Edible scaffolds and formulations for cultured meat

Abstract

This document describes cell culture scaffolds suitable for cultured meat. The scaffolds described herein can be made of cellulose or other edible and / or biocompatible polymeric materials and mechanically deformed to increase cell adhesion and / or proliferation. Mechanical deformation may include multiple indentations (e.g., grooves and channels) and / or multiple pores or openings introduced by mechanical perforation, and / or may produce cell culture scaffold particles / fibers. The scaffolds may contain one or more components that improve cell adhesion, proliferation, and sensory properties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelf life, culinary performance (e.g., promoting Maillard reactions), or any combination thereof. This document also describes methods for preparing the scaffolds and cultured meat products containing the scaffolds.

Description

[0001] This specification relates to cell culture scaffolds that have been mechanically deformed and modified to improve cell adhesion, proliferation, and / or mechanical and sensory properties. More specifically, this specification relates to scaffolds made of cellulose or other edible polymeric materials and biomolecules suitable for the production of cultured meat.

[0002] This manual cites several references, the contents of which are incorporated into this document in their entirety through citation. Background Technology

[0003] Cellular agriculture is an emerging field of biotechnology that involves producing agricultural products by culturing animal, plant, or microbial cells, rather than using animals or plants. Cultured meat is a form of cellular agriculture that offers an alternative to slaughtering animals for human consumption. Compared to traditional meat production, a major contributor to environmental damage, the field of cultured meat aims to cultivate animal muscle cells in bioreactors to produce a processed alternative to traditional meat for consumption. For cultured meat products to have a similar appearance, texture, and structure to traditional meat, the cultured cells must grow on a suitable scaffold material. Biocompatible scaffolds made from natural or synthetic materials have traditionally been developed for tissue engineering, regenerative medicine, or in vitro three-dimensional cell culture models. However, scaffolds developed for such purposes are generally unsuitable for cultured meat because they are typically expensive to manufacture, require sophisticated equipment, cannot be mass-produced, lack sufficient cell adhesion and / or mechanical properties suitable for bioreactor or fermenter culture, and are not suitable for large-scale human consumption. Therefore, a scaffold suitable for cultured meat is needed to address at least some of these drawbacks. Summary of the Invention

[0004] In a first aspect, this document describes a cell culture scaffold comprising edible and / or biocompatible structures, membranes, fibers, and / or particles having locally mechanically deformable structures that increase cell adhesion, proliferation, and / or mechanical properties compared to a corresponding undeformed cellulose membrane. In embodiments, the structures, membranes, fibers, and / or particles may be cellulose structures, membranes, fibers, and / or particles, or structures, membranes, fibers, and / or particles of other edible polymeric materials. In some embodiments, the mechanically deformed membrane may comprise multiple indentations (e.g., indentations, grooves, and / or trenches) introduced by mechanical compression, and / or multiple pores or openings introduced by mechanical puncture. In some embodiments, the cell culture scaffold may comprise or further comprise multiple cellulose particles and / or fibers small enough to produce a suspension, batter, or hydrogel when dispersed in an aqueous phase. In some embodiments, the particles may be prepared by mechanically disrupting a cellulose membrane through grinding, crushing, cutting, shearing, or other means.

[0005] On the other hand, this article describes a cultured meat product comprising a cell culture scaffold as described herein.

[0006] On the other hand, this article describes a method for preparing a cell culture scaffold, the method comprising providing an edible and / or biocompatible membrane, and introducing localized mechanical deformation in the membrane to increase cell adhesion, proliferation and / or mechanical properties compared to a corresponding undeformed membrane.

[0007] On the other hand, this article describes a method for preparing a cell culture scaffold, the method comprising providing an edible and / or biocompatible membrane, and grinding, crushing, cutting, shearing or otherwise mechanically breaking down the cellulose membrane into cellulose particles.

[0008] On the other hand, this article describes a method for producing a cultured meat precursor product, the method comprising culturing a cell culture scaffold described herein, inoculated with mammalian cells, in a bioreactor or fermenter for a sufficient time to obtain the cultured meat precursor product.

[0009] General definition Titles and other identifiers (e.g., (a), (b), (i), (ii), etc.) are provided solely for ease of reading the specification and claims. The use of titles or other identifiers in the specification or claims does not necessarily require that steps or elements be performed in alphabetical or numerical order or in the order in which they are presented.

[0010] When used in conjunction with the term "comprising" in the claims and / or description, the word "a (or an)" may mean "one," but may also mean "one or more," "at least one," and "one or more."

[0011] As used in this specification and claims, the terms “comprising” (and any of its forms, such as “comprise” and “comprises”), “having” (and any of its forms, such as “have” and “has”), “including” (and any of its forms, such as “includes” and “include”), or “containing” (and any of its forms, such as “contains” and “contain”) are inclusive or open-ended and do not exclude other unmentioned elements or method steps.

[0012] The term "about" is used to indicate that a value includes the standard deviation of the error of the apparatus or method used to determine that value. Typically, the term "about" is intended to indicate a possible variation of up to 10%. Therefore, the term "about" covers variations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% of the value. Unless otherwise stated, the use of the term "about" before a range applies to both ends of that range.

[0013] Other objectives, advantages, and features of this specification will become more apparent after reading the following non-limiting description of specific embodiments given by way of example only and with reference to the accompanying drawings. Attached Figure Description

[0014] In the attached diagram: Figure 1 A first prototype device designed and manufactured to compress a cellulose membrane and introduce aligned grooves is shown.

[0015] Figure 2 Representative images of MTT-labeled 3T3 fibroblasts cultured for 14 days on grooved cellulose scaffolds are shown, with these cells preferentially accumulating and orienting in aligned grooves.

[0016] Figure 3 Cell densities of viable 3T3 fibroblasts cultured for 14 days on compressed, ungrooved cellulose scaffolds (“flat”), compressed, grooved cellulose scaffolds (“grooved”), or compressed, grooved and porous cellulose scaffolds (“grooved + porous”) were compared.

[0017] Figure 4 A second prototype device is shown, designed and manufactured to mechanically introduce additional openings within a cellulose scaffold to create a “perforated” scaffold.

[0018] Figure 5 Representative images of MTT-labeled 3T3 fibroblasts cultured for 14 days on a grooved and perforated cellulose scaffold are shown, with these cells preferentially accumulating at high cell density within the introduced pores (as indicated by arrows).

[0019] Figure 6 Cell densities of viable 3T3 fibroblasts cultured for 14 days on grooved and porous cellulose scaffolds previously impregnated with chitosan / collagen solution (“chitosan + collagen”) or unimpregnated (“untreated”) were compared.

[0020] Figure 7 Representative images of MTT-labeled 3T3 fibroblasts cultured for 14 days on grooved and porous cellulose scaffolds previously impregnated with chitosan / collagen solution are shown.

[0021] Figure 8Cell densities of live C2C12 myoblasts cultured for 14 days on compressed grooved and porous cellulose scaffolds previously impregnated with chitosan / collagen solution (“chitosan + collagen”) or unimpregnated (“untreated”) were compared.

[0022] Figure 9 Representative images of MTT-labeled C2C12 myoblasts cultured for 14 days on grooved and porous cellulose scaffolds previously impregnated with chitosan / collagen solution are shown.

[0023] Figure 10 Cell densities of primary bovine cell heterogeneous populations cultured for 7 to 30 days on compressed, grooved and porous cellulose scaffolds previously impregnated with chitosan / collagen solution were compared in a stirred tank bioreactor (STB; 100 rpm, 37 °C, in humidified 5% CO2).

[0024] Figure 11 It shows Figure 10 A representative image of cells cultured for 14 days.

[0025] Figures 12A-12C Other prototype devices designed and manufactured to mechanically introduce additional openings within a cellulose scaffold to create a perforated scaffold are shown. Figure 12A This is a schematic diagram of the device, which includes a precisely aligned array of needles, an adjustable spring and plate system for stability, and a positioning grid for uniform perforation distribution. Figure 12B and Figure 12C The photo shows the complete and fully assembled device, which includes a micro-drilling module, adjustable control settings, and a robust frame, enabling consistent performance and precise drilling.

[0026] Figure 13A and 13B These are photographs of two embodiments of the mechanically deformable cellulose scaffold described in Example 13, including a circular cellulose scaffold sized to fit a 24-well plate. Figure 13A ) and cellulose scaffolds that are ground into particles to form suspensions, pastes / flour, or hydrogels when dispersed in an aqueous phase. Figure 13B ).

[0027] Figure 14 Representative photographs of the ground cellulose scaffold particles / fibers, taken under a stereomicroscope, are shown.

[0028] Figure 15A , 15B 15C is a photograph of cells grown on a mechanically deformed cellulose scaffold mixture, which were independently cultured, harvested, and shaped into discs resembling minced meat burger patties (“cultured meat patties”).

[0029] Figure 16 This is a photo of a hamburger patty made from ground beef from a regular slaughterhouse.

[0030] Figure 17 The image shows the color and texture changes of a raw cultured meat patty (left) after cooking (right).

[0031] Figure 18 It is an image of a regular butchered meat patty after cooking. Detailed Implementation

[0032] This article describes edible and / or biocompatible scaffolds suitable for the production of cultured meat and other applications. Cellulose membranes, as well as membranes based on other natural polymers, have previously been considered potential scaffold materials, but their poor cell adhesion and weak mechanical properties are significant drawbacks. Conventional attempts to address these issues have involved transforming or modifying the membranes, for example, through chemical or enzymatic crosslinking, or employing complex, multi-step processes requiring expensive and 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 result in membranes unsuitable for large-scale human consumption (potentially prohibitively expensive), compromised mechanical properties, and potentially unsuitable for large-scale production.

[0033] In a first aspect, this document describes a cell culture scaffold comprising edible and / or biocompatible structures (e.g., membranes, surfaces, fibers, and / or particles) that exhibit localized mechanical deformation that enhances cell adhesion, proliferation, and / or mechanical properties compared to their corresponding undeformed counterparts. In some embodiments, localized mechanical deformation can introduce additional surfaces within the membrane that facilitate cell adhesion, orientation, and / or proliferation, thereby enabling higher cell densities compared to their corresponding undeformed counterparts. In some embodiments, the locally mechanically deformed structures, membranes, fibers, and / or particles described herein may include a plurality of depressions or pits introduced through localized mechanical compression. In some embodiments, the plurality of depressions or pits may comprise a plurality of grooves. In some embodiments, at least some of the plurality of depressions, pits, or grooves may be aligned with each other, thereby promoting cell self-orientation in a specific direction. In some embodiments, the mechanically deformable membrane described herein can undergo localized mechanical compression to create subregions or sub-regions of structures, membranes, fibers, and / or particles that have a higher density compared to their corresponding uncompressed counterparts, which have a more uniform density. Unbound by theory, sub-regions or sub-areas with increased intramembrane density can advantageously improve the mechanical properties of the scaffold, particularly when the compression regions are aligned or patterned. In some embodiments, sub-regions or sub-areas with increased intramembrane density in the structure, membrane, fibers, and / or particles can act as ribs or other support structures, thereby improving the mechanical strength and / or stiffness (e.g., longitudinal strength and / or longitudinal stiffness) of the scaffold.

[0034] As used herein, the term "localized" or "local" generally refers to mechanical forces intentionally applied to a specific region (rather than the entire structure, membrane, fiber, and / or particle) of an edible and / or biocompatible structure, membrane, fiber, and / or particle to introduce persistent mechanical deformation. For example, localized mechanical deformation may include applying mechanical forces to a specific area of ​​a structure or membrane rather than other areas, or it may include applying larger mechanical forces to a specific area of ​​the membrane while smaller forces are applied to other areas. Furthermore, introducing localized mechanical deformation (e.g., by grinding, crushing, cutting, or otherwise mechanically disrupting an edible and / or biocompatible structure or membrane) can be used to produce smaller structures, membranes, fibers, and / or particles. In some embodiments, these smaller structures, membranes, fibers, and / or particles may themselves contain localized mechanical deformation.

[0035] In some embodiments, the locally mechanically deformable membrane described herein may comprise a plurality of pores or openings introduced by mechanical perforation. As used herein, the terms “pore” and “opening” refer to artificially created structures that are generally larger than the pores naturally present in edible and / or biocompatible membrane materials. In some embodiments, the plurality of pores or openings have a size (e.g., diameter and / or depth) sufficient to allow cell proliferation therein. In some embodiments, the average diameter of the plurality of pores or openings may be at least 200, 250, 300, 350, 400, or 450 micrometers. In some embodiments, the average diameter of the plurality of pores or openings may be 200 to 900, 250 to 850, 300 to 800, 350 to 750, or 400 to 700 micrometers. In some embodiments, the plurality of pores or openings may be present in the edible and / or biocompatible membrane at a density sufficient to allow increased cell proliferation compared to a membrane lacking corresponding pores or openings. In some implementations, the number of holes or openings can be 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 per cm. 2 The density exists within the cellulose membrane. In some embodiments, the number of pores or openings can be 10 to 150, 15 to 140, 20 to 130, or 20 to 120 / cm². 2 The density exists within the cellulose membrane.

[0036] In some embodiments, the cell culture scaffold described herein may comprise or further comprise multiple edible and / or biocompatible particles and / or fibers (e.g., cellulose particles and / or fibers). In some embodiments, the average diameter of 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 some embodiments, the particles and / or fibers may have different sizes and form different geometries, such as spherical, rod-shaped, fibrous, and other dispersed and irregular shapes. In some embodiments, the particles may be small enough to produce a suspension, paste / flour, or hydrogel when dispersed in an aqueous phase. In some embodiments, the aqueous phase may be a dispersion medium, such as water, buffer solution, or culture medium with or without serum or serum substitutes. In some embodiments, multiple cellulose particles and / or fibers may be produced by grinding, crushing, cutting, shearing, or otherwise mechanically disrupting the edible and / or biocompatible structure or membrane.

[0037] In some embodiments, the mechanically deformable edible and / or biocompatible structures, membranes, fibers, and / or particles described herein may be hydrated to levels below 95%, 90%, 85%, 80%, 75%, or 70% of their initial hydrated weight or maximum hydrated weight. In some embodiments, the mechanically deformable edible and / or biocompatible structures, membranes, fibers, and / or particles described herein may be hydrated to levels from 15% to 95%, 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75% of their initial hydrated weight or maximum hydrated weight. In some embodiments, localized mechanical deformation includes localized mechanical compression. In some embodiments, the mechanically deformable edible and / or biocompatible structures, membranes, fibers, and / or particles described herein may be subjected to compression or further compression to levels below 95%, 90%, 85%, 80%, 75%, or 70% of their initial or maximum hydrated weight, or to levels between 15% and 95%, 50% and 90%, 55% and 85%, 60% and 80%, or 65% and 75% of their initial or maximum hydrated weight.

[0038] In some embodiments, the edible and / or biocompatible membranes described herein may comprise or consist of cellulose membranes. As used herein, the term "cellulose membrane" refers to a material comprising cellulose as its primary structural component, including modified and unmodified cellulose from any source. In some embodiments, the 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 protein, pea protein, and / or rice protein), 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 unmodified materials (e.g., unmodified or natural cellulose). In some embodiments, the edible and / or biocompatible membranes described herein may comprise or consist of chemically or enzymatically modified materials (e.g., crosslinked, chemically modified) or consist of chemically or enzymatically modified materials (e.g., crosslinked, chemically modified).

[0039] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be impregnated or coated with one or more biomolecules, for example, to further enhance cell adhesion and / or proliferation. In some embodiments, the biomolecules described herein may comprise yeast-based products (e.g., yeast extracts, yeast peptones, inactivated yeast, etc.), peptones, proteins, polysaccharides, biopolymers, peptides (e.g., adhesion peptides), growth factors, or any combination thereof. In some embodiments, the biomolecules described herein may include: pectin, myoglobin or other heme-containing proteins, lignin, hemicellulose, chitosan, gelatin, fibronectin, laminin, collagen, glycoproteins, thromboretin, elastin, fibrils, mucopolysaccharides, glycolipids, keratin, glycosaminoglycans, glucomannan, hyaluronic acid, proteoglycans, hyaluronic acid, poly-D-lysine, RGD peptides, alginate, or any combination thereof. In specific 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.

[0040] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may undergo texture modification processing steps, for example, to improve mouthfeel and / or sensory properties. In some embodiments, the texture modification processing steps may include chemical or enzymatic modification or degradation. In a specific embodiment, when the edible and / or biocompatible membrane is a cellulose membrane, the membrane may be treated with cellulase to alter the texture of the scaffold.

[0041] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein are preferably suitable for human consumption, edible and / or biocompatible, biodegradable, or any combination thereof.

[0042] In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein may be colonized by animal or mammalian cells (e.g., cattle, bison, deer, pigs, goats, lambs, kangaroos, rabbits, chickens, turkeys, ducks, geese, emus, pheasants, quails, pigeons, fish, crabs, lobsters, shrimp, mussels, oysters, scallops, or clams). Unless otherwise stated, “cells” as used herein generally refers to adherent cells. In some embodiments, mammalian cells may include fibroblasts, muscle stem cells (e.g., satellite cells), muscle progenitor cells, fibro-adipocyte progenitor cells (FAP), adipocytes or other adipocyte-containing cells, or any combination thereof. In some embodiments, the cells described herein are cultured from biopsies (e.g., muscle biopsies) of animals (e.g., cattle, bison, deer, pigs, goats, lambs, kangaroos, rabbits, chickens, turkeys, ducks, geese, emus, pheasants, quails, pigeons, fish, crabs, lobsters, shrimp, mussels, oysters, scallops, clams). In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein can be colonized on the same scaffold by more than one cell type (e.g., on two different sides, surfaces, or sub-regions of the same scaffold). In some embodiments, this can be achieved by sequential cell seeding and adhesion cycling on different sides, surfaces, or sub-regions of the scaffold or membrane. In some embodiments, such co-culture settings can facilitate synergistic or cooperative growth (or synchronous growth) between different cell types, for example, when the first cell type secretes or expresses factors or signals (e.g., growth factors or signals) that promote the adhesion, proliferation, and / or maturation of the second cell type. In some embodiments, at least two, three, or four different cell types can be co-cultured on the same membrane. In some embodiments, co-culture can be achieved by co-culturing two or more different cell culture scaffolds described herein in the same bioreactor, fermenter, or container, wherein each scaffold is colonized with a different cell type. In some embodiments, the cell culture scaffolds or edible and / or biocompatible membranes described herein are used for the production of cultured meat products, tissue engineering, regenerative medicine, wound healing, or in vitro three-dimensional cell culture models. In some embodiments, the use of cell culture scaffolds or edible and / or biocompatible membranes as described herein for the production of cultured meat products, tissue engineering, regenerative medicine, wound healing, or in vitro three-dimensional cell culture models is described herein.

[0043] On the other hand, this document describes cultured meat products comprising the cell culture scaffolds or edible and / or biocompatible membranes described herein. In some embodiments, the cultured meat products do not contain animal-derived ingredients or molecules.

[0044] On the other hand, this document describes a method for preparing a cell culture scaffold, the method comprising providing an edible and / or biocompatible membrane, and introducing localized mechanical deformation into the edible and / or biocompatible membrane to increase cell adhesion, proliferation, and / or mechanical properties compared to a corresponding undeformed membrane. In some embodiments, the provided edible and / or biocompatible membrane is a hydrated membrane. In some embodiments, the edible and / or biocompatible membrane is mechanically deformed by compression between rigid surfaces to introduce multiple indentations in the membrane. In some embodiments, the edible and / or biocompatible membrane is mechanically deformed by perforation (e.g., partially or completely) with a device to introduce multiple pores or openings in the membrane. In some embodiments, the cell culture scaffold or edible and / or biocompatible membrane mentioned above is as defined herein.

[0045] In another embodiment, this document describes a method for preparing a cell culture scaffold, the method comprising providing a cellulose membrane and mechanically breaking down the cellulose membrane into cellulose particles and / or fibers by grinding, crushing, cutting, shearing, or otherwise altering it. In some embodiments, the particles and / or fibers may have different sizes and form different geometries, such as spherical, rod-shaped, fibrous, and other dispersed and irregular shapes. In some embodiments, the particles may be small enough to produce a suspension, paste / dough, or hydrogel when dispersed in an aqueous phase. In some embodiments, the average diameter of the particles may be less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm. In some embodiments, the average diameter of the particles is 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 some embodiments, the particles are small enough to produce a suspension, paste / dough, or hydrogel when dispersed in an aqueous phase.

[0046] On the other hand, this paper describes cell culture scaffolds produced by the methods described herein.

[0047] On the other hand, this document describes a method for producing a cultured meat precursor product, the method comprising providing a cell culture scaffold as described herein, and culturing the cell culture scaffold inoculated with mammalian cells in a bioreactor or fermenter for a sufficient time to obtain the cultured meat precursor product. In some embodiments, the method comprises co-culturing a mixture of culture scaffolds inoculated with different cell types (e.g., inoculated on the same scaffold and / or inoculated on different scaffolds). In some embodiments, mammalian cells are as described herein. In some implementations, 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, 5000, 5500, 6000, 6500, 7000, 8000, 9000, 10000, 20000, or 25000 L).

[0048] In some embodiments, the cultured meat precursor products described herein may contain one or more ingredients for improving sensory properties, appearance (e.g., color), texture, aroma, flavor, nutritional components, shelf life, culinary performance (e.g., promoting Maillard reactions), or any combination thereof. In some embodiments, the cultured meat precursor products described herein may contain one or more ingredients, such as yeast-based products (e.g., yeast extract, yeast peptone, inactivated yeast, etc.), metabolites, natural flavorings, natural colorings, dietary fiber (e.g., soluble fiber, insoluble fiber), emulsifiers, stabilizers, thickeners, and sulfur compounds (e.g., cysteine, acetylcysteine, cystine, taurine, thiamine, methionine, glutathione, allicin, biotin).

[0049] In some embodiments, the cultured meat precursor products described herein may contain one or more flavoring ingredients, such as sugars, sugar alcohols, sugar acids, sugar derivatives, oils, free fatty acids, amino acids or derivatives thereof, nucleosides, nucleotides, vitamins, acids, peptides, phospholipids, protein hydrolysates, yeast-based products (such as yeast extracts, yeast peptones, inactivated yeast, etc.) or mixtures thereof. In some implementations, the flavoring agent may comprise: glucose, fructose, ribose, arabinose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, inositol, maltose, sucrose, maltodextrin, glycogen, nucleotide-bound sugars, molasses, phospholipids, 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 seed oil, corn oil, soybean oil, palm fruit oil, palm kernel oil, safflower oil, flaxseed oil, rice bran oil, cottonseed oil, olive oil, and sunflower seeds. Oils, rapeseed oil, flaxseed oil, coconut oil, mango oil, free fatty acids, cysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, valine, arginine, histidine, alanine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, proline, serine, tyrosine, glutathione, amino acid derivatives, urea, pantothenic acid, ornithine, niacin, glycerol, citrulline, taurine, biotin, borage oil, fungal oil, blackcurrant oil, betaine, β-carotene, B vitamins, N-acetyl-L-cysteine, iron glutamate, peptone, or any combination thereof.

[0050] project In some implementations, this document describes one or more of the following items: 1. A cell culture scaffold comprising a cellulose structure (e.g., a cellulose membrane, a cellulose surface, cellulose fibers, and / or cellulose particles) having localized mechanical deformation that increases cell adhesion, proliferation, and / or mechanical properties compared to a corresponding undeformed cellulose membrane.

[0051] 2. The cell culture scaffold as described in Project 1, wherein the localized mechanical deformation includes multiple indentations introduced by mechanical compression.

[0052] 3. The cell culture scaffold as described in Project 2, wherein: (a) the mechanical compression creates a localized region of the cellulose membrane having an increased density compared to a corresponding uncompressed cellulose membrane; (b) the plurality of recesses comprise a plurality of grooves; (c) the plurality of recesses are aligned with each other; (d) the plurality of recesses have a depth sufficient to allow cell proliferation therein; or (e) any combination of (a) to (d).

[0053] 4. The cell culture scaffold as described in any one of items 1 to 3, wherein the local mechanical deformation includes a plurality of holes or openings introduced by mechanical perforation.

[0054] 5. The cell culture scaffold as described in Item 4, wherein: (a) the plurality of pores or openings have a size (e.g., diameter and / or depth) sufficient to allow cell proliferation therein; (b) the plurality of pores or openings have an average diameter of at least 200, 250, 300, 350, 400, or 450 micrometers, or an average diameter of 200 to 900, 250 to 850, 300 to 800, 350 to 750, or 400 to 700 micrometers; (c) the plurality of pores or openings are present in the cellulose membrane at a density sufficient to allow increased cell proliferation compared to a cellulose membrane lacking the corresponding pores or openings; (d) the plurality of pores or openings have 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 pores or openings / cm. 2 The density is present in the cellulose membrane, or in a concentration of 10 to 150, 15 to 140, 20 to 130, or 20 to 120 cm⁻¹. 2 The density exists in the cellulose membrane; or any combination of (e)(a) to (d).

[0055] 6. The cell culture scaffold as described in 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., with an average diameter less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 mm; having different sizes and / or geometries [e.g., spherical, rod-shaped, fibrous or other dispersed and irregular shapes]; or any combination thereof).

[0056] 7. The cell culture scaffold as described in Item 6, wherein the plurality of cellulose particles and / or fibers are small enough to produce a suspension, paste / flour or hydrogel when dispersed in an aqueous phase (e.g., in a dispersion medium such as water, buffer, culture medium with or without serum or serum substitute).

[0057] 8. The cell culture scaffold as described in item 6 or 7, wherein the plurality of cellulose particles / fibers are prepared by mechanically disrupting the cellulose membrane by grinding, crushing, cutting, shearing or other means.

[0058] 9. The cell culture scaffold as described in any one of items 1 to 8, wherein the mechanically deformed cellulose structure, membrane, fiber and / or particles are hydrated to a level below 95%, 90%, 85%, 80%, 75% or 70% of their initial hydrated weight or maximum hydrated weight, or the cellulose structure, membrane, fiber and / or particles are hydrated to a level from 45% to 95%, 50% to 90%, 55% to 85%, 60% to 80% or 65% to 75% of their initial hydrated weight or maximum hydrated weight.

[0059] 10. The cell culture scaffold as described in Item 9, wherein the localized mechanical deformation includes localized mechanical compression.

[0060] 11. The cell culture scaffold of any one of items 1 to 10, wherein the cellulose membrane comprises unmodified cellulose and / or chemically modified (e.g., cross-linked) cellulose or is composed of or made of said unmodified cellulose and / or chemically modified (e.g., cross-linked) cellulose.

[0061] 12. The cell culture scaffold as described in any one of items 1 to 11, wherein the cellulose membrane comprises or is composed of: microbial cellulose, bacterial cellulose, fungal cellulose, algal cellulose, plant-based cellulose, or any combination thereof.

[0062] 13. The cell culture scaffold as described in any one of items 1 to 12, wherein the cellulose membrane is impregnated with one or more biomolecules and / or components (e.g., for improving sensory properties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelf life, culinary performance (e.g., promoting Maillard reaction), or any combination thereof).

[0063] 14. The cell culture scaffold as described in Item 13, wherein the one or more biomolecules comprise yeast-based products (e.g., yeast extract, yeast peptone, inactivated yeast, etc.), peptone, proteins, polysaccharides, biopolymers, peptides (e.g., adhesion peptides), growth factors, or any combination thereof.

[0064] 15. The cell culture scaffold as described in Item 13 or 14, wherein the one or more biomolecules include: pectin, myoglobin or other heme-containing proteins, lignin, hemicellulose, chitosan, gelatin, fibronectin, laminin, collagen, glycoprotein, platelet-reactive protein, elastin, fibrinogen, mucopolysaccharide, glycolipid, keratin, glycosaminoglycan, glucomannan, hyaluronic acid, proteoglycan, hyaluronic acid, poly-D-lysine, RGD peptide, alginate, or any combination thereof.

[0065] 16. The cell culture scaffold of any one of items 1 to 15, wherein the cellulose membrane is treated with cellulase to alter the texture of the scaffold.

[0066] 17. The cell culture scaffold as described in any one of items 1 to 16, wherein the cell culture scaffold is suitable for human consumption, is biocompatible, is biodegradable, or any combination thereof.

[0067] 18. The cell culture scaffold as described in any one of items 1 to 17, wherein the cell culture scaffold is colonized with animal cells (e.g., domestic cattle, bison, deer, pigs, goats, lambs, kangaroos, rabbits, chickens, turkeys, ducks, geese, emus, pheasants, quails, pigeons, fish, crabs, lobsters, shrimp, mussels, oysters, scallops, or clams).

[0068] 19. The cell culture scaffold as described in Item 18, wherein the mammalian cells include primary cells, fibroblasts, muscle stem cells (e.g., satellite cells), muscle progenitor cells, fibro-adipocyte progenitor cells (FAP), endothelial cells, chondrocytes, adipocytes or other adipocytes, or any combination thereof.

[0069] 20. A cell culture scaffold as described in item 18 or 19, wherein more than one cell type is colonized on the same scaffold (e.g., on two different sides, surfaces or sub-regions of the same scaffold).

[0070] 21. A cell culture scaffold as defined in any of items 1 to 20, used for the production of cultured meat products, tissue engineering, regenerative medicine, wound healing, or in vitro three-dimensional cell culture models.

[0071] 22. A cultured meat product comprising a cell culture scaffold as defined in any one of items 1 to 20.

[0072] 23. The cultured meat products described in Project 18 do not contain any animal-derived ingredients.

[0073] 24. A method for preparing a cell culture scaffold, the method comprising providing a cellulose membrane, and: (a) introducing localized mechanical deformation in the cellulose membrane to increase cell adhesion, proliferation, and / or mechanical properties compared to a corresponding undeformed cellulose membrane; (b) grinding, crushing, cutting, shearing, or otherwise mechanically breaking down the cellulose membrane into cellulose particles / fibers (e.g., with an average diameter less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm; having different sizes and / or geometries [e.g., spherical, rod-shaped, fibrous, or other dispersed and irregular shapes]; or any combination thereof); or both (a) and (b).

[0074] 25. The method of item 24, wherein the provided cellulose membrane is a hydrated cellulose membrane.

[0075] 26. The method of item 24 or 25, wherein the cellulose membrane is mechanically deformed by compression between rigid surfaces to introduce a plurality of depressions in the cellulose membrane.

[0076] 27. The method of any one of items 24 to 26, wherein the cellulose membrane is mechanically deformed by partially or completely perforating the device to introduce a plurality of pores or openings in the cellulose membrane.

[0077] 28. 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.

[0078] 29. A cell culture scaffold, produced by any one of items 24 to 28.

[0079] 30. A method for producing a cultured meat precursor product, the method comprising providing a cell culture scaffold as defined in any one of items 1 to 20 or produced by the method of any one of items 24 to 28, and culturing the cell culture scaffold inoculated with mammalian cells in a bioreactor or fermenter for a sufficient time to produce the cultured meat precursor product.

[0080] 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 different scaffolds).

[0081] 32. The method as described in item 30 or 31, wherein the mammalian cell is as defined in item 18 or 19.

[0082] 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.

[0083] Example Example 1: Materials and Methods MTT assay The MTT assay is a colorimetric assay used as an indicator of cell viability, proliferation, and / or cytotoxicity. It is based on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan by oxidoreductases in metabolically active cells. Briefly, a stock solution of MTT in Hank's solution is prepared at a concentration of 5 mg / mL and diluted 1:10 in Hank's solution as needed to prepare a final reagent concentration (0.5 mg / mL). After transfer to 24-well plates, 800 µL of MTT reagent is added to each well to completely submerge the sample, and the sample is then incubated at 37°C, 5% CO2, and humidified conditions for 4 hours. The presence of metabolically active cells in the sample leads to the formation of colored formazan crystals, which are then observed using an optical stereomicroscope to qualitatively determine the presence of cells.

[0084] WST-1 determination The WST-1 assay measures cell proliferation, cell viability, and / or cytotoxicity. It is based on the cleavage of tetrazolium salt WST-1 into formazan by mitochondrial dehydrogenases. Briefly, all reagents are equilibrated to 37°C before starting the assay. Cell standards are prepared in 300 µL of medium at different cell densities for each cell type. Samples are transferred to 24-well plates and brought to a final volume of 400 µL with preheated DH medium. Then, 30 µL of WST-1 reagent is added to each well. The plates are mixed with a plate shaker for several minutes and then incubated at 37°C, 5% CO2, and humidified conditions for 2 or 4 hours. After incubation, small clumps in the wells are gently crushed to remove any retained, reacted WST-1 reagent. The plates are then mixed with a plate shaker for approximately 5 minutes, and 100 µL is transferred from each well to a 96-well plate. Spectral readings are taken at 440 or 450 nm, and cell growth rates are assessed using absorbance.

[0085] Example 2: Localized mechanical compression of cellulose scaffolds improves cell adhesion Biocompatible scaffolds made from natural or synthetic materials have traditionally been developed for tissue engineering, regenerative medicine, or in vitro three-dimensional cell culture models. The emerging field of cultured meat has recently generated a demand for a new generation of scaffolds, with requirements and / or limitations that differ from or exceed those of scaffolds developed for other applications. For example, scaffolds suitable for cultured meat should not only be biocompatible but also ideally inexpensive, mass-producible, biodegradable, suitable for large-scale human consumption, and ideally possess satisfactory sensory 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 involve, for example, chemical modification of cellulose (e.g., chemical or enzymatic crosslinking), and / or complex, multi-step cellulose modification processes requiring expensive and sophisticated equipment, neither of which are suitable for large-scale production and / or may render the processed cellulose membranes unsuitable for large-scale human consumption.

[0086] Cellulose is the most common polymer in nature, obtainable from plants and produced by fungi, algae, and certain microorganisms. Microbial cellulose, although chemically identical to plant cellulose, is particularly attractive for meat culture due to its biocompatibility, mechanical properties, structure, purity, reproducibility, and independence from plant or animal materials. Therefore, this study employed bacterial cellulose; however, it should be understood that the techniques described herein are applicable to cellulose from other sources.

[0087] Preliminary studies were conducted on various commercially available unmodified bacterial cellulose membranes to assess their ability to support mammalian cell growth. SWISS 3T3 fibroblasts were chosen for the preliminary studies because these cells grow rapidly, achieve high maximum density, tolerate variations in culture medium composition, and generally adhere firmly to all types of surfaces, but die once they cease adhesion. This allowed the experiments to distinguish successful attachment and proliferation from false positives or false negatives. 3T3 cells were directly seeded onto hydrated bacterial cellulose membranes obtained from different commercial suppliers and cultured in suitable media for up to 14 days. While some cell adhesion and growth were detected, even reaching up to approximately 1 x 103, some cell adhesion and growth were observed. 5 cells / cm 2While cell density was achieved, it was clear that the maximum cell density achievable using commercially available unmodified cellulose membranes was insufficient for the levels required for cultured meat products. Therefore, different methods of processing or modifying cellulose membranes were explored, ideally to improve cell adhesion and proliferation while maintaining or improving the membrane's mechanical properties. Interestingly, preliminary experiments showed that membranes with mechanical deformation (e.g., membranes inadvertently "damaged" due to localized compression) appeared to outperform "undamaged" membranes in both cell adhesion and proliferation. These observations were later validated by intentionally introducing mechanical deformation into commercially available cellulose membranes and evaluating their ability to support 3T3 cell growth in culture. Furthermore, sub-regional compression of the membrane was observed. Local Mechanical deformation not only increases cell adhesion and proliferation, but also has a positive impact on the mechanical structure of the membrane. Specifically, it produces... Aligned Mechanical deformation of compressed membrane subregions (e.g., aligned compression patterns, indentations, or grooves) appears to have a positive impact on membrane robustness / rigidity, while also improving cell adhesion and proliferation. Since grooves can be mass-produced using modified machinery, they were selected for further characterization in subsequent embodiments.

[0088] Example 3: Cellulose scaffolds with grooves created by compression can achieve higher cell densities. The first prototype was designed and manufactured, consisting of a pair of matching grooved metal plates. Figure 1 This allows the cellulose membrane to be compressed within it, thereby introducing aligned grooves into the scaffold structure in a consistent and repeatable manner. For hydrated membranes, compression was assessed by the level of dehydration after compression (e.g., 50–95% of the initial weight before compression). Interestingly, 3T3 cells were observed to preferentially accumulate and orient themselves in the aligned grooves and rough surfaces of the grooved cellulose scaffold, as seen in… Figure 2 The results show that metabolically active cells were labeled with MTT. Compared to a compressible scaffold (“flat”) without grooves, the use of a grooved cellulose scaffold (“grooved”) resulted in approximately a three-fold increase in 3T3 cell density after 14 days of culture. Figure 3 Furthermore, the grooves created by aligned compression advantageously affect the longitudinal mechanical properties of the membrane.

[0089] Example 4: Cellulose scaffolds with mechanically introduced openings further achieve higher cell densities. A second prototype was designed and manufactured, consisting of multiple regularly spaced pointed rods extending from a metal plate. Figure 4 This is done by mechanically introducing additional openings within a grooved cellulose scaffold, thereby creating a grooved and porous scaffold with pores typically 300-800 µm in diameter and a density of approximately 20-120 pores / cm³. 2 . Figure 4The second prototype shown introduces an opening or pore that traverses the membrane thickness, but other devices can be readily envisioned to introduce openings or pores that do not penetrate the membrane thickness. In this experiment, the first prototype was used to compress and introduce aligned grooves within a cellulose scaffold, followed by the second prototype to introduce additional openings or pores within the grooved cellulose scaffold. Interestingly, by applying these combined mechanical treatments to the scaffold, the density of 3T3 cells could be increased from 3 x 10⁻⁶ cells after 14 days of culture using only the grooved scaffold (“grooved”). 5 cells / cm 2 Add to 4 x 10 brackets with grooves and holes (“grooved + perforated”). 5 cells / cm 2 ( Figure 3 Unexpectedly, cells grown on grooved and perforated scaffolds were observed to accumulate at high density within the introduced openings, such as... Figure 5 The strong MTT staining (arrows) indicates that cells preferentially grow in the pores introduced by the mechanical introduction of the scaffold. Therefore, it can be envisioned that a higher pore density would further increase the total cell density of the scaffold, thereby allowing cells to penetrate and fill the entire scaffold.

[0090] Example 5: Collagen / chitosan-impregnated mechanically deformable cellulose scaffolds further enhance cell density. In this experiment, the above-described mechanical method was used to prepare grooved and porous cellulose scaffolds, which were then coated with a thin layer of collagen and chitosan solution (collagen:chitosan, 1:1 to 6:1 v / v) for approximately 1 hour to allow them to bind into the scaffold. Figure 6 As shown, by coating a grooved and porous cellulose membrane with biological material, the total 3T3 cell density can be increased from approximately 4 x 10⁻⁶ cells after 14 days of culture. 5 cells / cm 2 ("Unprocessed") increases to approximately 7 x 10 5 cells / cm 2 (Chitosan + Collagen). For example... Figure 7 As shown, cells accumulate at high density within the pores of the mechanically introduced collagen / chitosan impregnated scaffold, consistent with the results of Example 4.

[0091] Example 6: C2C12 myoblasts grown on mechanically deformed, collagen / chitosan-impregnated cellulose scaffolds Cell The experiment conducted in Example 5 with 3T3 cells was repeated using the myoblast cell line C2C12, which has been shown to rapidly differentiate into contractile myotubes and produce characteristic muscle proteins. Figure 8 and Figure 9 The results showed that C2C12 density increased when using collagen / chitosan-impregnated scaffolds ("chitosan + collagen") compared to uncoated scaffolds ("untreated").

[0092] Example 7: Primary bovine muscle cells can be mounted on a mechanically deformable, collagen / chitosan-impregnated cellulose scaffold. nourish A heterogeneous population of primary bovine cells, including fibroblasts, adipocytes, fibro-adipocyte progenitors (FAP), endothelial cells, chondrocytes, progenitor cells, and satellite cells, was obtained from fresh muscle tissue biopsies of living animals. Each cell type in the biopsy was independently characterized and cultured in vitro, then seeded onto mechanically deformable, collagen / chitosan-impregnated grooved and porous cellulose scaffolds and cultured as described in Example 5. Figure 10 As shown, different types of primary bovine cells were able to adhere to the scaffold and proliferate within 30 days, verifying the applicability of the cellulose scaffold described in this paper for cultured meat based on primary bovine cells.

[0093] Example 8: Mechanically deformable cellulose scaffolds suitable for cultivation in bioreactors / fermenters Although the experiments in Examples 2-7 were conducted under static culture conditions on a laboratory scale, industrial-scale bioreactors or fermenters will inevitably involve... dynamic The cultivation conditions impose additional stress on the mechanical structure of the cellulose membrane. The experiments in Example 8 were designed to evaluate... dynamic Effects of culture conditions on scaffold integrity and cell growth. Briefly, collagen / chitosan-impregnated grooved and porous cellulose scaffolds were prepared as described in Examples 4 and 5. The scaffolds were seeded with 3T3 cells under static conditions for a sufficient time to allow cell adhesion (at least 8 hours). Subsequently, some of the cell-loaded cellulose scaffolds were transferred to a 500 mL stirred tank bioreactor (STB) and 150 mL of DH medium containing sufficient sterile defoamer was added, while the control was cultured under the corresponding static conditions. Under dynamic culture conditions, the cellulose scaffolds remained intact, with cell densities up to 1 x 10⁻⁶ cells / year. 6 cells / cm 2 ,like Figure 10 As shown. Interestingly, the cell density achieved under dynamic culture conditions is equal to or higher than that achieved under corresponding static conditions, demonstrating the suitability of the cellulose scaffold described in this paper for large-scale bioreactor or fermenter culture.

[0094] Example 9: Construction and testing of other devices for introducing localized mechanical deformation into cellulose scaffolds In addition to those described in Examples 3 and 4, other prototype devices were designed, constructed, and tested to evaluate their ability to introduce different types of localized mechanical deformation within cellulose scaffolds of varying sizes, and to assess the effects of these localized mechanical deformations on cell adhesion, proliferation, and viable cell density. Overall, localized mechanical deformations used to increase the surface area of ​​the cellulose scaffold were observed to generally have a positive effect on cell adhesion, proliferation, and / or density, while those deformations leading to increased density in localized areas of the cellulose scaffold conferred favorable mechanical properties (e.g., increased mechanical strength and / or stiffness) onto the cellulose membrane. Figures 12A-12C The image shows an example of another prototype device that was designed, built, and tested. This other prototype device is based on... Figure 4 The device shown is employed in subsequent embodiments to mechanically introduce a specific ratio within the cellulose scaffold. Figure 4 The prototype device shown has a higher density of openings or perforations.

[0095] Example 10: Culturing on cellulose scaffolds impregnated with different combinations of chitosan, pectin, and yeast-based products Primary bovine muscle cells Mechanically deformed grooved and perforated cellulose scaffolds (sized to fit 24-well plates) were sterilized by autoclaving and then immersed in different solutions, each containing a different combination of chitosan, pectin, and yeast extract (as shown in conditions AD in Table 1), with gentle shaking at room temperature for 1 hour. Excess solution was removed, and the impregnated cellulose scaffolds were air-dried under aseptic conditions.

[0096] The primary bovine cell heterogeneous populations obtained as described in Example 7 were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine. Cells were then seeded onto impregnated cellulose scaffolds in 24-well plates and incubated at 37°C and 5% CO2 for 30 days. The number of viable cells per membrane was assessed at 40 hours, 15 days, and 30 days using the WST-1 assay as described in Example 1. The results shown in Table 1 are the average of three replicates. Data were analyzed using one-way ANOVA followed by Tukey post-hoc tests for pairwise comparisons, revealing significant differences (p < 0.05) between condition D and conditions A / B / C, and between condition B and conditions A / C, at day 30. Condition D produced the highest number of viable cells on each scaffold. Similar results were observed in C2C12 cells (data not shown). Furthermore, the presence of chitosan was associated with higher viable cell density at 40 hours (conditions A, B, and C), suggesting its beneficial effect on cell adhesion.

[0097] Table 1: Comparison of cells cultured on cellulose scaffolds impregnated with different combinations of chitosan, pectin, and yeast extract. Example 11: Primary bovine cells cultured on cellulose scaffolds impregnated with different concentrations of pectin and yeast extract. muscle cells The experiments performed in Example 10 were repeated using cellulose scaffolds impregnated for 2 hours 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.

[0098] Table 2: Comparison of cells cultured on cellulose scaffolds impregnated with different concentrations of pectin and yeast extract. The results in Table 2 indicate that pectin and yeast extract concentrations significantly affected cell adhesion (e.g., at 40 hours) and proliferation (e.g., at 15 and 30 days). The highest viable cell adhesion and density were observed on cellulose scaffolds impregnated with 0.5% pectin (conditions E, H, and K). In contrast, the lowest viable cell adhesion and density were observed on cellulose scaffolds impregnated with 3% pectin (conditions G, J, and M). Higher yeast extract concentrations were generally associated with higher cell density.

[0099] Example 12: Culturing on cellulose scaffolds impregnated with pectin and yeast extract (with or without RGD adhesion peptides) Primary bovine muscle cells The experiments performed as in Example 11 were repeated using cellulose scaffolds impregnated with 1% w / v pectin and 20 mg / mL yeast extract (with or without RGD adhesion peptide (0.35 mg / mL, "RGD")), as shown in Table 3.

[0100] Table 3: Comparison of cells cultured on cellulose scaffolds with and without RGD peptide impregnation Cells cultured on cellulose scaffolds impregnated with Condition O showed higher viable cell density at 40 hours, indicating that impregnation of cellulose scaffolds with adhesion peptides (e.g., RGD) can promote cell adhesion and initial growth.

[0101] Example 13: Primary bovine muscle cells cultured in a cellulose scaffold particle suspension yielded higher cell counts. density Mechanically deformed cellulose scaffolds with grooves and holes, 1–2 mm thick, were cut to fit 24-well plates (approximately 12 mm in diameter). Parallel, the cellulose scaffolds were ground into smaller cellulose scaffold particles using a laboratory coffee grinder, with an average diameter of approximately 0.3 mm and a particle weight of approximately 14.7 µg. Representative photographs of the complete cellulose scaffolds and the ground cellulose scaffold particles are shown below. Figure 13A and Figure 13BThe latter, when dispersed in an aqueous phase, forms a suspension, paste / flour, or hydrogel. Representative photographs of milled cellulose scaffold particles / fibers taken under a stereomicroscope are shown below. Figure 14 .

[0102] Intact cellulose scaffolds and cellulose scaffold particles / fibers were sterilized by autoclaving and impregnated with 1% chitosan solution. Primary bovine cell heterogeneous populations obtained as described in Example 7 were cultured in DH medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 5 ng FGF / mL. Cells were then seeded in 24-well plates onto impregnated intact cellulose scaffolds or impregnated cellulose scaffold particles at a concentration of 30% or 40% w / v. Cultures were incubated at 37°C and 5% CO2 for 48 and 96 hours with shaking (60 rpm), respectively, followed by viable cell density analysis using the WST-1 assay as described in Example 1. Viable cell density was analyzed as needed, using 2000-3000 cells per 1 ... g The impregnated cellulose scaffold particles were collected by centrifugation for culturing, washing, and live cell density determination. The results are shown in Table 4.

[0103] Table 4: Comparison of cells cultured on intact cellulose scaffolds versus in a slurry of cellulose scaffold particles Compared to unmilled (intact) cellulose scaffolds, cells cultured in suspensions, pastes / flocs, or hydrogels of cellulose scaffold particles / fibers achieved significantly higher cell densities. At 30% and 40% w / v cellulose scaffolds, relatively higher cell densities were also observed at 48 hours for cells cultured in suspensions, pastes / flocs, or hydrogels formed when cellulose scaffold particles / fibers were dispersed in an aqueous phase, even at higher oscillation speeds (e.g., 100 rpm) and without oscillation (static culture conditions) (data not shown).

[0104] Example 14: Texture Characteristics Analysis of Cultured Meat Patties and Conventionally Slaughtered Ground Beef Patties Primary bovine cells were cultured and inoculated onto various mechanically modified cellulose scaffold mixtures previously impregnated with pectin and yeast-based products (e.g., extracts, peptones, inactivated yeast, etc.) and cultured independently for approximately 30 days. The independently cultured mixtures of cell-colonized cellulose scaffolds were then harvested and shaped into discs resembling ground beef hamburger patties (“cultured meat patties”), for example... Figures 15A-15C As shown. To more closely resemble the bright red color of conventional freshly slaughtered meat, myoglobin and vitamin C (ascorbic acid) are added to the culture medium and / or the scaffolds colonized with cells harvested after culture to promote the presence and / or conversion of oxymyoglobin relative to methemoglobin.

[0105] Different sample meat patties, together with reference meat patties (“SGB”) made from conventionally slaughtered ground beef of similar size; Figure 16 Together with the samples, the cultured meat patties were sent to an independent laboratory for texture analysis using a texture analyzer. Physical properties evaluated in the blinded trials included hardness, elasticity, cohesiveness, adhesiveness, and chewiness. Overall, the cultured meat patties with a texture closer to that of conventional SGB contained a mixture of cell-colonized cellulose scaffolds of varying sizes, including intact (unmilled) cell-colonized cellulose scaffolds and milled cell-colonized cellulose scaffold particles. The former significantly contributed to the overall texture and consistency of the patties, while the latter also provided volume and increased cell density. The texture analysis results of cultured meat patties with texture characteristics closely similar to those of conventional SGB patties are shown in Table 5.

[0106] Table 5: Texture Characteristics Analysis of Cultured Meat Patties and Conventionally Slaughtered Ground Beef (SGB) Patties Example 15: Cultivating the cooking properties of meat patties Cultured meat patties were cooked to evaluate their performance compared to conventionally slaughtered ground beef. The cultured meat patties exhibited generally good cohesion after cooking and possessed a pleasant aroma profile similar to that produced by cooking conventionally slaughtered meat. Furthermore, the cultured meat patties achieved browning during cooking. Figure 17 This is similar to the Maillard reaction triggered by cooking conventionally slaughtered meat. Figure 18 ).

[0107] References

Claims

1. A cell culture scaffold comprising a cellulose structure (e.g., a cellulose membrane, a cellulose surface, cellulose fibers, and / or cellulose particles) having localized mechanical deformation that increases cell adhesion, proliferation, and / or mechanical properties compared to a corresponding undeformed cellulose membrane.

2. The cell culture scaffold of claim 1, wherein the localized mechanical deformation includes a plurality of indentations introduced by mechanical compression.

3. The cell culture scaffold as described in claim 2, wherein: (a) The mechanical compression creates a localized region of the cellulose membrane, which has an increased density compared to the corresponding uncompressed cellulose membrane; (b) The plurality of recesses include a plurality of grooves; (c) The plurality of recesses are aligned with each other; (d) The plurality of depressions have a depth sufficient to allow cells to proliferate therein; or Any combination of (e)(a) to (d).

4. The cell culture scaffold according to any one of claims 1 to 3, wherein the local mechanical deformation includes a plurality of holes or openings introduced by mechanical perforation.

5. The cell culture scaffold as described in claim 4, wherein: (a) The plurality of pores or openings have a size (e.g., diameter and / or depth) sufficient to allow cells to proliferate therein; (b) The plurality of holes or openings have an average diameter of at least 200, 250, 300, 350, 400 or 450 micrometers, or an average diameter of 200 to 900, 250 to 850, 300 to 800, 350 to 750 or 400 to 700 micrometers; (c) The plurality of pores or openings are present in the cellulose membrane to achieve an increased density of cell proliferation compared to a cellulose membrane lacking the corresponding pores or openings; (d) The plurality of holes or openings are arranged in quantities 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 per cm. 2 The density is present in the cellulose membrane, or in quantities of 10 to 150, 15 to 140, 20 to 130, or 20 to 120 cm⁻¹. 2 The density exists in the cellulose membrane; or Any combination of (e)(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., with an average diameter less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.5 mm; having different sizes and / or geometries [e.g., spherical, rod-shaped, fibrous or other dispersed and irregular shapes]; or any combination thereof).

7. The cell culture scaffold of claim 6, wherein the plurality of cellulose particles and / or fibers are small enough to produce a suspension, paste / flour or hydrogel when dispersed in an aqueous phase (e.g., in a dispersion medium such as water, 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 prepared by mechanically disrupting the cellulose membrane by grinding, crushing, cutting, shearing or other means.

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

10. The cell culture scaffold of claim 9, wherein the localized mechanical deformation includes localized mechanical compression.

11. The cell culture scaffold of any one of claims 1 to 10, wherein the cellulose membrane comprises unmodified cellulose and / or chemically modified (e.g., cross-linked) cellulose or is composed of said unmodified cellulose 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 is composed of the following: 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 biomolecules and / or components (e.g., for improving sensory properties, appearance (e.g., color), texture, aroma, flavor, nutritional content, shelf life, culinary performance (e.g., promoting Maillard reaction), or any combination thereof).

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

15. The cell culture scaffold of claim 13 or 14, wherein the one or more biomolecules comprise: Pectin, myoglobin or other heme-containing proteins, lignin, hemicellulose, chitosan, gelatin, fibronectin, laminin, collagen, glycoprotein, platelet-reactive protein, elastin, fibrin, mucopolysaccharides, glycolipids, keratin, glycosaminoglycans, glucomannan, hyaluronic acid, proteoglycans, hyaluronic acid, 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 is treated with cellulase to alter 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 for 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 colonized with animal cells (e.g., domestic cattle, bison, deer, pigs, goats, lambs, kangaroos, rabbits, chickens, turkeys, ducks, geese, emus, pheasants, quails, pigeons, fish, crabs, lobsters, shrimp, mussels, oysters, scallops, or clams).

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

20. The cell culture scaffold of claim 18 or 19, wherein more than one cell type is colonized on the same scaffold (e.g., on two different sides, surfaces or sub-regions 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 cultured meat products, tissue engineering, regenerative medicine, wound healing, or in vitro three-dimensional cell culture models.

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

23. The cultured meat product of claim 18, wherein it does not contain any animal-derived ingredients.

24. A method for preparing a cell culture scaffold, the method comprising providing a cellulose membrane, and: (a) introducing localized mechanical deformation in the cellulose membrane to increase cell adhesion, proliferation, and / or mechanical properties compared to a corresponding undeformed cellulose membrane; (b) grinding, crushing, cutting, shearing, or otherwise mechanically breaking down the cellulose membrane into cellulose particles / fibers (e.g., with an average diameter less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 mm; having different sizes and / or geometries [e.g., spherical, rod-shaped, fibrous, or other dispersed and irregular shapes]; or any combination thereof); or both (a) and (b).

25. The method of claim 24, wherein the provided cellulose membrane 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 depressions in 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 perforating the device to introduce a plurality of pores or openings in 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 cultured meat precursor product, the method comprising providing a cell culture scaffold as defined in any one of claims 1 to 20 or a cell culture scaffold produced by the method of any one of claims 24 to 28, and culturing the cell culture scaffold inoculated with mammalian cells in a bioreactor or fermenter for a period of time sufficient to produce the cultured 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 different scaffolds).

32. The method of claim 30 or 31, wherein the mammalian cell is 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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