Plant-based cell culture substrates

Abstract

Described in this specification are technologies including cell-culture substrates and tissue constructs formed therefrom (e.g., cultivated meats, cultivated leathers, cultivated furs, etc.) comprising configurations (e.g., nonwoven fabrics, battings, blends, meshes, yarns, etc.) and modifications (e.g., amphiphilic coatings to render said substrates substantially hydrophilic, etc.) of one or more coma bearing, pappus-bearing, or otherwise hairy plant seeds and their substantially hydrophobic hairy appendages. The technologies include systems and methods for manufacturing said cell-culture substrates and tissue constructs, as well as uses thereof.

Description

PLANT-BASED CELL CULTURE SUBSTRATESCROSS-REFERENCE

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 526,590, filed on July 13, 2023, entitled “Plant-based Cell Culture Substrates,” which is incorporated herein by reference in its entirety.BACKGROUND

[0002] In animal cell culture applications including tissue-engineering and cellular agriculture, there is a need for three-dimensional cell-culture substrates (e.g., scaffolds, etc.) capable of supporting cell attachment, survival, proliferation, differentiation, and tissue formation. In environmentally sustainable and animal-welfare conscious applications including, but not limited to, cultivated meat, cultivated leather, cultivated fur, etc., there is a further need for novel scaffolds comprised in part or wholly of plant materials.SUMMARY

[0003] Described in this specification are cell-culture substrates and tissue constructs formed therefrom (e.g., cultivated meats, cultivated leathers, cultivated furs, etc.) comprising configurations (e.g., nonwoven fabrics, battings, blends, meshes, yams, etc.) and modifications (e.g., amphiphilic coatings to render said substrates substantially hydrophilic, etc.) of one or more coma-bearing, pappus-bearing, or otherwise hairy plant seeds and their substantially hydrophobic hairy appendages.

[0004] Described in this specification are methods of manufacturing cell-culture substrates and tissue constructs formed therefrom (e.g., cultivated meats, cultivated leathers, cultivated furs, etc.) comprising configurations (e.g., nonwoven fabrics, battings, blends, meshes, yams, etc.) and modifications (e.g., amphiphilic coatings to render said substrates substantially hydrophilic, etc.) of one or more coma-bearing, pappus-bearing, or otherwise hairy plant seeds and their substantially hydrophobic hairy appendages.

[0005] The methods include providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs in a volume and processing the plant seeds into a cell-culture substrate. The methods include processing the plant seeds such that the hairs of a first seed overlaps with the hairs of at least a second seed and repeatedly inserting (punching) a needle into the volume, thereby entangling the hairs of the first seed with the hairs of the at least a second seed. The methods include providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; and forming, from the amount of seeds, a substrate by means of a non-woven wet-laid process, spunbond process, solvent bonding, thermal bonding, hydroentangling, calendaring, binding (e.g., gluing), or combination thereof. The methods include providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; spinning or otherwise forming the seeds into one or moremultifilament yams; and knitting or weaving the one or more yams into a scaffold. The methods include providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs such that the hairs are suspended from a fixed, movable, or floating substrate in or on a volume of fluid; and joining (entangling) the hairs through movement of the fluid. The methods include providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; and magnetizing or compartmentalizing the amount seeds within a substantially porous enclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The following figures are exemplary and are not intended to be limiting.

[0007] Fig. 1 illustrates schematically examples of seeds and seed hairs. Fig. 1A: Seed (1) and coma (i.e., seed hairs (2)) of common milkweed. Fig. IB: Achene (i.e., fruit containing a seed; (3)), pappus beak (4) and pappus (i.e., seed hairs; (5)) of common dandelion. Fig. 1C: Example of how a felting needle (6) may penetrate through a plurality of seed hairs to form a needle-punched nonwoven of seed hairs (7).

[0008] Fig. 2 illustrates examples of coma-bearing and pappus-bearing seeds and their substantially hydrophobic seed hairs. In particular, at the macro-scale: Fig. 2A: milkweed, Fig. 2B: kapok tree, Fig. 2C: dandelion, Fig. 2D: cottonwood tree, and Fig. 2E: cattail all exhibit a hairy structure, which can be observed microscopically (Fig. 2F-2J, respectively; scale bar = 200 microns).

[0009] Fig. 3 illustrates an example of: Fig. 3A: a needle-punched nonwoven cottonwood tree seed hair scaffold that was infiltrated with a suspension of bovine dermal fibroblasts suspended in a bovine collagen gel (Fig. 3B) and subjected to uniaxial tensile mechanical testing (Fig. 3C-3E). (Fig. 3B) shows an example of an approximately 7mm x 7mm test specimen, (Fig. 3C) and (Fig. 3D) show the specimen at the beginning and end of the uniaxial tensile test, respectively. (Fig. 3E) shows a representative force- displacement curve.

[0010] Fig. 4 illustrates a tissue construct (e.g., a cultivated meat) comprising a needle-punched nonwoven substrate of kapok tree seeds and seed hairs that was lecithin-treated and gelatin- coated prior to seeding with C2C12 muscle cells. Fig. 4A: Cells were observed to be attached to the hydrophilic-modified and cell-adhesive protein coated seed hairs at 1-day post-seeding (via Hoechst staining of cell nuclei; blue). Fig. 4B: Cells were observed to have proliferated on and between the hydrophilic-modified and cell-adhesive protein coated seed hairs at 7-days post- seeding (via Hoechst staining of cell nuclei; blue). Fig. 4C: Representative phase -contrast photomicrograph of C2C12 cells growing on the periphery of the lecithin-treated, gelatin- coated kapok tree seed hairs. Fig. 4D: Photo of a 14-day cultivated tissue construct comprising a needle-punched nonwoven substrate of kapok tree seeds and seed hairs that was lecithin- treated, gelatin-coated, and seeded with C2C12 muscle cells.

[0011] Fig. 5 provides results for lecithin-treated and gelatin-coated kapok tree seed and seed hair scaffold seeded with a combination of C2C12 muscle cells and dermal fibroblasts and cultivated for two weeks. In particular, Fig. 5A-5C shows phase-contrast light micrographs of cells attached andproliferating on the edge of scaffolds at days 1, 7, and 14 post-seeding, respectively (scale bar = 200 microns). Fig. 5D-5F show fluorescence micrographs of cells attached and proliferating on the kapok fibers at days 1, 7, and 14 post-seeding, respectively (Hoechst- stained cell nuclei; 4x). Fig. 5G shows DNA content (micrograms per gram wet weight) of cell-seeded kapok scaffolds at days 1, 7, and 14 post-seeding, showing quantitative evidence of cell proliferation consistent with micrographic observations. Fig. 5H shows Collagen content (micrograms per gram wet weight) of cell-seeded kapok scaffolds at days 1, 7, and 14 post-seeding, showing evidence of progressive accumulation of cell-synthesized collagen during prolonged culture. Fig. 51 shows uniaxial tensile force-displacement curves of cell-seeded kapok scaffolds at days 1, 7, and 14 post-seeding, showing evidence of changes in mechanical behavior in association with cell proliferation and collagen accumulation with prolonged culture. Fig. 5J-5L are photos captured of the cell-seeded kapok scaffold mechanical test specimens at the maximum displacement tested (approximately 50% strain; insets show photos of the same approximately 7mm x 7mm cell- seeded kapok scaffold mechanical test specimens prior to testing).

[0012] Fig. 6 provides results for lecithin-treated and gelatin-coated cottonwood tree seed and seed hair scaffolds seeded with a combination of C2C12 muscle cells and dermal fibroblasts and cultivated for two weeks. In particular, Fig. 6A-6C shows phase-contrast light micrographs of cells attached and proliferating on the edge of scaffolds at days 1, 7, and 14 post-seeding, respectively (scale bar = 200 microns). Fig. 6D-6F show fluorescence micrographs of cells attached and proliferating on the cottonwood fibers at days 1, 7, and 14 post-seeding, respectively (Hoechst-stained cell nuclei; 4x). Fig. 6G shows DNA content (micrograms per gram wet weight) of cell- seeded cottonwood scaffolds at days 1, 7, and 14 post-seeding, showing quantitative evidence of cell proliferation consistent with micrographic observations. Fig. 6H shows Collagen content (micrograms per gram wet weight) of cell-seeded cottonwood scaffolds at days 1, 7, and 14 post-seeding, showing evidence of progressive accumulation of cell-synthesized collagen during prolonged culture. Fig. 61 shows uniaxial tensile force-displacement curves of cell-seeded cottonwood scaffolds at days 1, 7, and 14 post-seeding, showing evidence of changes in mechanical behavior in association with cell proliferation and collagen accumulation with prolonged culture. Fig. 6J-6L are photos captured of the cell-seeded cottonwood scaffold mechanical test specimens at the maximum displacement tested (approximately 50% strain; insets show photos of the same approximately 7mm x 7mm cell-seeded cottonwood scaffold mechanical test specimens prior to testing).

[0013] Fig. 7 provides results for lecithin-treated and gelatin-coated milkweed seed and seed hair scaffolds seeded with a combination of C2C12 muscle cells and dermal fibroblasts and cultivated for two weeks. In particular, Fig. 7A-7C shows phase-contrast light micrographs of cells attached and proliferating on the edge of scaffolds at days 1, 7, and 14 post-seeding, respectively (scale bar = 200 microns). Fig. 7D-7F show fluorescence micrographs of cells attached and proliferating on the milkweed fibers at days 1, 7, and 14 post-seeding, respectively (Hoechst-stained cell nuclei; 4x). Fig.6G shows DNA content (micrograms per gram wet weight) of cell- seeded milkweed scaffolds at days 1, 7, and 14 post-seeding, showing quantitative evidence of cell proliferation consistent with micrographic observations. Fig. 7H shows Collagen content (micrograms per gram wet weight) of cell-seeded milkweed scaffolds at days 1, 7, and 14 post-seeding, showing evidence of progressive accumulation of cell-synthesized collagen during prolonged culture. Fig. 71 shows uniaxial tensile force-displacement curves of cell-seeded milkweed scaffolds at days 1, 7, and 14 post-seeding, showing evidence of changes in mechanical behavior in association with cell proliferation and collagen accumulation with prolonged culture. Fig. 7J-7L are photos captured of the cell-seeded milkweed scaffold mechanical test specimens at the maximum displacement tested (approximately 50% strain; insets show photos of the same approximately 7mm x 7mm cell-seeded milkweed scaffold mechanical test specimens prior to testing).

[0014] Fig. 8 provides results for lecithin-treated and gelatin-coated dandelion-cottonwood tree seed and seed hair blend scaffolds seeded with a combination of C2C12 muscle cells and dermal fibroblasts and cultivated for two weeks. In particular, Fig. 8A-8C shows phase-contrast light micrographs of cells attached and proliferating on the edge of scaffolds at days 1, 7, and 14 postseeding, respectively (scale bar = 200 microns). Fig. 8D-8F shows fluorescence micrographs of cells attached and proliferating on the dandelion and cottonwood fibers at days 1, 7, and 14 post-seeding, respectively (Hoechst-stained cell nuclei; 4x). Fig. 8G shows DNA content (micrograms per gram wet weight) of cell-seeded dandelion-cottonwood blend scaffolds at days 1, 7, and 14 post-seeding, showing quantitative evidence of cell proliferation consistent with micrographic observations. Fig. 8H shows Collagen content (micrograms per gram wet weight) of cell-seeded dandelion- cottonwood blend scaffolds at days 1, 7, and 14 post-seeding, showing evidence of progressive accumulation of cell-synthesized collagen during prolonged culture. Fig. 81 shows Uniaxial tensile force- displacement curves of cell-seeded dandelion-cottonwood blend scaffolds at days 1, 7, and 14 post-seeding, showing evidence of changes in mechanical behavior in association with cell proliferation and collagen accumulation with prolonged culture. Fig. 8J-8L are photos captured of the cell-seeded dandelion-cottonwood blend scaffold mechanical test specimens at the maximum displacement tested (approximately 50% strain; insets show photos of the same approximately 7mm x 7mm cell-seeded dandelion-cottonwood blend scaffold mechanical test specimens prior to testing).

[0015] Fig. 9 provides results for lecithin-treated and gelatin-coated cattail-cottonwood tree seed and seed hair blend scaffolds seeded with a combination of C2C12 muscle cells and dermal fibroblasts and cultivated for two weeks. In particular, Fig. 9A-9C shows phase-contrast light micrographs of cells attached and proliferating on the edge of scaffolds at days 1, 7, and 14 post-seeding, respectively (scale bar = 200 microns). Fig. 9D-9F show fluorescence micrographs of cells attached and proliferating on the cattail and cottonwood fibers at days 1, 7, and 14 post-seeding, respectively (Hoechst-stained cell nuclei; 4x). Fig. 9G shows DNA content (micrograms per gram wet weight) of cell-seeded cattail-cottonwood blend scaffolds at days 1, 7, and 14 post- seeding, showing quantitativeevidence of cell proliferation consistent with micrographic observations. Fig. 9H shows Collagen content (micrograms per gram wet weight) of cell-seeded cattail- cottonwood blend scaffolds at days 1, 7, and 14 post-seeding, showing evidence of progressive accumulation of cell-synthesized collagen during prolonged culture. Fig. 91 shows uniaxial tensile force- displacement curves of cell-seeded cattail-cottonwood blend scaffolds at days 1, 7, and 14 post- seeding, showing evidence of changes in mechanical behavior in association with cell proliferation and collagen accumulation with prolonged culture. Fig. 9J-9L are photos captured of the cell-seeded cattail-cottonwood blend scaffold mechanical test specimens at the maximum displacement tested (approximately 50% strain; insets show photos of the same approximately 7mm x 7mm cell-seeded cattail-cottonwood blend scaffold mechanical test specimens prior to testing).

[0016] Fig. 10A-C are photographs illustrating the fabrication and cell seeding of a cultivated fur on top of a cultivated skin. In particular, Fig. 10A shows the top (fur) and Fig. 10B shows the bottom (skin) of the scaffold, wherein (1) untreated, substantially hydrophobic fur hairs made of milkweed seed hairs have been incorporated by needle punching into a (2) lecithin-treated, gelatin-coated kapok tree seed and seed hair scaffold. Fig. 10C shows the autoclave-sterilized scaffold in a polypropylene container during the (3) lecithin treatment step, wherein the (4) kapok scaffold and (5) milkweed seed hairs are observable. Fig. 10D shows the scaffold in a polypropylene container during the (6) cell suspension seeding step, wherein the (7) kapok scaffold and (8) milkweed seed hairs are observable.

[0017] Fig. 11 demonstrates cell-mediated tissue formation, coloring and flavoring, and cooking of a dandelion seed hair-based cultivated meat product having colors and appearances substantially resembling those of bacon. Fig. 11A is a photograph of said cultivated meat comprising a lecithin- treated, gelatin-coated dandelion seed hair scaffold cultured with bovine dermal fibroblasts for 49 days (; shown in a standard 6-well tissue culture plate; scale bar = 1 cm). Fig. 11B is a phase-contrast light photomicrograph demonstrating bovine dermal fibroblasts substantially filling the scaffold pore space (a representative dandelion seed hair is indicated by white arrow; scale bar = 50 microns). Fig. 11C is a close-up photograph of said cultivated meat showing tissue variations (e.g., spatial variations in the fibrous appearance, thickness, curvature, and holes, etc.) associated with the substantially random orientations of the dandelion seed hairs comprising the scaffold and cell-mediated tissue formation processes (two representative achene associated with the dandelion seed hairs are indicated by white arrows; scale bar = 1 cm). Figs. 11D and HE are photographs of a first side (D) and opposite second side (E) of said cultivated meat after 10-hours refrigerated treatment in a solution comprising water and food-grade coloring (e.g., beet powder) and flavoring (e.g., yeast extracts) ingredients. Variations in the cultivated meat color (e.g., pink to red) and appearance (e.g., orientations, lengths, and widths of fibrous structures, tissue thickness, tissue curvature, and holes, etc.) (D, E; scale bars = 1cm) were substantially consistent with those of a slice of uncooked bacon. Fig. HF is a photograph of said cultivated meat on a digital scale showing the nominally 1.3-gram wet weight. Figs. 11G and 11H are photographs of a first side (G; scale bar = 1cm) and oppositesecond side (H; scale bar = 1 cm) of said colored and flavored cultivated after cooking (the sides and orientations of the cultivated meat in photos 11G and 11H correspond to those of D and E, respectively). Notably, upon cooking by frying in extra-virgin olive oil at a nominal temperature of 150-200 degrees Celsius for 10 minutes yielded colors (e.g., dark red, brown) and appearances (e.g., curled) substantially consistent with that of cooked bacon.

[0018] Fig. 12 demonstrates cell-mediated tissue formation, tanning, and dyeing of a kapok tree seed hair-based cultivated cow skin having colors and appearances substantially resembling those of leather. Fig. 12A is a fluorescence micrograph of bovine dermal fibroblasts isolated from a skin biopsy (lOOx original magnification; blue = cell nuclei; green = filamentous (F)-actin). Fig. 12B is a photograph of said cultivated cow skin comprising a lecithin-treated, gelatin-coated kapok tree seed hair scaffold cultured with said bovine dermal fibroblasts for 42 days ( adult hands shown for scale). Fig. 12C is a photograph of said cultivated cow skin being manually folded, demonstrating substantially tissue-like flexibility (adult hands shown for scale). Fig. 12D is a photograph of said cultivated cow skin following a vegetable-based tanning process, yielding a substantially flexible cultivated leather (adult hand shown for scale). Fig. 12E is a photograph of two strips of said cultivated leather demonstrating a substantially leather grain-like surface texture following dyeing either a saddle brown color (top-most strip) or a sunflower color (bottom-most strip) (strips cut to a nominal width (e.g., 22 mm) and nominal length (e.g., 17 cm) useful for watchbands; ruler shown for scale). Fig. 12F is a photograph of said two strips of dyed cultivated leather being folded, demonstrating substantial flexibility for applications such as watchstraps, wallets, clothing, upholstery, or any leather application wherein the ability of said leather to be bent, folded, or flexed, etc. is useful. Fig. 12G is a photograph of a tanned, undyed cultivated leather comprising a lecithin- treated, gelatin-coated milkweed seed hair scaffold cultured with said bovine dermal fibroblasts for 40 days (scale bar = 1 cm). Note that the surface texture of said milkweed seed hair-based cultivated leather (G) appears substantially different from that associated with the kapok tree seed hair-based scaffold (E, F).

[0019] Fig. 13 demonstrates visually observable changes in cultivated cow skin appearance, microscopic morphology, and collagen distribution in association with extended culture duration. Figs. 13A-C are photographs of said nominally 3 cm x 3 cm x 1-2 mm thick cultivated cow skins comprising lecithin-treated, gelatin-coated kapok tree seed hair scaffolds cultured with bovine dermal fibroblasts for 14 days (A; adult fingers shown for scale), 21 days (B; adult fingers shown for scale), and 28 days (C; adult fingers shown for scale). Following 14 days culture, said cultivated cow skin exhibited a glistening, substantially undulated (i.e., wavy) surface comprising a “ridge and valley”- like texture wherein the nominally beige color of the kapok tree seed hair was substantially visible (A). Following 21 days culture, said cultivated cow skin exhibited a substantially smooth, glistening surface wherein the color varied from substantially white to pink (B). Following 28 days culture, said cultivated cow skin exhibited substantial curling at the edges and bending, in association with cell-mediated traction forces typical of fibroblasts, wherein the color varied from substantially white to pink (C). Figs. 13D-F are Photomicrographs of cultivated cow skin tissues cultured for 14 days (D), 21 days (E), and 28 days (F) and histologically stained for cell nuclei and collagen (D-F; Masson’s trichrome stain; collagen = blue, cell nuclei = dark brown; scale bars = 250 microns). Cell nuclei and cell-secreted collagen were evident throughout the 1-2 mm thickness of said cultivated cow skins, with substantially more collagen (darker blue) evident toward the outer periphery at all time points. Substantially more collagen was evident by way of a thicker, darker-blue outer periphery of said tissues following 21 days (E) and 28 days (F) culture relative to that at 14 days culture (D). Close-up photomicrographs showed evidence of the substantially circular cross-section, hollow kapok tree seed hairs (black arrows in D-F insets; inset scale bar = 50 microns).

[0020] Fig. 14 is a graph illustrating collagen assay results for cultivated cow skins including lecithin-treated, gelatin-coated kapok tree seed hair scaffolds cultured with bovine dermal fibroblasts for 14 days, 21 days, and 28 days (average of n=3 samples per culture duration condition; error bars = standard deviation). Said quantitative collagen concentration data was consistent with the qualitative observations of increased collagen demonstrated histologically in Fig. 13.DESCRIPTION

[0021] Described in this specification are cell-culture substrates and tissue constructs formed therefrom (e.g., cultivated meats, cultivated leathers, cultivated furs, macro-carriers, packed- bed bioreactor substrates, tissue-engineered constructs for biomedical applications, in vitro diagnostics, organotypic in vitro models (e.g., organ-on-a-chip devices), etc.) comprising configurations (e.g., nonwoven fabrics, battings, blends, meshes, yams, etc.) and modifications (e.g., amphiphilic coatings to render said substrates substantially hydrophilic, etc.) of one or more coma-bearing, pappus-bearing, or otherwise hairy plant seeds and their substantially hydrophobic hairy appendages. Said hairy appendages are hereafter referred to as seed hairs.

[0022] In some implementations said seeds exhibiting intrinsically hydrophobic seed hairs include, but are not limited to, those of the kapok tree (Ceiba pentandra), eastern cottonwood tree (Populus deltoides), common dandelion (Taraxacum officinale), common milkweed (Asclepias syriaca), common cattail (Typha latifolia), and sow thistle (Sonchus oleraceus), etc., to name a few. These exemplary seeds and others have seed hairs covered in an outer layer containing substantially hydrophobic molecules (e.g., waxes) capable of substantially repelling water. It is understood and disclosed here that while said coma, pappus, or other types of seed hairs have been colloquially and often interchangeably referred to as “hair,” “fiber,” “tuft,” “fluff,” etc., said seed hairs comprise specialized plant seed cell extrusions which are distinct from other types of plant or animal fibers and, by virtue of their intrinsic substantial hydrophobicity, offer unique and nonobvious attributes and functionalities for use in cell-culture substrate and associated tissue construct applications.

[0023] In some implementations, the seeds may be substantially separated from their associated substantially hydrophobic seed hairs (e.g., by mechanical (e.g., ginning) or chemical means or combinations thereof). In some implementations, the seeds may be retained in their substantially natural state, wherein their seed hairs remain substantially intact and substantially connected to said seeds.

[0024] In some implementations, all or a portion of said intrinsically hydrophobic seed hairs may be modified to render one or more of them partially or entirely substantially hydrophilic (e.g., by coating with an amphiphile (e.g., a solution of lecithin in ethanol, etc.)). In some implementations, said hydrophilic modifications may be for the purpose of enhancing aqueous fluid wetting of a cell-culture substrate formed therefrom (e.g., by culture media), enhancing animal cell attachment (e.g., as mediated by a cell-adhesive peptide or protein coating provided via an aqueous buffer, etc.), enabling aqueous fluid transport (e.g., as pumped through a fluid channel comprising a wetted portion of cellculture substrate, etc.). In some implementations, all or a portion of said intrinsically hydrophobic seed hairs may be left naturally substantially hydrophobic, particularly in blends or in other configurations with hydrophilic-modified seed hairs. In some implementations, said intrinsically hydrophobic portions may be for the purpose of enhancing gas transport (e.g., by providing substantially unwetted channels capable of mediating oxygen or carbon dioxide transport, etc.), enabling non-aqueous or oily fluid flow (e.g., of a plant oil or melted fat into said substantially hydrophobic portions of said cell-culture substrates or tissue constructs made therefrom, to mimic conventional meat marbling in a cultivated meat product, etc.), etc.

[0025] In some implementations, cell-culture substrates formed from one or more coma-bearing, pappus-bearing, or otherwise hairy seeds and their substantially hydrophobic seed hairs are directed toward cell expansion applications (e.g., as macro-carriers, packed-bed bioreactor substrates, mist bioreactor substrates, etc.), wherein following modification to promote anchorage-dependent animal cell attachment, said substrates are seeded with cells at a relatively low density (e.g., between 1000 and 10,000 cells / cm2, e.g., 5000 cells / cm2of substrate surface area), said cells attach, and said cells proliferate to a relatively higher density (e.g., between 10,000 and 100,000 cells / cm2, e.g., 50,000 cells / cm2of substrate surface area) prior to being harvested from said substrate (e.g., by enzymatic dissociation (e.g., trypsin, etc.), mechanical agitation, or any other means) for subsequent downstream processing. In some implementations, cells can be seeded at densities of between 500 cells / cm2and 50,000,000 cells / cm2of substrate surface area, and proliferate to a relatively higher density of between 5,000 and 500,000 cells / cm2of substrate surface area prior to being harvested from said substrate. The technologies described in this specification can be used with any animal cell. Example animal cell comprise cells from a mammal (including a human), a bird, a fish, a crustacean, a reptile, an amphibian, an invertebrate, or a combination thereof. The animal can be a farm animal, a laboratory animal, or animal residing at an animal sanctuary. When cells derived from an animal residing at an animal sanctuary are used, the technologies described in this specification can beparticularly useful for improving animal welfare and reducing the environmental and societal impact of meat and leather production.

[0026] In some implementations, said cell-culture substrates are directed toward use as three- dimensional scaffolds for cell attachment, survival proliferation, differentiation, and tissue formation for cultivated meat, cultivated leather, or cultivated fur production, to name a few applications. Some implementations of the technologies disclosed herein are principally directed toward cellular agriculture applications, including cultivated meat, cultivated leather, and cultivated fur production. It is understood by one skilled in the art that other applications for said coma-bearing, pappus-bearing, or otherwise hairy plant seeds and their substantially hydrophobic seed hairs and scaffolds made therefrom may include biomedical tissue engineering, in vitro diagnostics, or any other anchoragedependent animal cell-culture substrate application. In some implementations said cell-culture substrates serve both as macro -carriers and as scaffolds for subsequent tissue formation.

[0027] In some implementations, a nonwoven scaffold may be fabricated by needle-punching comabearing, pappus-bearing, or otherwise hairy seeds and their substantially hydrophobic seed hairs (e.g., those of the kapok tree, cottonwood tree, milkweed, dandelion, cattail, thistle, or any other plant seed of the same or any other species, genus, or family bearing substantially similar hydrophobic hairy appendages). It is understood by one skilled in the art that a nonwoven of said exemplary seed hairs may be formed by any of a number of means, including wet-laid, spunbond, solvent bonding, thermal bonding, hydroentangling, calendaring, binding (e.g., gluing), etc. In some implementations any one or more methods may be utilized alone or in combination to form a nonwoven scaffold.

[0028] In some implementations, coma-bearing, pappus-bearing, or otherwise hairy seeds and their substantially hydrophobic seed hairs may be spun or otherwise formed into multifdament yam and knitted or woven to form scaffolds. In some implementations, said coma-bearing, pappus-bearing, or otherwise hairy seeds and their seed hairs or yams made therefrom may be suspended in a fluid media (e.g., culture media) from a fixed, movable, or floating substrate (e.g., a piece of cork, foam, etc.) such that said seed hairs dangle or float in said fluid medium. In some implementations said seed hairs may be moved (e.g., pulled, flexed, compressed, buckled, etc.) by movement of said fluid medium.

[0029] In some implementations, coma-bearing, pappus-bearing, or otherwise hairy seeds and their substantially hydrophobic seed hairs may be configured into substantially porous, substantially three- dimensional scaffolds by any means, including but not limited to the abovementioned conventional texture fabrication processes, gluing, magnetization, compartmentalization within a substantially porous enclosure (e.g., by filling a porous mesh bag, such as a mesh laundry bag, with one or a plurality of seeds and seed hairs). In some implementations, all or a portion of said intrinsically hydrophobic seed hairs may be rendered substantially hydrophilic (e.g., by coating with an amphiphile (e.g., a solution of lecithin in ethanol, etc.)) prior to forming into a cell-culture substrate or after forming into a cell-culture substrate.

[0030] In some implementations, cell-culture substrates are prepared from one or a plurality of comabearing, pappus-bearing, or otherwise hairy seeds and their substantially hydrophobic seed hairs (e.g., those of kapok tree, cottonwood tree, milkweed, dandelion, cattail, and thistle, etc.), modified to promote aqueous wettability (e.g., by amphiphilic coating all or a portion of said intrinsically hydrophobic seed hairs or scaffolds fabricated therefrom (e.g., by a solution of lecithin in ethanol)), and further treated to support anchorage-dependent animal cell attachment (e.g., by coating all or a portion of said seed hairs or cell-culture substrates formed therefrom with one or more cell-adhesive proteins or peptides (e.g., by a solution of gelatin in water or aqueous buffer solution, by a solution of zein protein in nominally 90% ethanol and 10% water, etc.).

[0031] In some implementations, cell-culture substrates are prepared by blending two or more types of coma-bearing, pappus-bearing, or otherwise hairy seeds and substantially hydrophobic seed hairs in blends comprising two or more types, wherein some implementations any proportion (e.g., mass ratio, numerical ratio (i.e., the number of hair-bearing seeds or seed hairs, etc.), layer thickness, or hair orientations (i.e., angular orientation relative to any axis of the scaffold) of any one to any other type of seed hair comprising the blend may be utilized to achieve certain performance characteristics.

[0032] In some implementations, the intrinsically hydrophobic nature of the native coma-bearing, pappus-bearing, or otherwise hairy seeds and seed hairs may be leveraged to form scaffolds comprising blends and configurations of substantially hydrophobic and substantially hydrophilic elements. For example, in some implementations hydrophilic seed hair elements (e.g., formed by amphiphile coating of one or more seed hairs prior to scaffold fabrication, etc.) may be needle- punched or otherwise bonded to hydrophobic seed hair elements. Said hydrophilic seed hair elements may be designed and configured for use in supporting cell attachment, proliferation, and tissue formation whilst one or more substantially distinct hydrophobic seed hair elements may be designed and configured for perfusion or filling with hydrophobic constituents (e.g., oxygen-carrying perfluorocarbons for tissue oxygenation, oils or fats for creating marbling in cultivated meat applications, polymers for strengthening or otherwise modifying the mechanical or textural properties of the cultivated product, etc.).

[0033] In some implementations, such blends and configurations of substantially hydrophobic and substantially hydrophilic seed hair elements may be for the purpose of modifying the buoyancy of the composite scaffold constructed thereby, such as to enable transient or continuous, free or restricted, floatation of all or a portion of said scaffold for any purpose useful in forming or processing of a cultivated product, including but not limited to gas or nutrient transport, harvesting macro-carriers, mechanical stimulation of cells (e.g., by way of mediating any contact- or non-contact-mediated movement or deformation of said scaffolds, e.g., as induced by solid or fluid mechanical means, electrical means, magnetic means, etc.).

[0034] In some implementations, said hydrophobic seed hair elements may be designed and configured to serve as one or more substantially air-filled or gas-filled compartments within or on theperiphery of said scaffolds, thereby serving to promote gas (e.g., oxygen, carbon dioxide) or vapor (e.g., water vapor) exchange to and from the cells adhered to the hydrophilic seed hair elements. For example, in some implementations said substantially gas-fdled hydrophobic seed hair elements may serve in the capacity of “snorkels,” whereby top portions of said hydrophobic seed hair elements may be configured to reside substantially in the gaseous headspace of a bioreactor system (e.g., analogous to snorkels), thereby providing active or passive transport of headspace gases (e.g., oxygen, carbon dioxide, etc.) to and from scaffold elements configured below the gas-liquid interface between the headspace and the culture media, etc.

[0035] In some implementations, a plurality of substantially hydrophobic seed hairs may be configured to provide a fur-like layer on the surface of an underlying skin-like layer. For example, in some implementations a plurality of substantially hydrophobic, relatively long seed hairs (e.g., the nominally 5 to 50 mm long hairs comprising the coma of milkweed seeds) may be either inserted, needle -punched, glued, or integrated by any method onto the surface of a like (e.g., milkweed seed hair) or dissimilar (e.g., kapok seed hair) element. In some implementations a reverse felting needle (i.e., one designed to pull fibers as opposed to pushing fibers) may be utilized to pull a plurality of relatively hydrophobic fibers into fur-like projections substantially normal to the plane of the underlying relatively hydrophilic seed hair element.

[0036] In some implementations, seed hairs may be treated with one or more enzymes (e.g., cellulase, hemicellulase, pectinase, amylase, ligninase, lipase, xylanase, etc.), sequentially or in combination, for the purpose of substantially modifying, degrading, removing, etc. one or more constituents of said seed hairs (e.g., lignin). It is understood by one skilled in the art that modification, degradation, removal, etc. of one or more seed hair constituents (e.g., lignin) may render said enzyme- treated seed hairs substantially smaller in dimension (e.g., diameter), weaker (e.g., in tension), more flexible, more biodegradable, etc.

[0037] In some implementations, the plants from which coma-bearing or pappus-bearing seeds and their associated hairy appendages are collected may be cross-bred or genetically engineered to express desirable characteristics for cell-culture substrate production. In some implementations, genetic engineering may be employed to express animal cell gene products in the seeds or their hairy appendages (e.g., growth factors, cell-adhesive proteins, etc.). In some implementations, crossbreeding or genetic engineering may be employed to optimize or facilitate hairy seed production or collection from said plants (e.g., to increase the seed yield per plant, to facilitate seed collection by strengthening or weakening the connection between the seed and the plant, to reduce or increase the dimensions of the plant, to reduce the water, sun, or nutrient requirements of said plants, etc.). In some implementations, said genetically engineered seeds and their associated hairy appendages may be crushed (e.g., by calendar rolling) or otherwise rendered substantially powdered or dissolvable to facilitate delivery of expressed growth factors, etc. to the attached animal cells, etc.

[0038] In some implementations, any of the broad variety of plant seeds having substantially hydrophobic hairy appendages (e.g., in the form of coma, pappi, etc.) may be utilized as described herein to form cell-culture substrates and tissue constructs formed therefrom (e.g., cultivated meats, cultivated leathers, cultivated furs, macro-carriers, engineered tissues for biomedical applications, etc.). It is understood by one skilled in the art that the example seeds and associated seed hairs cited and tested herein (e.g., that of kapok, cottonwood, milkweed, dandelion, and cattail) are intended as representative examples of windbome seeds that exhibit substantially hydrophobic seed hairs and are not intended to limit in any way the disclosures or claims made herein. For example, other members of the daisy plant family Asleraceae (which includes the genus Taraxacum and, therein, the common dandelion species Taraxacum officinale), exhibit substantially hydrophobic hairy appendages (e.g., pappi) similar to that of the common dandelion. For example, the various Cat’s Ear species belonging to the Hypochaeris genus also exhibit pappi and, by virtue, are commonly referred to as false dandelions.

[0039] In some implementations, a cell-culture substrate or tissue construct formed therefrom may be made by combining (e.g., by needling, binding, infdtrating, etc.) any of the plant seeds and substantially hydrophobic hairy appendages disclosed herein, to name a few, with any synthetic material (e.g., polyesters (e.g., poly(lactic acid), poly(caprolactone), etc.), silicones (e.g., poly(dimethyl siloxane)), etc.). In some implementations, any of the exemplary plant seeds and substantially hydrophobic hairy appendages disclosed herein, to name a few, may be combined with inorganic materials (e.g., by deposition (e.g., sputter coating) of silver or gold onto said hairy appendages, etc.). In some implementations, any of the plant seeds and substantially hydrophobic hairy appendages disclosed herein, to name a few, may be combined with any naturally derived animal or plant material (e.g., collagen gel, grass jelly, com silk, coconut coir, etc.).

[0040] In some implementations, said seeds may be germinated, or other seeds may be incorporated into the cell-culture substrate and germinated (e.g., yielding a sprout, flower, etc.).

[0041] A broad variety of animal cell types, including those derived from muscle, fat, skin, bone, blood vessels, and connective tissues, exhibit the phenomenon of anchorage dependence, wherein said cells require attachment to a substantially solid surface (aka “substrate”) in order to survive, proliferate, differentiate, and express their phenotype (e.g., synthesizing extracellular matrix and other factors, etc.). It is understood by one skilled in the art that said animal cell attachment is generally mediated via transmembrane integrin receptors on the cell surface and cell-adhesive proteins or peptides on the substrate surface. In conventional two-dimensional animal cell culture, including that of mammals (including humans), birds, fish, crustaceans, reptiles, and others, anchorage-dependent animal cells isolated therefrom are generally propagated on sterile, cell-adhesive protein- or peptide- coated plastic surfaces (e.g., within gamma- sterilized flasks comprising a serum protein-coated, tissue culture -treated polystyrene surface, etc.).

[0042] In substantially three-dimensional animal cell culture applications such as biomedical tissue engineering (e.g., blood vessels, etc.), organotypic tissue models (e.g., for use in research or diagnostics, etc.), and cellular-agriculture applications (e.g., cultivated meat, cultivated leather, and cultivated fur, etc.), etc., suspensions of anchorage -dependent animal cells in fluid culture media or buffer solution are generally dispersed (aka “seeded”) onto substantially porous materials referred to as scaffolds, wherein the surfaces of said scaffolds, either intrinsically cell adhesive (e.g., gelatin, collagen, etc.) or coated to promote cell adhesion (e.g., gelatin-coated, collagen-coated, etc.) enable not only cell attachment and proliferation on said surfaces but also promote cell growth into and fdling of the scaffold void space. Said substantially porous scaffolds (e.g., textiles, foams, sponges, etc.) may be substantially biodegradable (e.g., poly(glycolic acid) nonwovens) or substantially non- degradable (e.g., polyethylene terephthalate (PET) mesh) and generally exhibit several features rendering them suitable for their function as scaffolds, including a high surface area-to-volume ratio, high porosity (e.g., greater than 90% void space, etc.), substantial pore interconnectivity, and structural elements (e.g., fibers) having surface properties amenable to cell-adhesive protein or peptide adsorption (e.g., hydrophilicity, charge, etc.) or covalent grafting (e.g., functional groups) as well as pore and structural element dimensions (e.g., pore size, fiber diameter) and morphology (e.g., surface topography) amenable to cell attachment and subsequent elaboration of cellular behavior. Further, it is generally desirable that said substantially porous materials have structural-mechanical properties consistent with or tunable to mimicking those of a tissue of interest (e.g., muscle), either on their own or in combination with the structural- mechanical properties of the seeded cells and extracellular matrix (i.e., as a composite material) (Ref. 1). Said anchorage-dependent animal cell seeded scaffolds may be referred to as tissue constructs.

[0043] In developing scaffolds for cellular agriculture applications such as cultivated meat, cultivated leather, and cultivated fur production (to name a few), additional considerations in materials sourcing and processing include environmental sustainability, naturally sourced (e.g., in raw form, organic, minimally processed, non-GMO, etc.), fair trade, cost of goods, animal welfare impact, edibility and nutrition, taste, texture, color, compatibility with tanning (e.g., in the case of cultivated leather), and scalability. Thus, in cellular agriculture in particular, there is a need for scaffolds that optimize for parameters beyond those directly related to their function as cell-culture substrates.

[0044] Deriving from numerous members of the plant kingdom, wind-dispersed plant seeds comprising a tuft of substantially hydrophobic, aerodynamically drag -producing hairs (e.g., coma, pappus, etc.) represent a heretofore unexplored category of materials for use as tissue-engineering scaffolds (Fig. 1). In particular, by virtue of the established edibility and nutrient density of several seeds of this category (e.g., dandelion seeds, kapok tree seeds, etc.), such materials may be particularly useful in fabricating scaffolds for cultivated meat applications and other environmentally sustainable and non-toxic applications (e.g., cultivated leather, cultivated fur, etc.). By contrast,certain plant seeds, such as that of cotton, are not edible because they contain toxins (e.g., gossypol in the case of cotton), thereby rendering said seeds generally unsuitable for food applications.

[0045] Referring to Fig. 2 and further to the utility of certain plant seeds and their associated seed hairs (e.g., common milkweed (Fig. 2A,F), kapok tree (Fig. 2B,G), common dandelion (Fig. 2C,H), eastern cottonwood tree (Fig. 2D, I), and common cattail (Fig. 2E,J)), we found that the dimensions of said coma and pappus hairs (e.g., reported as 8.7 ± 5.7 microns for poplar (cottonwood) seed comose hairs and 16.5 ± 2.4 microns for kapok seed comose hairs, etc. (Ref. 2,3)) are consistent with those of synthetic textile fibers utilized in tissue-engineering applications (e.g., poly(glycolic acid) fibers of nominally 10-15 microns in diameter, etc.; Ref. 1).

[0046] Further to the utility of certain plant seeds and their associated hairy appendages, we found that, by virtue of their having evolved to enable wind-borne seed dispersal, the hairs of said comabearing and pappus-bearing seeds exhibit exceptionally low densities, thereby enabling said materials to be incorporated into cellular agriculture products such as cultivated meats and leathers at low mass fraction whilst serving in the function of providing a substrate for cell attachment, proliferation, differentiation, and tissue formation. For example, the hairs of kapok seeds are hollow and have a reported density of 0.29 g / cm3(Ref. 3). Indeed, a useful metric is the ratio of substrate surface area to mass, wherein a high ratio of surface area to mass is particularly desirable in cellular agriculture applications in which a relatively low percentage of plant-based ingredients and, by extension, a high percentage of animal cell and tissue is desirable. To provide a particular surface area of circular crosssection substrate for cell attachment, the surface area can be calculated by multiplying the circumference of the substrate by the total length of substrate. For example, poly(glycolic acid) fibers having an average density 1.53 g / cm3would be expected to provide significantly less surface area per unit weight than hollow kapok fibers of similar diameter.

[0047] Referring to Fig. 3 and further to the utility of certain plant seeds and their seed hairs, we disclose here that cell-culture substrates (e.g., needle-punched nonwovens such as of eastern cottonwood tree seed and seed hairs; Fig. 3A) of said hairs may be mechanically tested (e.g., uniaxial tensile testing of a needle-punched nonwoven eastern cottonwood tree seed and seed hair scaffold; Fig. 3B-E). Said cell-culture substrates can have mechanical properties (e.g., tensile, compressive, or flexural stiffness, strength, strain-to-failure, etc.) consistent with their application, either alone (i.e., as a single hair species, such as for example cottonwood tree seed and seed hairs, etc.) or in blends with other seed hairs (e.g., blends of kapok tree seed and seed hairs and eastern cottonwood tree seed and seed hairs, etc.), to contribute as components of a cultivated meat, leather, or fur product in substantially mimicking the structural, mechanical and textural properties of conventional meat, leather or fur products or entirely novel products.

[0048] Further to the utility of certain plant seeds and their associated hairy appendages, we found that, by virtue of the hollow hairs of certain wind-dispersed seeds (e.g., those of kapok tree,cotonwood tree, etc.), cultivated animal tissue constituents (e.g., cells, extracellular matrix, etc.), coatings (e.g., gelatin, collagen, zein protein, etc.) may substantially infdtrate and substantially fill said hollow hairs. This unique aspect of said hairy seed appendages may enable an increased mass, volume or area fraction of cell and tissue components relative to those of the seed components (i.e., the scaffold) as well as enable continuity of cell and tissue structures between the surface and lumen (i.e., interior) of said same hollow hairs and neighboring hairs, thereby providing enhanced structural integrity and texture to the cultivated product.

[0049] Beyond their abovementioned utility in adherent animal cell culture applications, a technical barrier to utilizing a broad variety of wind-dispersed plant seed coma hairs and pappus hairs in aqueous cell culture applications is their intrinsic hydrophobicity, which has been demonstrated to be atributable to their waxy coating. Indeed, the comose hairs of kapok seeds have been widely explored for use in oil absorption (e.g., for oil spill cleanup, etc.). Such hydrophobic surface properties are inconsistent with requirements for tissue-engineering scaffold materials of construction, because scaffolds need to wet with aqueous solutions (e.g., cell culture media, cell-adhesive protein coating solutions, etc.) in order to enable robust protein coating, cell atachment, nutrient transport, etc. The technologies described in this specification address this problem, e.g., by treating one or more of intrinsically hydrophobic seed hairs with an amphiphilic substance to render them hydrophilic.

[0050] The following examples are representative of the natural-product fibers, scaffolds prepared therefrom, and methods disclosed herein and are not meant to be limiting in any way.

[0051] Example 1 overview. Referring to Fig. 3, we provide a demonstration of scaffold fabrication (Fig. 3A), cell seeding (delivered via collagen gel; specimen of which depicted in Fig. 3B), and associated mechanical properties (as assessed by uniaxial tensile testing; Fig. 3C-3E). A scaffold comprising cotonwood seed and associated substantially hydrophobic hairy appendages was fabricated by needle punching using a 36-gauge felting needle.

[0052] Example 1 methods. C2C12 and CRL-1213 cells were grown in a culture media comprising Dulbecco’s Modification of Eagle’s Medium (DMEM), 20% fetal bovine serum (FBS), and 1% antibiotic-antimycotic. The above-mentioned four different types of needle-punched nonwoven scaffolds were seeded with a mix of C2C12s (immortalized mouse myoblasts, ATCC CRL-1772, Lot # 70052825) and rat skin fibroblasts (ATCC CRL-1213, Lot # 70036801)) using an approximately 6 mg / mL bovine type I collagen gel as a carrier (TeloCol-6 Type I Collagen; Product # 5225, Advanced BioMatrix, Inc., Carlsbad, CA). On ice, 1.5 mL of C2C12 in culture media was combined with 1 mL of rat skin fibroblasts in culture media and gently mixed with 16 mL of ice-cold collagen solution and 2 mL of ice-cold neutralization solution, yielding about 20.5 mL of ice-cold cell-collagen solution. Approximately 5 mL of said cell-collagen solution was pipeted onto each of the four scaffolds. Of note, due to the substantial intrinsic hydrophobicity of the untreated scaffolds, the cell-collagen solution was pipeted several times through the scaffold in order to facilitate penetration through the thickness. Gelation of the neutralized collagen-cell suspension was initiated by placing the cell-seededscaffolds in a 37°C, 5% CO2 incubator for about 1 hour prior to adding culture media. Cell-seeded scaffolds were henceforth cultivated in the above-mentioned media for 4 days.

[0053] Example 1 results. On Day 4 post-seeding, samples of the above-mentioned cell-seeded scaffolds were dissected from their respective frames and prepared for tensile testing (Fig. 3C-3E). In brief, approximately 7 mm x 7 mm samples (nominally about 2-mm thick) were cut using a scalpel and dissecting scissors and subjected to tensile testing (Fig. 3E).

[0054] Example 2 overview. Referring to Fig. 4, needle-punched nonwoven kapok tree seed and seed hair was pre -treated with an amphiphile (i.e., lecithin), coated with gelatin, and subsequently wetted by an aqueous cell suspension and demonstrated cell attachment (Day 1 post-seeding) and proliferation (Days 7 and 14 post-seeding).

[0055] Example 2 methods. In this example, nominally 3 cm wide x 3 cm long x 2 mm thick nonwoven scaffolds were prepared by needle-punching kapok tree seed coma (i.e., the tuft of hairs, an aerodynamic drag promoting appendage, attached to the end of the kapok tree seeds) and subsequently placed in a lidded polypropylene container and treated by immersion in 10 ml of a solution of nominally 5% (w / v) sunflower lecithin (a food-safe amphiphile) in 200-proof ethanol. Said lecithin-treated kapok scaffolds were allowed to dry overnight within a biological safety cabinet and then autoclave sterilized (gravity cycle with 15 min dwell time). Said scaffolds were then coated for about 2 hours with a sterile solution of 0.1% (w / v) gelatin in water solution (i.e., a food-safe celladhesive protein). Said lecithin-treated, gelatin-coated scaffolds were then seeded with C2C12 cells at a density of nominally 500,000 cells per square centimeter of planar scaffold area (i.e., nominally 4,500,000 cells resuspended in a volume of 2.5 mb of culture media) in an aqueous culture media comprising Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) antibiotic-antimycotic. It is notable that said lecithin-treated kapok scaffolds wetted immediately and readily with the aqueous cell suspension (i.e., the aqueous cell suspension was wicked into the lecithin-treated Kapok scaffold). Said cell-seeded lecithin-treated, nonwoven kapok seed coma scaffolds were incubated 37 degrees Celsius and 5% CO2 in a cell culture incubator for about 3 hours to allow time for cells to attach prior to adding an additional 10 mb of culture media. The next day (i.e., Day 1 post-seeding) and at days 7 and 14 post-seeding, cell- seeded scaffold samples were fixed in 10% neutral -buffered formalin and then stained with a 2% (v / v) solution of Hoechst 33342 (to stain cell nuclei) and imaged on an EVOS fluorescence microscope.

[0056] Example 2 results. Referring to Fig. 4, on Day 1 post-seeding, C2C12 cells were readily observed attached along the length of and spanning between the lecithin-treated, gelatin- coated kapok tree seed hairs comprising the scaffold (Fig. 4A). By Day 7 post-seeding, C2C12 cells had proliferated and begun to fill regions of the pore space between the lecithin-treated, gelatin-coated kapok tree seed hairs (Fig. 4B). By Day 14 post-seeding, C2C12 cells were readily observable by phase-contrast microscopy attached to and spanning the kapok tree seed hairs (Fig. 4C). A photograph of the Day- 14 tissue construct is shown in Fig. 4D.

[0057] Example 3 overview. Referring to Figs. 5-9, we demonstrate herein that amphiphile (i.e., lecithin) treatment of multiple types of substantially hydrophobic seed hairs renders said seed hairs and scaffolds formed therefrom amenable to cell attachment, proliferation, and collagenous tissue formation, with associated changes in tissue construct tensile mechanical behavior.

[0058] Example 3 methods. In this example, nominally 3 cm wide x 3 cm long x 2 mm thick nonwoven scaffolds prepared by needle-punching the following representative examples of seeds and seed hairs: (a) kapok tree (Fig. 5); (b) cottonwood tree (Fig. 6); (c) common milkweed (Fig. 7); (d) common dandelion and cottonwood tree blend (Fig. 8); and (e) common cattail and cottonwood tree blend (Fig. 9) and subsequently placed in a lidded polypropylene container and treated by immersion in 10 ml of a solution of nominally 5% (w / v) sunflower lecithin (a food-safe amphiphile) in 200-proof ethanol. Said lecithin-treated scaffolds were allowed to dry overnight within a biological safety cabinet and then autoclave sterilized (gravity cycle with 15 min dwell time). Said scaffolds were then coated for about 2 hours with a sterile solution of 0.1% (w / v) gelatin in water solution (i.e., a foodsafe cell-adhesive protein). Said lecithin- treated, gelatin-coated scaffolds were then seeded with a nominally 50:50 blend of C2C12 cells and rat dermal fibroblasts at a density of nominally 500,000 cells per square centimeter of planar scaffold area (i.e., nominally 4,500,000 cells resuspended in a volume of 2.5 m of culture media) in an aqueous culture media comprising Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) antibiotic- antimycotic. It is notable that said lecithin-treated scaffolds wetted immediately and readily with the aqueous cell suspension (i.e., the aqueous cell suspension was wicked into the lecithin- treated Kapok scaffold). Said cell-seeded lecithin-treated, nonwoven seed hair scaffolds were incubated 37 degrees Celsius and 5% CO2 in a cell culture incubator for about 3 hours to allow time for cells to attach prior to adding an additional 10 mb of culture media. The next day (i.e., Day 1 postseeding) and at days 7, 14, and 21 post-seeding, cell-seeded scaffold samples were fixed in 10% neutral-buffered formalin and then stained with a 2% (v / v) solution of Hoechst 33342 (to stain cell nuclei) and imaged on an EVOS M5000 fluorescence microscope. Samples were subject to uniaxial tensile testing as well as biochemical quantification of DNA (picogreen) and collagen (Sircol assay) content using established methods.

[0059] Example 3 results. As evident in Figs. 5-9, on Day 1 post-seeding, C2C12 muscle cells and dermal fibroblasts were readily observed attached along the length of and spanning between the lecithin-treated, gelatin-coated seed hairs comprising all scaffolds. By Day 7 and Day 14 postseeding, cells had progressively proliferated and filled regions of the pore space between the lecithin- treated, gelatin-coated seed hairs, with commensurate increases in DNA and collagen content and changes in tensile mechanical properties.

[0060] Example 4. Referring to Fig. 10, A blend of lecithin-treated coma-bearing seed hairs and intrinsically hydrophobic, non-treated coma-bearing seed hairs were capable of generating a cultivated fur. Fig. 10 illustrates the fabrication and cell seeding of a cultivated fur on top of acultivated skin. In particular, (A) shows the top (fur) and (B) shows the bottom (skin) of the scaffold, wherein (1) untreated, substantially hydrophobic fur hairs made of milkweed seed hairs have been incorporated by needle punching into a (2) lecithin-treated, gelatin-coated kapok tree seed and seed hair scaffold. (C) shows the autoclave-sterilized scaffold in a polypropylene container during the (3) lecithin treatment step, wherein the (4) kapok scaffold and (5) milkweed seed hairs are observable. (D) shows the scaffold in a polypropylene container during the (6) cell suspension seeding step, wherein the (7) kapok scaffold and (8) milkweed seed hairs are observable.

[0061] Example 5 overview. Referring to Fig. 11, we demonstrate herein that a scaffold comprising lecithin-treated dandelion seed hairs can be fabricated, sterilized, seeded with bovine dermal fibroblasts, cultured in-vitro, colored and flavored using food-grade ingredients, and cooked (e.g., fried in oil) to yield an edible cultivated meat substantially resembling a piece of bacon.

[0062] Example 5 methods and results. Referring to Fig. 11, dandelion seed hair-based scaffolds were fabricated by needle-punching, lecithin treated, and gelatin coated as described in Example 3. Bovine dermal fibroblast cells (Black Angus breed cow) were isolated using standard enzymatic dissociation methods from a veterinarian-performed 8 mm skin punch biopsy. In brief, the epidermis was aseptically dissected from the dermis using a sterile scalpel, and nominally 1 gram of dermis tissue was added to and incubated with gentle mixing (Labquake shaker rotisserie; Bamstead Thermolyne) for 6 hours at 37 degrees Celsius and 5% CO2 in a solution of 0.2% (w / v) collagenase (Collagenase from Clostridium histolyticum, type I; C-0130, Sigma-Aldrich) in Hank’s balanced salt solution. Said isolated bovine dermal cells had atypical fibroblastic appearance and were propagated by serial passaging in culture media comprising Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 20% (v / v) fetal bovine serum and 1% (v / v) antibiotic-antimycotic. Bovine dermal fibroblasts were utilized at passage numbers p6 to p9. Lecithin-treated, gelatin-coated scaffolds were autoclave -sterilized and seeded with said bovine dermal fibroblasts as described in Example 3. Said bovine dermal fibroblast-seeded scaffolds were cultured for 49 days in a lidded polypropylene container prior to placing in a 6-well polystyrene tissue culture plate (Fig. 11 A) prior to phase -contrast light microscopy imaging (Fig. 11B) and harvesting (Fig. 11C). Said lecithin-treated dandelion seed hair-based cultivated bovine tissues (nominally 1.3 grams; Fig. HF) were rinsed 3x with phosphate buffered saline without calcium or magnesium and refrigerated at 2-8 degrees Celsius for 4 hours prior to further processing. A food-grade coloring and flavoring solution (i.e., a marinade) was prepared by adding 1.3 grams of Maxivor Rye D (DSM; Heerlen, Netherlands), 0.7 grams of Multirome LS powder yeast extract (DSM; Heerlen, Netherlands), 0.7 grams of Maxivor White Meat yeast extract (DSM; Heerlen, Netherlands), and 0.7 grams of red beet powder (Suncore Foods; Irvine, CA) to 70 mb of tap water. The cultivated bovine tissue (Fig. HD) was placed in the 70 mb of said marinade solution in a polypropylene food container and placed in a refrigerator at 2-8 degrees Celsius for 10 hours. Following said marinade solution treatment, the colored and flavored cultivated bovine tissue (“cultivated meat”; Figs. 11E-G) had the gross color (e.g., pink and red) and grossappearance of a thin cut of meat (e.g., bacon). Said colored and flavored cultivated bovine tissue was cooked by frying in extra-virgin olive oil at nominally 150-200 degrees Celsius for 10 minutes, resulting in a gross appearance substantially resembling that of cooked bacon (Fig. 11G, 11H).

[0063] Example 6 overview. Referring to Fig. 12, we demonstrate herein that a scaffold comprising lecithin-treated kapok tree seed hairs can be fabricated, sterilized, seeded with bovine dermal fibroblasts, cultured in-vitro, and tanned and dyed by conventional tanning methods to yield a cultivated leather substantially resembling conventional (i.e., deceased animal hide) leather.

[0064] Example 6 methods and results. Referring to Fig. 12, kapok tree seed hair-based scaffolds were fabricated by needle-punching, lecithin treated, and gelatin coated as described in Example 3. Bovine dermal cells (Fig. 12A) were isolated and propagated as described in Example 5. Bovine dermal fibroblasts were utilized at passage numbers p6 to p9. Lecithin-treated, gelatin-coated scaffolds were autoclave -sterilized and seeded with said bovine dermal fibroblasts as described in Example 3. Said bovine dermal fibroblast-seeded scaffolds were cultured for 42 days in a lidded polypropylene container prior to harvest (Fig. 12B, 12C). Said lecithin-treated kapok tree seed hairbased cultivated bovine tissues were rinsed 3x with phosphate buffered saline without calcium or magnesium and transported wet on ice to a tannery (Pergamena; Montgomery, NY) for further processing. In brief, said cultivated bovine tissues were pickled using a sodium chloride (NaCl) salt and formic acid solution using standard methods known in the field of leather tanning, bringing the pH down to nominally 2.2. Said pickled cultivated bovine tissue was then tanned using standard methods of vegetable-based tanning known in the field of leather tanning (e.g., using a water-based solution of vegetable derived tannins, for example catechol tannins from the black wattle tree, pyrogallol tannins from the chestnut tree, etc.) (Fig. 12D). Said vegetable-tanned cultivated leather was then dyed using standard methods known in the field of leather tanning, using saddle brown and sunflower colored dyes (Fig. 12E, 12F). Additional cultivated leathers were made using the same methods from other seed hair-based scaffolds, such as milkweed seed hairs (Fig. 12G).

[0065] Example 7 overview. Referring to Fig. 13 and Fig. 14, we demonstrate herein that a scaffold comprising lecithin-treated kapok tree seed hairs can be fabricated, sterilized, seeded with bovine dermal fibroblasts, and cultured in-vitro for different durations, resulting in visually observable changes in cultivated cow skin appearance, microscopic morphology, and collagen distribution in association with extended culture duration.

[0066] Example 7 methods and results. Referring to Fig. 13, kapok tree seed hair-based scaffolds were fabricated by needle-punching, lecithin treated, and gelatin coated as described in Example 3. Bovine dermal cells were isolated and propagated as described in Example 5. Bovine dermal fibroblasts were utilized at passage numbers p6 to p9. Lecithin-treated, gelatin-coated scaffolds were autoclave-sterilized and seeded with said bovine dermal fibroblasts as described in Example 3. Said bovine dermal fibroblast-seeded scaffolds were cultured for 14 days, 21 days, and 28 days in lidded polypropylene containers prior to harvest (Fig. 13A-13C). Said lecithin-treated kapok tree seed hair-based cultivated bovine tissues were rinsed 3x with phosphate buffered saline without calcium or magnesium, and samples were cut and fixed in 10% neutral buffered formalin prior to shipping to a histology service provider (Histoserv, Inc.; Germantown, MD). Said formalin-fixed cultivated bovine tissues were processed using standard histology methods known in the field of histology, including paraffin embedding, microtome sectioning, slide mounting, and histological staining by the hematoxylin and eosin (H&E) method as well as the Masson’s trichrome method (Fig. 13D-13F). Additional samples of said cultivated bovine tissues were cut and processed using standard methods known in the field of chemical assays to measure collagen content (Fig. 14). In brief, nominally 0.1- 0.3 grams wet weight tissue samples were extracted in an aqueous solution of 0.5 molar acetic acid and 1 mg / mL pepsin for nominally 48 hours and collagen content was measured per manufacturer’s recommendations using the Sircol collagen assay kit (Biocolor; Carrickfergus, UK).REFERENCES

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[0069] (3) Meiwu et al. “The Fine Structure of Kapok Fiber.” Tex Res J. 2010 Jan; 80(2): 159-165.Itemized ImplementationsItem 1. A construct comprising a cell-culture substrate comprising hairy plant seeds having intrinsically hydrophobic seed hairs.Item 2. The construct of item 1, wherein said seeds are coma-bearing or pappus-bearing.Item 3. The construct of item 1, wherein said seeds and seed hairs are derived from kapok tree, eastern cottonwood tree, common dandelion, common milkweed, common cattail, sow thistle, Asteraceae, Taraxacum officinale, Hypochaeris, or a combination thereof.Item 4. The construct as in any one of items 1-3, wherein the seeds are substantially separated from their associated substantially hydrophobic seed hairs.Item 5. The construct as in any one of items 1-4, wherein said intrinsically hydrophobic seed hairs are processed into a nonwoven material.Item 6. The construct as in any one of items 1-5, wherein at least a portion of said intrinsically hydrophobic seed hairs are modified to render them substantially hydrophilic.Item 7. The construct of item 6, wherein at least a portion of said intrinsically hydrophobic seed hairs are treated with an amphiphilic substance to render them hydrophilic.Item 8. The construct of item 7, wherein the amphiphilic substance comprises lecithin.Item 9. The construct of item 8, wherein at least a portion of said lecithin-treated seed hairs are modified by coating the lecithin-treated seed hairs with a cell-adhesive protein or peptide.Item 10. The construct of item 9, wherein the coating comprises gelatin, collagen, zein protein, or a combination thereof.Item 11. The construct as in any one of items 1-10, comprising a mixture of two, three, four, or more different types of intrinsically hydrophobic seed hairs.Item 12. The construct as in any one of items 1-11, comprising at least one unmodified intrinsically hydrophobic seed hair and at least one intrinsically hydrophobic seed hair that has been modified to render it substantially hydrophilic.Item 13. The construct as in any one of items 1-11, comprising at least one type of unmodified intrinsically hydrophobic seed hair and at least one type of intrinsically hydrophobic seed hair that has been modified to render it substantially hydrophilic.Item 14. The construct as in any one of items 12-13, wherein the unmodified intrinsically hydrophobic seed hairs form one or more hydrophobic portions in the substrate.Item 15. The construct of item 14, wherein the one or more hydrophobic portions in are configured to provide gas transport through at least a portion of the substrate.Item 16. The construct as in any one of items 14-15, wherein the one or more hydrophobic portions are configured to provide transport of non-aqueous or oily fluid through at least a portion of the substrate.Item 17. The construct as in any one of items 14-16, wherein the one or more hydrophobic portions contain fat.Item 18. The construct as in any one of items 14-17, wherein the one or more hydrophobic portions are disposed on the periphery of the substrate.Item 19. The construct as in any one of items 14-18, wherein the one or more hydrophobic portions at least partially surround or encapsulate one or more hydrophilic portions.Item 20. The construct as in any one of items 1-19, the substrate having hydrophobic seed hairs inserted in or through a non-woven pad-like portion of the substrate, thereby providing a fur-like appearance.Item 21. The construct of item 20, wherein the pad-like portion is hydrophilic.Item 22. The construct as in any one of items 1-19, wherein the seed hairs have been treated with one or more enzymes.Item 23. The construct of item 22, wherein the one or more enzymes comprise cellulase, hemicellulase, pectinase, amylase, ligninase, lipase, or a combination thereof.Item 24. The construct as in any one of items 1-23, wherein the seeds are derived from one or more plants genetically modified to express desirable characteristics for use of the substrate for cell-culture. Item 25. The construct of item 24, wherein the genetic modification results in the expression animal cell gene products in the seeds or the hairs.Item 26. The construct of item 25, wherein the gene products comprise growth factors or cell-adhesive proteins, or both.Item 27. The construct of as in any one of items 1-26, wherein the construct is a tissue construct.Item 28. The construct as in any one of items 1-26, comprising one or more animal cells, the animal cells comprising cells from a mammal, a bird, a fish, a crustacean, a reptile, an amphibian, an invertebrate, or a combination thereof.Item 29. The construct of item 28, wherein the one or more animal cells comprise fibroblasts, muscle cells, adipocytes, nerve cells, vascular cells, or a combination thereof.Item 30. The construct as in any one of items 27-29, wherein the construct is or comprises cultivated meat, cultivated leather, or cultivated fur.Item 31. The construct as in any one of items 27-30, wherein the construct is configured for use in biomedical tissue engineering, in vitro diagnostics, or any other anchorage-dependent cell-culture substrate application.Item 32. The construct as in any one of items 27-31, wherein the construct comprises a synthetic material.Item 33. The construct of item 32, wherein the synthetic material comprises polyester or silicone, or a combination thereof.Item 34. The construct in any one of items 27-33, wherein the construct further comprises a naturally derived animal material or plant material, or a combination thereof.Item 35. The construct in any one of items 27-34, wherein the construct comprises a coloring agent, a tanning agent, a flavoring agent, or a combination thereof.Item 36. The construct as in any one of items 34-35, wherein the naturally derived animal material or plant material comprises collagen gel, grass jelly, com silk, coconut coir, or a combination thereof. Item 37. The construct as in any one of items 27-36, wherein the substrate has been seeded with animal cells at between 500 cells / cm2and 50,000,000 cells / cm2of substrate surface area.Item 38. A method of manufacturing a construct, the method comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs in a volume and processing the plant seeds into a cell-culture substrate.Item 39. The method of item 38, wherein the construct is a construct as in any one of items 1-38.Item 40. The method as in any one of items 38-39, comprising processing the plant seeds such that the hairs of a first seed overlaps with the hairs of at least a second seed; and repeatedly inserting (punching) a needle into the volume, thereby entangling the hairs of the first seed with the hairs of the at least a second seed.Item 41. The method as in any one of items 38-40, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; and forming, from the amount of seeds, a substrate by means of a non-woven wet-laid process, spunbond process, solvent bonding, thermal bonding, hydroentangling, calendaring, binding, or combination thereof.Item 42. The method as in any one of items 38-41, comprising:providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; spinning or otherwise forming the seeds into one or more multifilament yams; and knitting or weaving the one or more yams into a scaffold.Item 43. The method as in any one of items 38-42, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs such that the hairs are suspended from a fixed, movable, or floating substrate in or on a volume of fluid; and joining (entangling) the hairs through movement of the fluid.Item 44. The method as in any one of items 38-43, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; and magnetizing or compartmentalizing the amount seeds within a substantially porous enclosure. Item 45. The method as in any one of items 38-44, wherein said seeds are coma-bearing or pappusbearing.Item 46. The method as in any one of items 38-45, wherein said seeds and seed hairs are derived from kapok tree, eastern cottonwood tree, common dandelion, common milkweed, common cattail, sow thistle, Asteraceae, Taraxacum officinale, Hypochaeris, or a combination thereof.Item 47. The method as in any one of items 38-46, comprising substantially separating seeds from their associated substantially hydrophobic seed hairs.Item 48. The method as in any one of items 38-47, comprising processing the intrinsically hydrophobic seed hairs into a nonwoven material.Item 49. The method as in any one of items 38-48, comprising modifying at least a portion of said intrinsically hydrophobic seed hairs to render them substantially hydrophilic.Item 50. The method as in any one of items 38-49, comprising treating at least a portion of the seeds with an amphiphilic substance prior to forming the substrate.Item 51. The method as in any one of items 38-50, comprising treating at least a portion of the seeds with an amphiphilic substance after forming the substrate.Item 52. The method as in any one of items 50-51, wherein the amphiphilic substance comprises lecithin.Item 53. The method as in any one of items 38-52, comprising treating the substrate with one or more cell-adhesive proteins or peptides.Item 54. The method of item 53, comprising treating the substrate with gelatin, collagen, zein protein, or a combination thereof.Item 55. The method as in any one of items 38-54, comprising blending two or more types of hairy plant seeds.Item 56. The method as in any one of items 38-55, comprising blending an amount of seeds treated with the amphiphilic substance with an amount of untreated seeds.Item 57. The method of item 56, comprising forming one or more hydrophobic portions in the substrate.Item 58. The method as in any one of items 38-57, comprising treating the cell hairs with one or more enzymes.Item 59. The method of item 58, wherein the one or more enzymes comprise cellulase, hemicellulase, pectinase, amylase, ligninase, lipase, xylanase, or a combination thereof.What is claimed is:

Claims

CLAIMS1. A construct comprising a cell-culture substrate comprising hairy plant seeds having intrinsically hydrophobic seed hairs.

2. The construct of claim 1, wherein said seeds are coma-bearing or pappus-bearing.

3. The construct of claim 1, wherein said seeds and seed hairs are derived from kapok tree, eastern cottonwood tree, common dandelion, common milkweed, common cattail, sow thistle, Asteraceae, Taraxacum officinale, Hypochaeris, or a combination thereof.

4. The construct as in any one of claims 1-3, wherein the seeds are substantially separated from their associated substantially hydrophobic seed hairs.

5. The construct as in any one of claims 1-4, wherein said intrinsically hydrophobic seed hairs are processed into a nonwoven material.

6. The construct as in any one of claims 1-5, wherein at least a portion of said intrinsically hydrophobic seed hairs are modified to render them substantially hydrophilic.

7. The construct of claim 6, wherein at least a portion of said intrinsically hydrophobic seed hairs are treated with an amphiphilic substance to render them hydrophilic.

8. The construct of claim 7, wherein the amphiphilic substance comprises lecithin.

9. The construct of claim 8, wherein at least a portion of said lecithin-treated seed hairs are modified by coating the lecithin-treated seed hairs with a cell-adhesive protein or peptide.

10. The construct of claim 9, wherein the coating comprises gelatin, collagen, zein protein, or a combination thereof.

11. The construct as in any one of claims 1-10, comprising a mixture of two, three, four, or more different types of intrinsically hydrophobic seed hairs.

12. The construct as in any one of claims 1-11, comprising at least one unmodified intrinsically hydrophobic seed hair and at least one intrinsically hydrophobic seed hair that has been modified to render it substantially hydrophilic.

13. The construct as in any one of claims 1-11, comprising at least one type of unmodified intrinsically hydrophobic seed hair and at least one type of intrinsically hydrophobic seed hair that has been modified to render it substantially hydrophilic.

14. The construct as in any one of claims 12-13, wherein the unmodified intrinsically hydrophobic seed hairs form one or more hydrophobic portions in the substrate.

15. The construct of claim 14, wherein the one or more hydrophobic portions in are configured to provide gas transport through at least a portion of the substrate.

16. The construct as in any one of claims 14-15, wherein the one or more hydrophobic portions are configured to provide transport of non-aqueous or oily fluid through at least a portion of the substrate.

17. The construct as in any one of claims 14-16, wherein the one or more hydrophobic portions contain fat.

18. The construct as in any one of claims 14-17, wherein the one or more hydrophobic portions are disposed on the periphery of the substrate.

19. The construct as in any one of claims 14-18, wherein the one or more hydrophobic portions at least partially surround or encapsulate one or more hydrophilic portions.

20. The construct as in any one of claims 1-19, the substrate having hydrophobic seed hairs inserted in or through a non-woven pad-like portion of the substrate, thereby providing a fur- like appearance.

21. The construct of claim 20, wherein the pad-like portion is hydrophilic.

22. The construct as in any one of claims 1-19, wherein the seed hairs have been treated with one or more enzymes.

23. The construct of claim 22, wherein the one or more enzymes comprise cellulase, hemicellulase, pectinase, amylase, ligninase, lipase, or a combination thereof.

24. The construct as in any one of claims 1-23, wherein the seeds are derived from one or more plants genetically modified to express desirable characteristics for use of the substrate for cell-culture.

25. The construct of claim 24, wherein the genetic modification results in the expression animal cell gene products in the seeds or the hairs.

26. The construct of claim 25, wherein the gene products comprise growth factors or cell-adhesive proteins, or both.

27. The construct of as in any one of claims 1-26, wherein the construct is a tissue construct.

28. The construct as in any one of claims 1-26, comprising one or more animal cells, the animal cells comprising cells from a mammal, a bird, a fish, a crustacean, a reptile, an amphibian, an invertebrate, or a combination thereof.

29. The construct of claim 28, wherein the one or more animal cells comprise fibroblasts, muscle cells, adipocytes, nerve cells, vascular cells, or a combination thereof.

30. The construct as in any one of claims 27-29, wherein the construct is or comprises cultivated meat, cultivated leather, or cultivated fur.

31. The construct as in any one of claims 27-30, wherein the construct is configured for use in biomedical tissue engineering, in vitro diagnostics, or any other anchorage -dependent cell-culture substrate application.

32. The construct as in any one of claims 27-31, wherein the construct comprises a synthetic material.

33. The construct of claim 32, wherein the synthetic material comprises polyester or silicone, or a combination thereof.

34. The construct in any one of claims 27-33, wherein the construct further comprises a naturally derived animal material or plant material, or a combination thereof.

35. The construct in any one of claims 27-34, wherein the construct comprises a coloring agent, a tanning agent, a flavoring agent, or a combination thereof.

36. The construct as in any one of claims 34-35, wherein the naturally derived animal material or plant material comprises collagen gel, grass jelly, com silk, coconut coir, or a combination thereof.

37. The construct as in any one of claims 27-36, wherein the substrate has been seeded with animal cells at between 500 cells / cm2and 50,000,000 cells / cm2of substrate surface area.

38. A method of manufacturing a construct, the method comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs in a volume and processing the plant seeds into a cell-culture substrate.

39. The method of claim 38, wherein the construct is a construct as in any one of claims 1-38.

40. The method as in any one of claims 38-39, comprising processing the plant seeds such that the hairs of a first seed overlaps with the hairs of at least a second seed; and repeatedly inserting (punching) a needle into the volume, thereby entangling the hairs of the first seed with the hairs of the at least a second seed.

41. The method as in any one of claims 38-40, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; and forming, from the amount of seeds, a substrate by means of a non-woven wet-laid process, spunbond process, solvent bonding, thermal bonding, hydroentangling, calendaring, binding, or combination thereof.

42. The method as in any one of claims 38-41, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; spinning or otherwise forming the seeds into one or more multifilament yams; and knitting or weaving the one or more yams into a scaffold.

43. The method as in any one of claims 38-42, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs such that the hairs are suspended from a fixed, movable, or floating substrate in or on a volume of fluid; and joining (entangling) the hairs through movement of the fluid.

44. The method as in any one of claims 38-43, comprising: providing an amount of hairy plant seeds having intrinsically hydrophobic seed hairs; and magnetizing or compartmentalizing the amount seeds within a substantially porous enclosure.

45. The method as in any one of claims 38-44, wherein said seeds are coma-bearing or pappusbearing.

46. The method as in any one of claims 38-45, wherein said seeds and seed hairs are derived from kapok tree, eastern cottonwood tree, common dandelion, common milkweed, common cattail, sow thistle, Asteraceae, Taraxacum officinale, Hypochaeris, or a combination thereof.

47. The method as in any one of claims 38-46, comprising substantially separating seeds from their associated substantially hydrophobic seed hairs.

48. The method as in any one of claims 38-47, comprising processing the intrinsically hydrophobic seed hairs into a nonwoven material.

49. The method as in any one of claims 38-48, comprising modifying at least a portion of said intrinsically hydrophobic seed hairs to render them substantially hydrophilic.

50. The method as in any one of claims 38-49, comprising treating at least a portion of the seeds with an amphiphilic substance prior to forming the substrate.

51. The method as in any one of claims 38-50, comprising treating at least a portion of the seeds with an amphiphilic substance after forming the substrate.

52. The method as in any one of claims 50-51, wherein the amphiphilic substance comprises lecithin.

53. The method as in any one of claims 38-52, comprising treating the substrate with one or more cell-adhesive proteins or peptides.

54. The method of claim 53, comprising treating the substrate with gelatin, collagen, zein protein, or a combination thereof.

55. The method as in any one of claims 38-54, comprising blending two or more types of hairy plant seeds.

56. The method as in any one of claims 38-55, comprising blending an amount of seeds treated with the amphiphilic substance with an amount of untreated seeds.

57. The method of claim 56, comprising forming one or more hydrophobic portions in the substrate.

58. The method as in any one of claims 38-57, comprising treating the cell hairs with one or more enzymes.

59. The method of claim 58, wherein the one or more enzymes comprise cellulase, hemicellulase, pectinase, amylase, ligninase, lipase, xylanase, or a combination thereof.

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