0283] As exemplified in the Examples section herein, the inventors surprisingly identified that the cellular composite of the present invention has physical properties (for example, ECM composition, hydrolysed amino acid profile, and thermal stability) similar to and / or enhanced over natural animal skin (such as porcine dermis) used for producing leather.The Examples study the free amino acid content, hydrolysed amino acid content and lipid content of the cellular composites of the present invention, for example compared to porcine dermis. As outlined herein, the inventors observed that the cellular composites of the invention have enhanced lipid content in comparison to natural animal skin (porcine dermal tissue). Suitably, the cellular composite may have a lipid content of at least about 20 mg / g dry tissue, at least about 25 mg / g dry tissue, at least about 30 mg / g dry tissue, at least about 35 mg / g dry tissue, at least about 40 mg / g dry tissue, at least about 45 mg / g dry tissue, at least about 50 mg / g dry tissue, at least about 55 mg / g dry tissue, at least about 60 mg / g dry tissue, at least about 65 mg / g dry tissue, at least about 70 mg / g dry tissue, at least about 80 mg / g dry tissue, at least about 90 mg / g dry tissue, or at least about 100 mg / g dry tissue.

[0284] Suitably, the cellular composite may have a lipid content of at least about 45 mg / g dry tissue. For example, in a 25-cell layer cellular composite.

[0285] Suitably, the cellular composite may have a lipid content of at least about 60 mg / g dry tissue. For example, in a 50-cell layer cellular composite.

[0286] As used herein, the term “free amino acid content” refers to the determination of the type and amount of individual, unbound amino acids present in a sample. As used herein, the term “hydrolysed amino acid content” refers to the determination of the type and amount of amino acids comprised within a protein or peptide structure present in a sample. As used herein, the term “lipid content” refers to the determination of the type and amount of lipids, including triglycerides, phospholipids, sterols, waxes, and fatty acids present in a sample.

[0287] The Examples also study the ultrastructure of the cellular composites of the present invention. Ultrastructure is the architecture of cells and ECM in tissues, identifiable at magnifications higher than those from standard optical light microscopy, with resolutions typically within the nanometre (nm) range.

[0288] Suitably, after culturing, the cellular composite comprises soluble collagen, insoluble collagen, elastin, sGAGs (sulphated glucosaminoglycans), O-GAGs, N-GAGs, HA (Hyaluronan), and water. As shown in the examples herein, the concentration of soluble collagen, insoluble collagen, elastin, sGAGs, O-GAGs, N-GAGs, and HA will depend on the number of cell layers the cellular composite was produced from.

[0289] Suitably, the cellular composite of the present invention comprises at least a similar concentration of sGAGs, O-GAGs and N-GAGs to porcine dermis. Suitably, the cellular composite of the present invention comprises an enhanced concentration of elastin to porcine dermis.

[0290] Suitably, a cellular composite produced from 25 cell layers comprises about 50 mg / g dry tissue insoluble collagen, about 300 mg / g dry tissue soluble collagen, about 125 mg / g dry tissue elastin, about2.5 mg / g dry tissue total sGAGs (about 1.3 mg / g dry tissue O-GAGs and about 1.3 mg / g dry tissue N-GAGs), and less than about 0.05 mg / g dry tissue HA.

[0291] Suitably, a cellular composite produced from 50 cell layers comprises about 36 mg / g dry tissue insoluble collagen, about 335 mg / g dry tissue soluble collagen, about 125 mg / g dry tissue elastin, about 3.6 mg / g dry tissue total sGAGs (about 2.5 mg / g dry tissue O-GAGs and about 1.1 mg / g dry tissue N-GAGs), and less than about 0.05 mg / g dry tissue HA.

[0292] As outlined herein, the inventors surprisingly identified that the cellular composites (and therefore the cultivated leather products produced therefrom) have significantly higher concentrations of elastin than porcine dermis. Without wishing to be bound by theory, the inventors believe that because elastin is more thermostable than collagen, higher concentrations might account for the higher onset temperatures observed for cellular composites and cultivated leather produced therefrom.

[0293] Suitably, the cellular composite may have a reduced, similar, or enhanced concentration of ECM proteins in comparison to animal dermis. Suitably, the animal dermis is porcine dermis.

[0294] Suitably, the cellular composite may have an enhanced concentration of total elastin in comparison to animal dermis. Suitably, the animal dermis is porcine dermis.

[0295] Suitably, the cellular composite may have a total elastin concentration of at least about 50 mg / g dry tissue, at least about 60 mg / g dry tissue, at least about 70 mg / g dry tissue, at least about 80 mg / g dry tissue, at least about 90 mg / g dry tissue, at least about 100 mg / g dry tissue, at least about 110 mg / g dry tissue, at least about 120 mg / g dry tissue, at least about 130 mg / g dry tissue, at least about 140 mg / g dry tissue, or at least about 150 mg / g dry tissue . Suitably, the cellular composite may have a total elastin concentration of at least about 120 mg / g dry tissue.

[0296] Suitably, the cellular composite may have total elastin concentrations of at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, or at least about 5-fold of that of animal dermis. Suitably, the cellular composite may have total elastin concentrations of at least about 4-fold of animal dermis. Suitably, the animal dermis is porcine dermis. Suitably, the cellular composite may have a similar concentration of sGAGs to animal dermis. Suitably, the animal dermis is porcine dermis.

[0297] Suitably, the cellular composite may have a total sGAGs concentration of at least about 1 mg / g dry tissue, at least about 1.5 mg / g dry tissue, at least about 2 mg / g dry tissue, at least about 2.5 mg / g dry tissue, at least about 3 mg / g dry tissue, or at least about 3.5 mg / g dry tissue. Suitably, the cellular composite may have a total sGAGs concentration of at least about 2.5 mg / g dry tissue (25-layer cellular composite). Suitably, the cellular composite may have a total sGAGs concentration of at least about 3.5 mg / g dry tissue (50-layer cellular composite).As shown by the inventors herein, the cellular composite produced by the methods of the present invention displays a hydrolysed amino acid profde comparable to that of porcine dermis. Suitably, the cellular composite of the invention may comprise a hydrolysed amino acid profde similar to natural animal skin, such as porcine dermis.

[0298] Furthermore, as shown by the inventors herein, the diameter of collagen fibres from cellular composites was consistent between cellular composites and comparable with that from control dermis samples. Without being bound by theory, the inventors believe it is the similarities in collagen fibre morphology and organisation of the cellular composites of the invention to natural animal skin that allows the cellular composites to have a similar response to tanning as well as similar physical properties after tanning.

[0299] Suitably, the cellular composite of the present invention may comprise collagen fibres with a diameter of at least about 180 nm, at least about 181 nm, at least about 182 nm, at least about 183 nm, at least about 184 nm, at least about 185 nm, at least about 186 nm, at least about 187 nm, at least about 188 nm, or at least about 189 nm. Suitably, the cellular composite of the present invention may comprise collagen fibres with a diameter of at least about 189 nm.

[0300] Suitably, the cellular composite of the present invention may comprise collagen fibres with a diameter of at most about 200 nm, at most about 199 nm, at most about 198 nm, at most about 197 nm, at most about 196 nm, at most about 195 nm, at most about 194 nm, at most about 193 nm, or at most about 192 nm. Suitably, the cellular composite of the present invention may comprise collagen fibres with a diameter of at most about 192 nm.

[0301] Tanning

[0302] Suitably, the cellular composite produced by the invention is tanned to produce cultivated leather. Suitably, the cellular composite produced by the invention is tanned to produce thin cultivated leather. Suitably, the primary cell layer and / or the cellular composite produced by the invention is tanned to produce ultra-thin cultivated leather. Suitably, the cellular composite produced by the invention is tanned to produce ultra-thin cultivated leather. Suitably, the primary cell layer produced by the invention is tanned to produce ultra-thin cultivated leather.

[0303] As used herein, the term “tanning” refers to the process typically applied to natural animal skin to produce leather. During the tanning process, cross linking agents such as chromium sulphate or vegetable tannins are introduced to increase the integrity of the collagen matrix by cross-linking collagen. Methods of tanning are very well known in the art.

[0304] In the context of the present invention, the tanning of primary cell layers and / or cellular composites produces cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather. The aim of tanning is to produce durable cultivated leather, and / or ultra-thin cultivated leather, that is resistant towater, heat, abrasion, and putrefaction. The tanning method can be tuned to control the physical and organoleptic properties of the cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather.

[0305] Each tanning method utilises different tanning agents to achieve particular physical and / or organoleptic properties. Various tanning methods may be combined in order to achieve certain properties of the cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather.

[0306] Suitably, the tanning of the primary cell layer and / or the cellular composite is selected from chrome tanning, vegetable tanning, and chrome-free tanning, or a combination thereof. Suitably, the tanning of the primary cell layer is selected from chrome tanning, vegetable tanning, and chrome-free tanning, or a combination thereof. Suitably, the tanning of the cellular composite is selected from chrome tanning, vegetable tanning, and chrome-free tanning, or a combination thereof.

[0307] Typically, tanning involves the following reagents: a pickling solution (for example, 6% (w / v) NaCl in water adjusted for pH 2.6-2.8 with formic acid), chromium taming solution (for example, 6% (w / v) Chromium III / VI salts in water) and / or vegetable tanning solution (for example, 25% (w / v) Mimosa ME in water), MGO fixing solution (for example, 0.5% (w / v) Magnesium Oxide in water, pH 4), Sodium Bicarbonate fixing solution, pH 4, Formic Acid fixing solution, pH 4, Sodium Bicarbonate / Sodium Formate neutralisation solution, pH 4.5-4.8, re-tanning solution (for example, 5% (w / v / ) Mimosa ME and 3% (w / v) dye in water), and fatliquoring solution (for example, 10% (v / v) Trupon LP2 in water).

[0308] Suitably, tanning the cellular composite to produce cultivated leather, thin cultivated leather, and / or ultra-thin cultivated leather comprises the following reagents: a pickling solution (for example, 6% (w / v) NaCl in water adjusted for pH 2.6-2.8 with formic acid), chromium tanning solution (for example, 6% (w / v) Chromium III / VI salts in water) and / or vegetable tanning solution (for example, 25% (w / v) Mimosa ME in water), MGO fixing solution (for example, 0.5% (w / v) Magnesium Oxide in water, pH 4), Sodium Bicarbonate fixing solution, pH 4, Formic Acid fixing solution, pH 4, Sodium Bicarbonate / Sodium Formate neutralisation solution, pH 4.5-4.8, re-tanning solution (for example, 5% (w / v / ) Mimosa ME and 3% (w / v) dye in water), and fatliquoring solution (for example, 10% (v / v) Trupon LP2 in water).

[0309] As used herein, the term “chrome tanning” refers to the process of producing leather comprising the use of chromium salts.

[0310] Suitably, chrome tanning may comprise the steps of pickling the cellular composite for 7 days at room temperature, followed by adding chromium tanning solution for 2 h followed by MGO fixing solution overnight followed by Sodium Bicarbonate fixing solution for 1 h, water wash for 15 min, Sodium Bicarbonate / Sodium Formate neutralisation solution for 1 h, water wash for 15 min, re-tanning solutionovernight followed by Formic Acid fixing solution overnight, water wash for 15 min, fatliquoring solution overnight followed by Formic Acid fixing solution for 1 h, and finally water wash for 15 min. As used herein, the term “vegetable tanning” refers to the process of producing leather comprising the use of tannins. Tannins (or tannoids) are a class of astringent, polyphenolic biomolecules that bind to and precipitate proteins and various other organic compounds including amino acids and alkaloids. Non-limiting examples of tannin sources comprise chestnut, oak, redoul, tanoak, hemlock, quebracho, mangrove, wattle, and myrobalans from Terminalici spp., such as Terminalici chebula.

[0311] Suitably, vegetable tanning may comprise the steps of pickling the cellular composite for 7 days at room temperature, followed by adding vegetable tanning solution for 2 h followed by Formic Acid fixing solution overnight, water wash for 15 min, Sodium Bicarbonate / Sodium Formate neutralisation solution for 1 h, water wash for 15 min, re-tanning solution overnight followed by Formic Acid fixing solution overnight, water wash for 15 min, fatliquoring solution overnight followed by Formic Acid fixing solution for 1 h, and finally water wash for 15 min.

[0312] As described in the examples herein, the inventors identified that even a single cell layer (without a scaffold) can be subjected to pickling solution and remain intact. Without wishing to be bound by theory, the inventors believe this may be due to the physical properties of the cell layer as provided by the method of the invention described herein.

[0313] As used herein, the term “chrome-free tanning” refers to any process of producing leather using crosslinking reagents in the place of chromium salts or tannins. Non-limiting examples of cross-linking reagents utilised in the chrome-free tanning process comprise aldehydes, titanium, iron salts, zirconium, aluminium, oxazolidine, or tetrakis hydroxymethyl phosphonium sulfate (THPS), or a combination thereof. Natural taming, synthetic taming and tawing are chrome-free tanning techniques. Suitably, the tanning of the primary cell layer and / or the cellular composite may be selected from natural tanning, synthetic tanning, and tawing, or a combination thereof.

[0314] As appreciated by the person skilled in the art, there are several methods of tanning in addition to those outlined above. Therefore, the method of tanning of the present invention will not be restricted to the above methods but will encompass other methods suitable for tanning primary cell layers and / or cellular composites of the invention in order to produce cultivated leather, thin cultivated leather and / or ultrathin cultivated leather.

[0315] The cultivated leather, thin and / or ultra-thin cultivated leather may be subjected to further processes. Suitably, the further processes may be selected from 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, tumbling and my combination thereof.Suitably, the method for producing cultivated leather, thin cultivated leather and / or ultra-thin cultivated leather further comprises splitting cultivated leather or thin cultivated leather to produce at least two independent tanned tissue sheets.

[0316] Suitably, the tanned tissue sheets are distinct from tanned primary cell layers as the tanned tissue sheets are produced by splitting a cultivated leather produced from tanning cellular composites. A cultivated leather, thin cultivated leather or ultra-thin cultivated leather of the invention or produced by a method of the invention, may be split (or separated or divided) into at least two independent (or distinct or separate) tanned tissue sheets, which are robust and able to hold their structure independently of each other. The independent tanned tissue sheets produced by splitting cultivated leather are thinner than the original cultivated leather and this method can also be used to produce thin or ultra-thin cultivated leather.

[0317] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skills in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provides those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Aspects of the invention are demonstrated by the following non-limiting examples.

[0318] EXAMPLES

[0319] Example 1: Proliferation of cells for producing scaffold-free animal tissue in serum-free conditions

[0320] The inventors set out to evaluate the ability of serum-free medium supplemented with macromolecular crowding agents to expand animal cells in mono- and co-cultures. These serum-free cultures matched or even surpassed the total number of cells accumulating at and beyond confluence obtained with serumcontaining media.

[0321] Materials and MethodsMedia formulation

[0322] Cells were expanded and maintained with serum-free proliferation medium (SPM) comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 5% (v / v) Proliferum -M™ growth factor cocktail, and 4% (v / v) London-Mix™ macromolecular crowding solution. Serum-containing medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 1% (v / v) Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) solution, and 5% (v / v) foetal bovine serum (FBS) was used as control for cell proliferation.

[0323] Cell proliferation in mono- and co-cultures

[0324] E. Derm (NBL-6) horse dermis fibroblasts were seeded on polystyrene tissue culture plates alone (mono-culture) or combined with CPF4-A porcine hypodermal pre-adipocytes mixed in a 95:5 percent ratio (co-culture) at a density of 1 x 104cells / cm2. Cells were grown for 14 days with serum-free or FBS-containing proliferation media (“w / FBS”) to compare the ability of serum-free medium to sustain cell proliferation. Cells were incubated at 37°C and 5% CO2 in a humidified environment during cultures. Media was exchanged every 3-4 days of culture. All experiments were performed using three independent replicates.

[0325] Quantification of cell proliferation

[0326] Cell proliferation was evaluated at day 7 and 14 of culture via quantification of viable cells using resazurin metabolic analysis, with cell numbers calculated by interpolation using a fluorescence standard curve. In resazurin metabolic analysis, resazurin conversion to resorufm in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. All experiments were performed using three independent replicates, with proliferation expressed as the normalised percentage of FBS control.

[0327] Statistical analysis

[0328] Data was normalised against that of day control i.e., monoculture in FBS -containing on day 7 and on day 14. Error bars represent the standard deviation of the mean. Differences between groups were determined using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between groups was established for p < 0.05 (4-).

[0329] Results and Discussion

[0330] E. Derm fibroblasts grown in mono-culture with serum-free proliferation medium (SPM) were shown to proliferate to levels comparable to those of cells grown with serum-containing medium, with total cell numbers corresponding to 94±3% and 105±6% of the w / FBS mono-culture control at day 7 and 14, respectively (Fig. 2; M). In absolute terms, cell number increased both in SPM and w / FBS conditionsfrom day 7 to 14, indicating that, similarly to what previously observed for the serum -containing medium, SPM provides a suitable medium environment for cells to proliferate beyond confluence and stratification.

[0331] Similarly to what was observed in mono-culture, co-cultures maintained with SPM grew to levels comparable to those from w / FBS co-cultures (Fig. 2; C). In addition, E.Derm fibroblasts grown in coculture with CPF4-A pre-adipocyte cells with SPM were shown to proliferate at levels significantly higher than those from mono-culture counterparts, and corresponding to 119±9% and 114±3% of total cells from w / FBS mono-culture control at day 7 and 14, respectively (Fig. 2).

[0332] Together, these results demonstrate that SPM represents a medium formulation that allows animal cells to proliferate in serum -free conditions at the high levels expected from serum -containing, gold-standard conditions, and at equivalent (or lower) costs. Moreover, this illustrates that SPM allows the cell proliferation step of the process to be performed to produce cultivated leather (Fig. 1) without the need for animal-derived ingredients. These three features are very relevant in the cultivated leather space, where high efficiency, low cost, and high sustainability and ethical standards are paramount.

[0333] Example 2: Tissue deposition for producing scaffold-free animal tissue in serum-free conditions The inventors set out to evaluate the ability of their serum-free tissue production medium supplemented with macromolecular crowding agents to maintain animal cells in long-term mono- and co-cultures, while matching or even increasing the deposition of tissue-specific ECM components compared to animal cell cultures obtained with serum-containing media.

[0334] Materials and Methods

[0335] Media formulation

[0336] Cells were expanded with SPM comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 5% (v / v) Proliferum-M™ growth factor cocktail, and 4% (v / v) London-Mix™ macromolecular crowding solution. Tissue was deposited by cells maintained with serum-free tissue production medium (STPM) comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution. Serum-containing medium comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 1% (v / v) Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) solution, 8% (v / v) London-Mix™ macromolecular crowding solution, and 0.5% (v / v) FBS was used as control fortissue deposition.

[0337] Production of tissue from mono- and co-culturesE. Derm (NBL-6) horse dermis fibroblasts were seeded on polystyrene tissue culture plates alone (mono-culture) or combined with CPF4-A porcine hypodermal pre-adipocytes mixed in a 95:5 percent ratio (co-culture) at a density of 1 x 104cells / cm2, and grown for 14 days with SPM at 37°C and 5% CO2 in a humidified environment during cultures. Media was exchanged every 3-4 days of culture. Subsequently, mono- or co-cultures were maintained with STPM or FBS -containing medium for another 14 days to compare the ability of serum -free medium to sustain the formation of cultivated animal tissue. Cultures were incubated at 37°C and 5% CO2 in a humidified environment during the culture period. Media was exchanged every 3-4 days of culture. All experiments were performed using three independent replicates.

[0338] Quantification of tissue production

[0339] Total production of cultivated animal tissue was evaluated via i) quantification of the wet weight of tissues formed by cells in different conditions after 28 days in culture, using a high-precision analytical balance and ii) Picrosirius Red analysis, with tissue samples fixed with 70% ethanol for 2h at -20°C, washed with PBS for 15 min, incubated with a mix of Direct Red 80 dye diluted at 1 mg / mL in a 1.3% Picric Acid saturated solution for 4 h at room temperature, washed repeatedly with 5% (v / v) Acetic Acid for 15 min and finally with water to remove excess dye, and incubated with 1 M NaOH solution for 30 min, which was then sampled and analysed for absorbance at 490 nm. All experiments were performed using three independent replicates, with tissue biomass expressed as normalised percentage of FBS control.

[0340] Quantification of cell layer area shrinkage

[0341] Cultivated cell layers produced in serum-free or FBS -containing media was imaged before and after recovery from their culture substrate, with cell layer size quantified from the photographs of at least 20 individual tissues. Cell layer shrinkage was calculated as the average ± standard deviation of the ratio between cell layer area after recovery and cell layer area before recovery.

[0342] Statistical analysis

[0343] Data was normalised against that of day control i.e., monoculture in FBS -containing on day 7 and of day 14. Error bars represent the standard deviation of the mean. Differences between groups were determined using one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison post hoc tests. Significance between groups was established for p < 0.05 and 0.01

[0344]

[0345] and 4-. respectively).

[0346] Results and Discussion

[0347] E. Derm cells grown and maintained in mono-culture with serum-free tissue production medium (STPM) were shown to produce collagen-rich cell layers at levels significantly higher than those of cells grown with serum-containing medium, with corresponding total collagen production increasing on average by 23% and 11% over the w / FBS mono-culture control at day 7 and 14 of the STPM or FBSculture (after an initial 14 day culture in SPM), respectively (Fig. 3; M). In absolute terms, collagen deposition increased both in STPM and w / FBS conditions from day 7 to 14, indicating that, similarly to what previously observed for the serum-containing medium, STPM provides a suitable medium environment for cells to continue producing, depositing and accumulating cell-specific ECM components, including fibrillar collagens, into forming multi-stratified, structured cell layers in the absence of scaffolds.

[0348] Similarly to what was observed in mono-culture, E.Derm cells maintained in co-culture with CPF4-A cells with STPM produced collagen-rich cell layers at levels significantly higher than those from w / FBS conditions (Fig. 3; C), with corresponding total collagen production increasing on average by 32% and 38% over the mono-culture control, and by 14% and 18% over co-culture control at day 7 and 14, respectively (Fig. 3).

[0349] In addition, total wet weight of cell layers produced with STPM was greater than that of cell layers produced with serum -containing medium, independently if such cell layers were produced in mono- or co-culture (Table 1). Specifically, cell layers produced from mono- or co-cultures with STPM showed to weigh on average 18% or 25% more than their w / FBS counterparts. Furthermore, STPM resulted in more consistent, reproducible outputs, with the scaffold-free cell layers produced in serum-free conditions showing a lower range of variation in wet weight and production yield (Table 1). This could be in part attributable to lot variations from serum, a well-known feature affecting consistency in cell performance and tissue culture production. The presence of serum in media is also expected to increase variation in yield by promoting spontaneous cell contraction that leads to cell and tissue loss during culture. In contrast, cell layers produced with STPM failed to show signs of contraction (Table 2). Condition Wet weight (mg / cm2) Production yield (% of control) w / FBS, Day 14 (M) 1.80 ± 0.30 100 ± 17

[0350] STPM, Day 14 (M) 2.10 ± 1.25 118 ± 7

[0351] w / FBS, Day 14 (C) 2.20 ± 1.90 122 ± 11

[0352] STPM, Day 14 (C) 2.75 ± 1.50 153 ± 8

[0353]

[0354] Table 1. Production yield of scaffold-free cell layers in serum -free and serum -containing media, evaluated as tissue wet weight after 14 days of mono- and co-culture of E. Derm cells (after an initial 14-day culture in SPM).

[0355] Condition Cell layer area shrinkage (%)

[0356] Tissue (M) produced in serum-free medium 0.9±0.1%

[0357] Tissue (M) produced in serum-containing medium 6.5±0.2%

[0358] Tissue (in literature1) in serum-containing medium 15-20%

[0359]

[0360] Table 2. Cell layer shrinkage in serum -free versus serum -containing medium.

[0361] These results demonstrate that STPM represents a medium formulation that allows animal cells to deposit ECM and produce tissue (cell layers) in serum-free conditions at levels comparable to or even surpassing those expected from serum-containing, gold-standard conditions, and at equivalent (or lower) costs. Together with SPM, STPM allows the production of all the essential raw materials to make cultivated leather (Fig. 1). Moreover, by resulting in denser, non-contractile tissues, STPM allows such tissues to be grown and later recovered from culture without the need of scaffolds or other supporting materials not derived from the cells themselves. These scaffold-free cell layers aim at facilitating the production of cultivated leather by making this process more efficient, less expensive, more sustainable, ethical, and resulting in a final leather product with better performance and without additional natural or synthetic components.

[0362] Example 3: Production of scaffold-free animal tissues and their assembly into cellular composites in serum-free conditions

[0363] The inventors set out to demonstrate that their tissue production process is able to generate scaffold-free cell layers capable of withstanding extensive handling and manipulation without compromising their integrity and viability either in isolation or after being stacked and assembled into thicker scaffold-free cellular composites. Moreover, the inventors set out to characterise the structural and compositional properties of these scaffold-free cellular composites.

[0364] Materials and Methods

[0365] Media formulation

[0366] Cells were expanded with SPM comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 0.25 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 5% (v / v) Proliferum-M™ growth factor cocktail, and 4% (v / v) London-Mix™ macromolecular crowding solution. Tissues was deposited by cells maintained with STPM comprising DMEM / F12 basal medium supplemented with 5 mM L-alanyl-L-glutamine dipeptide, 1 mM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 0.5% (v / v) Proliferum-M™ growth factor cocktail, and 8% (v / v) London-Mix™ macromolecular crowding solution. Scaffold-free cell layers were assembled and fused together into cellular composites with serum-free tissue assembly medium (STAM) comprising STPM supplemented with 0.1% (w / v) riboflavin.

[0367] Scaffold-free tissue production and recovery

[0368] E. Derm (NBL-6) horse dermis fibroblasts were seeded with SPM on polystyrene tissue culture plates at a density of lx 104cells / cm2, grown for 14 days with SPM, and subsequently maintained with STPM for another 14 days to sustain the formation of cell layers. Cell layers were then recovered by mechanical means (i.e., using tweezers) and maintained in PBS until further use e.g. assembly ofcellular composite. Cells and tissues were incubated at 37°C and 5% CO2 in a humidified environment during the culture period. Media was exchanged every 3-4 days of culture. All experiments were performed using three independent replicates.

[0369] Cellular composite assembly

[0370] The scaffold-free cell layers were assembled within a culture dish by consecutive stacking of 50 or 25 individual cell layers (comprising a primary cell layer and further cell layers), to form cellular composites which may go on to produce thin or ultra-thin cultivated leather, respectively. Stacked cell layers were subsequently cultured with STPM for 7 days, with the stack kept in place by a glass plate presser. At the end of this period, the cellular composites comprising stacked tissues were exposed to 8 rounds of riboflavin / UV treatment on each side of the cellular composite for a total of 16 rounds, with each round consisting of addition of STAM to cover the cellular composite, incubation at 37°C for 30 min in the dark, exposure to UVA light for 150 s, removal of medium, and wash with excess sterile PBS for 2 min to remove media residues. Scaffold-free cellular composites were subsequently maintained in PBS until use for analysis. All experiments were performed using three independent replicates.

[0371] Evaluation of cell viability within scaffold-free cell layers

[0372] Cell viability of scaffold-free cell layers after recovery and after assembly into cellular composites was evaluated via fluorescence microscopy analysis using the Calcein AM (live) / Propidium iodide (dead) double cell staining, after incubating tissues with a 0.1% (v / v) Calcein AM and PI solution in PBS for 30 min, followed by washing twice with PBS for 5 min. All experiments were performed using three independent replicates.

[0373] Evaluation of integrity of scaffold-free cellular composites

[0374] The integrity of scaffold-free cell layers after recovery and after assembly into cellular composites was tested by submerging tissues in 12 volumes of pickling solution comprising acid water (pH = 2.5-3, adjusted using formic acid) supplemented with 10% (w / v) of NaCl for 1 day at room temperature and under constant orbital agitation at 100 rpm, and subsequently analysed for signs of tissue delamination. All experiments were performed using three independent replicates.

[0375] Evaluation of structure and composition of cellular composites

[0376] Samples of the scaffold-free cellular composites of approximately 1 cm2were collected and analysed for their composition, including the quantification of total and free amino acid content and lipid content via hydrolysis followed by LC-MS / MS, quantification of collagen, elastin, glucosaminoglycan (GAG), and hyaluronic acid (HA) content via hydrolysis followed by Picrosirius Red, Fastin, Blyscan, and Purple-Jelly colorimetric assays, respectively, and for their structure, including ultrastructuralcharacteristics via scanning electron microscopy (SEM). Porcine dermal tissue samples were used as controls of natural animal skin. All experiments were performed using three independent replicates.

[0377] Results and Discussion

[0378] Scaffold-free cell layers produced by culturing E.Derm cells for 2 weeks with serum-free proliferation medium (SPM) followed by another 2 weeks with serum-free tissues production medium (STPM) were recovered from their growth surface (polystyrene tissue culture plates) by minimal mechanical handling, and then successfully assembled into stacks of 25 or 50 layers. Further culture of the assembled cellular composites for 1 week with STPM and a final treatment with serum-free assembly medium (STAM) and UVA radiation allowed cells comprising these tissues to produce additional ECM, depositing both within and between each tissue layer, and create a biological glue capable of converting the layered stacks into single, thicker, scaffold-free cellular composites of different thickness (Fig. 4a and 4b). These scaffold-free cellular composites were easily handled both inside and outside a liquid environment, where they maintained their general shape, size, and integrity, even after considerable manipulation (Fig. 4c). Importantly, the maintenance of the original dimensions of the cellular composites independently of time, environment, or degree of handling were the result of the inherently more robust structural / mechanical properties of the scaffold-free cell layers that originated them, as exemplified by the considerable shrinkage observed in less dense; less robust tissues before and after assembly, as described in the literature1of the field.

[0379] The viability of the scaffold-free animal tissues and of their resulting cellular composites were further analysed using a Calcein AM / Propidium iodide Live / Dead double cell staining (Fig. 5, top panels). The former showed that that the overall majority of cells within both the 50- and 25 -layer scaffold-free cellular composites and cell layers were stained positively for Calcein AM, and only a small fraction of cell nuclei were Pl-positive (Fig. 5, top panels), indicating that the processes of tissue recovery and assembly did not meaningfully result in increased cell death. This is relevant, as it confirms that cells are still viable and capable of producing ECM during the assembly step and beyond, without the deleterious effects of widespread cell death and the consequent release of cellular contents, including proteases that might affect the structure and integrity of tissues post-cellular composite assembly. This ability to retain integrity was further demonstrated by a picking and agitation assay (Fig. 5, bottom panels). Results showed that both the 50- and 25-layer scaffold-free cellular composites and scaffold-free cell layers remained intact after extensive shear in the acidic saline, further confirming that such cellular composites and cell layers are resistant to a type of tissue preservation commonly used in the tanning industry to avoid spoilage of natural animal hides and skin.

[0380] The ability of cellular composites to react to chemical tanning the way natural animal skin does and thus achieving equivalent performance is not only dependent on their structure but also on their biochemical composition. To understand how the scaffold-free cellular composites compared withnatural animal skin, their composition was analysed in detail. Firstly, this analysis showed that, with the exception of glutamine, scaffold-free cellular composites evidenced lower levels of free amino acids compared with that of porcine dermis (Fig. 6), but no differences were observed between the 25- and 50-layer cellular composites. Free amino acids are not bound in the form of proteins, and in natural skin their availability is determined by the primary structure of the most expressed proteins i.e. collagens and other proteins of connective tissue. The discrepancies in free amino acid content between the scaffold-free cellular constructs and porcine dermis was probably mostly due to non-optimal levels of amino acids in STPM. The content profde of hydrolysed amino acids from scaffold-free cellular constructs was comparable to that of porcine dermis (Fig. 7), suggesting that both cultivated and natural tissues are composed of broadly the same type of proteins, and in comparable proportions.

[0381] The scaffold-free cellular composites showed higher levels of lipid content compared to porcine dermis (Table 3), with 25- and 50- layer cellular composites containing approximately 3- and 4-fold higher concentrations than that of natural animal tissue. This difference is probably due to the higher density of cells in the cellular composites versus the relatively low cell number in animal dermis.

[0382] Lipid content (mg / g dry tissue)

[0383] Scaffold-free cellular composites Porcine dermal tissue 25 layers 50 layers Average ± S.D

[0384] 44.5 63.0 53.8 ± 13.1 14.80

[0385]

[0386] Table 3. Comparative analysis of lipid content from scaffold-free cellular constructs and porcine dermis.

[0387] The scaffold-free cellular composites were shown to comprise soluble collagen, hyaluronic acid, insoluble collagen, and GAGs (Table 4). Compared with porcine dermis, the scaffold-free cellular composites showed to comprise considerably lower amounts of soluble collagen (on average, only 32% of control) and hyaluronic acid (on average, less than 7% of control), and slightly lower amounts of insoluble collagen (on average, only 67% of control) and GAGs (on average, only 76% of control) (Table 4). In contrast, cellular composites were surprisingly enriched in elastin (on average, 4-fold that of control levels) and had higher hydration (approximately 90% in comparison with 68% of control) (Table 4). Overall, these values indicate that the scaffold-free cellular composites, despite comprising dermis-characteristic ECM components, are nonetheless sparser than natural animal skin, with most of their volume comprising water.

[0388] Without wishing to be bound by theory, the inventors believe that the lower levels of sGAGs and HA are an advantage as it may simplify the tanning process, as they need to be removed during tanning in any case.Component Scaffold-free cellular constructs Porcine dermis 25 layers 50 layers

[0389] Soluble collagen (mg / g dry tissue) 48.8 ± 3 36 ± 0 131.9 ± 44.0 Insoluble collagen (mg / g dry tissue) 299.7 ± 16.6 334.4 ± 5.2 472.0 ± 83.8 Total collagen (mg / g dry tissue) 348.5 ± 16.3 370.4 ± 5.2 603.9 ± 43.4 Total elastin (mg / g dry tissue) 124.1 ± 16.9 122.6 ± 18.2 29.9 ± 1.1 Total sGAGs (mg / g dry tissue) 2.5 ± 0.4 3.6 ± 0.2 4.0 O-GAG (mg / g dry tissue) 1.3 ± 0.4 2.51 ± 0.1 2.15 N-GAG (mg / g dry tissue) 1.3 ± 0.1 1.1 ± 0.4 1.9 HA (mg / g dry tissue) <0.05 <0.05 0.7 ± 0.2 Water (%) 89.7 ± 0.8 68.2 ± 1.6

[0390]

[0391] Table 4. Quantitative analysis of the characteristics and composition of cellular composites and Porcine dermis.

[0392] The ultrastructure of scaffold-free cellular composites, and how it compares with porcine dermis, was analysed in greater detail via SEM (Fig. 8). Both the 25- and 50-layer cellular composites were shown to be formed by very dense layers of collagen fibrils arranged in loose bundles (Fig. 8a and 8b, respectively) with orientations comparable to those from models of mammalian dermis (Fig. 8c). The individual layers of cells and matrix of cellular composites were shown to be fused together, forming compacted cellular composites of different thickness (i.e., 0.19 or 0.36 mm, respectively). The average thickness of each layer was 6.3 ± 0.4 pm and 5.9 ± 0.2 pm for the thinner and thicker cellular composites, respectively (Fig. 8a and 8b); in contrast, the collagen fibers forming porcine dermis are arranged in long, highly-compacted, regionally-aligned, and considerably irregular bundles (Table 8) 13 ± 3 pm in thickness (Fig. 8f).

[0393] Importantly, the diameter of collagen fibers from scaffold-free cellular composites was consistent between tissues and comparable with that from control dermis samples (Fig. 8). Furthermore, it was similar to what was described in the literature for collagen from the reticular dermis. Collagen fiber diameter is specific to different tissue types, being dependent on the complex biochemical environment created by the mixture of other ECM components surrounding the fibers. The slightly higher-than-expected range of fiber diameter in cellular constructs, i.e. 189-192 run compared with 146 nm from porcine dermis, and 60-130 nm in horse skin, may be associated with its GAG and HA content (Table 4). This feature plays a fundamental role in defining the biophysical and tensile properties of each tissue; therefore, the similarities in collagen fiber morphology and organization equally suggests that thescaffold-free cellular composite have similar response to tanning as well as similar performance after processing.

[0394] The space between collagen fibers was also reproducible in 25- and 50-layer cellular composites and shown to be about 2.5-fold bigger than those from porcine dermis (Fig. 8). This is consistent with chemical analysis data (Table 4) showing that cellular composites are more hydrated and just over half as dense as porcine dermis. Interestingly, in areas where the tissues are denser, possibly due to greater accumulation / assembly of collagen fibers and other matrix components, the size of open spaces between ECM components (“pores”) is comparable between cellular composites (namely in the 50-layer sample) and porcine dermis (Fig. 8e and 8f).

[0395] Together, these results indicate a close similarity in structural and compositional characteristics between the scaffold-free cellular composites and natural animal dermis, a conclusion that supports the notion that the cellular composites produced using the methods described herein represent a good equivalent of animal hide / skin, and therefore will be expected to perform just as well as a raw material for leather production.

[0396] Example 4: Performance of thin and ultra-thin leather resulting from chemical tanning of scaffold-free cellular composites

[0397] The inventors set out to demonstrate that scaffold-free cell layers and the corresponding assembled cellular composites are feasible materials for chemical tanning, and that, once treated, the resulting cultivated leathers have ultrastructural, compositional, tensile and imputrescible properties comparable to those of natural animal leather, as well as equivalent organoleptic performance.

[0398] Materials and Methods

[0399] Reagents

[0400] Chemical tanning involved the use of a pickling solution (6% (w / v) NaCl in water adjusted for pH 2.6-2.8 with formic acid), chromium tanning solution (6% (w / v) Chromium III / VI salts in water), vegetable tanning solution (25% (w / v) Mimosa ME in water), MGO fixing solution (0.5% (w / v) Magnesium Oxide in water, pH 4), Sodium Bicarbonate fixing solution, pH 4, Formic Acid fixing solution, pH 4, Sodium Bicarbonate / Sodium Formate neutralisation solution, pH 4.5-4.8, re-tanning solution (5% (w / v / ) Mimosa ME and 3% (w / v) dye in water), and fatliquoring solution (10% (v / v) Trupon LP2 in water).

[0401] Chromium and Vegetable tanning

[0402] Scaffold-free single cell layers and scaffold-free cellular composites comprising 12, 25, 50 or 100 layers of scaffold-free cell layers were preserved in pickling solution for up to 7 days at room temperature, and subsequently chromium-tanned by consecutive treatments with chromium tanning solution for 2 h followed by MGO fixing solution overnight followed by Sodium Bicarbonate fixing solution for 1 h,water wash for 15 min, Sodium Bicarbonate / Sodium Formate neutralisation solution for 1 h, water wash for 15 min, re-tanning solution overnight followed by Formic Acid fixing solution overnight, water wash for 15 min, fatliquoring solution overnight followed by Formic Acid fixing solution for 1 h, and finally water wash for 15 min; or vegetable-tanned by consecutive treatments with vegetable tanning solution for 2 h followed by Formic Acid fixing solution overnight, water wash for 15 min, Sodium Bicarbonate / Sodium Formate neutralisation solution for 1 h, water wash for 15 min, re-tanning solution overnight followed by Formic Acid fixing solution overnight, water wash for 15 min, fatliquoring solution overnight followed by Formic Acid fixing solution for 1 h, and finally water wash for 15 min. Tanned single cell layers and tanned cellular composites, i.e. the resulting thin and ultra-thin cultivated leather, were subsequently air-dried and stored until analysis. Natural animal skin was also chromium-tanned as a control. All experiments were performed using three independent replicates.

[0403] Compositional analysis of thin and ultra-thin cultivated leather

[0404] The cultivated leather made from scaffold-free cellular composites was stained with Picrosirium Red and Sudan IV for the detection of ECM proteins (most specifically collagen) and of lipids, respectively. Briefly, for Picrosirium Red staining, 0.3 cm2button samples of cultivated leather were re-hydrated with PBS for 15 min, incubated with 0.1% (w / v) of Direct Red 80 dye diluted in a 1.3% (v / v) Picric Acid-saturated solution for 4 h at room temperature, washed repeatedly with 5% (v / v) Acetic Acid for 15 min and finally with water to remove excess dye; for Sudan IV staining, thin sections were de-waxed, rinsed in 70% ethanol for 5 min, incubated with 0.1% (w / v) of Sudan IV dye in an acetone : ethanol : water (50:35: 15 v / v ratio) solution for 6 min, washed repeatedly with 80% (v / v) ethanol and finally with water to remove excess dye.

[0405] Structural analysis of thin and ultra-thin cultivated leather

[0406] The cultivated leather made from scaffold-free single cell layers and scaffold-free cellular composites was evaluated for overall weight and dimensions, including thickness, using a high-precision digital balance and calipers, respectively. The stability of the cultivated leather was evaluated by differential scanning calorimetry (DSC) to measure how the physical properties change with temperature against time. The ultrastructure of the cultivated leather was analysed by scanning electron microscopy (SEM). Briefly, cultivated leather samples were transferred to a culture dish, freeze-dried at -35 °C for 26 h, mounted onto a conductive stub, coated by low temperature plasma irradiation and low gas pressure, and then imaged using a TESCAN VEGA3 scanning electron microscope. Scaffold-free cellular composites without preservation (raw) and tanning (pickled) were also analysed, alongside natural animal leather.

[0407] Mechanical analysis of thin and ultra-thin cultivated leather

[0408] Ultra-thin cultivated leather specimens were cut into dogbone shapes using a scalpel and custom-made stencil, and the average thickness on the shoulder and gauge regions was measured using digital calipers(dimensions and measurement locations described in Fig. 10). The shoulder regions of specimens were then glued to thick, high-grammage (250 gsm) paper to protect from damage and ensure good grip, and then secured into the clamps of Shimadzu Autograph AGS-X tensile tester. Tensile properties were evaluated at a constant speed of 100 mm / min.

[0409] Results and Discussion

[0410] Scaffold-free cell layers were assembled in 12-, 25-, 50- and 100-layer stacks to create cellular composites with a defined thickness (Fig. 9a). These scaffold-free cellular composites were then successfully transformed into strong, imputrescible leather using either chromium-based or vegetablebased chemical tanning protocols (Fig. 9b) while avoiding using any animal-derived ingredient (i.e., a truly cultivated product). Remarkably, the resulting cultivated leather presented many of the hallmarks of natural animal leather, including its characteristic smooth feel, warm, earthy smell, pliable, elastic strength, velvety suppleness, and a dense, fibrous appearance (Fig. 9b). Importantly, this tissue production method allowed to establish the close direct correlation between the number of layers in a stack and an average thickness of the resulting cellular composites, which consequently defined the thickness of the final cultivated leather product. In other words, these results demonstrated the ability of producing thin and ultra-thin leather using a rational, bottom-up approach, where the number of scaffold-free cell layers used determines the thickness of the final cultivated leather product.

[0411] The structural and compositional properties of the produced cultivated leather were further characterised in detail. Firstly, the histochemical analysis of cultivated leather produced from 50- and 25 -layer scaffold-free cellular composites showed that both strongly stained positively with Picrosirius Red (Fig. 9c), as expected for tissues mostly comprising fibrillar collagens, and with Sudan IV (Fig.

[0412] 9d), indicating that both were successfully enriched with lipids during the fatliquoring step of chemical tanning, independently of their thickness. The distribution of Sudan IV staining in cultivated leather was more even throughout the tissue than in natural animal leather, with the latter having lipids mostly accumulating within spaces previously occupied by vasculature.

[0413] Secondly, the stability of cultivated leather and of the scaffold-free cellular composites (formed from 100 cell layers) that originated it was analysed by differential scanning calorimetry (DSC) (Table 5). Interestingly, these results showed that raw and picked cellular composites presented higher onset, peak, and endset temperature values compared with porcine dermis samples, indicating that, even without tanning, these cellular composites have improved thermal stability compared with their natural counterparts (Table 5). In addition, cultivated leather made from such cellular composites presented calorimetry profiles at the higher range of that of natural animal leather, which typically have onset values of 95-105°C or 70-80°C when chromium- or vegetable-tanned, respectively. This observation again supports the notion that cultivated leather produced by the abovementioned methods have a stability performance comparable to that of natural animal leather of the highest quality.Sample Temp, range (°C) Onset (°C) Peak (°C) Endset (°C) Natural Skin #1 (Raw) 25-100@5 52.30 53.50 57.40

[0414] Natural Skin #2 (Raw) 25-100@5 55.60 60.40 63.60

[0415] Natural Skin #3 (Raw) 25-100@5 56.60 61.20 68.60

[0416] SCL #1 (Raw) 25-100@5 76.12 77.50 79.14

[0417] SCC #1 (Raw) 25-100@5 76.31 77.99 79.89

[0418] SCC #2 (Raw) 25-100@5 75.26 77.19 79.35

[0419] SCC #3 (Raw) 25-100@5 74.88 76.67 78.98

[0420] Natural Skin#l (Pickled) 25-100@5 60.06 63.65 67.95

[0421] Natural Skin #2 (Pickled) 25-100@5 67.30 70.24 73.16

[0422] Natural Skin #3 (Pickled) 25-100@5 59.13 61.50 65.86

[0423] SCL #1 (Pickled) 25-100@5 70.06 71.72 73.96

[0424] SCC #1 (Pickled) 25-100@5 69.20 71.31 74.46

[0425] SCC #2 (Pickled) 25-100@5 69.11 71.31 73.90

[0426] SCC #3 (Pickled) 25-100@5 69.32 71.09 73.39

[0427] Natural leather #1 (C) 25-100@5 97.03 104.42 106.46

[0428] Natural leather #2 (C) 25-100@5 98.83 104.90 108.05

[0429] Natural leather #3 (C) 25-100@5 101.55 104.71 107.22

[0430] Cultivated leather #1 (C) 25-150@5 102.17 106.58 113.08

[0431] Cultivated leather #2 (C) 25-150@5 101.20 105.69 108.68

[0432] Cultivated leather #3 (C) 25-150@5 103.24 106.69 112.63

[0433] Cultivated leather #4 (C) 25-150@5 104.21 107.58 113.33

[0434] Cultivated leather #1 (V) 25-100@5 80.25 85.91 89.39

[0435] Cultivated leather #2 (V) 25-100@5 79.79 84.58 88.87

[0436] Cultivated leather #3 (V) 25-100@5 81.21 87.02 90.72

[0437] Cultivated leather #4 (V) 25-100@5 81.74 86.59 90.46

[0438]

[0439] Table 5. Hydrothermal stability measured using a DSC method. The onset temperature is usually considered the shrinkage temperature. SCL (single cell layer) was analysed as raw or picked tissues, and after tanning (Cultivated leather #1), and SCCs (scaffold-free cellular composites comprising 100cell layers) were analysed as raw or pickled tissued and after tanning (Cultivated leather #2, #3, and #4); C, chromium tanning; V, vegetable tanning.

[0440] The strong performance of cultivated leather was probably derived from a combination between its collagen- and elastin-rich composition (Table 4) and very dense structure (Fig. 10). Compared with the structure of full-grain natural animal leather (i.e., made from both a loosely-arranged papillary dermis and an highly-organised reticular dermis), the layered structure of cultivated leather results in a denser matrix (Fig. 10a and 10b). However, the chemical tanning equally provided additional cross-linking of the fibres comprising cultivated leather of different thickness (Fig. 10c and lOd) over that of untanned cellular composites (Fig. lOe), and comparable to that of natural animal leather (Fig. 1 Of).

[0441] Leather is an elastic material, as demonstrated by the high maximum displacement and percentage elongation observed in the sample of natural animal leather under applied force (Fig. 11). However, these material properties depend on multiple factors, including animal species, area of the body, leather type (full grain, genuine, etc.), tanning process and operator, tissue thickness, etc. Ultra-thin cultivated leather samples produced from 12- and 25-layer cellular composites were subjected to tensile testing. When comparing cultivated leather to natural animal leather — both tanned using the same technique in the same tannery (Fig. 11b) — the maximum force the former can withstand is lower. This is expected, as cultivated leather samples tested were significantly thinner. For example, the 0.109 (produced from 25 -cell layer cellular composite) and 0.045 mm (produced from 12-cell layer cellular composite) cultivated leather samples (Fig. 11, i and ii, respectively) are approximately 8 and 16 times thinner than the natural animal leather tested and withstand ~ 5 and 18 times less force. However, when accounting for thickness, the tensile strength of cultivated leather samples is comparable to that of natural animal leather. The small increase in tensile strength between the 0.045 mm and 0.109 mm cultivated leather samples suggests that further thickness increases could enhance tensile strength beyond that of natural animal leather. It is noteworthy that cultivated leather met the minimum tensile strength requirements (>10 N / mm2) for natural animal leather applications such as making watch straps. The stress-strain data showed that both cultivated leather and natural animal leather exhibited similar material behaviour (Fig. 11c), with an elastic region showing a nearly linear increase in strain with increased stress in the initial phase. Both cultivated leather samples had larger yielding and plastic deformation phases than their natural counterpart (i.e., longer region of the curve where when strain increases, the rate of stress increase decreases). The yielding phase of a material marks the transition from elastic (reversible) to plastic (permanent) deformation, where the material begins to deform permanently under stress, even after the load is removed. The differences in peak stress (tensile strength also referred to as tensile stress herein) were also evident, whereby the peak for the thicker cultivated leather sample is at a higher tensile strength point and lower strain compared with that of the thinner sample (Fig. 11c, i and ii, respectively), indicating that increased cultivated leather thickness increasesmaterial strength but decreases elasticity. Elongation of cultivated leather was also lower than that of natural animal leather (Fig. 1 Id), as expected from the former’s denser, layered ultrastructure (Fig. 10). Unlike tensile strength, percentage elongation in cultivated leather decreased with increasing thickness (Fig. 11c and 1 Id). While this may be a normal variation, it also suggests that increased thickness could make the material more brittle. Leather elongation is not typically used as an industry benchmark, using standard flexibility resistance tests instead. Moreover, leather flexibility is highly dependent on the tanning and finishing processes, and these can be optimised to improve this aspect of cultivated leather’s performance.

[0442] Cultivated leather Processing Average onset Average tensile temperature (°C) strength (N / mm2)

[0443] Cultivated leather of the None 75±1 (100 cell layers) n.a.

[0444] present invention

[0445] Pickling 69±0 (100 cell layers) n.a.

[0446] Chrome tanning 102±l (100 cell layers) 11 (25 layers) Vegetable tanning 81±1 (100 cell layers) n.a.

[0447] Natural skin (results None 55±6 n.a.

[0448] obtained in the Examples

[0449] herein and described in Pickling 62±6 n.a.

[0450] the literature2-5)

[0451] Chrome tanning 100±5 8-40

[0452] Vegetable tanning 78±5 7-28

[0453] Jakab et al. 2019 (results None n.a. n.a.

[0454] described in this paper)

[0455] Pickling n.a. n.a.

[0456] Chrome tanning 96±2 19

[0457] Vegetable tanning 79±1 12

[0458] WO 2017 / 193058 Al None n.a. n.a.

[0459] (results described in this

[0460] patent) Pickling n.a. n.a.

[0461] Chrome tanning n.a. 2.5

[0462] Vegetable tanning n.a. n.a.

[0463]

[0464] Table 6. Comparison of thermal stability and tensile performance between thin and ultra-thin cultivated leather produced via methods described in this patent, and natural animal leather and other cultivated leathers.

[0465] The data in Table 6 shows that the cultivated leather of the present invention performs better than other leathers (both natural and cultivated) described in the literature, in particular as the cultivated leather of the present invention has high thermal stability, with onset or heat shrinkage temperature >74°C for raw cellular constructs, and >100°C and >80°C for chrome-tanned and veg-tanned cultivated leather, respectively.

[0466] The reader's attention is directed to all papers and documents which are fded concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0467] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0468] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0469] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0470] REFERENCES

[0471] 1. K. Jakab, et. al. Non-medical applications of tissue engineering: biofabrication of a leather-like material, Materials Today Sustainability, Volume 5, 2019, 100018, ISSN 2589-2347

[0472] 2. Claire Chahine, Changes in hydrothermal stability of leather and parchment with deterioration: a DSC study, Thermochimica Acta, Volume 365, Issues 1-2, 2000, Pages 101-110

[0473] 3. Z. Sebestyen, et al., Thermal degradation study of vegetable tannins and vegetable tanned leathers, Journal of Analytical and Applied Pyrolysis, Volume 138, 2019, Pages 178-187

[0474] 4. Virgilijus Valeika, Low-pickle Processing of Leather: Assessment of Leather Tanning Quality by Methods of Thermal Analysis, Vol. 26 No. 3 (2020): Materials Science

[0475] 5. Zhang, Yi., et al. High-Efficiency Chrome Tanning Using Pre-Treatments: Synchrotron Saxs And Dsc Study, IULTCS congress Dresden, 2019.

Claims

CLAIMS1. A method for producing thin cultivated leather, the method comprising:(a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) repeating step (a) at least once to independently produce further cell layers;(c) contacting the primary cell layer with the further cell layers to produce a cellular composite; (d) culturing the cellular composite in serum-free medium; and(e) tanning the cellular composite to produce thin cultivated leather; wherein the cellular composite is between about 0.2 mm and 0.8 mm thick and wherein the thin cultivated leather is at most 0.4 mm thick.

2. The method of claim 1, the method comprising a further step (f), wherein the thin cultivated leather produced in step (e) is split to produce at least two independent tanned tissue sheets.

3. A method for producing ultra-thin cultivated leather, the method comprising:(a) culturing a composition comprising one or more cells in serum-free medium to produce a primary cell layer, wherein the one or more cells comprise fibroblasts and / or fibroblast progenitor cells; (b) optionally repeating step (a) at least once to independently produce further cell layers and contacting the primary cell layer with the further cell layers to produce a cellular composite;(c) culturing the primary cell layer or the cellular composite in serum-free medium; and (d) tanning the primary cell layer or the cellular composite to produce ultra-thin cultivated leather; wherein the primary cell layer is between about 0.005 mm and 0.2 mm thick or the cellular composite is between about 0.01 mm and 0.4 mm thick and the ultra-thin cultivated leather is less than 0.2 mm thick.

4. The method according to any preceding claim, wherein the primary cell layer and / or the cellular composite and / or the cultivated leather does not comprise a scaffold.

5. The method according to any preceding claim, wherein the fibroblasts are dermal fibroblasts.

6. The method according to any preceding claim, wherein the composition further comprises adipocytes and / or adipose progenitor cells,7. The method according to claim 6, wherein the adipose progenitor cells are pre-adipocytes, optionally wherein the pre-adipocytes are hypodermal pre-adipocytes.

8. The method according to any preceding claim, wherein the serum -free medium comprises one or more macromolecular crowding (MMC) agents.

9. The method according to claim 8, wherein the MMC is selected from: PEG8, PEG35, PVP40, PVP360, carrageenan, dextran sulphate, Ficoll® 70, Ficoll® 400, and / or PSS; or combinations thereof.

10. The method according to any preceding claim, wherein the culturing step to produce a primary cell layer is for at least about 7 days, preferably at least about 14 days.

11. The method according to any preceding claim, wherein the culturing of the cellular composite is for at least about 7 days.

12. The method according to any preceding claim, wherein the tanning is selected from chrome tanning, vegetable tanning, or chrome-free tanning.

13. A thin cultivated leather obtained by a method according to any preceding claim, wherein the thin cultivated leather has an average thickness of between 0.2 and 0.4 mm, wherein the onset temperature of the leather is more than 80°C.

14. An ultra-thin cultivated leather obtained by a method according to any preceding claim, wherein the ultra-thin cultivated leather has an average thickness of less than 0.2 mm, wherein the onset temperature of the leather is more than 80°C.

15. The cultivated leather of any one of claims 13 or 14, wherein the cultivated leather does not comprise a scaffold.

16. The cultivated leather of any one of claims 13-15, wherein the onset temperature of the leather is more than 85°C, more than 90°C, more than 95°C, more than 100°C, or more than 105°C.

17. The cultivated leather of any one of claims 13-16, wherein the leather has a tensile strength of >10 N / mm2.

18. A thin cultivated leather wherein the thin cultivated leather has an average thickness of between 0.2 and 0.4 mm, wherein the onset temperature of the leather is more than 80°C.

19. An ultra-thin cultivated leather wherein the ultra-thin cultivated leather has an average thickness of less than 0.2 mm, wherein the onset temperature of the leather is more than 80°C.

20. The leather according to any one of claims 18 or 19, wherein the cultivated leather does not comprise a scaffold.

21. The leather of claim 19, wherein the leather has an average thickness of 0.109 mm.

22. The leather of claim 19, wherein the leather has an average thickness of less than 0.051 mm.

23. The leather of claim 19, wherein the leather has an average thickness of 0.045 mm.

24. The leather according to any one of claims 18-23, wherein the onset temperature of the leather is more than 85°C, more than 90°C, more than 95°C, more than 100°C, or more than 105°C.

25. The leather according to any one of claims 18-24, wherein the leather has a tensile strength of >10 N / mm2.

26. The leather according to any one of claims 13-25, wherein the leather does not comprise one or more of the basal laminin components Laminin-332, Tenascin-C, and / or Periostin.

27. A cellular composite wherein the cellular composite has an average thickness of at most 0.8 mm, wherein the onset temperature of the cellular composite is more than 69°C when pickled and / or more than 74°C when raw.

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