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WO2025015242A2 - Substrats de culture cellulaire à base de plantes - Google Patents

Substrats de culture cellulaire à base de plantes Download PDF

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Publication number
WO2025015242A2
WO2025015242A2 PCT/US2024/037722 US2024037722W WO2025015242A2 WO 2025015242 A2 WO2025015242 A2 WO 2025015242A2 US 2024037722 W US2024037722 W US 2024037722W WO 2025015242 A2 WO2025015242 A2 WO 2025015242A2
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WIPO (PCT)
Prior art keywords
construct
hairs
seed
seeds
cell
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WO2025015242A3 (fr
WO2025015242A9 (fr
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George Carl ENGELMAYR
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Vasavance Inc
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Vasavance Inc
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Publication of WO2025015242A9 publication Critical patent/WO2025015242A9/fr
Publication of WO2025015242A3 publication Critical patent/WO2025015242A3/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants

Definitions

  • cell-culture substrates and tissue constructs formed therefrom e.g., cultivated meats, cultivated leathers, cultivated furs, etc.
  • configurations e.g., nonwoven fabrics, battings, blends, meshes, yams, etc.
  • modifications e.g., amphiphilic coatings to render said substrates substantially hydrophilic, etc.
  • 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 more multifilament 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.
  • 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).
  • Fig. 2 illustrates examples of coma-bearing and pappus-bearing seeds and their substantially hydrophobic seed hairs.
  • Fig. 2A milkweed
  • Fig. 2B kapok tree
  • Fig. 2C dandelion
  • Fig. 2D cottonwood tree
  • 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 shows the specimen at the beginning and end of the uniaxial tensile test, respectively.
  • Fig. 3E shows a representative force- displacement curve.
  • 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. 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 nucle
  • 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.
  • 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.
  • 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. 5D-5F show fluorescence micrographs of cells attached and proliferating on the kapok
  • 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.
  • 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).
  • 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.
  • 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. 6D-6F show fluorescence micrographs of cells attached and proliferating on the cottonwood fiber
  • 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.
  • 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).
  • 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.
  • 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. 7D-7F show fluorescence micrographs of cells attached and proliferating on the milkweed fibers
  • 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.
  • 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).
  • 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.
  • 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. 8D-8F shows fluorescence micrographs of cells attached
  • 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).
  • 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.
  • 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. 9D-9F show fluorescence micrographs of cells attached and pro
  • 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 quantitative evidence 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.
  • 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).
  • FIG. 10A-C are photographs illustrating the fabrication and cell seeding of a cultivated fur on top of a cultivated skin.
  • Fig. 10A shows the top (fur)
  • 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. 10A shows the top (fur)
  • 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
  • tissue variations e.g., spatial variations in the fibrous appearance, thickness, curvature, and holes, etc.
  • the sides and orientations of the cultivated meat in photos 11G and 11H correspond to those of D and E, respectively.
  • 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.
  • 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. 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. 12B is a photograph of said cultivated cow skin comprising a lecithin-treated, gelatin
  • 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. 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).
  • said cultivated cow skin 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.
  • 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.
  • 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.
  • 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).
  • the seeds may be retained in their substantially natural state, wherein their seed hairs remain substantially intact and substantially connected to said seeds.
  • 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.)).
  • an amphiphile e.g., a solution of lecithin in ethanol, etc.
  • 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.).
  • 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.
  • 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.
  • gas transport e.g., by providing substantially unwetted channels capable of mediating oxygen or carbon dioxide transport, etc.
  • 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.
  • cells can be seeded at densities of between 500 cells/cm 2 and 50,000,000 cells/cm 2 of substrate surface area, and proliferate to a relatively higher density of between 5,000 and 500,000 cells/cm 2 of 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 be particularly useful for improving animal welfare and reducing the environmental and societal impact of meat and leather production.
  • 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.
  • said cell-culture substrates serve both as macro -carriers and as scaffolds for subsequent tissue formation.
  • 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.
  • 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.
  • 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.
  • a fluid media e.g., culture media
  • a fixed, movable, or floating substrate e.g., a piece of cork, foam, etc.
  • said seed hairs may be moved (e.g., pulled, flexed, compressed, buckled, etc.) by movement of said fluid medium.
  • 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).
  • 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.
  • an amphiphile e.g., a solution of lecithin in ethanol, etc.
  • 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.).
  • 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.
  • any proportion e.g., mass ratio, numerical ratio (i.e., the number of hair-bearing seeds or seed hairs, etc.)
  • layer thickness i.e., angular orientation relative to any axis of the scaffold
  • hair orientations i.e., angular orientation relative to any axis of the scaffold
  • 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.
  • hydrophilic seed hair elements e.g., formed by amphiphile coating of one or more seed hairs prior to scaffold fabrication, etc.
  • 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.).
  • 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.
  • 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.).
  • 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 the periphery 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.
  • gas e.g., oxygen, carbon dioxide
  • vapor e.g., water vapor
  • 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.
  • headspace gases e.g., oxygen, carbon dioxide, etc.
  • 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.
  • 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
  • a like e.g., milkweed seed hair
  • dissimilar e.g., kapok seed hair
  • a reverse felting needle i.e., one designed to pull fibers as opposed to pushing fibers
  • a reverse felting needle 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.
  • 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).
  • one or more constituents of said seed hairs e.g., lignin
  • modification, degradation, removal, etc. of one or more seed hair constituents may render said enzyme- treated seed hairs substantially smaller in dimension (e.g., diameter), weaker (e.g., in tension), more flexible, more biodegradable, etc.
  • 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.
  • 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.).
  • 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.).
  • 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.
  • any of the broad variety of plant seeds having substantially hydrophobic hairy appendages 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.).
  • example seeds and associated seed hairs cited and tested herein 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.
  • 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
  • the various Cat’s Ear species belonging to the Hypochaeris genus also exhibit pappi and, by virtue, are commonly referred to as false dandelions.
  • 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.).
  • any synthetic material e.g., polyesters (e.g., poly(lactic acid), poly(caprolactone), etc.), silicones (e.g., poly(dimethyl siloxane)), etc.).
  • 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.).
  • 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.).
  • 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.).
  • 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.).
  • substrate substantially solid surface
  • 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.
  • 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.).
  • 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.
  • intrinsically cell adhesive e.g., gelatin, collagen, etc.
  • coated to promote cell adhesion e.g., gelatin-coated, collagen-coated, etc.
  • Said substantially porous scaffolds 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.
  • substantially biodegradable e.g., poly(glycolic acid) nonwoven
  • 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.
  • 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).
  • tissue-engineering scaffolds Fig. 1
  • 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.).
  • 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.
  • Fig. 2 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).
  • tissue-engineering applications e.g., poly(glycolic acid) fibers of nominally 10-15 microns
  • 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.
  • the hairs of kapok seeds are hollow and have a reported density of 0.29 g/cm 3 (Ref. 3).
  • 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.
  • 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/cm 3 would be expected to provide significantly less surface area per unit weight than hollow kapok fibers of similar diameter.
  • cell-culture substrates e.g., needle-punched nonwovens such as of eastern cottonwood tree seed and seed hairs; Fig. 3A
  • Fig. 3A cell-culture substrates
  • Fig. 3B-E uniaxial tensile testing of a needle-punched nonwoven eastern cottonwood tree seed and seed hair scaffold
  • 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.
  • mechanical properties e.g., tensile, compressive, or flexural stiffness, strength, strain-to-failure, etc.
  • 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.
  • 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.
  • aqueous solutions e.g., cell culture media, cell-adhesive protein coating solutions, 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.
  • Example 1 Example 1 overview.
  • Fig. 3A 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 Lactam seed and associated substantially hydrophobic hairy appendages was fabricated by needle punching using a 36-gauge felting needle.
  • 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.
  • DMEM Dulbecco’s Modification of Eagle’s Medium
  • FBS fetal bovine serum
  • antibiotic-antimycotic 1% antibiotic-antimycotic.
  • 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).
  • Example 2 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).
  • an amphiphile i.e., lecithin
  • Example 2 methods.
  • 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.
  • DMEM Dulbecco’s Modification of Eagle’s Medium
  • 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.
  • 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.
  • 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.
  • Example 3 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.
  • amphiphile i.e., lecithin
  • 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.
  • DMEM Dulbecco’s Modification of Eagle’s Medium
  • 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.
  • 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.
  • 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.
  • 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 a cultivated skin.
  • (A) shows the top (fur)
  • (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.
  • Example 5 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.
  • 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.
  • 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.
  • collagenase Collagenase from Clostridium histolyticum, type I; C-0130, Sigma-Aldrich
  • 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.
  • DMEM Dulbecco
  • 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
  • a food-grade coloring and flavoring solution i.e., a marinade
  • the cultivated bovine tissue 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.
  • the colored and flavored cultivated bovine tissue (“cultivated meat”; Figs.
  • 11E-G had the gross color (e.g., pink and red) and gross appearance 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).
  • Example 6 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.
  • Example 6 methods and results.
  • 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.
  • 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).
  • Example 7 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.
  • 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).
  • Item 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/cm 2 and 50,000,000 cells/cm 2 of 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.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

La présente invention concerne des technologies comprenant des substrats de culture cellulaire et des constructions tissulaires formées à partir de ceux-ci (par exemple, des viandes cultivées, des cuirs cultivés, des fourrures cultivées, etc.) comprenant des configurations (par exemple, des tissus non tissés, des nappes pour ouatinage, des mélanges, des maillages, des fils, etc.) et des modifications (par exemple, des revêtements amphiphiles pour rendre lesdits substrats sensiblement hydrophiles, etc.) d'une ou de plusieurs graines de plantes porteuses de coma, de pappe, ou autrement chevelues et leurs appendices chevelus sensiblement hydrophobes. Les technologies comprennent des systèmes et des procédés de fabrication desdits substrats de culture cellulaire et des constructions tissulaires, ainsi que leurs utilisations.
PCT/US2024/037722 2023-07-13 2024-07-12 Substrats de culture cellulaire à base de plantes Pending WO2025015242A2 (fr)

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US202363526590P 2023-07-13 2023-07-13
US63/526,590 2023-07-13

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WO2025015242A2 true WO2025015242A2 (fr) 2025-01-16
WO2025015242A9 WO2025015242A9 (fr) 2025-03-06
WO2025015242A3 WO2025015242A3 (fr) 2025-04-03

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Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7691472B2 (en) * 2005-06-23 2010-04-06 The Procter & Gamble Company Individualized seed hairs and products employing same
US8790921B2 (en) * 2007-02-14 2014-07-29 Drexel University Alimentary protein-based scaffolds (APS) for wound healing, regenerative medicine and drug discovery
ES2673726T3 (es) * 2010-01-05 2018-06-25 Cell Constructs I, Llc Biomateriales fabricados a partir de cabello humano
WO2011087975A1 (fr) * 2010-01-14 2011-07-21 The Procter & Gamble Company Structures fibreuses molles et solides et procédés de fabrication de celles-ci
US9814802B2 (en) * 2012-04-30 2017-11-14 The University Of Kansas Method for promoting hair growth comprising implanting a tissue scaffold comprising CK-19 positive cells derived from Wharton's jelly mesenchymal stromal cells

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