Edible scaffolds and formulations for cultivated meat

Abstract

Cell culture scaffolds suitable for cultivated meat are described herein. The scaffolds may be made from cellulose or other edible and / or biocompatible polymeric materials and are mechanically deformed to increase cell adhesion and / or proliferation are described herein. The mechanical deformations may include a plurality of recessions (e.g., grooves and channels) and / or holes or openings introduced by mechanical puncturing, and / or may produce cell culture scaffold particles / fibers. The scaffolds may contain one or more ingredients for improving cellular adhesion, proliferation, as well as organoleptic properties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelf-life, cooking performance (e.g., to promote the Maillard reaction), or any combination thereof. Also described herein are method of preparing the scaffolds and cultivated meat products comprising the scaffolds.

Description

[0001] EDIBLE SCAFFOLDS AND FORMULATIONS FOR CULTIVATED MEAT

[0002] The present description relates to cell culture scaffolds mechanically-deformed and modified to improve cell adhesion, proliferation, and / or mechanical and organoleptic properties. More specifically, the present description relates to scaffolds made from cellulose or other edible polymeric materials and biomolecules, which are suitable for use in the production of cultivated meat.

[0003] The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Cellular agriculture is an emerging area of biotechnology that involves the creation of agricultural goods by culturing animal, plant, or microorganism cells, rather than using animals or plants. Cultivated meat is a form of cellular agriculture that provides an alternative to slaughtering animals for human consumption. In contrast to traditional meat production, which is one of the main causes of environmental destruction, the field of cultivated meat aims to grow animal muscle cells in a bioreactor to generate a product that can be processed to replace conventional meat for consumption. For a cultivated meat product to have an appearance, texture, and structure resembling that of traditional meat, the cultivated cells must be grown on a proper scaffolding material. Biocompatible scaffolds made from natural or synthetic materials have traditionally been developed for the purposes of tissue engineering, regenerative medicine, or for in vitro three-dimensional cell culture models. However, scaffolds developed for such purposes are generally unsuitable for cultivated meat, as they are typically expensive to manufacture, require sophisticated equipment, are not mass-producible, lack sufficient cell-adherence and / or mechanical properties suitable for bioreactor or fermenter cultivation, and are unsuitable for human consumption in large quantities. Therefore, there is a need for scaffolds suitable for cultivated meat that address at least some of the aforementioned drawbacks.

[0006] SUMMARY

[0007] In a first aspect, described herein is a cell culture scaffold comprising an edible and / or biocompatible structure, membrane, fibers, and / or particles harbouring localized mechanical deformations to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding nondeformed cellulose membrane. In embodiments, the structure, membrane, fibers, and / or particles may be a cellulose structure, membrane, fibers, and / or particles or a structure, membrane, fibers, and / or particles of another edible polymeric material. In some embodiments, the mechanically deformed membranes may comprise a plurality of recessions (e.g., indentations, grooves and / or channels) introduced by mechanical compression, and / or a plurality of holes or openings introduced by mechanical puncturing. In some embodiments, the cell culture scaffolds may comprise or further comprise a plurality of cellulose particles and / or fibers sufficiently small to produce a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase. In some embodiments, the particles may be produced by grinding, milling, cutting, shearing, or other mechanical disruption of a cellulose membrane.

[0008] In a further aspect, described herein is a cultivated meat product comprising the cell culture scaffold as described herein.

[0009] In a further aspect, described herein is a method for preparing a cell culture scaffold, the method comprising providing an edible and / or biocompatible membrane, and introducing localized mechanical deformations into the membrane to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding non-deformed membrane.

[0010] In a further aspect, described herein is a method for preparing a cell culture scaffold, the method comprising providing an edible and / or biocompatible membrane, and grinding, milling, cutting, shearing, or otherwise mechanically disrupting the cellulose membrane into cellulose particles.

[0011] In a further aspect, described herein is a method for producing a cultivated meat precursor product, the method comprising cultivating cell culture scaffolds as described herein seeded with mammalian cells in a bioreactor or fermenter for a sufficient time to yield the cultivated meat precursor product.

[0012] General Definitions

[0013] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0014] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0015] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0016] The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0017] Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the appended drawings:

[0020] Fig- 1 shows a first prototype device designed and manufactured to compress cellulose membranes and introduce aligned grooves.

[0021] Fig- 2 shows a representative image of MTT-labeled 3T3 fibroblasts cultivated for 14 days on grooved cellulose scaffolds, which preferentially accumulate and orient in the aligned channels.

[0022] Fig- 3 compares the cell density of viable 3T3 fibroblasts cultivated for 14 days on scaffolds of compressed cellulose lacking grooves (“flat”), compressed grooved cellulose (“grooved”), or compressed grooved and holed cellulose (“grooved + holed”).

[0023] Fig. 4 shows a second prototype device designed and manufactured to mechanically introduce additional openings within cellulose scaffolds to create a “holed” scaffold.

[0024] Fig. 5 shows a representative image of MTT-labeled 3T3 fibroblasts cultivated on grooved and holed cellulose scaffolds for 14 days, which preferentially accumulate at high cell densities within the introduced holes (indicated by arrows).

[0025] Fig. 6 compares the cell density of viable 3T3 fibroblasts cultivated for 14 days on grooved and holed cellulose scaffolds previously impregnated (“chitosan + collagen”) or not (“No treatment”) with a chitosan / collagen solution.

[0026] Fig. 7 shows a representative image of MTT-labeled 3T3 fibroblasts cultivated for 14 days on grooved and holed cellulose scaffolds previously impregnated with a chitosan / collagen solution.

[0027] Fig. 8 compares the cell density of viable C2C12 myoblasts cultivated for 14 days on compressed grooved and holed cellulose scaffolds previously impregnated (“chitosan + collagen”) or not (“No treatment”) with a chitosan / collagen solution.

[0028] Fig. 9 shows a representative image of MTT-labeled C2C12 myoblasts cultivated for 14 days on grooved and holed cellulose scaffolds previously impregnated with a chitosan / collagen solution.

[0029] Fig. 10 compares the cell density of a heterogenous population of primary bovine cells cultivated from a fresh muscle tissue biopsy of a live animal from 7 to 30 days on compressed grooved and holed cellulose scaffolds previously impregnated with a chitosan / collagen solution in a stirred tank bioreactor (STB; 100 rpm, 37 °C in a humidified 5% CO2).

[0030] Fig. 11 shows a representative image of the cells of Fig. 10 cultivated for 14 days.

[0031] Figs. 12A-12C shows a further prototype device designed and manufactured to mechanically introduce additional openings within cellulose scaffolds to create perforated scaffolds. Fig. 12 A is a schematic representation of the device, which includes a matrix of precision-aligned needles, an adjustable spring and plate system for stability, and a positioning grid for uniform perforation distribution. Figs. 12B and 12C are photographs of the completed and fully-assembled device, which incorporates a micro-drilling module, adjustable control settings, and a robust frame enabling consistent performance and precise perforations.

[0032] Fig. 13A and 13B are photographs of two embodiments of mechanically-deformed cellulose scaffolds described in Example 13, including circular cellulose scaffolds sized to fit in a 24-well plate (Fig. 13A) and cellulose scaffolds that were grinded into particles to form a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase (Fig. 13B).

[0033] Fig. 14 shows a representative photograph of the grinded cellulose scaffold particles / fibers taken under a stereoscope.

[0034] Fig. 15A, 15B and 15C are photographs of cells grown on mixtures of mechanically-deformed cellulose scaffolds that were independently cultivated, harvested, and formed into disks resembling ground meat hamburger patties (“cultivated meat patties”).

[0035] Fig. 16 is a photograph of a hamburger patty made from conventional slaughtered ground beef.

[0036] Fig. 17 shows the color and texture changes of a raw cultivated meat patty (left image) upon cooking (right image).

[0037] Fig. 18 is an image of a cooked conventional slaughtered meat patty.

[0038] DETAILED DESCRIPTION

[0039] Described herein are edible and / or biocompatible scaffolds suitable for the production of cultivated meat and other applications. Cellulose membranes, and membranes based on other natural polymers, have been previously considered as potential scaffold material, but their poor cell adhesion and weak mechanical properties represented significant drawbacks. Conventional attempts to address these issues have involved transforming or modifying the membranes, for example, by chemical or enzymatic cross-linking, or employing complex multistep processes that require costly sophisticated equipment (Charest et al., 2006; Hu et al., 2019; Liu et al., 2023; Norris et al., 2022; Olyveira et al., 2013; Wang et al., 2017; Xiong et al. 2013). Such modifications may render the membranes unsuitable for human consumption in large quantities, may be cost-prohibitive, mechanically weaken the membranes, and may not be suitable for mass production.

[0040] In a first aspect, described herein is a cell culture scaffold comprising an edible and / or biocompatible structure (e.g., membrane, surface, fibers, and / or particles) harbouring localized mechanical deformations to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding non-deformed membrane. In some embodiments, the localized mechanical deformations may introduce additional surfaces within the membrane that facilitate cell adhesion, orientation, and / or proliferation, thereby enabling higher cell densities to be achieved, as compared to a corresponding non-deformed membrane. In some embodiments, localized mechanically deformed structures, membranes, fibers, and / or particles described herein may comprise a plurality of recessions or depressions introduced by localized mechanical compression. In some embodiments, the plurality of recessions or depressions may comprise a plurality of grooves. In some embodiments, at least some of the plurality of recessions, depressions, or grooves may be aligned with respect to one another, thereby facilitating cells to orient themselves in a particular direction. In some embodiments, mechanically deformed membranes described herein may be subjected to localized mechanical compression to create subareas or subregions of the structure, membrane, fibers, and / or particles having increased density as compared to a corresponding uncompressed counterparts, which would have a more uniform density. Without being bound by theory, the subareas or subregions of increased density within the membranes may desirably improve the mechanical properties of the scaffold, particularly when the compressed areas are aligned or patterned. In some embodiments, the subareas or subregions of increased density within the structures, membranes, fibers, and / or particles may act as ribs or other support structures, thereby improving the mechanical strength and / or rigidity (e.g., longitudinal strength and / or longitudinal rigidity) of the scaffold.

[0041] As used herein, the term “localized” or “local” generally refers to a mechanical force that is deliberately applied to specific regions of the edible and / or biocompatible structures, membranes, fibers, and / or particles (as opposed to the structures, membrane, fibers, and / or particles as a whole) in order to introduce a lasting mechanical deformation. For example, a localized mechanical deformation may include applying a mechanical force to specific regions of a structure or membrane and not others, or may include applying a greater mechanical force to specific regions of the membrane and a lesser force to other regions. Also, introducing localized mechanical deformations such as by grinding, milling, cutting, or otherwise mechanically disrupting an edible and / or biocompatible structure or membrane, may be used to produce smaller structures, membranes, fibers, and / or particles. In some embodiments, these smaller structures, membranes, fibers, and / or particles may themselves comprise localized mechanical deformations. In some embodiments, localized mechanically deformed membranes described herein may comprise a plurality of holes or openings introduced by mechanical puncturing. As used herein, the terms “holes” and “openings” refer to artificially created structures that are typically larger than the pores natively present in the edible and / or biocompatible membrane materials. In some embodiments, the plurality of holes or openings are of sufficient size (e.g., diameter and / or depth) to enable cell proliferation therein. In some embodiments, the plurality of holes or openings may have an average diameter of at least 200, 250, 300, 350, 400, or 450 microns. In some embodiments, the plurality of holes or openings may have an average diameter between 200 to 900, 250 to 850, 300 to 800, 350 to 750, or 400 to 700 microns. In some embodiments, the plurality of holes or openings may be present in the edible and / or biocompatible membrane at a density sufficient to enable increased cell proliferation as compared to a corresponding membrane lacking the holes or openings. In some embodiments, the plurality of holes or openings may be present in the cellulose membrane at a density of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 holes or openings / cm2. In some embodiments, the plurality of holes or openings may be present in the cellulose membrane at a density of 10 to 150, 15 to 140, 20 to 130, or 20 to 120 cm2.

[0042] In some embodiments, the cell culture scaffolds described herein may comprise or further comprise a plurality of edible and / or biocompatible particles and / or fibers (e.g., cellulose particles and / or fibers). In some embodiments, the particles and / or fibers may be less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm in average diameter. In some embodiments, the particles and / or fibers can be of different dimensions and form different geometries, e.g., spherical, rod- like, fibers, and other scattered and irregular forms. In some embodiments, the particles may be sufficiently small to produce a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase. In some embodiments, the aqueous phase may be a dispersing medium such as water, a buffer, culture medium with or without serum or serum substitutes. In some embodiments, the plurality of cellulose particles and / or fibers may be produced by grinding, milling, cutting, shearing, or other mechanical disruption of an edible and / or biocompatible structure or membrane.

[0043] In some embodiments, the mechanically-deformed edible and / or biocompatible structures, membranes, fibers, and / or particles described herein may be hydrated to a level less than 95%, 90%, 85%, 80%, 75%, or 70% of their initial hydration weight or of their weight at maximum hydration. In some embodiments, the mechanically-deformed edible and / or biocompatible structures, membranes, fibers, and / or particles described herein may be hydrated to a level 15% to 95%, 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75% of their initial hydration weight or weight at maximum hydration. In some embodiments, the localized mechanical deformation comprises localized mechanical compression. In some embodiments, the mechanically- deformed edible and / or biocompatible structures, membranes, fibers, and / or particles described herein may be subjected to compression or further compression to a level less than 95%, 90%, 85%, 80%, 75%, or 70% of their initial hydration weight or of their weight at maximum hydration, or to a level 15% to 95%, 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75% of their initial hydration weight or weight at maximum hydration.

[0044] In some embodiments, the edible and / or biocompatible membranes described herein may comprise or consist of cellulose membranes. As used herein, the expression “cellulose membrane” refers to materials comprising cellulose as their main structural component, including modified and unmodified celluloses, from any source. In some embodiments, cellulose membranes described herein may comprise or consist of microbial cellulose (e.g., bacterial cellulose), fungal cellulose, algal cellulose, plant-based cellulose, or any combination thereof. In some embodiments, the edible and / or biocompatible membranes described herein may comprise or consist of other natural polymers, such as proteins (e.g., silk, collagen, gelatin, fibrinogen, elastin, keratin, actin, myosin, zein, soy, pea, and / or rice), polysaccharides (e.g., chitosan, amylose, dextran, chitin, and / or glycosaminoglycans), or polynucleotides (e.g., DNA and / or RNA). In some embodiments, the edible and / or biocompatible membranes described herein may comprise or consist of an unmodified material (e.g., unmodified or native cellulose). In some embodiments, the edible and / or biocompatible membranes described herein may comprise or consist of a material that was chemically or enzymatically modified (e.g., cross-linked, chemically modified).

[0045] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be impregnated or coated with one or more biological molecules, for example to further enhance cell adhesion and / or proliferation. In some embodiments, the biological molecules described herein may comprise a yeast-based product (e.g., yeast extract, yeast peptone, inactivated yeasts, etc.), peptone, a protein, a polysaccharide, a biological polymer, a peptide (e.g., an adhesion peptide), a growth factor, or any combination thereof. In some embodiments, the biological molecules described herein may comprise: pectin, myoglobin or other heme-containing proteins, lignin, hemicellulose, chitosan, gelatin, fibronectin, laminin, collagen, glycoprotein, thrombospondin, elastin, fibrillin, mucopolysaccharide, glycolipid, keratin, glycosaminoglycan, glucomannan, hyaluronic acid, proteoglycan, vitronectin, poly-D-lysine, RGD peptide, alginate, or any combination thereof. In particular embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be impregnated or coated with chitosan, with or without collagen. In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be impregnated or coated with recombinant collagen.

[0046] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be subjected to a texture -modifying processing step, for example to improve mouthfeel and / or organoleptic properties. In some embodiments, the texture-modifying processing step may comprise chemical or enzymatic modification or degradation. In particular embodiments where the edible and / or biocompatible membrane is a cellulose membrane, the membrane may be subjected to cellulase treatment to modify the texture of the scaffold.

[0047] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein are preferably suitable or human consumption, edible and / or biocompatible, biodegradable, or any combination thereof.

[0048] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be populated with animal or mammalian cells (e.g., bovine, bison, deer, porcine, goat, lamb, kangaroo, rabbit, chicken, turkey, duck, goose, emu, pheasant, quail, squab, fish, crab, lobster, prawns, mussels, oysters, scallops, or clams). Unless otherwise stated, “cells” referred to herein generally means adherent cells. In some embodiments, the mammalian cells may comprise fibroblasts, muscle stem cells (e.g., satellite cells), muscle progenitor cells, fibro-adipogenic progenitors (FAP), adipocytes or other fat-containing cells, or any combination thereof. In some embodiments, the cells described herein are cultured from a biopsy (e.g., muscle biopsy) of an animal (e.g., bovine, bison, deer, porcine, goat, lamb, kangaroo, rabbit, chicken, turkey, duck, goose, emu, pheasant, quail, squab, fish, crab, lobster, prawns, mussels, oysters, scallops, clams). In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be populated with more than one cell type on the same scaffold (e.g., on two different sides, surfaces, or subregions of the same scaffold). In some embodiments, this may be accomplished by sequential cell seeding and adherence cycles on different sides, surfaces, or subregions of the scaffold or membrane. In some embodiments, such co-culture set-ups may be advantageous to facilitate synergistic or cooperative growth (or growth in concert) between different cell types, such as when a first cell type secretes or expresses factors or signals (e.g., growth factors or signals) that promote adherence, proliferation, and / or maturation of a second cell type. In some embodiments, at least two, at least three, or at least four different cell types may be co-cultured on the same membrane. In some embodiments, synergistic co-culture may be achieved by co-cultivating, in the same bioreactor, fermenter, or vessel, two or more different cell culture scaffolds described herein, wherein each scaffold is populated with a different cell type. In some embodiments, the cell culture scaffold or edible and / or biocompatible membrane described herein is for use in the production of a cultivated meat product, in tissue engineering, in regenerative medicine, in wound healing, or in an in vitro three-dimensional cell culture model. In some embodiments, described herein is the use of a cell culture scaffold or edible and / or biocompatible membrane as described herein for the production of a cultivated meat product, in tissue engineering, in regenerative medicine, in wound healing, or in an in vitro three-dimensional cell culture model. In a further aspect, described herein is a cultivated meat product comprising a cell culture scaffold or edible and / or biocompatible membrane described herein. In some embodiments, the cultivated meat product does not comprise an animal- derived ingredient or molecule.

[0049] In a further aspect, described herein is a method for preparing a cell culture scaffold, the method comprising providing an edible and / or biocompatible membrane, and introducing localized mechanical deformations into the edible and / or biocompatible membrane to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding non-de formed membrane. In some embodiments, the edible and / or biocompatible membrane provided is a hydrated membrane. In some embodiments, the edible and / or biocompatible membrane is mechanically deformed by compression between rigid surfaces to introduce a plurality of recessions into the membrane. In some embodiments, the edible and / or biocompatible membrane is mechanically deformed by puncturing (e.g., partially or completely) with a device to introduce a plurality of holes or openings into the membrane. In some embodiments, the cell culture scaffold or the edible and / or biocompatible membrane referred to above is as defined herein.

[0050] In a further aspect, described herein is a method for preparing a cell culture scaffold, the method comprising providing a cellulose membrane, and grinding, milling, cutting, shearing, or otherwise mechanically disrupting the cellulose membrane into cellulose particles and / or fibers. In some embodiments, particles and / or fibers may be of different dimensions and form different geometries, e.g., spherical, rod-like, fibers, and other scattered and irregular forms. In some embodiments, the particles may be sufficiently small to produce a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase. In some embodiments, the particles are less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm in average diameter. In some embodiments, the particles are about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm in average diameter. In some embodiments, the particles are sufficiently small to produce a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase.

[0051] In a further aspect, described herein is a cell culture scaffold produced by a method described herein.

[0052] In a further aspect, described herein is a method for producing a cultivated meat precursor product, the method comprising providing cell culture scaffolds as described herein and cultivating the cell culture scaffolds seeded with mammalian cells in a bioreactor or fermenter for a sufficient time to yield the cultivated meat precursor product. In some embodiments, the method comprises co-culturing a mixture of cell culture scaffolds seeded with different cell types (e.g., seeded on the same scaffold and / or seeded on distinct scaffolds). In some embodiments, the mammalian cells are as described herein. In some embodiments, the bioreactor or fermenter is a stirred tank bioreactor or fermenter, or any other type of bioreactor or fermenter (e.g., an industrial scale bioreactor or fermenter of at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000, 5500, 6000, 6500, 7000, 8000, 9000, 10 000, 20 000, or 25 000 L).

[0053] In some embodiments, the cultivated meat precursor products described herein may comprise one or more ingredients for improving organoleptic properties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelf-life, cooking performance (e.g., to promote the Maillard reaction), or any combination thereof. In some embodiments, the cultivated meat precursor products described herein may comprise one or more ingredients such as yeast-based products (e.g., yeast extracts, yeast peptone, inactivated yeasts, etc.), metabolites, natural flavors, natural pigments, dietary fiber (e.g., soluble fiber, insoluble fiber), emulsifiers, stabilizers, thickeners, and sulfur compounds (e.g., cysteine, acetyl cysteine, cystine, taurine, thiamine, methionine, glutathione, alliin, biotin).

[0054] In some embodiments, the cultivated meat precursor products described herein may comprise one or more ingredients flavoring agents such as a sugar, a sugar alcohol, a sugar acid, a sugar derivative, an oil, a free fatty acid, an amino acid or derivative thereof, a nucleoside, a nucleotide, a vitamin, an acid, a peptide, a phospholipid, a protein hydrolysate, a yeast-based products (e.g., yeast extracts, yeast peptone, inactivated yeasts, etc.), or a mixture thereof. In some embodiments, the flavoring may comprise: glucose, fructose, ribose, arabinose, glucose-6-phosphate, fructose 6-phosphate, fructose 1 ,6-diphosphate, inositol, maltose, sucrose, maltodextrin, glycogen, nucleotide-bound sugars, molasses, a phospholipid, a lecithin, inosine, inosine monophosphate (IMP), guanosine monophosphate (GMP), pyrazine, adenosine monophosphate (AMP), lactic acid, succinic acid, glycolic acid, thiamine, creatine, pyrophosphate, vegetable oil, algal oil, sunflower oil, com oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, rice bran oil, cottonseed oil, olive oil, sunflower oil, canola oil, flaxseed oil, coconut oil, mango oil, a free fatty acid, cysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, alanine, asparagine, aspartate, glutamate, glutamine, glycine, proline, serine, tyrosine, glutathione, an amino acid derivative, urea, pantothenic acid, ornithine, niacin, glycerol, citrulline, taurine, biotin, borage oil, fungal oil, blackcurrant oil, betaine, beta carotene, B- vitamins, N- Acetyl L-cysteine, iron glutamate, a peptone, or any combination thereof.

[0055] ITEMS

[0056] In some embodiments, described herein are one of more of the following items:

[0057] 1. A cell culture scaffold comprising a cellulose structure (e.g., cellulose membrane, cellulose surface, cellulose fibers, and / or cellulose particles) harbouring localized mechanical deformations to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding non-deformed cellulose membrane. The cell culture scaffold of item 1, wherein the localized mechanical deformations comprise a plurality of recessions introduced by mechanical compression. The cell culture scaffold of item 2, wherein: (a) said mechanical compression creates localized areas of the cellulose membrane having increased density as compared to a corresponding uncompressed cellulose membrane; (b) the plurality of recessions comprises a plurality of grooves; (c) the plurality of recessions are aligned with respect to one another; (d) the plurality of recessions are of sufficient depth to enable cell proliferation therein; or (e) any combination of (a) to (d). The cell culture scaffold of any one of items 1 to 3, wherein the localized mechanical deformations comprise a plurality of holes or openings introduced by mechanical puncturing. The cell culture scaffold of item 4, wherein: (a) the plurality of holes or openings are of sufficient size (e.g., diameter and / or depth) to enable cell proliferation therein; (b) the plurality of holes or openings have an average diameter of at least 200, 250, 300, 350, 400, or 450 microns, or an average diameter between 200 to 900, 250 to 850, 300 to 800, 350 to 750, or 400 to 700 microns; (c) the plurality of holes or openings are present in the cellulose membrane at a density sufficient to enable increased cell proliferation as compared to a corresponding cellulose membrane lacking said holes or openings; (d) the plurality of holes or openings are present in the cellulose membrane at a density of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 holes or openings / cm2, or are present in the cellulose membrane at a density of 10 to 150, 15 to 140, 20 to 130, or 20 to 120 cm2; or (e) any combination of (a) to (d). The cell culture scaffold of any one of items 1 to 5, wherein the cell culture scaffold comprises or further comprises a plurality of cellulose particles and / or fibers (e.g., less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm in average diameter; having different dimensions and / or geometries [e.g., spherical, rod-like, fibers, or other scattered and irregular forms]; or any combination thereof). The cell culture scaffold of item 6, wherein the plurality of cellulose particles and / or fibers are sufficiently small to produce a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase (e.g., in a dispersing medium such as water, a buffer, culture medium with or without serum or serum substitutes). The cell culture scaffold of item 6 or 7, wherein the plurality of cellulose particles / fibers are produced by grinding, milling, cutting, shearing, or other mechanical disruption of a cellulose membrane. The cell culture scaffold of any one of items 1 to 8, wherein the mechanically-deformed cellulose structure, membrane, fibers, and / or particles is / are hydrated to a level less than 95%, 90%, 85%, 80%, 75%, or 70% of their initial hydration weight or weight at maximum hydration, or the cellulose structure, membrane, fibers, and / or particles is / are hydrated to a level between 45 to 95%, 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75% of their initial hydration weight or weight at maximum hydration. The cell culture scaffold of item 9, wherein said localized mechanical deformation comprises localized mechanical compression. The cell culture scaffold of any one of items 1 to 10, wherein the cellulose membrane comprises or consists of unmodified and / or chemically-modified (e.g., cross-linked) cellulose. The cell culture scaffold of any one of items 1 to 11, wherein the cellulose membrane comprises or consists of microbial cellulose, bacterial cellulose, fungal cellulose, algal cellulose, plant-based cellulose, or any combination thereof. The cell culture scaffold of any one of items 1 to 12, wherein the cellulose membrane is impregnated with one or more biological molecules and / or ingredients (e.g., for improving organoleptic properties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelflife, cooking performance (e.g., to promote the Maillard reaction), or any combination thereof). The cell culture scaffold of item 13, wherein the one or more biological molecules comprise a yeast-based product (e.g., yeast extract, yeast peptone, inactivated yeast, etc.), peptone, a protein, a polysaccharide, a biological polymer, a peptide (e.g., an adhesion peptide), a growth factor, or any combination thereof. The cell culture scaffold of item 13 or 14, wherein the one or more biological molecules comprise: pectin, myoglobin or other heme-containing proteins, lignin, hemicellulose, chitosan, gelatin, fibronectin, laminin, collagen, glycoprotein, thrombospondin, elastin, fibrillin, mucopolysaccharide, glycolipid, keratin, glycosaminoglycan, glucomannan, hyaluronic acid, proteoglycan, vitronectin, poly-D-lysine, RGD peptide, alginate, or any combination thereof. The cell culture scaffold of any one of items 1 to 15, wherein the cellulose membrane was subjected to cellulase treatment to modify the texture of the scaffold. The cell culture scaffold of any one of items 1 to 16, wherein the cell culture scaffold is suitable or human consumption, biocompatible, biodegradable, or any combination thereof. The cell culture scaffold of any one of items 1 to 17, wherein the cell culture scaffold is populated with animal cells (e.g., bovine, bison, deer, porcine, goat, lamb, kangaroo, rabbit, chicken, turkey, duck, goose, emu, pheasant, quail, squab, fish, crab, lobster, prawns, mussels, oysters, scallops, or clams). The cell culture scaffold of item 18, wherein the mammalian cells comprise primary cells, fibroblasts, muscle stem cells (e.g., satellite cells), muscle progenitor cells, fibro-adipogenic progenitors (FAP), endothelial cells, chondrocytes, adipocytes, or other fat-containing cells, or any combination thereof. The cell culture scaffold of item 18 or 19, which is populated with more than one cell type on the same scaffold (e.g., on two different sides, surfaces, or subregions of the same scaffold). The cell culture scaffold as defined in any one of items 1 to 20 for use in the production of a cultivated meat product, in tissue engineering, in regenerative medicine, in wound healing, or in an in vitro three-dimensional cell culture model. A cultivated meat product comprising the cell culture scaffold as defined in any one of items 1 to 20. The cultivated meat product of item 18, which does not comprise an animal-derived ingredient. A method for preparing a cell culture scaffold, the method comprising providing a cellulose membrane, and: (a) introducing localized mechanical deformations into the cellulose membrane to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding non-deformed cellulose membrane; (b) grinding, milling, cutting, shearing, or otherwise mechanically disrupting the cellulose membrane into cellulose particles / fibers (e.g., less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm in average diameter; having different dimensions and / or geometries [e.g., spherical, rod-like, fibers, or other scattered and irregular forms]; or any combination thereof); or both (a) and (b). The method of item 24, wherein the cellulose membrane provided is a hydrated cellulose membrane. The method of item 24 or 25, wherein the cellulose membrane is mechanically deformed by compression between rigid surfaces to introduce a plurality of recessions into the cellulose membrane. The method of any one of items 24 to 26, wherein the cellulose membrane is mechanically deformed by partially or completely puncturing with a device to introduce a plurality of holes or openings into the cellulose membrane. The method of any one of items 24 to 27, wherein the cell culture scaffold or the cellulose membrane is as defined in any one of items 1 to 20. A cell culture scaffold produced by the method of any one of items 24 to 28. A method for producing a cultivated meat precursor product, the method comprising providing cell culture scaffolds as defined in any one of items 1 to 20, or produced by the method of any one of items 24 to 28, and cultivating the cell culture scaffolds seeded with mammalian cells in a bioreactor or fermenter for a sufficient time to yield the cultivated meat precursor product. 31. The method of item 30, wherein the method comprises co-culturing a mixture of cell culture scaffolds seeded with different cell types (e.g., seeded on the same scaffold and / or seeded on distinct scaffolds).

[0058] 32. The method of item 30 or 31 , wherein the mammalian cells are as defined in item 18 or 19.

[0059] 33. The method of any one of items 30 to 32, wherein the bioreactor or fermenter is a stirred tank bioreactor or fermenter, or any other type of bioreactor or fermenter.

[0060] EXAMPLES

[0061] Example 1: Materials and methods

[0062] MTT assay

[0063] The MTT assay is a colorimetric assay used as an indicator of cell viability, proliferation, and / or cytotoxicity based on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan by oxidoreductase enzymes in metabolically active cells. Briefly, a stock solution of MTT at a concentration of 5 mg / mL in Hank’ s solution was prepared and diluted 1 : 10 in Hank’ s solution as needed to create the final reagent concentration (0.5 mg / mL). Following transfer to a 24-well plate, 800 pL of MTT reagent was added to each well to completely submerge the samples, which were then incubated at 37 °C and 5% CO2 under humidified conditions for 4 h. Metabolically active cells present in the samples result in development of colored formazan crystals, which were then observed with a light stereoscope to qualify the presence of cells.

[0064] WST-1 assay

[0065] The WST-1 assay measures cell proliferation, cell viability, and / or cytotoxicity based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Briefly, all reagents were equilibrated to 37 °C before starting manipulations. Cell standards were prepared in 300 pL of media and were done for each cell type for various cell densities. Samples were transferred to a 24- well plate and volumes were completed to 400 pL with tempered DH media, before adding 30 pL of WST-1 reagent to each well. Plates were mixed with a plate shaker for a few minutes before incubating for 2 or 4 hours at 37 °C and 5% CO2 under humidified conditions. Following incubation, small pellets inside the wells were gently crushed to expel the trapped reacted WST-1 reagent. Plates were then mixed with a plate shaker for about 5 minutes before 100 pL from each well was transferred to a 96-well plate. Spectroscopy readings were taken at 440 or 450 nm and cell growth rates were evaluated using absorbance. Example 2: Localized mechanical compression of cellulose scaffolds improves cellular adhesion

[0066] Biocompatible scaffolds made from natural or synthetic materials have traditionally been developed for the purposes of tissue engineering, regenerative medicine, or for in vitro three-dimensional cell culture models. The emerging field of cultivated meat has created a recent demand for a new generation of scaffolds having requirements and / or limitations that differ from, or go beyond, those developed for other applications. For example, scaffolds suitable for cultivated meat should not only be biocompatible, but ideally inexpensive, mass-producible, biodegradable, suitable for human consumption in large quantities, and ideally possess desirable organoleptic properties. Cellulose-based materials have been explored as potential scaffolds, but their poor cell adhesion and weak mechanical properties are significant drawbacks. Conventional attempts to address these issues have involved, for example, chemically modifying the cellulose (e.g., chemical or enzymatic cross-linking), and / or complex multistep cellulose-modifying processes requiring costly sophisticated equipment, which are not suitable for mass production and / or may render the processed cellulose membranes unsuitable for human consumption in large quantities.

[0067] Being the most common polymeric substances found in nature, cellulose is obtainable from plants or can be produced by fungi, algae, and certain microorganisms. Although chemically identical to plant cellulose, microbially-produced cellulose is particularly attractive for cultivated meat purposes due to its biocompatibility, mechanical properties, structure, purity, reproducibility, and non-reliance on plant or animal material. Accordingly, the present study employs bacterial cellulose with the understanding that the technologies described herein may be applicable to celluloses from other sources.

[0068] Preliminary studies on different commercially available unmodified bacterial cellulose membranes were performed to evaluate their ability to support the growth of mammalian cells. SWISS 3T3 fibroblast cells were selected for initial studies, as these cells grow quickly, achieve high maximum densities, are tolerant to changes in the composition of the medium, generally attach strongly to all types of surfaces, but die as soon as they do not adhere. This allows for an experiment that can distinguish between successful attachment and proliferation versus a false positive or negative. 3T3 cells were seeded directly on hydrated bacterial cellulose membranes obtained from different commercial suppliers and cultivated for up to 14 days in suitable media. While some cell adherence and growth were detected, even reaching a cell density of up to about 1 x 105cells / cm2, it became clear that the maximum cell densities attainable using commercially available unmodified cellulose membranes would not meet the levels required for cultivated meat products. Different methods of processing or modifying the cellulose membranes were thus explored that could ideally improve cell adherence and proliferation, while maintaining or improving the mechanical properties of the membranes. Interestingly, preliminary experiments revealed that membranes having mechanical deformations (e.g., membranes inadvertently “damaged” by localized compressions) appeared to out-perform “undamaged” membranes both in terms of cell adherence and proliferation. These observations were later validated by deliberately introducing mechanical deformations into the commercially-obtained cellulose membranes and evaluating their capabilities to support growth of 3T3 cells in culture. Furthermore, it was observed that localized mechanical deformations that resulted in compression of subregions of the membranes not only increased cell adherence and proliferation, but also positively impacted the mechanical structure of the membranes. In particular, mechanical deformations that resulted in aligned compression membrane subregions (e.g., aligned compression patterns, indentations, or grooves) seemed to have a positive impact on membrane robustness / rigidity, while improving cell adherence and proliferation. Because of their ability to be mass- produced using adapted machinery, grooves were selected for further characterization in subsequent Examples.

[0069] Example 3: Cellulose scaffolds with compression-induced grooves enable higher cell densities

[0070] A first prototype consisting of a pair of matching grooved metal plates (Fig. 1) was designed and manufactured to enable the cellulose membranes to be compressed therebetween to introduce aligned grooves in the scaffold’s structure in a consistent and reproducible manner. For hydrated membranes, compression was assessed by the level of water elimination post-compression (e.g., 50-95% of initial weight, pre-compression). Interestingly, 3T3 cells were observed to preferentially accumulate and orient in the aligned channels and rough surfaces of the grooved cellulose scaffold, as shown after MTT labeling of metabolically active cells in Fig. 2. The use of the grooved cellulose scaffold (“grooved”) allowed for about a three-fold increase in the density of 3T3 cells after 14 days in culture, as compared to a comparatively compressed scaffold lacking the introduced grooves (“flat”) (Fig. 3). Furthermore, the aligned compression-induced grooves favourably affected the longitudinal mechanical properties of the membranes.

[0071] Example 4: Cellulose scaffolds with mechanically-introduced openings further enable higher cell densities

[0072] A second prototype consisting of a plurality of regularly-spaced sharp-tipped rods extending from a metal plate (Fig. 4) was designed and manufactured to mechanically introduce additional openings within grooved cellulose scaffolds to create grooved and holed scaffolds, the holes generally having diameters between 300-800 pm and a density of about 20-120 holes / cm2. The second prototype shown in Fig. 4 introduced openings or holes that traverse the thickness of the membranes, but other devices may be readily conceived to introduce openings or holes that stop short thereof. In this experiment, the first prototype was used to compress and introduce aligned grooves in a cellulose scaffold, and subsequently, the second prototype was used to introduce additional openings or holes within the grooved cellulose scaffold. Interestingly, by applying these combined mechanical treatments to the scaffold, it was possible to increase the density of 3T3 cells from 3 x 105cells / cm2using only the grooved scaffold (“grooved”), to 4 x 105cells / cm2with the grooved and holed scaffold (“grooved + holed”) after 14 days of culture (Fig. 3). Unexpectedly, the cells grown on the grooved and holed scaffold were observed to accumulate at high densities within the introduced openings, as shown by the intense MTT straining in Fig. 5 (arrows), suggesting that the cells grew preferentially in the mechanically-introduced holes of the scaffold.

[0073] Therefore, higher hole densities may be envisioned to further increase the overall cell density of the scaffold, thereby allowing cells to penetrate and populate the entire scaffold.

[0074] Example 5: Collagen / chitosan-impregnated mechanically-deformed cellulose scaffolds further improve cell densities

[0075] In this experiment, the mechanical methods described above were performed to produce a grooved and holed cellulose scaffold, which was subsequently covered with a thin later of a collagen and chitosan solution (collagen : chitosan, 1 : 1 to 6: 1 v / v) for about 1 hour to allow incorporation into the scaffold. As shown in Fig. 6, by coating the grooved and holed cellulose membrane with biomaterials, it was possible to increase overall 3T3 cell density from about 4 x 105cells / cm2(“No treatment”) to about 7 x 105cells / cm2(“Chitosan + collagen”) after 14 days of culture. As shown in Fig. 7, cells accumulated at high densities within the mechanically-introduced holes of the collagen / chitosan-impregnated scaffold, consistent with the results in Example 4.

[0076] Example 6: C2C12 myoblasts grown on mechanically-deformed, collagen / chitosan-impregnated cellulose scaffolds

[0077] The experiments performed in Example 5 with 3T3 cells were repeated with the myoblast cell line C2C12, which has been shown to differentiate rapidly to form contractile myotubes and produce characteristic muscle proteins. The results in Fig. 8 and Fig. 9 show an increase in C2C12 density using collagen / chitosan-impregnated scaffold (“Chitosan + collagen”) as compared to an uncoated scaffold (“No treatment”).

[0078] Example 7: Primary bovine muscle cells are cultivatable on mechanically-deformed, collagen / chitosan-impregnated cellulose scaffolds

[0079] A heterogenous population of primary bovine cells was obtained from a fresh muscle tissue biopsy of a live animal, which included fibroblasts, adipocytes, fibro-adipogenic progenitors (FAP), endothelial cells, chondrocytes, progenitor and satellite cells. Individual cell types contained in the biopsy were independently characterized and cultivated in vitro before being separately seeded on mechanically- deformed, collagen / chitosan-impregnated grooved and holed cellulose scaffolds and cultured as described in Example 5. As shown in Fig. 10, primary bovine cells of different types were able to adhere to the scaffolds and proliferate over the course of 30 days, validating the suitability of the cellulose scaffolds described herein for cultivated meat based on primary bovine cells.

[0080] Example 8: Mechanically-deformed cellulose scaffolds are suitable for cultivation in a bioreactor / fermentor

[0081] While the experiments in Examples 2-7 were performed under lab-scale static culture conditions, industrial-scale bioreactors or fermenters necessarily involve dynamic culture conditions that place additional strain on the mechanical structure of the cellulose membranes. The experiments in Example 8 aimed to evaluate the effect of dynamic culture conditions on scaffold integrity and cell growth. Briefly, collagen / chitosan-impregnated grooved and holed cellulose scaffolds were prepared as described in Examples 4 and 5. The scaffolds were seeded with 3T3 cells under static conditions for sufficient time to allow the cells to adhere (at least 8 h). Subsequently, some cell-bearing cellulose scaffolds were then transferred to a 500-mL stirred tank bioreactor (STB) and 150 mL of DH medium with enough sterile antifoam were added, while controls were cultured under corresponding static conditions. Cellulose scaffolds remained intact under dynamic culture conditions, with cell densities reaching up to 1 x 106cells / cm2, as shown in Fig. 10. Interestingly, the cell densities reached under dynamic culture conditions were equivalent to or higher than those reached under corresponding static conditions, demonstrating the suitability of the cellulose scaffolds described herein for large-scale bioreactor or fermenter cultivation.

[0082] Example 9: Construction and testing of further devices to introduce localized mechanical deformations into cellulose scaffolds

[0083] Further prototype devices, in addition to those described in Examples 3 and 4, were designed, constructed, and tested for their abilities to introduce different types of localized mechanical deformations into different sizes of cellulose scaffolds and to evaluate the effect of the localized mechanical deformations on cellular adhesion, proliferation, and viable cell density. In general, it was observed that localized mechanical deformations that served to increase the surface area of the cellulose scaffolds had generally a positive impact on cellular adhesion, proliferation, and / or density, while those that resulted in the creation of localized areas of the cellulose scaffolds having increased density imparted favorable mechanical properties (e.g., increased mechanical strength and / or rigidity) to the cellulose membranes. An example of a further prototype device that was designed, constructed, and tested is shown Figs. 12A- 12C. This further prototype device, based on the one shown in Fig. 4, was employed in subsequent Examples to mechanically introduce openings or perforations within the cellulose scaffolds at a greater density than the prototype device shown in Fig. 4.

[0084] Example 10: Primary bovine muscle cells cultivated on cellulose scaffolds impregnated with different combinations of chitosan, pectin, and yeast-based products

[0085] Mechanically-deformed “grooved + holed” cellulose scaffolds, sized to be placed in 24-well plates, were sterilized by autoclaving and then immersed with distinct solutions, each solution containing different combinations of chitosan, pectin, and yeast extract (as shown in Conditions A-D shown in Table 1), for 1 hour at room temperature with gentle agitation. Excess solutions were removed and the impregnated cellulose scaffolds were air-dried under sterile conditions.

[0086] A heterogenous population of primary bovine cells obtained as described in Example 7 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine. Cells were then seeded on the impregnated cellulose scaffolds placed in 24-well plates and cultures were incubated at 37°C and 5% CO2 for 30 days. The number of viable cells per membrane was evaluated at 40 h, 15 days, and 30 days by WST-1 assay as described in Example 1 and the results shown in Table 1 represent means of experiments performed in triplicate. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test for pairwise comparisons and differences at 30 days between Condition D vs A / B / C and between Condition B vs. A / C were found to be significant (p < 0.05). Condition D resulted in the highest number of viable cells per scaffold. Similar results were observed in C2C12 cells (data now shown). Furthermore, the presence of chitosan was associated with a higher viable cell density at 40 h (Conditions A, B and C), suggesting a benefit for cellular adherence.

[0087] Table 1: Comparison of cells cultivated on cellulose scaffolds impregnated with different combinations of chitosan, pectin, and yeast extract

[0088] Viable cells per scaffold

[0089] Condition Biological molecules (w / v) 40 h 15 days 30 days

[0090] A 0.5% chitosan 51,330 72,797 314,016

[0091] B 0.5% chitosan + 1% pectin + 4% yeast extract 48,830 74,218 369,122

[0092] C 0.5% chitosan + 1% pectin, followed by 4% yeast extract 51,401 47,353 139,202

[0093] D 1 % pectin + 4% yeast extract 30,751 83,759 415,665

[0094] Example 11: Primary bovine muscle cells cultivated on cellulose scaffolds impregnated with different concentrations of pectin and yeast extract

[0095] Experiments as performed in Example 10 were repeated with cellulose scaffolds impregnated for 2 h with different concentrations of low-methoxyl pectin (0.5%, 2%, and 3% w / v) and yeast extract (2%, 4%, and 6% w / v), as shown in Table 2. Table 2: Comparison of cells cultivated on cellulose scaffolds impregnated with different concentrations of pectin and yeast extract

[0096] Viable cells per scaffold

[0097] Condition Biological molecules (w / v) 40 h 15 days 30 days

[0098] E 0.5% pectin + 2% yeast extract 19,298 102,216 424,089

[0099] F 2% pectin + 2% yeast extract 8,315 85,008 211,475

[0100] G 3% pectin + 2% yeast extract 8,498 19,110 81,932

[0101] H 0.5% pectin + 4% yeast extract 19,420 120,336 455,122

[0102] I 2% pectin + 4% yeast extract 18,120 83,287 329,717

[0103] J 3% pectin + 4% yeast extract 17,836 29,030 133,017

[0104] K 0.5% pectin + 6% yeast extract 21,693 116,590 483,290

[0105] L 2% pectin + 6% yeast extract 13,898 48,972 276,564

[0106] M 3% pectin + 6% yeast extract 8,680 37,230 102,939

[0107] The results in Table 2 demonstrate that pectin and yeast extract concentrations may significantly influence cell attachment (e.g., at 40 h) and proliferation (e.g., at 15 and 30 days). Highest viable cell adherence and densities were observed on cellulose scaffolds impregnated with 0.5% pectin (Conditions E, H, and K). In contrast, lowest viable cell adherence and densities were observed on cellulose scaffolds impregnated with 3% pectin (Conditions G, J, and M). Higher concentrations of yeast extract were generally associated with higher cell densities.

[0108] Example 12: Primary bovine muscle cells cultivated on cellulose scaffolds impregnated with pectin, yeast extract, with or without RGD adhesion peptide

[0109] Experiments as performed in Example 11 were repeated with cellulose scaffolds impregnated with 1% w / v of pectin, 20 mg / mL yeast extract with or without an RGD adhesion peptide (0.35 mg / mL, “RGD”), as shown in Table 3.

[0110] Table 3: Comparison of cells cultivated on cellulose scaffolds impregnated with or without RGD peptide

[0111] Viable cells per scaffold

[0112] Condition Biological molecules (w / v) 40 h 10 days 20 days 30 days

[0113] N 1 % pectin + 20 mg / mL yeast extract 20,482 134,096 361,326 577,024

[0114] O 1% pectin + 20 mg / mL yeast extract + RGD 36,240 151,623 365,630 525,675

[0115] Higher viable cell density at 40 h was observed for cells cultivated on cellulose scaffolds impregnated with Condition O, suggesting that impregnating cellulose scaffolds with an adhesion peptide (e.g., RGD) may facilitate cellular adhesion and initial growth. Example 13: Primary bovine muscle cells cultivated in a suspension of cellulose scaffold particles achieved higher cell densities

[0116] Mechanically-deformed “grooved + holed” cellulose scaffolds of 1 -2 mm in thickness were sized to be placed in 24-well plates (approximately 12 mm in diameter). In parallel, the cellulose scaffolds were also grinded using a laboratory coffee grinder into smaller cellulose scaffold particles having an average diameter of approximately 0.3 mm and an approximate weight of 14.7 pg per particle. Representative photographs of the intact cellulose scaffolds and the grinded cellulose scaffold particles are shown in Figs. 13A and 13B, respectively, with the latter forming a suspension, paste / batter, or hydrogel when dispersed in aqueous phase. A representative photograph of grinded cellulose scaffold particles / fibers taken under a stereoscope is shown in Fig. 14.

[0117] The intact cellulose scaffolds and cellulose scaffold particles / fibers were both sterilized by autoclaving and impregnated with a 1% chitosan solution. A heterogenous population of primary bovine cells obtained as described in Example 7 was cultured in DH medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 5 ng FGF / mL. Cells were then seeded on the impregnated intact cellulose scaffolds or impregnated cellulose scaffold particles at concentrations of 30% or 40% w / v placed in 24- well plates. Cultures were incubated at 37°C and 5% CO2 for 48 and 96 h with shaking (60 rpm) prior to viable cell density analysis by WST-1 assay as described in Example 1. The impregnated cellulose scaffold particles were collected by centrifugation at 2000-3000g for cultivation, washing, and viable cell density assays, as needed. Results are shown in Table 4.

[0118] Table 4: Comparison of cells cultivated on intact cellulose scaffolds vs. slurry of cellulose scaffold particles

[0119] Cellulose Viable cells per 100 mg scaffold scaffold (w / v) Intact vs particles At seeding 48 h

[0120] 30% Intact membrane 26,173 35,850

[0121] Particles 42,278 88,902

[0122] 40% Intact membrane 23,195 29,218

[0123] Particles 38,286 97,744

[0124] Strikingly higher cell densities were achieved for cells cultivated in the suspension, paste / batter, or hydrogel of cellulose scaffold particles / fibers as compared to unground (intact) cellulose scaffolds. Comparatively higher cell densities at 48 h were also observed for cells cultivated in a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase of cellulose scaffold particles / fibers at higher agitation speeds (e.g., 100 rpm) and without agitation (static culture conditions) at 30% and 40% w / v of cellulose scaffolds (data not shown). Example 14: Texture profile analysis of cultivated meat patty vs. conventional slaughtered ground beef patty

[0125] Primary bovine cells were cultured and seeded on different mixtures of mechanically-deformed cellulose scaffolds previously impregnated with pectin and yeast-based products (e.g., extract, peptone, inactivated yeasts, etc.) and independently cultivated for about 30 days. The different mixtures of cell- populated cellulose scaffolds that were independently cultivated were then harvested and formed into disks resembling ground meat hamburger patties (“cultivated meat patties”), as shown for example in Fig. 15A-15C. To more closely resemble the bright red color of fresh conventional slaughtered meat, myoglobin was added to the culture medium and / or to the harvested cell-populated scaffolds postcultivation along with vitamin C (ascorbic acid) to facilitate the presence and / or conversion of oxymyoglobin over metmyoglobin.

[0126] The different sample patties, along with a reference consisting of a similar-sized patty made from conventional slaughtered ground beef (“SGB”; Fig. 16), were sent to an independent lab for texture profile analysis with a texturometer. The physical properties that were evaluated in blinded experiments included hardness, springiness, cohesiveness, gumminess, and chewiness. Overall, it was found that the cultivated meat patties that more closely resembled the texture of conventional SGB contained a mixture of cell-populated cellulose scaffolds of different sizes, including cell-populated intact (unground) cellulose scaffolds and cell-populated ground cellulose scaffold particles. The former valuably contributed to the overall texture and consistency of the patties, while the latter also provided bulk and increased cell density. The texture profile analysis results of a cultivated meat patty that closely resembled the texture profile of a conventional SGB patty are shown Table 5.

[0127] Table 5: Texture profile analysis of a cultivated meat patty vs. conventional slaughtered ground beef (SGB) patty

[0128] Example 15: Cooking performance of cultivated meat patties

[0129] Cultivated meat patties were cooked to evaluate their performance compared to conventional slaughtered ground beef. The cultivated meat patties exhibited generally good cohesiveness after cooking and had a pleasant odor profile similar to that generated by cooking conventional slaughtered meat. Furthermore, browning was achieved upon cooking of the cultivated meat patties (Fig. 17) in a manner similar to the Maillard reaction triggered by cooking conventional slaughtered meat (Fig. 18). REFERENCES

[0130] Charest et al., “Combined microscale mechanical topography and chemical patterns on polymer cell culture substrates.” Biomaterials. 2006; 27(11):2487-94.

[0131] Hu et al., “Surface engineering of spongy bacterial cellulose via constructing crossed groove / column micropattem by low-energy CO2 laser photolithography toward scar- free wound healing.” Mater Sei Eng C Mater Biol Appt. 2019; 99:333-343.

[0132] Liu et al., “Optimization of Surface-Engineered Micropattems on Bacterial Cellulose for Guided Scar- Free Skin Wound Healing.” Biomolecules. 2023; 13(5):793.

[0133] Norris et al., “Emulsion-templated microparticles with tunable stiffness and topology: Applications as edible microcarriers for cultured meat.” Biomaterials. 2022; 287: 121669.

[0134] Olyveira et al., “Physically Modified Bacterial Cellulose as Alternative Routes for Transdermal Drug Delivery.” Journal of Biomaterials and Tissue Engineering, 2013; 3, 227-232.

[0135] Wang et al., “Fabrication of novel surface-imprinted magnetic graphene oxide-grafted cellulose nanocrystals for selective extraction and fast adsorption of fluoroquinolones from water.” Anal Bioanal Chem. 2017; 409(28):6643-6653.

[0136] Xiong et al., “Novel in Vitro Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of Breast Cancer Cells.” Journal of Biomaterials and Nanobiotechnology. 2013; 4, 316- 326.

Claims

CLAIMS1. A cell culture scaffold comprising a cellulose structure (e.g., cellulose membrane, cellulose surface, cellulose fibers, and / or cellulose particles) harbouring localized mechanical deformations to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding nondeformed cellulose membrane.

2. The cell culture scaffold of claim 1, wherein the localized mechanical deformations comprise a plurality of recessions introduced by mechanical compression.

3. The cell culture scaffold of claim 2, wherein:(a) said mechanical compression creates localized areas of the cellulose membrane having increased density as compared to a corresponding uncompressed cellulose membrane;(b) the plurality of recessions comprises a plurality of grooves;(c) the plurality of recessions are aligned with respect to one another;(d) the plurality of recessions are of sufficient depth to enable cell proliferation therein; or(e) any combination of (a) to (d).

4. The cell culture scaffold of any one of claims 1 to 3, wherein the localized mechanical deformations comprise a plurality of holes or openings introduced by mechanical puncturing.

5. The cell culture scaffold of claim 4, wherein:(a) the plurality of holes or openings are of sufficient size (e.g., diameter and / or depth) to enable cell proliferation therein;(b) the plurality of holes or openings have an average diameter of at least 200, 250, 300, 350, 400, or 450 microns, or an average diameter between 200 to 900, 250 to 850, 300 to 800, 350 to 750, or 400 to 700 microns;(c) the plurality of holes or openings are present in the cellulose membrane at a density sufficient to enable increased cell proliferation as compared to a corresponding cellulose membrane lacking said holes or openings;(d) the plurality of holes or openings are present in the cellulose membrane at a density of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 holes or openings / cm2, or are present in the cellulose membrane at a density of 10 to 150, 15 to 140, 20 to 130, or 20 to 120 cm2; or(e) any combination of (a) to (d).

6. The cell culture scaffold of any one of claims 1 to 5, wherein the cell culture scaffold comprises or further comprises a plurality of cellulose particles and / or fibers (e.g., less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm in average diameter; having different dimensions and / or geometries [e.g., spherical, rod-like, fibers, or other scattered and irregular forms]; or any combination thereof).

7. The cell culture scaffold of claim 6, wherein the plurality of cellulose particles and / or fibers are sufficiently small to produce a suspension, paste / batter, or hydrogel when dispersed in an aqueous phase (e.g., in a dispersing medium such as water, a buffer, culture medium with or without serum or serum substitutes).

8. The cell culture scaffold of claim 6 or 7, wherein the plurality of cellulose particles / fibers are produced by grinding, milling, cutting, shearing, or other mechanical disruption of a cellulose membrane.

9. The cell culture scaffold of any one of claims 1 to 8, wherein the mechanically-deformed cellulose structure, membrane, fibers, and / or particles is / are hydrated to a level less than 95%, 90%, 85%, 80%, 75%, or 70% of their initial hydration weight or weight at maximum hydration, or the cellulose structure, membrane, fibers, and / or particles is / are hydrated to a level between 45 to 95%, 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75% of their initial hydration weight or weight at maximum hydration.

10. The cell culture scaffold of claim 9, wherein said localized mechanical deformation comprises localized mechanical compression.

11. The cell culture scaffold of any one of claims 1 to 10, wherein the cellulose membrane comprises or consists of unmodified and / or chemically-modified (e.g., cross-linked) cellulose.

12. The cell culture scaffold of any one of claims 1 to 11, wherein the cellulose membrane comprises or consists of microbial cellulose, bacterial cellulose, fungal cellulose, algal cellulose, plant-based cellulose, or any combination thereof.

13. The cell culture scaffold of any one of claims 1 to 12, wherein the cellulose membrane is impregnated with one or more biological molecules and / or ingredients (e.g., for improving organolepticproperties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelf-life, cooking performance (e.g., to promote the Maillard reaction), or any combination thereof).

14. The cell culture scaffold of claim 13, wherein the one or more biological molecules comprise a yeast-based product (e.g., yeast extract, yeast peptone, inactivated yeast, etc.), peptone, a protein, a polysaccharide, a biological polymer, a peptide (e.g., an adhesion peptide), a growth factor, or any combination thereof.

15. The cell culture scaffold of claim 13 or 14, wherein the one or more biological molecules comprise: pectin, myoglobin or other heme-containing proteins, lignin, hemicellulose, chitosan, gelatin, fibronectin, laminin, collagen, glycoprotein, thrombospondin, elastin, fibrillin, mucopolysaccharide, glycolipid, keratin, glycosaminoglycan, glucomannan, hyaluronic acid, proteoglycan, vitronectin, poly-D- lysine, RGD peptide, alginate, or any combination thereof.

16. The cell culture scaffold of any one of claims 1 to 15, wherein the cellulose membrane was subjected to cellulase treatment to modify the texture of the scaffold.

17. The cell culture scaffold of any one of claims 1 to 16, wherein the cell culture scaffold is suitable or human consumption, biocompatible, biodegradable, or any combination thereof.

18. The cell culture scaffold of any one of claims 1 to 17, wherein the cell culture scaffold is populated with animal cells (e.g., bovine, bison, deer, porcine, goat, lamb, kangaroo, rabbit, chicken, turkey, duck, goose, emu, pheasant, quail, squab, fish, crab, lobster, prawns, mussels, oysters, scallops, or clams).

19. The cell culture scaffold of claim 18, wherein the mammalian cells comprise primary cells, fibroblasts, muscle stem cells (e.g., satellite cells), muscle progenitor cells, fibro-adipogenic progenitors (FAP), endothelial cells, chondrocytes, adipocytes, or other fat-containing cells, or any combination thereof.

20. The cell culture scaffold of claim 18 or 19, which is populated with more than one cell type on the same scaffold (e.g., on two different sides, surfaces, or subregions of the same scaffold).

21. The cell culture scaffold as defined in any one of claims 1 to 20 for use in the production of a cultivated meat product, in tissue engineering, in regenerative medicine, in wound healing, or in an in vitro three-dimensional cell culture model.

22. A cultivated meat product comprising the cell culture scaffold as defined in any one of claims 1 to 20.

23. The cultivated meat product of claim 18, which does not comprise an animal-derived ingredient.

24. A method for preparing a cell culture scaffold, the method comprising providing a cellulose membrane, and: (a) introducing localized mechanical deformations into the cellulose membrane to increase cell adhesion, proliferation, and / or mechanical properties as compared to a corresponding nondeformed cellulose membrane; (b) grinding, milling, cutting, shearing, or otherwise mechanically disrupting the cellulose membrane into cellulose particles / fibers (e.g., less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm in average diameter; having different dimensions and / or geometries [e.g., spherical, rod-like, fibers, or other scattered and irregular forms]; or any combination thereof); or both (a) and (b).

25. The method of claim 24, wherein the cellulose membrane provided is a hydrated cellulose membrane.

26. The method of claim 24 or 25, wherein the cellulose membrane is mechanically deformed by compression between rigid surfaces to introduce a plurality of recessions into the cellulose membrane.

27. The method of any one of claims 24 to 26, wherein the cellulose membrane is mechanically deformed by partially or completely puncturing with a device to introduce a plurality of holes or openings into the cellulose membrane.

28. The method of any one of claims 24 to 27, wherein the cell culture scaffold or the cellulose membrane is as defined in any one of claims 1 to 20.

29. A cell culture scaffold produced by the method of any one of claims 24 to 28.

30. A method for producing a cultivated meat precursor product, the method comprising providing cell culture scaffolds as defined in any one of claims 1 to 20, or produced by the method of any one ofclaims 24 to 28, and cultivating the cell culture scaffolds seeded with mammalian cells in a bioreactor or fermenter for a sufficient time to yield the cultivated meat precursor product.

31. The method of claim 30, wherein the method comprises co-culturing a mixture of cell culture scaffolds seeded with different cell types (e.g., seeded on the same scaffold and / or seeded on distinct scaffolds).

32. The method of claim 30 or 31 , wherein the mammalian cells are as defined in claim 18 or 19.

33. The method of any one of claims 30 to 32, wherein the bioreactor or fermenter is a stirred tank bioreactor or fermenter, or any other type of bioreactor or fermenter.

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