Expanded animal component free scaffolds for cultivated meat production

Abstract

A cellular scaffold for cultivated meat production containing at least one nanofiber arranged to provide cellular attachment, the at least one nanofiber forming a three-dimensional porous structure, wherein the three-dimensional porous structure is formed by gas expansion of a two-dimensional nanofiber mat, the gas expansion creating volumetric expansion and increased porosity within the nanofiber structure, and wherein the cellular scaffold is edible and animal-component free. Methods of making the cellular scaffold include forming nanofibers into a two-dimensional mat by electrospinning, electrospray, or extrusion, immersing the mat in an effervescent system to generate gas and induce volumetric expansion, and stabilizing the three-dimensional porous scaffold by freeze-drying. Methods of culturing cells for cultivated meat production using the cellular scaffold in a bioreactor are also provided.

Description

EXPANDED ANIMAL COMPONENT FREE SCAFFOLDS FOR CULTIVATED MEAT PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 765,104, titled "Expanded Scaffolds", filed February 28, 2025, which is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0002] This application relates to the fields of nanofibers, nanofiber structures and cell culture scaffolds, aerogels and combinations of these that are safe and suitable for consumption as a food ingredient or additive, and more particularly to edible, animalcomponent free scaffolds formed by gas expansion of nanofiber mats for use in cultivated meat production. In application further relates to methods and systems used to increase 3D thickness, texture, flavor and protein content in cellular products used for consumption as food by humans and animals.BACKGROUND

[0003] Non-human cellular agriculture is a promising technology for producing animalbased proteins to address problems associated with farming live animals. However, high costs are associated with cell culture techniques for cellular agriculture, including expensive cell culture media and bioreactors. These costs may hinder the economically viable production of products using cellular agriculture at commercial levels.

[0004] In cellular agriculture, cells can proliferate and differentiate in bioreactors, and in some designs, the cells are harvested and assembled into products. Cells may be grown in suspension, static under fluid flow or agitation, in cell culture media on microcarriers, or fixed to scaffold mats. One factor affecting commercial viability is the quality of the cellular biomass produced. Cells cultured in suspension processes have a different protein expression profile than cells in native muscle tissue, which can be more accurately recapitulated in adherent cells cultured on scaffolds. Equally relevant is the ability to reduce the cost of the product, scale production efficiently, and achieve higher volumetric productivity of biomass while also achieving a taste profile that more closely mimics animal-derived meat products. One option is to increase cell density, for example to millions of cells per milliliter, and increase the cell-to-scaffold ratio.

[0005] US 11,946,164 B2, assigned to University of Nebraska, is directed to nanofiber structures using gas expanded nanofiber scaffolds used in regenerative medicine. One aspect of this disclosure includes scaffold structures wherein the nanofiber structures comprise an expanded nanofiber structure comprising a plurality of nanofibers. In a particular embodiment, the nanofiber structure has been expanded by exposure to a subcritical fluid such as subcritical CO2 and then depressurized within a container. The nanofiber structure may comprise a plurality' of electrospun nanofibers including uniaxially aligned, random, entangled, and / or electrospun fibers.

[0006] CN 114317394, assigned to Shanghai Shiwei Biotechnology Co. Ltd., is directed to a microcarrier for three-dimensional cell culture, and a preparation method and application thereof. The method comprises directly mixing edible plant-derived ionic crosslinked geltype polysaccharide and edible non-animal-derived protein to prepare a mixed solution, then preparing the mixed solution into liquid drops through extrusion and cartying out crosslinking reaction to obtain the microcarrier. The publication also discloses a method for carrying out surface modification on the microcarrier to obtain a modified microcarrier.

[0007] US 11,976,302, assigned to Upside Foods Inc., is directed to increasing the culture density and thickness of cellular biomass using cells cultured in vitro. Production ofthese products has been projected to require fewer resources, convert biomass at a higher caloric efficiency, and result in reduced environmental impacts relative to conventional in vivo methods.

[0008] However, increasing cell densify can be challenging, as higher cell densities result in higher consumption of nutrients and production of wastes. Accordingly, bioreactors are often in need of constant media exchange or recycling. In addition, increases in media usage may be economically unfavorable. Current limitations in cultivated meat often stem from the inability to create large, contiguous muscle tissues. Typical hydrogel or porous scaffolds lack the porosity of structure and permeability of growth surfaces that nanofiber-based scaffolds can produce. Mass transfer is insufficient and inconsistent in culture scaffolds greater than a few hundred micrometers in thickness, resulting in cell necrosis, an undesirable low ratio of cellular product to scaffold, and an economically non-viable low utilization of bioreactor volume. Additionally, cell migration is inhibited or prevented by scaffold structures that lack sufficient interconnectivity and topology similar to the channels found in native muscle tissue.

[0009] Current approaches to cultivated meat are focused on one of several approaches, each with inherent limitations. Single cell suspension approaches result in less myogenic protein formation, lack of structure, and are incompatible with traditional meat products, requiring addition of large mass fractions of non-cultivated cell material such as plants, fungus, or synthetic materials to create texture. Cell sheet approaches have proven costly to scale up due to high cost of bioreactors and low volumetric productivity. Cell clusters in suspension have similar challenges as single cell suspension. Solid microcarriers have relatively low mass fraction of cultivated meat and tissues, typically less than thirty percent. Porous scaffolds that are currently available do not permit cell infiltration beyond several hundred micrometers because of mass transfer limitations and do not provide the correct mechanical properties to support myogenic differentiation.

[0010] In view of the aforementioned technologies, there remains a desire for scaffolds that can be used for research and consumer products with organoleptic qualities includingtaste and texture closer to that of a whole cut of conventional meat, along with higher volumetric productivity. Additionally, there is a desire for a non-animal scaffold that is edible and can optionally be used to enhance food products.SUMMARY

[0011] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summan' is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0012] In one embodiment, a cellular scaffold is provided. In this embodiment, the cellular scaffold comprises at least one fiber arranged to provide cellular attachment, thereby forming the cellular scaffold having a first surface and a second surface.

[0013] In another embodiment, a cellular scaffold system is provided. In this embodiment, the cellular scaffold system comprises a first cellular scaffold and a second cellular scaffold, wherein the first cellular scaffold and the second cellular scaffold comprise a structural construct providing for cell transfer between the first and second cellular scaffold.

[0014] In yet another embodiment, a method of making a cellular scaffold is provided. In this embodiment, the method comprises the steps of preparation of at least one fiber and forming the at least one fiber into a sheet, thereby forming a cellular scaffold.

[0015] In another embodiment, a method of using a cellular scaffold is provided. In this embodiment, the method comprises combining the cellular scaffold with a bioreactor.

[0016] In yet another embodiment, a cellular scaffold for cultivated meat production is provided. In this embodiment, the cellular scaffold comprises at least one nanofiber arranged to provide cellular attachment, the at least one nanofiber forming a three-dimensional porous structure. The three-dimensional porous structure is formed by gas expansion of a two-dimensional nanofiber mat, the gas expansion creating volumetric expansion and increasedporosity within the nanofiber structure. The cellular scaffold is edible and animal-component free.

[0017] In another embodiment, a method of making a cellular scaffold for cultivated meat production is provided. In this embodiment, the method comprises forming at least one nanofiber into a two-dimensional mat by electrospinning, electrospray, or extrusion. The method further comprises immersing the two-dimensional mat in an effervescent system to generate gas within the mat, thereby inducing volumetric expansion and creating a three-dimensional porous scaffold. The method further comprises stabilizing the three-dimensional porous scaffold by freeze-drying. The cellular scaffold is edible and animal-component free.

[0018] In yet another embodiment, a method of culturing cells for cultivated meat production is provided. In this embodiment, the method comprises providing a cellular scaffold comprising at least one nanofiber arranged in a three-dimensional porous structure formed by gas expansion, wherein the cellular scaffold is edible and animal-component free. The method further comprises introducing the cellular scaffold into a bioreactor containing cell culture media. The method further comprises seeding cells onto the cellular scaffold. The method further comprises culturing the cells on the cellular scaffold to produce cultivated meat tissue.

[0019] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplar}' aspects of the teachings of this disclosure and are not restrictive.BRIEF DESCRIPTION OF FIGURES

[0020] A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below. Nonlimiting and non-exhaustive examples are described with reference to the following figures.

[0021] FIG. 1 depicts an expanded scaffold floating at the surface of a liquid following gas expansion, according to aspects of the present disclosure.

[0022] FIG. 2 depicts the expanded scaffold of FIG. 1 positioned prior to freeze-dry ing, according to an embodiment.DETAILED DESCRIPTION

[0023] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of cellular biology', tissue engineering, and cultivated meat production.

[0024] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0025] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified. Reference will now be made in detail to each embodiment of the present invention. Such embodiments are provided by way of explanation of the present invention, which is not intended to be limited thereto. In fact, those of ordinary skill in the art may appreciate upon reading the present specification and viewing the present drawings that various modifications and variations can be made thereto.

[0026] The present disclosure relates to cellular scaffolds for cultivated meat production. A cellular scaffold as described herein may comprise a three-dimensional porous structure formed from nanofibers that have undergone gas expansion. The cellular scaffold may' beedible and animal-component free, making the cellular scaffold suitable for incorporation into food products intended for human or animal consumption.

[0027] Cellular scaffolds as described herein may be configured as high-density cellular scaffolds (HDCS) or medium-density cellular scaffolds (MDCS). A high-density cellular scaffold may have a surface-to-volume ratio optimized to allow high-density cell culture at approximately 1><1O10cells / mL. A medium-density cellular scaffold (MDCS) may have a surface-to-volume ratio optimized to allow low density cell culture at approximately IxlO6cells / mL. The selection between high-density and medium-density configurations may depend on the particular application, cell type, and desired characteristics of the cultivated meat product.

[0028] The cellular scaffolds described herein address challenges in cultivated meat production by providing structures that support cell adhesion, proliferation, and differentiation while maintaining properties suitable for food consumption. By utilizing gas expansion techniques applied to two-dimensional nanofiber mats, the cellular scaffolds achieve three-dimensional porous architectures that permit mass transfer of nutrients and oxygen throughout the scaffold volume. The animal-component free nature of the cellular scaffolds eliminates reliance on animal-derived materials such as fetal bovine serum during the scaffold fabrication process, thereby addressing ethical concerns and reducing production costs associated with conventional cell culture approaches.

[0029] The edible nature of the cellular scaffolds allows the scaffolds to remain incorporated w ithin the final cultivated meat product without requiring removal or separation prior to consumption. Materials used in the cellular scaffolds may be selected from foodgrade polymers and compounds that contribute to or do not detract from the organoleptic qualities of the finished cultivated meat product.Definitions

[0030] The following definitions apply throughout the present specification and claims.

[0031] A "nanofiber" is a fiber with a diameter in the nanometer range, typically between about 1 nanometer and about 1000 nanometers. Nanofibers are produced by electrospinning, electrospray, or extrusion processes. Nanofibers form the structural basis of the cellular scaffolds described herein and provide texture at the cellular scale to promote cell adhesion and spreading, as well as mechanical strength and integrity to the scaffold structure.

[0032] In an embodiment, the nanofiber has a diameter of 1 to 50 nanometers. In an embodiment, the nanofiber has a diameter of 50 to 100 nanometers. In an embodiment, the nanofiber has a diameter of 100 to 250 nanometers. In an embodiment, the nanofiber has a diameter of 250 to 500 nanometers. In an embodiment, the nanofiber has a diameter of 500 to 1000 nanometers.

[0033] A "high-density cellular scaffold" (HDCS) is a scaffold with a surface-to-volume ratio optimized to allow high-density cell culture at approximately 1 xlO10cells / mL. High-density cellular scaffolds are configured to support the production of cultivated meat products with high cellular content relative to scaffold mass.

[0034] A "medium-density cellular scaffold" (MDCS) is a scaffold with a surface-to-volume ratio optimized to allow low-density cell culture at approximately l*106cells / mL. Medium-density cellular scaffolds are suitable for applications where lower cell densities are acceptable or desired.

[0035] A "monolithic structure" is a scaffold of size greater than 300 micrometers in any of the X, Y, or Z dimensions. Monolithic scaffolds are either fixed in place relative to an agitation mechanism in a bioreactor, immobilized in place, or rest in the bioreactor during cell culture. In an embodiment, a monolithic scaffold has dimensions similar to the dimensions of a whole meat product, such as approximately 200 mm length x 200 mm width x 20 mm thickness.

[0036] In an embodiment, the monolithic structure has a dimension of 300 micrometers to 1 mm. In an embodiment, the monolithic structure has a dimension of 1 mm to 10 mm. Inan embodiment, the monolithic structure has a dimension of 10 mm to 50 mm. In an embodiment, the monolithic structure has a dimension of 50 mm to 100 mm. In an embodiment, the monolithic structure has a dimension of 100 mm to 200 mm.

[0037] A "microcam er" is a scaffold with dimensions on the order of less than 300 micrometers that moves freely inside a bioreactor via fluid flow or energy imparted by an agitation device, impeller, or similar mechanism. Microcarriers are cultured in well plates, shake flasks, spinner flasks for process development, and then transferred to bioreactors for scale-up and manufacturing.

[0038] "Cultivated meat" (CM) is meat produced by culturing animal cells in vitro rather than from slaughtered animals. Cultivated meat production involves proliferation of cells acquired from an animal in bioreactors or other cell culture environments in the presence of oxygen-rich cell culture medium containing nutrients such as amino acids, glucose, vitamins, inorganic salts, protein supplements, and growth factors. The proliferation and growth of cells takes place on structural supports called scaffolds, whose role is to mimic the three-dimensional growth environment of tissue.

[0039] A "serum-free growth medium" is cell culture media that does not contain animal serum such as fetal bovine serum (FBS). Serum-free growth medium eliminates the need for animal serum, which is often derived from animal fetuses, animal blood, or animal tissue, thereby reducing reliance on animal slaughter while also lowering production costs and addressing ethical concerns associated with using animal-derived components in cell culture processes.

[0040] "Animal-component free" refers to materials that do not contain any ingredients derived from animals. Animal-component free scaffolds are fabricated without the use of animal-derived polymers, proteins, or other substances, making such scaffolds suitable for cultivated meat production processes that seek to minimize or eliminate animal inputs.

[0041] A "three-dimensional porous structure" is a scaffold architecture characterized by volumetric expansion and interconnected porosity that permits cell infiltration, mass transferof nutrients and oxygen, and waste removal throughout the scaffold volume. Three-dimensional porous structures are formed by gas expansion of two-dimensional nanofiber mats, wherein gas generation within the fibrous network induces rapid volumetric expansion and increased porosity7.

[0042] "Gas expansion" is a process wherein a two-dimensional nanofiber mat is converted into a three-dimensional porous scaffold via controlled gas generation within the fibrous network. Gas expansion is achieved by immersing the nanofiber mat in an effervescent system, such as an aqueous system generated by reacting dilute acetic acid with sodium bicarbonate to produce carbon dioxide in situ, or by immersing the nanofiber mat in an aqueous sodium borohydride solution to generate hydrogen gas.Nanofiber Structure and Composition

[0043] The cellular scaffolds described herein comprise at least one nanofiber arranged to provide cellular attachment. The nanofibers form the structural basis of the scaffold and provide texture at the cellular scale that promotes cell adhesion, spreading, and growth. The nanofibers also provide mechanical strength and integrity to the scaffold structure.Sufficiently stiff substrates are beneficial for the differentiation of many cell types, including the differentiation of myoblasts into myotubes, which is an aspect of cultivated meat production.

[0044] The at least one nanofiber comprises zein. Zein is a plant-derived protein obtained from com that is edible and animal-component free. Zein provides suitable mechanical properties for cell culture applications while maintaining food-grade status. The use of zein as a nanofiber material allows the cellular scaffold to remain incorporated within the final cultivated meat product without requiring removal prior to consumption.

[0045] In an embodiment, the at least one nanofiber further comprises cellulose acetate. Cellulose acetate is a plant-derived polymer that enhances the mechanical properties andstability of the nanofiber structure. The combination of zein and cellulose acetate in the nanofiber composition provides a scaffold material with suitable stiffness for myogenic differentiation while maintaining edibility and animal-component free status. A zein / cellulose acetate nanofiber composition supports cell adhesion, proliferation, and maturation under dynamic culture conditions.

[0046] The at least one nanofiber is formed by a process selected from the group consisting of electrospinning, electrospray, and extrusion. Electrospinning is a spinning process that uses electrostatic forces to produce fibrous scaffolds from biocompatible polymers. Electrospinning is versatile and cost effective, resulting in fibers with high surface area to volume ratios and tunable porosities that are beneficial for cell proliferation, differentiation, and migration. Electrospray is a related technique that produces fibers or particles using electrostatic forces applied to polymer solutions. Extrusion involves forcing polymer solutions through a die or nozzle to form continuous fibers. Each of these nanofiber formation processes produces nanofibers suitable for cellular scaffold fabrication.

[0047] The nanofibers formed by electrospinning, electrospray, or extrusion are arranged into two-dimensional mats. The two-dimensional mats comprise a plurality of nanofibers that are uniaxially aligned, random, and / or entangled. The arrangement of nanofibers within the mat affects the mechanical properties and cell behavior on the scaffold surface.

[0048] The at least one nanofiber is crosslinked with a crosslinking agent to confer stability when used in common cell culture conditions. Crosslinking stabilizes the nanofiber structure and prevents degradation or dissolution during cell culture processes that involve chelators, such as when detaching or recovering cells and eluting or washing media components.

[0049] The crosslinking agent is selected from the group consisting of genipin, transglutaminase, glutaraldehyde, citric acid, tannic acid, succinic anhydride, epigallocatechin. tyrosinase, phosphoryl chloride, sodium trimetaphosphate, sodium tripolyphosphate, plant-derived proanthocyanidins, plant-derived epigallocatechin gallate, and mixtures thereof. Genipin is a naturally derived crosslinking agent obtained fromgardenia fruit that provides biocompatible crosslinking. Transglutaminase is an enzyme that catalyzes crosslinking between protein chains and is commonly used in food applications. Glutaraldehyde is a chemical crosslinking agent that forms stable crosslinks between amine groups. Citric acid and tannic acid are organic acids that provide crosslinking through ester bond formation or hydrogen bonding interactions. Succinic anhydride reacts with hydroxyl and amine groups to form crosslinks. Epigall ocatechin and plant-derived epigallocatechin gallate (EGCG) are polyphenolic compounds that crosslink proteins through oxidative mechanisms. Tyrosinase is an enzyme that catalyzes oxidative crosslinking of phenolic compounds. Phosphoryl chloride (POCI3), sodium trimetaphosphate (STMP), and sodium tripolyphosphate (STPP) are phosphate-based crosslinking agents, and mixtures of STMP with STPP provide effective crosslinking for polysaccharide and protein-based materials. Plant-derived proanthocyanidins are polyphenolic compounds that crosslink collagen and other proteins through hydrogen bonding and covalent interactions.Three-Dimensional Porous Structure Formation

[0050] The cellular scaffolds described herein comprise at least one nanofiber forming a three-dimensional porous structure. The three-dimensional porous structure is formed by gas expansion of a two-dimensional nanofiber mat. The gas expansion creates volumetric expansion and increased porosity within the nanofiber structure, transforming an originally flat, dense nanofiber mat into a voluminous scaffold with interconnected pores suitable for cell infiltration and mass transfer.

[0051] The two-dimensional nanofiber mat serves as the starting material for three-dimensional porous structure formation. The two-dimensional nanofiber mat is produced by electrospinning, electrospray, or extrusion of polymer solutions containing zein, cellulose acetate, or combinations thereof, as described above. The two-dimensional nanofiber mat is cut to desired dimensions prior to the gas expansion process.

[0052] Gas expansion converts the two-dimensional nanofiber mat into the three-dimensional porous structure through controlled gas generation within the fibrous network. Gas formation within the nanofiber mat induces rapid volumetric expansion as gas bubbles nucleate and grow between and within the nanofiber layers. The volumetric expansion separates adjacent nanofibers and creates space between layers of the nanofiber mat, resulting in increased porosity throughout the scaffold structure.

[0053] The increased porosity within the nanofiber structure provides several functional benefits for cell culture applications. The porous architecture permits cell infiltration beyond the surface layers of the scaffold, allowing cells to populate the interior volume of the three-dimensional porous structure. The interconnected pore network enables mass transfer of nutrients, oxygen, and growth factors to cells throughout the scaffold volume while facilitating removal of metabolic waste products. The porosity also provides channels for cell migration within the scaffold structure, supporting the formation of contiguous tissue constructs.

[0054] The gas expansion process is controlled to achieve desired levels of volumetric expansion and porosity. In an embodiment, the degree of volumetric expansion is adjusted by varying the concentration of gas-generating reagents, the duration of exposure to the effervescent system, and the temperature at which gas expansion occurs. Higher concentrations of gas-generating reagents and longer exposure times produce greater volumetric expansion and higher porosity levels.

[0055] The three-dimensional porous structure retains the nanofibrous texture of the original two-dimensional nanofiber mat at the cellular scale. The nanofibers within the expanded scaffold maintain their diameter and surface characteristics, providing the surface texture that promotes cell adhesion and spreading. The gas expansion process increases the macroscopic dimensions and porosity of the scaffold without altering the nanoscale features that support cellular attachment.

[0056] Providing a cellular scaffold comprising at least one nanofiber arranged in a three-dimensional porous structure formed by gas expansion enables the production of thickcultivated meat products. The three-dimensional porous structure supports high-density cell culture by providing sufficient surface area for cell attachment while maintaining mass transfer throughout the scaffold volume. The gas-expanded scaffold architecture addresses limitations of conventional porous scaffolds that do not permit cell infiltration beyond several hundred micrometers due to mass transfer constraints.

[0057] The volumetric expansion achieved through gas expansion transforms scaffolds from thin, sheet-like structures into thick, three-dimensional constructs suitable for whole-cut cultivated meat applications. In an embodiment, the gas expansion process increases the thickness of the nanofiber mat by a factor of ten or more, creating scaffolds with dimensions suitable for producing cultivated meat products that mimic the size and structure of conventional meat cuts.Gas Expansion Methods and Effervescent Systems

[0058] The gas expansion that converts a two-dimensional nanofiber mat into a three-dimensional porous scaffold is produced by various methods that generate gas within the fibrous network. The gas expansion induces volumetric expansion and creates a three-dimensional porous scaffold with interconnected porosity suitable for cell culture applications.

[0059] In an embodiment, the gas expansion is produced by reacting an acid with sodium bicarbonate to generate carbon dioxide in situ within the two-dimensional nanofiber mat. The reaction between the acid and sodium bicarbonate produces carbon dioxide gas that nucleates and expands within the nanofiber network, separating adjacent fibers and creating porous channels throughout the scaffold structure. The carbon dioxide generation occurs rapidly upon contact betw een the acid and sodium bicarbonate, inducing volumetric expansion of the nanofiber mat.

[0060] The acid comprises acetic acid. Acetic acid is a food-grade organic acid that reacts with sodium bicarbonate according to the following reaction to produce carbon dioxide, water, and sodium acetate. Acetic acid is provided in the form of dilute acetic acid, such as vinegar, which contains approximately 4-8% acetic acid by volume. The use of acetic acid maintains the edible and animal-component free status of the cellular scaffold, as both acetic acid and the reaction products are food-safe compounds.

[0061] Referring to FIG. 1, an expanded scaffold floats at the surface of liquid following gas expansion. The liquid comprises an effervescent aqueous system generated by reacting dilute acetic acid with sodium bicarbonate. The buoyancy of the expanded scaffold at the liquid surface indicates the presence of entrapped gas within the porous structure, demonstrating successful volumetric expansion of the nanofiber mat. The expanded scaffold exhibits a three-dimensional configuration with increased thickness and volume compared to the original two-dimensional nanofiber mat.

[0062] The effervescent system comprises an aqueous solution of acetic acid reacted with sodium bicarbonate to generate carbon dioxide in situ within the two-dimensional mat. In an embodiment, the sodium bicarbonate concentration is approximately 1 % weight per volume (w / v) in the aqueous solution. The two-dimensional nanofiber mat, cut to desired dimensions, is briefly immersed in the effervescent aqueous system. Carbon dioxide formation within the fibrous network induces rapid volumetric expansion and increased porosity, thereby inducing volumetric expansion and creating a three-dimensional porous scaffold.

[0063] The immersion time in the effervescent system is controlled to achieve the desired degree of volumetric expansion. Shorter immersion times produce moderate expansion, while longer immersion times allow for greater gas generation and more extensive volumetric expansion. The immersion duration is adjusted based on the thickness of the starting two-dimensional nanofiber mat and the target porosity of the three-dimensional porous scaffold.

[0064] In an embodiment, the gas expansion is produced by immersing the two-dimensional nanofiber mat in an aqueous sodium borohydride solution to generate hydrogen gas within the nanofiber mat. Sodium borohydride reacts with water to produce hydrogen gas, which nucleates and expands within the nanofiber network to create the three-dimensional porous structure. The aqueous sodium borohydride solution has a concentration of approximately 0.15 M. The two-dimensional nanofiber mat is immersed in the aqueous sodium borohydride solution for a controlled period sufficient to induce hydrogen gas formation and volumetric expansion within the fibrous network.

[0065] Following gas expansion using the sodium borohydride method, the expanded scaffold is removed from the aqueous sodium borohydride solution and thoroughly rinsed with deionized water to eliminate residual reagents. The rinsing step removes sodium borohydride and reaction byproducts from the scaffold structure, ensuring that the final cellular scaffold is suitable for cell culture applications and food consumption.

[0066] Both the acid / sodium bicarbonate method and the sodium borohy dride method produce three-dimensional porous scaffolds with similar structural characteristics, including increased thickness, volumetric expansion, and interconnected porosity. The selection between these gas expansion methods depends on the specific nanofiber composition, desired expansion characteristics, and processing considerations. The acid / sodium bicarbonate method utilizes food-grade reagents throughout the process, while the sodium borohydride method requires thorough rinsing to remove residual chemicals prior to use in cell culture or food applications.

[0067] In an embodiment, generation of gas within and / or adjacent to the nanofiber structure induces volumetric expansion that may be described as fluffing of the nanofiber mat into a three-dimensional porous architecture. In an embodiment, gas generation and / or foaming reagents are introduced during forming and shaping operations, including during joining or lamination of multiple nanofiber sheets, such that the structure expands while being conformed to a desired geometry. In an embodiment, the scaffold and / or a precursor composition is co-extruded with a gas-generating composition and / or foaming agent toproduce a porous, expanded structure. In an embodiment, the resulting scaffold format comprises a foamed microcarrier and / or a foamed scaffold fragment suitable for suspension culture, aggregation, or post-culture assembly into larger cultivated meat constructs.Additional Foaming Agents and Stabilizers

[0068] In addition to the gas expansion methods described above utilizing acid / sodium bicarbonate reactions and sodium borohydride solutions, the cellular scaffolds described herein are fabricated using additional foaming agents and stabilizers that create volumetric expansion and porous architectures within the nanofiber structures. These foaming agents function as emulsifiers and stabilizers that generate and maintain foam structures during scaffold fabrication. The performance of foaming agents depends on various factors such as temperature, pH, pressure, and additional ingredients present in the scaffold formulation.

[0069] In an embodiment, a foaming agent comprises Sodium Stearoyl Lactylate. Sodium Stearoyl Lactylate functions as an emulsifier and stabilizer of foams within the scaffold structure. Sodium Stearoyl Lactylate is commonly used in bakery and dairy applications and is available in both synthetic and non-synthetic forms. The emulsifying properties of Sodium Stearoyl Lactylate stabilize gas bubbles within the nanofiber matrix during the foaming process, maintaining the porous architecture of the expanded scaffold.

[0070] In an embodiment, a foaming agent comprises Mono / Di-glycerides. Mono / Di-glycerides function as emulsifiers and stabilizers of foams within the scaffold structure. Mono / Di-glycerides are commonly used in bakery’ and dairy applications and are available in both synthetic and non-synthetic forms. The emulsifying properties of Mono / Di-glycerides promote the formation and stabilization of gas bubbles within the nanofiber network during scaffold expansion.

[0071] In an embodiment, a foaming agent comprises Aquafaba derived from chickpea water. Aquafaba is the viscous liquid obtained from cooking or canning chickpeas andcontains proteins and carbohydrates that provide foaming and emulsifying properties.Aquafaba generates stable foam structures when agitated or whipped, and the foaming properties of Aquafaba are utilized to create volumetric expansion within nanofiber scaffolds. The plant-derived nature of Aquafaba maintains the animal-component free status of the cellular scaffold.

[0072] In an embodiment, a foaming agent comprises Sucrose Ester. Sucrose Ester is particularly suitable for molecular gastronomy applications where precise control over foam structure and stabi 1 i ty is desired. Sucrose Ester provides emulsifying and foaming properties that create fine, stable foam structures within the scaffold matrix. The food-grade status of Sucrose Ester maintains the edible nature of the cellular scaffold.

[0073] In an embodiment, a foaming agent comprises Saponins derived from soybeans and other plants. Saponins are glycosidic compounds that exhibit surfactant properties and generate stable foams when agitated in aqueous solutions. In an embodiment, Saponins are combined with gums to improve foaming performance and foam stability . The addition of gums enhances the viscosity of the foaming solution and stabilizes the foam structure during scaffold fabrication.

[0074] In an embodiment, a foaming agent comprises Soy Lecithin derived from soybeans. Soy Lecithin is a phospholipid mixture that provides emulsifying and foaming properties. Soy Lecithin stabilizes gas-liquid interfaces and promotes the formation of foam structures within the nanofiber scaffold. The plant-derived nature of Soy Lecithin maintains the animal-component free status of the cellular scaffold.

[0075] The foaming agents described above are used individually or in combination with one another to achieve desired foam characteristics and scaffold properties. Combinations of foaming agents provide synergistic effects that enhance foam stability', pore size distribution, and overall scaffold architecture.

[0076] To utilize the effectiveness of the foaming agents, the foaming agents are melted in with fat during scaffold fabrication. The incorporation of foaming agents into a fat phaseenhances the dispersion and activity of the foaming agents within the scaffold formulation. The fat phase provides a medium for dissolving lipophilic foaming agents and facilitates uniform distribution of the foaming agents throughout the nanofiber matrix during the expansion process.Scaffold Stabilization by Freeze-Drying

[0077] The three-dimensional porous structure is stabilized by freeze-drying following the gas expansion. Freeze-drying, also referred to as lyophilization, preserves the expanded porous architecture of the scaffold by removing water through sublimation under controlled conditions. The freeze-drying process locks the volumetric expansion and interconnected porosity achieved during gas expansion into a stable, dry scaffold structure that retains the three-dimensional configuration upon subsequent handling and storage.

[0078] Following gas expansion, the expanded scaffold is rinsed with deionized water prior to freeze-drying. Rinsing the three-dimensional porous scaffold with deionized water removes residual reagents from the gas expansion process, including unreacted acid, sodium bicarbonate, sodium acetate byproducts, or sodium borohydride and associated reaction products depending on the gas expansion method employed. The rinsing step ensures that the final cellular scaffold is free from chemical residues that could interfere with cell culture applications or compromise the edible and animal-component free status of the scaffold.

[0079] Referring to FIG. 2, an expanded three-dimensional scaffold structure is positioned on a metal surface or holder after rinsing with deionized water prior to freeze-drying. The scaffold is photographed within a freezer or cold storage environment, as evidenced by the frost-covered surroundings and green container visible in the background. The expanded scaffold exhibits a pale yellow to cream coloration and displays a distinctly porous, foam-like architecture with visible layered striations along the cross-section. The layered appearance visible along the edge of the scaffold indicates the multi-layered fibrousstructure that has been expanded to create space between the nanofiber sheet layers through the gas expansion process.

[0080] With continued reference to FIG. 2, the expanded scaffold demonstrates significant three-dimensional thickness, showing the volumetric expansion achieved through the gas foaming process. The scaffold appears to be in a frozen state, consistent with the freeze-drying step wherein the expanded scaffold is frozen at approximately negative eighty degrees Celsius prior to lyophilization. The freezing step solidifies the water within the scaffold structure, maintaining the expanded porous architecture during the subsequent sublimation phase of freeze-drying.

[0081] The freeze-drying process is conducted under controlled conditions for a duration sufficient to remove substantially all water from the scaffold structure. In an embodiment, the freeze-drying is conducted for at least 24 hours to ensure complete sublimation of ice from the scaffold matrix. The controlled conditions of freeze-drying include reduced pressure that facilitates sublimation of ice directly from the solid phase to the vapor phase without passing through a liquid phase. This direct sublimation prevents collapse of the porous structure that would occur if the ice melted to liquid water before evaporation.

[0082] Stabilizing the three-dimensional porous scaffold by freeze-drying yields a stabilized three-dimensional scaffold with preserved expanded porous architecture. The freeze-dried scaffold maintains the volumetric expansion, interconnected porosity, and nanofibrous texture achieved during gas expansion. The stabilized scaffold is stored in dry conditions until use in cell culture applications, and the scaffold is rehydrated prior to introduction into a bioreactor or cell culture environment.

[0083] The cellular scaffold produced through gas expansion and freeze-drying stabilization is edible and animal-component free, as described previously. The materials used in scaffold fabrication, including zein, cellulose acetate, and food-grade reagents for gas expansion, maintain the food-safe status of the final scaffold product. The freeze-drying process does not introduce any animal-derived components and preserves the edible nature of the scaffold materials.Scaffold Configurations and Dimensions

[0084] The cellular scaffolds described herein are configured in two primary' forms based on dimensional characteristics: monolithic structures and microcarriers. The selection between monolithic structures and microcarriers depends on the intended application, bioreactor configuration, and desired characteristics of the cultivated meat product.

[0085] The cellular scaffold is a monolithic structure having a dimension of at least 300 micrometers in each of an X, Y, and Z dimension. Monolithic structures are fixed in place relative to an agitation mechanism in a bioreactor, immobilized in place, or rest in the bioreactor during cell culture. The three-dimensional nature of monolithic structures with dimensions exceeding 300 micrometers in all three spatial dimensions provides sufficient volume for high-density cell culture and the production of thick cultivated meat products. Monolithic scaffolds support the cultivation of cells on a single large scaffold that has dimensions similar to the dimensions of the whole meat product being produced.

[0086] The cellular scaffold is a microcarrier having a dimension of less than 300 micrometers in each of an X, Y, and Z dimension. Microcarriers move freely inside a bioreactor via fluid flow or energy imparted by an agitation device, impeller, or similar mechanism. The small dimensions of microcarriers allow the microcarriers to remain suspended in cell culture media during agitation, providing high surface area for cell attachment relative to the volume of the bioreactor. Microcarriers are cultured in well plates, shake flasks, and spinner flasks for initial process development, and then transferred to bioreactors for scale-up and manufacturing.

[0087] In an embodiment, the scaffold is sized to produce whole cuts of approximately 200 mm length x 200 mm width x 20 mm thickness. These dimensions result in cuts equivalent to a Top Sirloin Steak weighing approximately 225 grams. In an embodiment, cuts equivalent to a Top Sirloin Steak are also included and include filet / fillet, ribeye, strip steak, or other whole-cut portions. The monolithic scaffold configuration supports the production ofwhole-cut cultivated meat products where the cultivated meat is grown on a single large scaffold having dimensions similar to the final meat product. The scaffold and cells are cultured in a bioreactor, and the scaffold is moved through the cell culture media in the bioreactor, or held at rest in the bioreactor while culture media fluid circulates through the scaffold.

[0088] Multiple pieces of scaffolds and microcarriers with cultivated meat grown on the scaffolds and microcarriers are combined together outside of the bioreactor to form pieces of cultivated meat of various sizes. The combination of multiple scaffold pieces and microcarriers enables the production of cultivated meat products ranging from 5 grams to 350 kilograms. Smaller cultivated meat products are formed by combining a limited number of scaffold pieces or microcarriers, while larger products are assembled from numerous scaffold pieces and microcarriers to achieve the target mass. The combination of scaffold pieces occurs after harvesting from the bioreactor, allow ing flexibility in the size and shape of the final cultivated meat product.

[0089] Tn an embodiment, the cultivated meat product has a mass of 5 grams to 50 grams. In an embodiment, the cultivated meat product has a mass of 50 grams to 250 grams. In an embodiment, the cultivated meat product has a mass of 250 grams to 1 kilogram. In an embodiment, the cultivated meat product has a mass of 1 kilogram to 10 kilograms. In an embodiment, the cultivated meat product has a mass of 10 kilograms to 100 kilograms. In an embodiment, the cultivated meat product has a mass of 100 kilograms to 350 kilograms.

[0090] The ability to combine multiple scaffold pieces and microcarriers provides manufacturing flexibility for producing cultivated meat products of arbitrary size. Small scaffold pieces and microcarriers are combined with adhesives or binders such as starch, pectin, transglutaminase, or methylcellulose to create stable, multilayered tissue constructs. The combination process incorporates layers of hydrogel, cell-laden hydrogel, pure cell layers, or additional microcarriers to form structured, multicellular constructs. The scaffold pieces maintain structural integrity during the combination process, and the nanofibrous texture of the scaffold surfaces promotes adhesion between adjacent scaffold pieces.Scaffold Layer Assembly and Porosity

[0091] The cellular scaffolds described herein comprise multiple layers of nanofiber sheets that are assembled and joined to form three-dimensional scaffold structures. The scaffold layers are joined via mechanical, chemical, thermal, or enz matic means to create stable, multilayered constructs suitable for cell culture applications.

[0092] Mechanical joining of scaffold layers involves physical interlocking or compression of adjacent nanofiber sheets. In an embodiment, mechanical joining comprises pressing multiple nanofiber sheets together under controlled pressure to create contact points between the fibrous networks of adjacent layers. The entanglement of nano fibers at the interface between layers provides mechanical stability to the assembled scaffold structure. In an embodiment, mechanical joining involves stitching or suturing adjacent scaffold layers together using biocompatible threads or fibers that maintain the edible and animal-component free status of the scaffold.

[0093] Chemical joining of scaffold layers involves the application of chemical crosslinking agents that form covalent or ionic bonds between adjacent nanofiber sheets. The crosslinking agents described previously, including genipin, transglutaminase, glutaraldehyde, citric acid, tannic acid, succinic anhydride, epigallocatechin, tyrosinase, phosphoryl chloride, sodium trimetaphosphate, sodium tripolyphosphate, plant-derived proanthocyanidins, and plant-derived epigallocatechin gallate, are applied at the interface between scaffold layers to create chemical bonds that join the layers together. The chemical crosslinking provides stable attachment between adjacent layers that withstands the mechanical stresses encountered during cell culture and bioreactor agitation.

[0094] Thermal joining of scaffold layers involves the application of heat to fuse adjacent nanofiber sheets together. In an embodiment, thermal joining comprises heating the interface between scaffold layers to a temperature sufficient to partially melt or soften the nanofiber material, allowing the fibers from adjacent layers to intermingle and fuse uponcooling. The thermal joining temperature is controlled to achieve fusion at the layer interface without degrading the bulk nanofiber structure or compromising the edible nature of the scaffold materials. In an embodiment, thermal joining involves the application of localized heat using heated plates, ultrasonic welding, or laser heating to create discrete fusion points between adjacent scaffold layers.

[0095] Enzymatic joining of scaffold layers involves the application of enzymes that catalyze bond formation between adjacent nanofiber sheets. Transglutaminase is an enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues in protein-based nanofibers such as zein. The application of transglutaminase at the interface between scaffold layers creates enzymatic crosslinks that join the layers together. Enzymatic joining provides a food-safe method for assembling scaffold layers, as transglutaminase is commonly used in food processing applications.

[0096] Porosity is added to the scaffold via punching or laser cutting. Punching involves the use of mechanical punches or dies to create holes or perforations through the scaffold structure. The punched holes provide channels for mass transfer of nutrients, oxygen, and waste products through the scaffold thickness. The size, shape, and distribution of punched holes are controlled to achieve desired porosity levels and mass transfer characteristics. In an embodiment, punching creates a regular array of holes with uniform spacing throughout the scaffold surface.

[0097] Laser cutting involves the use of focused laser energy to ablate or vaporize scaffold material, creating holes or channels through the scaffold structure. Laser cutting provides precise control over the size, shape, and position of the created openings. In an embodiment, laser cutting creates microchannels with diameters ranging from tens of micrometers to several millimeters. The laser cutting parameters, including laser power, pulse duration, and scanning speed, are adjusted to achieve the desired hole dimensions and edge quality. Laser cutting enables the creation of complex porosity patterns that are difficult to achieve through mechanical punching methods.

[0098] The scaffold is formed by chopping, mincing, cuting, grinding, or extruding nanofiber sheets into smaller fragments and combining the fragments together. Chopping involves cuting nanofiber sheets into discrete pieces using blades or cuting implements. Mincing involves reducing nanofiber sheets into fine particles through repeated cuting or shearing actions. Cuting involves dividing nanofiber sheets into defined shapes or sizes using scissors, blades, or dies. Grinding involves reducing nanofiber sheets into particles through abrasive or compressive forces. Extruding involves forcing nanofiber material through a die or orifice to create shaped fragments or strands.

[0099] The smaller fragments produced by chopping, mincing, cuting, grinding, or extruding are combined together to form scaffold structures. In an embodiment, the fragments are combined with themselves to create homogeneous scaffold constructs composed entirely of nanofiber material. The fragments are compressed, bonded, or otherwise assembled to create three-dimensional scaffold structures with desired dimensions and porosity characteristics. The combination of nanofiber fragments provides a method for creating scaffolds with controlled density7and pore size distribution.

[0100] The scaffold is formed as a multi-component hybrid aggregate with a second phase such as a hydrogel. The multi-component hybrid aggregate comprises nanofiber fragments combined with a hydrogel phase that fills the spaces between the nanofiber fragments and provides additional structural support. The hydrogel phase comprises biocompatible, edible hydrogel materials such as alginate, pectin, gellan gum, or other polysaccharide-based gels. The hydrogel phase is crosslinked to provide mechanical stability7to the hybrid aggregate scaffold.

[0101] In an embodiment, the multi-component hybrid aggregate is formed by dispersing nanofiber fragments in a hydrogel precursor solution, followed by gelation of the hydrogel phase to encapsulate the nanofiber fragments within the gel matrix. The nanofiber fragments provide mechanical reinforcement and cell atachment surfaces within the hydrogel matrix, while the hydrogel phase provides a hydrated environment that supports cell viability7andnutrient diffusion. The ratio of nanofiber fragments to hydrogel phase is adjusted to achieve desired mechanical properties and cell culture performance.

[0102] In an embodiment, the multi-component hybrid aggregate is formed by layering nanofiber sheets with hydrogel layers to create a laminated structure. The alternating layers of nanofiber sheets and hydrogel provide a scaffold architecture with distinct regions for cell attachment on the nanofiber surfaces and nutrient diffusion through the hydrogel layers. The laminated structure is assembled by sequential deposition of nanofiber sheets and hydrogel layers, with each layer bonded to adjacent layers through chemical, thermal, or enzymatic means as described above.Additives and Flavorings

[0103] The cellular scaffolds described herein include flavorings and additives for enhanced sensory or nutritional properties. The flavorings and additives are incorporated into the scaffold structure through various methods including encapsulation, dissolution, mixing or emulsification, embedding, and coating. The incorporation method is selected based on the physical and chemical properties of the flavoring or additive, the desired release profile during cooking or consumption, and compatibility with the scaffold fabrication process.

[0104] Encapsulated flavorings and additives are contained within protective shells or matrices that control the release of the encapsulated material. Encapsulation protects sensitive flavorings and additives from degradation during scaffold fabrication, storage, and cell culture processes. The encapsulating material comprises food-grade polymers, lipids, or carbohydrates that release the encapsulated contents upon exposure to specific conditions such as elevated temperature during cooking, changes in pH, or mechanical disruption during chewing. Encapsulated fats are incorporated into the scaffold to provide lipid content that contributes to the mouthfeel and flavor profile of the cultivated meat product.

[0105] Dissolved flavorings and additives are incorporated into the scaffold by dissolving the flavoring or additive in a solvent that is compatible with the scaffold fabrication process. The dissolved material is distributed throughout the scaffold matrix during nanofiber formation or during subsequent processing steps. Water-soluble flavorings and additives are dissolved in aqueous solutions used during scaffold fabrication, while lipid-soluble materials are dissolved in oil phases that are incorporated into the scaffold structure.

[0106] Mixed or emulsified flavorings and additives are incorporated into the scaffold through mechanical mixing or emulsification processes. Emulsification creates stable dispersions of immiscible phases, allowing the incorporation of both hydrophilic and hydrophobic flavorings and additives into the scaffold structure. The emulsified materials are distributed throughout the scaffold matrix, providing uniform distribution of flavorings and additives throughout the cultivated meat product.

[0107] Embedded flavorings and additives are physically incorporated within the scaffold structure during fabrication. Embedding involves the incorporation of solid particles, droplets, or other discrete units of flavoring or additive material within the nanofiber matrix. The embedded materials are retained within the scaffold structure throughout cell culture and are released during cooking or consumption of the cultivated meat product.

[0108] Coated flavorings and additives are applied to the surface of the scaffold following fabrication. Coating involves the deposition of a layer of flavoring or additive material onto the scaffold surface through spraying, dipping, or other application methods. The coated materials are positioned at the scaffold surface where the coated materials interact with cells during culture and contribute to the sensory properties of the final cultivated meat product.

[0109] The scaffold includes fungal and microbial purified or fermented components. Fungal components are derived from edible fungi and provide protein, fiber, and flavor compounds that enhance the nutritional and sensory properties of the cultivated meat product. Microbial components are derived from bacteria, yeast, or other microorganisms through purification or fermentation processes. Fermented components provide flavor compounds.enzymes, and other bioactive substances that contribute to the taste profile of the cultivated meat product. The fungal and microbial components are incorporated into the scaffold through encapsulation, dissolution, mixing, embedding, or coating as described above.

[0110] The scaffold includes microbial polysaccharides that provide structural, textural, and functional properties. Microbial polysaccharides are carbohydrate polymers produced by bacteria, fungi, or other microorganisms through fermentation processes. The microbial polysaccharides are incorporated into the scaffold structure to modify the mechanical properties, water-holding capacity7, and texture of the cultivated meat product.[OHl] In an embodiment, the scaffold includes Pullulan as a microbial polysaccharide. Pullulan is a water-soluble polysaccharide produced by the fungus Aureobasidium pullulans. Pullulan provides film-forming properties and is used to create coatings or encapsulation matrices for flavorings and additives within the scaffold structure. Pullulan is edible and biodegradable, maintaining the food-safe status of the cellular scaffold.

[0112] In an embodiment, the scaffold includes gums as microbial polysaccharides. Gums are polysaccharide compounds that provide thickening, gelling, and stabilizing properties. Microbial gums are produced through fermentation processes and include xanthan gum, gellan gum, and welan gum. Xanthan gum is produced by the bacterium Xanthomonas campestris and provides viscosity7and stability7to aqueous systems. Gellan gum is produced by the bacterium Sphingomonas elodea and forms gels in the presence of cations. Welan gum is produced by Alcaligenes species and provides viscosity and suspension properties. The gums are incorporated into the scaffold structure to modify the rheological properties and texture of the cultivated meat product.

[0113] In an embodiment, the scaffold includes Curdlan as a microbial polysaccharide. Curdlan is a linear beta- 1,3 -glucan produced by the bacterium Alcaligenes faecalis. Curdlan forms thermally reversible gels when heated and provides textural properties that contribute to the mouthfeel of the cultivated meat product.

[0114] In an embodiment, the scaffold includes Gellan as a microbial polysaccharide. Gellan is an anionic polysaccharide produced by the bacterium Sphingomonas elodea. Gellan forms gels in the presence of divalent cations such as calcium and provides structural support within the scaffold matrix.

[0115] In an embodiment, the scaffold includes Konjac as a component. Konjac glucomannan is a polysaccharide derived from the konjac plant and provides gelling and thickening properties. Konjac glucomannan is incorporated into the scaffold to modify the texture and water-holding capacity of the cultivated meat product.

[0116] The microbial polysaccharides described above are used individually or in combination with one another to achieve desired textural and functional properties in the cellular scaffold. Combinations of microbial polysaccharides provide synergistic effects that enhance gel strength, water retention, and overall scaffold performance during cell culture and in the final cultivated meat product.Sterilization Methods

[0117] The cellular scaffolds described herein are sterilized prior to use in cell culture applications. Sterilization eliminates microorganisms, including bacteria, fungi, viruses, and spores, from the scaffold structure to prevent contamination during cell culture processes. The sterilization method is selected based on the scaffold composition, the sensitivity' of scaffold materials to the sterilization process, and the requirements of the cell culture application.

[0118] In an embodiment, the scaffold is sterilized using UV illumination for in vitro experiments. UV illumination exposes the scaffold to ultraviolet light, typically in the UV-C wavelength range of approximately 200 to 280 nanometers. UV-C light damages the DNA and RNA of microorganisms, rendering the microorganisms incapable of reproduction and thereby achieving sterilization. The scaffold is positioned within a UV sterilization chamberor biosafety cabinet equipped with UV lamps, and the scaffold is exposed to UV illumination for a duration sufficient to achieve sterilization of the scaffold surfaces. UV illumination provides a non-contact sterilization method that does not introduce chemical residues onto the scaffold and does not require elevated temperatures that could degrade temperaturesensitive scaffold materials.

[0119] In an embodiment, the scaffold is sterilized using ethylene oxide gas for in vitro experiments. Ethylene oxide is a cyclic ether compound that functions as a chemical sterilant by alky lating proteins and nucleic acids of microorganisms. The scaffold is placed within an ethylene oxide sterilization chamber, and the chamber is evacuated and filled with ethylene oxide gas at controlled temperature and humidity conditions. The scaffold is exposed to ethylene oxide gas for a duration sufficient to achieve sterilization throughout the scaffold structure, including the interior porous regions of the three-dimensional scaffold. Following ethylene oxide exposure, the scaffold undergoes an aeration period to allow residual ethylene oxide to dissipate from the scaffold material. Ethylene oxide sterilization is effective for sterilizing porous scaffolds where penetration of the sterilant throughout the scaffold volume is required.

[0120] In an embodiment, the scaffold is sterilized using cold gasses. Cold gas sterilization utilizes gaseous sterilants at temperatures below those used in steam sterilization, making cold gas sterilization suitable for temperature-sensitive scaffold materials. Cold gas sterilants include vaporized hydrogen peroxide, ozone, and chlorine dioxide. Vaporized hydrogen peroxide sterilization exposes the scaffold to hydrogen peroxide vapor, which oxidizes and destroys microorganisms on the scaffold surfaces and within the porous structure. Ozone sterilization exposes the scaffold to ozone gas. which is a strong oxidizing agent that inactivates microorganisms through oxidation of cellular components. Chlorine dioxide sterilization exposes the scaffold to chlorine dioxide gas, which penetrates the scaffold structure and destroys microorganisms through oxidative mechanisms.

[0121] In an embodiment, the scaffold is sterilized using chemical methods. Chemical sterilization involves the application of liquid or gaseous chemical sterilants that destroymicroorganisms through chemical reactions with cellular components. Chemical sterilants include peracetic acid, glutaraldehyde, and formaldehyde solutions. Peracetic acid is an oxidizing agent that sterilizes scaffolds through immersion in peracetic acid solutions at controlled concentrations and exposure times. Glutaraldehyde is an aldehyde compound that crosslinks proteins and nucleic acids of microorganisms, achieving sterilization through immersion in glutaraldehyde solutions. Formaldehyde is an aldehyde compound that sterilizes scaffolds through exposure to formaldehyde vapor or immersion in formaldehyde solutions. Following chemical sterilization, the scaffold is rinsed with sterile water or buffer solutions to remove residual chemical sterilants prior to use in cell culture applications.

[0122] In an embodiment, the scaffold is sterilized using steam. Steam sterilization, also referred to as autoclaving, exposes the scaffold to saturated steam under pressure at elevated temperatures, typically 121 degrees Celsius for 15 to 30 minutes or 134 degrees Celsius for shorter durations. The combination of heat and moisture denatures proteins and destroys microorganisms throughout the scaffold structure. Steam sterilization is effective for scaffolds composed of materials that withstand the elevated temperatures and moisture conditions of the autoclaving process. The scaffold is placed within autoclave-compatible containers or wrapped in autoclave-compatible materials prior to steam sterilization.

[0123] In an embodiment, the scaffold is sterilized using gamma irradiation. Gamma irradiation exposes the scaffold to ionizing radiation from a gamma-emitting source, ty pically cobalt-60 or cesium-137. Gamma radiation penetrates the scaffold structure and damages the DNA of microorganisms, achieving sterilization throughout the scaffold volume including the interior porous regions. The scaffold is exposed to a radiation dose sufficient to achieve the desired sterility assurance level, typically in the range of 15 to 35 kiloGray for medical and cell culture applications. Gamma irradiation provides a terminal sterilization method that sterilizes the scaffold within sealed packaging, maintaining sterility until the packaging is opened for use.

[0124] In an embodiment, the scaffold is exposed to a gamma irradiation dose of 15 to 20 kiloGray. In an embodiment, the scaffold is exposed to a gamma irradiation dose of 20 to25 kiloGray. In an embodiment, the scaffold is exposed to a gamma irradiation dose of 25 to 30 kiloGray. In an embodiment, the scaffold is exposed to a gamma irradiation dose of 30 to 35 kiloGray.

[0125] The scaffolds are sterilized prior to introduction into a bioreactor, or the scaffolds are sterilized in situ within the bioreactor. Sterilization prior to bioreactor introduction involves sterilizing the scaffold using one of the methods described above, followed by aseptic transfer of the sterile scaffold into a pre-sterilized bioreactor. Sterilization in situ involves introducing the scaffold into the bioreactor and sterilizing the scaffold and bioreactor together, such as through steam sterilization of the assembled bioreactor system. The selection between pre-sterilization and in situ sterilization depends on the bioreactor configuration, scaffold material compatibility with the sterilization method, and the requirements of the cell culture process.Multilayer Tissue Assembly and Adhesive Binders

[0126] The cellular scaffolds described herein are used for spontaneous microtissue adhesion to create structured, multicellular constructs. Spontaneous microtissue adhesion occurs when scaffold surfaces and cellular components adhere to one another through natural biological and physicochemical interactions without requiring external mechanical fixation. The nanofibrous texture of the scaffold surfaces promotes adhesion between adjacent scaffold pieces and between scaffolds and cellular components, enabling the formation of stable, multilayered tissues suitable for cultivated meat production.

[0127] Multilayered tissue constructs are formed by incorporating layers of hydrogel between scaffold layers. Hydrogel layers provide a hydrated matrix that supports cell viability, nutrient diffusion, and mechanical integration between adjacent scaffold layers. The hydrogel is deposited onto a scaffold surface, and a second scaffold layer is placed onto the hydrogel layer to create a laminated structure. The hydrogel adheres to the nanofibrous surfaces of both scaffold layers, creating a stable bond between the layers. In an embodiment,the hydrogel comprises alginate, pectin, gellan gum, or other polysaccharide-based gels that are crosslinked to provide mechanical stability to the multilayered construct.

[0128] Multilayered tissue constructs are formed by incorporating layers of cell-laden hydrogel between scaffold layers. Cell-laden hydrogel comprises hydrogel material containing suspended cells that are distributed throughout the gel matrix. The cell-laden hydrogel is prepared by mixing cells with a hydrogel precursor solution prior to gelation, resulting in a hydrogel layer with cells uniformly distributed throughout the gel volume. The cell-laden hydrogel layer is deposited onto a scaffold surface, and a second scaffold layer is placed onto the cell-laden hydrogel layer. The cells within the hydrogel layer proliferate and migrate during culture, integrating with cells on the adjacent scaffold surfaces to form contiguous tissue structures spanning multiple scaffold layers.

[0129] Multilayered tissue constructs are formed by incorporating pure cell layers between scaffold layers. Pure cell layers comprise concentrated cell suspensions or cell sheets that are deposited directly onto scaffold surfaces w ithout a hydrogel carrier. The pure cell layer is deposited onto a scaffold surface by pipetting a concentrated cell suspension onto the scaffold, by transferring a pre-formed cell sheet onto the scaffold surface, or by allowing cells to settle onto the scaffold surface from a cell suspension. A second scaffold layer is placed onto the pure cell layer, sandwiching the cells between the two scaffold surfaces. The cells adhere to both scaffold surfaces and proliferate to fill the space betw een the scaffold layers, creating a tissue layer that mechanically integrates the adjacent scaffold layers.

[0130] Multilayered tissue constructs are formed by incorporating microcarriers between scaffold layers. Microcarriers with cells cultured on the microcarrier surfaces are deposited onto a scaffold surface, and a second scaffold layer is placed onto the microcarrier layer. The microcarriers provide additional surface area for cell attachment within the multilayered construct and contribute to the cellular content of the final cultivated meat product. The cells on the microcarrier surfaces proliferate and migrate onto the adjacent scaffold surfaces, integrating the microcarriers with the scaffold layers to form a cohesive tissue structure.

[0131] Additional adhesives or binders are used to create structured multicellular constructs with enhanced mechanical stability. The adhesives or binders are applied at the interfaces between scaffold layers, between scaffolds and hydrogel layers, or between scaffolds and cellular components to promote adhesion and maintain structural integrity7during cell culture and subsequent processing.

[0132] In an embodiment, starch is used as an adhesive or binder for creating structured multicellular constructs. Starch is a polysaccharide composed of glucose units that provides adhesive properties when hydrated and gelatinized. Starch is applied as a paste or solution at the interface between scaffold layers, where the starch adheres to the nanofibrous surfaces and creates a bond between adjacent layers upon drying or cooling. The edible nature of starch maintains the food-safe status of the cellular scaffold and contributes to the carbohydrate content of the final cultivated meat product.

[0133] In an embodiment, pectin is used as an adhesive or binder for creating structured multicellular constructs. Pectin is a polysaccharide found in plant cell walls that forms gels in the presence of calcium ions or under acidic conditions with high sugar concentrations. Pectin is applied as a solution at the interface between scaffold layers, and gelation is induced by the addition of calcium ions or by adjusting the pH and sugar content. The pectin gel adheres to the scaffold surfaces and creates a stable bond between adjacent layers. Pectin provides gelling and adhesive properties while maintaining the edible and animal-component free status of the cellular scaffold.

[0134] In an embodiment, transglutaminase is used as an adhesive or binder for creating structured multicellular constructs. Transglutaminase is an enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues in proteins.Transglutaminase is applied at the interface between scaffold layers containing protein-based nanofibers such as zein, where the enzyme catalyzes crosslinking reactions that covalently bond adjacent scaffold layers together. The enzymatic crosslinking provides strong, stable bonds between scaffold layers that withstand mechanical stresses during cell culture andbioreactor agitation. Transglutaminase is commonly used in food processing applications and maintains the food-safe status of the cellular scaffold.

[0135] In an embodiment, methylcellulose is used as an adhesive or binder for creating structured multicellular constructs. Methylcellulose is a cellulose derivative that dissolves in cold water and forms gels upon heating. Methylcellulose is applied as a cold solution at the interface between scaffold layers, and the assembly is heated to induce gelation of the methylcellulose layer. The methylcellulose gel adheres to the scaffold surfaces and creates a thermally reversible bond between adjacent layers. The thermal gelation behavior of methylcellulose allows for controlled assembly of multilayered constructs, as the bond forms upon heating and the construct is disassembled by cooling if repositioning of layers is required.

[0136] The adhesives and binders described above are used individually or in combination with one another to achieve desired adhesive strength and structural properties in the multilayered tissue construct. Combinations of adhesives and binders provide synergistic effects that enhance bond strength, flexibility, and stability of the assembled construct. In an embodiment, transglutaminase is combined with starch or pectin to provide both enzymatic crosslinking and polysaccharide-based adhesion at the interface between scaffold layers.

[0137] The multilayered tissue constructs formed using the scaffold, hydrogel layers, cell-laden hydrogel layers, pure cell layers, microcarriers, and adhesive binders described herein provide structured cultivated meat products with controlled layer composition and thickness. The layered architecture enables the creation of cultivated meat products that mimic the layered structure of native muscle tissue, with distinct regions of muscle cells, fat cells, and connective tissue components distributed throughout the product thickness.Scaffold Storage and Shipping

[0138] The cellular scaffolds described herein are shipped and stored under various conditions depending on the scaffold composition and the additional ingredients incorporated into the scaffold fibers. The shipping and storage conditions are selected to maintain the structural integrity, sterility', and functional properties of the scaffold until use in cell culture applications.

[0139] In an embodiment, the scaffold is shipped without temperature controls. Scaffolds composed of stable materials such as zein and cellulose acetate, particularly when crosslinked and freeze-dried, withstand ambient temperature conditions during shipping without degradation of the scaffold structure or loss of functional properties. The absence of temperature control requirements during shipping reduces logistics costs and simplifies the supply chain for scaffold distribution. Scaffolds shipped without temperature controls are packaged in moisture-resistant containers to prevent rehydration during transit and to maintain the dry, stabilized state achieved through freeze-drying.

[0140] In an embodiment, the scaffold is shipped at 0-4 degrees Celsius for temperature-controlled storage. Temperature-controlled shipping at refrigerated conditions preserves scaffold properties for scaffolds containing temperature-sensitive components such as encapsulated flavorings, bioactive compounds, or labile additives. Refrigerated shipping maintains the scaffold at temperatures that slow degradation reactions and preserve the activity of incorporated bioactive substances. Scaffolds shipped under temperature-controlled conditions are packaged with insulated containers and cooling elements to maintain the 0-4 degrees Celsius temperature range throughout the shipping duration.

[0141] Additional factors that influence shipping requirements include the need for light protection, the use of bioprocess bags, transfer bottles, and food-grade shipping containers. Scaffolds containing light-sensitive components are packaged in opaque or light-blocking containers to prevent photodegradation during shipping and storage. Bioprocess bags provide sterile, flexible containers for shipping scaffolds that have been pre-sterilized and require maintenance of sterility until use. Transfer bottles provide rigid containers for shipping scaffolds in fluid storage conditions. Food-grade shipping containers ensure that the scaffoldpackaging materials are compatible with food safety requirements and do not introduce contaminants that would compromise the edible status of the scaffold.

[0142] The scaffold is stored in dry storage conditions. Dry' storage involves maintaining the freeze-dried scaffold in a low-humidity environment to prevent rehydration and maintain the stabilized three-dimensional porous architecture. Dry-stored scaffolds are packaged in sealed containers with desiccants to absorb residual moisture and maintain low humidity within the storage container. Dry’ storage provides extended shelf life for the scaffold, as the absence of water inhibits microbial grow th and slows chemical degradation reactions. Dry-stored scaffolds are rehydrated prior to use in cell culture applications.

[0143] The scaffold is stored in fluid storage conditions. Fluid storage involves maintaining the scaffold in a liquid medium that preserves the scaffold structure and properties. Fluid storage is suitable for scaffolds that have been rehydrated following freeze-drying or for scaffolds that are fabncated and stored in a hydrated state without undergoing freeze-dry ing. The fluid storage medium is selected based on compatibility w ith the scaffold materials and the requirements of the subsequent cell culture application. Fluid-stored scaffolds are maintained under sterile conditions to prevent microbial contamination during storage.

[0144] The scaffold is stored with hydration buffer options that provide controlled hydration and stabilization of the scaffold structure. Hydration buffers are aqueous solutions containing salts, buffers, and other components that maintain the scaffold in a hydrated state while preserving structural integrity and functional properties. The hydration buffer composition, storage duration, and storage temperature are selected based on the scaffold composition and the requirements of the cell culture application.

[0145] In an embodiment, the scaffold is hydrated in a storage buffer comprising buffered saline and calcium chloride. Buffered saline provides an isotonic aqueous environment that maintains the hydration state of the scaffold without causing osmotic stress to the scaffold materials. Calcium chloride provides calcium ions that crosslink and stabilize scaffold polymers containing anionic groups, such as alginate components within the scaffoldstructure. The calcium ions form ionic crosslinks with carboxylate groups on alginate chains, creating a crosslinked gel network that provides mechanical stability to the scaffold. The scaffold hydrated in buffered saline and calcium chloride storage buffer is stored at room temperature or refrigerated depending on the storage duration and scaffold composition.

[0146] In an embodiment, the scaffold hydrated in storage buffer is stored at room temperature. Room temperature storage is suitable for scaffolds with stable compositions that do not require refrigeration to maintain structural integrity and functional properties. Room temperature storage simplifies storage logistics and reduces energy7requirements compared to refrigerated storage. The storage duration at room temperature depends on the scaffold composition, the presence of preservatives or antimicrobial agents in the storage buffer, and the sterility requirements of the cell culture application.

[0147] In an embodiment, the scaffold hydrated in storage buffer is stored refrigerated. Refrigerated storage at temperatures between 0 and 4 degrees Celsius extends the storage duration by slowing degradation reactions and inhibiting microbial growth. Refrigerated storage is suitable for scaffolds containing temperature-sensitive components or for extended storage durations where room temperature storage would result in unacceptable degradation of scaffold properties.

[0148] Sterile hydration buffer is added into a sterile transport container, and hydration takes place inside the transport container. The scaffold is introduced into the sterile transport container containing the hydration buffer, and the scaffold absorbs the buffer solution to achieve the hydrated state. Hydration within the transport container maintains sterility throughout the hydration process and eliminates the need for aseptic transfer of the hydrated scaffold to a separate storage container.

[0149] Hydration takes place inside the bioreactor after the addition of the scaffold. The dry7scaffold is introduced into the bioreactor, and hydration buffer or cell culture media is added to the bioreactor to hydrate the scaffold in situ. Hydration within the bioreactor eliminates the need for separate hydration and transfer steps, reducing the risk ofcontamination during scaffold handling. The hydration duration within the bioreactor is controlled to ensure complete hydration of the scaffold structure prior to cell seeding.

[0150] Hydration is performed before or after the sterilization step. In an embodiment, hydration is performed after the sterilization step to maintain the sterility achieved during sterilization. Hydration after sterilization involves the use of sterile hydration buffers and aseptic handling techniques to prevent contamination of the sterile scaffold. In an embodiment, hydration is performed before the sterilization step, such as in the case of steam sterilization where the hydrated scaffold is sterilized by autoclaving. Steam sterilization of the hydrated scaffold achieves sterilization while maintaining the hydrated state of the scaffold.

[0151] The scaffold is hydrated directly with cell culture media if the system and media formulation are compatible. Direct hydration with cell culture media eliminates the need for a separate hydration buffer and allows the scaffold to equilibrate to the cell culture conditions during the hydration process. The cell culture media provides the nutrients, growth factors, and buffering capacity required for subsequent cell culture while serving as the hydration medium for the scaffold. Direct hydration with cell culture media is suitable for scaffolds composed of materials that are stable in the cell culture media formulation and do not require ionic crosslinking or other stabilization treatments prior to cell seeding.

[0152] Hydration is performed with a fluid containing cations or anions to crosslink and stabilize certain scaffold polymers. The cations or anions form ionic crosslinks with charged groups on the scaffold polymer chains, creating a crosslinked network that provides mechanical stability and resistance to dissolution in the cell culture environment. The ionic crosslinking stabilizes scaffold polymers such as alginates that contain carboxylate groups capable of forming ionic bonds with divalent or trivalent cations.

[0153] In an embodiment, hydration is performed with a fluid containing calcium salts at a concentration of 0.1 micromolar to 1 millimolar. Calcium ions are divalent cations that crosslink alginate and other anionic polysaccharides through ionic interactions with carboxylate groups. The calcium salt concentration is selected to achieve the desired degreeof crosslinking without causing excessive gelation that would impede cell infdtration or nutrient diffusion within the scaffold. Lower calcium concentrations within the 0.1 micromolar to 1 millimolar range provide moderate crosslinking that maintains scaffold flexibility, while higher concentrations within this range provide stronger crosslinking and increased mechanical stability.

[0154] In an embodiment, the calcium salt concentration is 0.1 micromolar to 1 micromolar. In an embodiment, the calcium salt concentration is 1 micromolar to 10 micromolar. In an embodiment, the calcium salt concentration is 10 micromolar to 100 micromolar. In an embodiment, the calcium salt concentration is 100 micromolar to 1 millimolar.

[0155] The fluid containing cations or anions for ionic crosslinking is incorporated into the cell culture media, or the fluid is drained and removed and exchanged with cell culture media prior to cell culture. Incorporation of the ionic crosslinking fluid into the cell culture media provides continuous exposure of the scaffold to crosslinking ions throughout the cell culture process, maintaining the crosslinked state of the scaffold polymers. Draining and exchanging the ionic crosslinking fluid with cell culture media removes excess crosslinking ions that could interfere with cell behavior or media composition while retaining the crosslinked scaffold structure achieved during the initial hydration step.

[0156] The addition of proteins to the hydration fluid aids in functionalization of the scaffold surface. Proteins such as fibronectin, laminin, collagen, or recombinant cell adhesion proteins adsorb onto the scaffold surface during hydration, providing cell attachment sites that promote cell adhesion and spreading on the scaffold. The protein-containing hydration fluid is incorporated into the cell culture media, or the fluid is drained and exchanged with cell culture media prior to cell seeding while retaining the adsorbed protein layer on the scaffold surface.Bioreactor Systems and Configurations

[0157] The cellular scaffolds described herein are cultured in bioreactor systems that provide controlled environments for cell proliferation, differentiation, and tissue formation. Bioreactor systems maintain the temperature, pH, dissolved oxygen, and nutrient concentrations required for cell culture while providing mixing or agitation to ensure uniform distribution of nutrients and oxygen throughout the culture volume. The bioreactor configuration is selected based on the scaffold format, the scale of production, and the requirements of the cell culture process.

[0158] In an embodiment, the bioreactor is a spinner flask ty pe. Spinner flask bioreactors comprise a vessel with a central magnetic stir bar or suspended impeller that provides gentle agitation of the culture medium. The agitation maintains scaffolds and microcarriers in suspension and promotes mass transfer of nutrients and oxygen to cells on the scaffold surfaces. Spinner flask bioreactors are suitable for initial process development and small-scale production of cultivated meat on cellular scaffolds.

[0159] In an embodiment, the bioreactor is a stirred tank ty pe. Stirred tank bioreactors comprise a vessel with one or more impellers mounted on a central shaft that rotates to provide mixing of the culture medium. The impeller design, rotation speed, and vessel geometry7are configured to achieve desired mixing characteristics and shear stress profiles within the culture volume. Stirred tank bioreactors provide scalable platforms for cultivated meat production, with designs available across a range of working volumes from laboratory' scale to industrial production scale.

[0160] In an embodiment, the bioreactor is a packed bed ty pe. Packed bed bioreactors comprise a column or vessel filled with scaffold material through which culture medium is perfused. The scaffolds remain stationary' within the packed bed while culture medium flows through the interstitial spaces between scaffold pieces, delivering nutrients and oxygen to cells on the scaffold surfaces and removing metabolic waste products. Packed bed bioreactors provide high surface area for cell attachment relative to the bioreactor volume and are suitable for monolithic scaffold configurations where the scaffold is fixed in place during culture.

[0161] In an embodiment, the bioreactor is a fluidized bed type. Fluidized bed bioreactors comprise a column or vessel in which scaffolds or microcarriers are suspended by upward flow of culture medium. The flow rate is controlled to maintain the scaffolds or microcarriers in a fluidized state where the particles are suspended but not carried out of the vessel. Fluidized bed bioreactors provide efficient mass transfer and uniform exposure of scaffold surfaces to culture medium while allowing for continuous or semi-continuous operation.

[0162] In an embodiment, the bioreactor is an airlift type. Airlift bioreactors comprise a vessel with a central draft tube or riser section through which gas bubbles are introduced. The rising gas bubbles create circulation of culture medium within the vessel, with medium flowing upward through the riser section and downward through the surrounding downcomer section. Airlift bioreactors provide gentle mixing with low shear stress, making airlift bioreactors suitable for culturing shear-sensitive cells on scaffolds. The gas bubbles also provide oxygenation of the culture medium.

[0163] In an embodiment, the bioreactor is a rotating w all type. Rotating wall bioreactors comprise a cylindrical vessel that rotates around a horizontal axis, creating a low-shear environment where scaffolds and cells experience simulated microgravity conditions. The rotation of the vessel wall suspends scaffolds and microcarriers in the culture medium while minimizing mechanical stress on the cells. Rotating wall bioreactors promote three-dimensional tissue formation and are suitable for culturing cells on scaffolds where low shear stress is beneficial for cell differentiation and tissue maturation.

[0164] In an embodiment, the bioreactor is a spinning basket type. Spinning basket bioreactors comprise a vessel with a rotating basket or cage that holds scaffolds within the culture medium. The rotation of the basket provides mixing of the culture medium and exposes the scaffold surfaces to fresh medium while retaining the scaffolds within the basket structure. Spinning basket bioreactors are suitable for monolithic scaffold configurations where the scaffolds are too large to remain suspended by fluid flow alone.

[0165] In an embodiment, the bioreactor is a rotary wheel type. Rotary wheel bioreactors comprise a wheel-shaped vessel that rotates around a horizontal axis, with scaffolds attached to the inner surface of the wheel or suspended within the wheel volume. The rotation of the wheel alternately exposes the scaffolds to culture medium and gas phases, providing oxygenation and nutrient delivery to cells on the scaffold surfaces. Rotary wheel bioreactors are suitable for culturing cells on scaffolds where alternating exposure to liquid and gas phases promotes tissue development.

[0166] In an embodiment, the bioreactor is a hollow fiber type. Hollow fiber bioreactors comprise a bundle of semi -permeable hollow fibers through which culture medium is perfused. Cells are cultured on the outer surfaces of the hollow fibers or within the extracapillary space surrounding the fiber bundle. The hollow fiber membranes provide a large surface area for nutrient and oxygen exchange while separating the cells from the bulk culture medium. Hollow fiber bioreactors are suitable for high-density cell culture applications where efficient mass transfer is required.

[0167] In an embodiment, the bioreactor is a shake flask type. Shake flask bioreactors comprise flasks placed on orbital shaker platforms that provide agitation through circular motion. The orbital shaking creates swirling of the culture medium within the flask, maintaining scaffolds and microcarriers in suspension and promoting mass transfer. Shake flask bioreactors are suitable for initial process development, screening of culture conditions, and small-scale production of cultivated meat on cellular scaffolds.

[0168] In an embodiment, the bioreactor is a rocking motion wave bag type. Rocking motion wave bag bioreactors comprise a flexible bag placed on a rocking platform that tilts back and forth to create wave-like motion of the culture medium within the bag. The rocking motion provides gentle mixing with low shear stress and promotes oxygenation through the liquid-gas interface at the medium surface. Wave bag bioreactors are disposable systems that eliminate the need for cleaning and sterilization between batches, reducing turnaround time and contamination risk.

[0169] The bioreactor is small, medium, or large in size depending on the scale of cultivated meat production. Small bioreactors have a working volume of less than 10 liters and are suitable for research and development, process optimization, and small-scale production. Medium bioreactors have a working volume of 10 to 200 liters and are suitable for pilot-scale production, early -stage manufacturing, and supplying small-scale product for research and product design. Large bioreactors have a working volume of 200 to 250,000 liters and are suitable for commercial-scale manufacturing of cultivated meat products.

[0170] The bioreactor is reusable or disposable depending on the production requirements and operational considerations. Reusable bioreactors comprise vessels constructed from stainless steel, glass, or other durable materials that are cleaned and sterilized between batches. Reusable bioreactors provide lower per-batch costs for high-volume production but require cleaning validation and sterilization infrastructure. Disposable bioreactors comprise single-use vessels, bags, or containers that are discarded after each batch. Disposable bioreactors eliminate cleaning and sterilization requirements, reduce contamination risk, and provide flexibility for multi-product facilities. In an embodiment, disposable bioreactors comprise pre-sterilized bags or containers that are supplied ready for use and are disposed of following harvest of the cultivated meat product.

[0171] In an embodiment, the large bioreactor has a working volume of 200 to 1,000 liters. In an embodiment, the large bioreactor has a working volume of 1,000 to 10,000 liters. In an embodiment, the large bioreactor has a working volume of 10,000 to 50,000 liters. In an embodiment, the large bioreactor has a working volume of 50,000 to 100,000 liters. In an embodiment, the large bioreactor has a working volume of 100,000 to 250,000 liters.

[0172] Combinations of bioreactor types are used in cultivated meat production processes. In an embodiment, microcarriers are cultured in spinner flasks or shake flasks for initial process development, then transferred to stirred tank bioreactors for scale-up and manufacturing. In an embodiment, monolithic scaffolds are cultured in packed bed or spinning basket bioreactors where the scaffolds remain fixed in place while culture medium circulates through the scaffold structure. The selection of bioreactor type and size is based onthe scaffold format, cell type, production scale, and desired characteristics of the cultivated meat product.Scaffold Movement and Positioning in Bioreactors

[0173] The cellular scaffolds described herein are positioned and moved within bioreactors according to various configurations that support cell culture and tissue formation. The scaffold is moved through the cell culture media in the bioreactor, or the scaffold is held static while culture media fluid circulates through the scaffold. The selection between moving and static scaffold configurations depends on the scaffold format, bioreactor type, and requirements of the cell culture process.

[0174] In an embodiment, the scaffold is moved through the cell culture media in the bioreactor. Moving the scaffold through the culture media exposes the scaffold surfaces to fresh nutrients and oxygen while facilitating removal of metabolic waste products from the vicinity of the cells. The movement of the scaffold is achieved through impeller-driven agitation, rocking motion, rotation of the bioreactor vessel, or fluid flow within the bioreactor. The rate and pattern of scaffold movement are controlled to achieve desired mass transfer characteristics while maintaining shear stress levels that are compatible with cell viability and differentiation. Microcarrier scaffolds with dimensions less than 300 micrometers move freely within the bioreactor via fluid flow or energy imparted by the agitation device, impeller, or similar mechanism.

[0175] In an embodiment, the scaffold is held static while culture media fluid circulates through the scaffold. Static scaffold positioning maintains the scaffold in a fixed location within the bioreactor while culture media is perfused through the scaffold structure or circulated around the scaffold surfaces. Perfusion of culture media through the scaffold delivers nutrients and oxygen to cells within the interior regions of the three-dimensional porous structure while removing metabolic waste products. The perfusion rate is controlled to achieve adequate mass transfer throughout the scaffold volume without generating excessiveshear stress on the cells. Static scaffold positioning is suitable for monolithic scaffold configurations where the scaffold dimensions are similar to the dimensions of the whole meat product being produced.

[0176] The scaffold is fixed in place relative to the agitation mechanism in the bioreactor. Fixing the scaffold in place involves securing the scaffold to a support structure, holder, or fixture within the bioreactor that maintains the scaffold position during agitation of the culture media. The support structure positions the scaffold within the flow field generated by the agitation mechanism, ensuring that culture media circulates around and through the scaffold structure. In an embodiment, the scaffold is attached to a mesh, frame, or basket that holds the scaffold within the bioreactor while allowing culture media to flow through the scaffold. The fixed positioning of the scaffold relative to the agitation mechanism provides consistent exposure of the scaffold surfaces to culture media flow throughout the culture period.

[0177] The scaffold is immobilized in place within the bioreactor. Immobilization involves securing the scaffold to prevent movement during cell culture without necessarily positioning the scaffold relative to a specific agitation mechanism. In an embodiment, the scaffold is immobilized by attachment to the bioreactor vessel wall, by placement within a compartment or chamber within the bioreactor, or by compression between support surfaces that hold the scaffold in position. Immobilization of the scaffold prevents damage to the scaffold structure and developing tissue that could result from collision with bioreactor components or other scaffolds during agitation.

[0178] The scaffold rests freely in the bioreactor. Resting freely involves placing the scaffold within the bioreactor without attachment to support structures or immobilization fixtures, allowing the scaffold to settle to the bottom of the bioreactor vessel or to float within the culture media depending on the scaffold density and buoyancy. In an embodiment, monolithic scaffolds with dimensions greater than 300 micrometers rest at the bottom of the bioreactor vessel while culture media is circulated through the vessel by agitation or perfusion. The scaffold remains stationary' due to the scaffold mass and dimensions whileculture media flows around and through the scaffold structure. Resting freely provides a simple scaffold positioning approach that does not require specialized fixtures or attachment mechanisms.

[0179] The scaffold is cultured in well plates for initial process development. Well plate culture involves seeding cells onto scaffolds placed within the wells of multi-well culture plates, such as 6-well, 12-well, 24-well, or 96-well plates. Well plate culture provides a platform for screening scaffold compositions, cell seeding densities, culture media formulations, and other process parameters at small scale before committing to larger-scale bioreactor culture. The small culture volumes in well plates reduce the consumption of cells and culture media during process development while enabling parallel testing of multiple conditions.

[0180] The scaffold is cultured in shake flasks for initial process development. Shake flask culture involves placing scaffolds and culture media in flasks positioned on orbital shaker platforms that provide agitation through circular motion. Shake flask culture provides larger culture volumes than well plates while maintaining the simplicity and low cost of flask-based culture systems. The orbital shaking maintains microcarrier scaffolds in suspension and promotes mass transfer of nutrients and oxygen to cells on the scaffold surfaces.

[0181] The scaffold is cultured in spinner flasks for initial process development. Spinner flask culture involves placing scaffolds and culture media in vessels equipped with magnetic stir bars or suspended impellers that provide gentle agitation. Spinner flask culture provides controlled agitation conditions that are more representative of stirred tank bioreactor environments than static well plate culture or orbital shaking. The agitation in spinner flasks maintains microcarrier scaffolds in suspension and provides mixing that promotes uniform distribution of nutrients and oxygen throughout the culture volume.

[0182] Following initial process development in well plates, shake flasks, or spinner flasks, the scaffold is transferred to a bioreactor for scale up. Transfer to a bioreactor involves moving the scaffold, along with attached cells and developing tissue, from the processdevelopment culture vessel to a larger bioreactor system for continued culture at increased scale. In an embodiment, microcarrier scaffolds cultured in spinner flasks are transferred to stirred tank bioreactors by aseptically pumping or pouring the microcarrier suspension into the bioreactor vessel. In an embodiment, monolithic scaffolds cultured in well plates or shake flasks are transferred to packed bed or spinning basket bioreactors by aseptically placing the scaffold into the bioreactor scaffold holder or basket. The transfer to bioreactor enables scale up of cultivated meat production from laboratory-scale process development to pilot-scale and commercial-scale manufacturing.Cell Culture Methods and Media

[0183] The cellular scaffolds described herein are used in methods of culturing cells for cultivated meat production. A method of culturing cells for cultivated meat production comprises providing a cellular scaffold comprising at least one nanofiber arranged in a three-dimensional porous structure formed by gas expansion, wherein the cellular scaffold is edible and animal-component free as described previously. The method further comprises introducing the cellular scaffold into a bioreactor containing cell culture media, seeding cells onto the cellular scaffold, and culturing the cells on the cellular scaffold to produce cultivated meat tissue.

[0184] Introducing the cellular scaffold into a bioreactor containing cell culture media establishes the culture environment for cell attachment and proliferation. The bioreactor is pre-sterilized and filled with cell culture media prior to scaffold introduction, or the cell culture media is added to the bioreactor following scaffold introduction. The cell culture media provides nutrients, growth factors, and buffering capacity required for cell viability and proliferation. The scaffold equilibrates to the cell culture media conditions, including temperature, pH, and osmolarity7, prior to cell seeding. In an embodiment, the scaffold is hydrated with cell culture media during introduction into the bioreactor, allowing the scaffold to absorb media and reach equilibrium with the culture environment.

[0185] Seeding cells onto the cellular scaffold involves introducing cells into the bioreactor and distributing the cells onto the scaffold surfaces. The cells are delivered directly onto the scaffold by pipetting or dispensing a cell suspension onto the scaffold surface, or the cells are introduced into the bioreactor and distributed onto the scaffolds via fluid flow or mixing. The cell seeding density is selected based on the scaffold surface area, the cell type, and the desired rate of tissue formation. Higher seeding densities result in faster confluence and tissue formation, while lower seeding densities allow for greater cell proliferation before confluence is achieved. In an embodiment, components are added in a temporal manner to induce cell adhesion following cell seeding, promoting attachment of the seeded cells to the scaffold surfaces.

[0186] Culturing the cells on the cellular scaffold to produce cultivated meat tissue involves maintaining the seeded scaffold in the bioreactor under conditions that support cell proliferation, differentiation, and tissue maturation. The culture conditions include controlled temperature, typically 37 degrees Celsius for mammalian cells, controlled pH maintained through buffering and carbon dioxide supplementation, and controlled dissolved oxygen levels maintained through aeration or oxygenation of the culture media. The culture duration depends on the cell type, scaffold configuration, and desired characteristics of the cultivated meat tissue. Culture durations range from several days for initial cell attachment and proliferation to several weeks for tissue maturation and differentiation.

[0187] The cells comprise one or more cell types selected from the group consisting of myoblasts, mesangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, and endothelial cells. The selection of cell types depends on the desired composition and characteristics of the cultivated meat tissue.

[0188] In an embodiment, the cells seeded onto and cultured on the cellular scaffold comprise a co-culture of two or more cell types. By way of example, the co-culture may comprise myoblasts and adipocytes, myoblasts and fibroblasts, myoblasts and endothelial cells, or combinations thereof, thereby enabling formation of cultivated meat tissues havingmuscle, fat, and / or connective tissue features and improved structure and organoleptic properties.

[0189] Myoblasts are muscle precursor cells that proliferate and differentiate into myocytes and myotubes, which are the primary cellular components of skeletal muscle tissue. Myoblasts fuse to form multinucleated myotubes that express muscle-specific proteins including myosin heavy chain, which contributes to the texture and protein content of cultivated meat products. The differentiation of myoblasts into myotubes on the cellular scaffold produces muscle tissue that mimics the structure and composition of native meat.

[0190] Mesangioblasts are vessel-associated stem cells with myogenic potential that differentiate into skeletal muscle cells. Mesangioblasts provide an alternative source of muscle precursor cells for cultivated meat production and contribute to the regenerative capacity of the developing tissue.

[0191] Myofibroblasts are cells with characteristics of both fibroblasts and smooth muscle cells that produce extracellular matrix components and provide contractile function. Myofibroblasts contribute to the structural integrity and texture of cultivated meat tissue through the production of collagen and other matrix proteins.

[0192] Mesenchymal stem cells are multipotent stromal cells that differentiate into multiple cell lineages including adipocytes, chondrocytes, and osteoblasts. Mesenchymal stem cells provide a versatile cell source for cultivated meat production, as the differentiation of mesenchymal stem cells is directed toward specific lineages through the application of appropriate culture conditions and differentiation factors.

[0193] Hepatocytes are liver parenchymal cells that perform metabolic functions and produce proteins. In an embodiment, hepatocytes are included in cultivated meat tissue to provide specific nutritional or functional properties associated with liver tissue.

[0194] Fibroblasts are connective tissue cells that produce extracellular matrix components including collagen, elastin, and glycosaminoglycans. Fibroblasts contribute tothe structural framework of cultivated meat tissue and provide mechanical support for muscle and fat cells within the tissue construct.

[0195] Pericytes are cells associated with blood vessel walls that have stem cell-like properties and contribute to tissue regeneration and vascular function. Pericytes differentiate into multiple cell ty pes and contribute to the cellular diversity of cultivated meat tissue.

[0196] Adipocytes are fat cells that store lipids and contribute to the flavor, texture, and mouthfeel of meat products. The inclusion of adipocytes in cultivated meat tissue provides the marbling and fat content that characterize high-quality' meat products. Adipocytes are derived from preadipocytes or from mesenchymal stem cells induced to undergo adipogenic differentiation.

[0197] Epithelial cells are cells that form the lining of tissues and organs and provide barrier and secretory functions. In an embodiment, epithelial cells are included in cultivated meat tissue to provide specific structural or functional properties.

[0198] Chondrocytes are cartilage cells that produce the extracellular matrix of cartilage tissue. In an embodiment, chondrocytes are included in cultivated meat tissue to provide cartilaginous components that contribute to the texture and structure of certain meat products.

[0199] Osteoblasts are bone-forming cells that produce the mineralized matrix of bone tissue. Osteoclasts are bone-resorbing cells that break down bone matrix. In an embodiment, osteoblasts and osteoclasts are included in cultivated meat tissue to provide bone-like components for specific meat product applications.

[0200] Pluripotent cells are cells with the capacity to differentiate into any' cell type of the body. Pluripotent cells include embryonic stem cells and induced pluripotent stem cells. Pluripotent cells provide a source of cells that are directed to differentiate into specific cell ty pes for cultivated meat production through the application of appropriate differentiation protocols.

[0201] Somatic stem cells are tissue-resident stem cells with the capacity’ to differentiate into the cell types of the tissue in which the somatic stem cells reside. Somatic stem cellsinclude satellite cells of skeletal muscle, which are muscle-resident stem cells that proliferate and differentiate into myoblasts and myocytes. Somatic stem cells provide a source of tissuespecific progenitor cells for cultivated meat production.

[0202] Endothelial cells are cells that line blood vessels and provide vascular function. In an embodiment, endothelial cells are included in cultivated meat tissue to provide vascular structures that support nutrient delivery and tissue organization within thick tissue constructs.

[0203] The cells cultured on the scaffold are from livestock, poultry, game, and aquatic animal species including vertebrate and invertebrate species. Livestock species include cattle, pigs, sheep, and goats, which provide cells for the production of beef, pork, lamb, and goat meat products. Poultry species include chickens, turkeys, ducks, and geese, which provide cells for the production of poultry meat products. Game species include deer, elk, bison, wild boar, and other wild animals, which provide cells for the production of game meat products. Aquatic animal species include fish such as salmon, tuna, tilapia, and catfish, as well as shellfish such as shrimp, crab, lobster, and mollusks, which provide cells for the production of seafood products.

[0204] Vertebrate species that provide cells for cultivated meat production include mammals, birds, reptiles, amphibians, and fish. Mammalian cells are obtained from livestock and game species as described above. Avian cells are obtained from poultry and game bird species. Fish cells are obtained from freshwater and marine fish species. In an embodiment, cells from reptile or amphibian species are used for cultivated meat production in regions where such meat products are consumed.

[0205] Invertebrate species that provide cells for cultivated meat production include crustaceans, mollusks, and insects. Crustacean cells are obtained from shrimp, crab, lobster, and crayfish species. Mollusk cells are obtained from oysters, mussels, clams, scallops, squid, and octopus species. In an embodiment, insect cells are used for cultivated meat production, providing protein-rich tissue for food applications.

[0206] The cells are obtained from donor animals through biopsy or tissue sampling procedures that do not require slaughter of the donor animal. The obtained cells are expanded in culture to generate sufficient cell numbers for cultivated meat production. The expanded cells are cryopreserved to create cell banks that provide a consistent source of cells for ongoing production. The use of cells from diverse animal species enables the production of cultivated meat products that replicate the taste, texture, and nutritional profile of conventional meat from the corresponding species.Serum-Free and Animal Component Media Options

[0207] The cell culture media used with the cellular scaffolds described herein is a serum-free growth medium. Serum-free growth medium eliminates the need for animal serum such as fetal bovine serum (FBS), which is derived from animal fetuses, animal blood, or animal tissue. The use of serum-free growth medium reduces reliance on animal slaughter, lowers production costs, and addresses ethical concerns associated with using animal-derived components in cell culture processes for cultivated meat production. Serum-free growth medium formulations contain defined components including amino acids, glucose, vitamins, inorganic salts, protein supplements, and growth factors that support cell attachment, proliferation, and differentiation on the cellular scaffold without requiring animal serum supplementation.

[0208] Serum-free grow th medium is formulated to provide the nutrients and signaling molecules that cells require for viability and function. The defined composition of serum-free growth medium provides batch-to-batch consistency that is difficult to achieve with serumcontaining media, as serum composition varies between lots and sources. The consistent composition of serum-free growth medium enables reproducible cell culture processes and cultivated meat production outcomes. Serum-free growth medium formulations are optimized for specific cell types and culture applications, with formulations available for myoblastproliferation, myogenic differentiation, adipocyte differentiation, and other cell culture processes relevant to cultivated meat production.

[0209] The cellular scaffolds described herein support cell adhesion in serum-free growth medium. Serum normally contains a mixture of proteins including fibronectin, vitronectin, and other cell adhesion molecules that promote cell attachment to culture surfaces. In the absence of serum, the scaffold surface provides cell attachment sites through the nanofibrous texture and surface chemistry of the scaffold materials, or through the addition of cell adhesion factors to the scaffold surface as described previously. The functionalization of the scaffold surface with cell adhesion factors enables cell attachment and spreading in serum-free growth medium, supporting cultivated meat production processes that do not rely on animal-derived serum components.

[0210] In an embodiment, the scaffold is used with animal component-containing growth medium including serum or other animal denved materials for research and development purposes. Animal component-containing grow th medium provides a well-characterized culture environment that supports robust cell growth and is suitable for initial process development, optimization of culture parameters, and characterization of scaffold performance. The use of animal component-containing growth medium during research and development enables the establishment of baseline cell culture conditions and scaffold performance metrics before transitioning to serum-free grow th medium formulations for commercial cultivated meat production.Cultivated Meat Tissue Composition and Density

[0211] The cultivated meat tissue produced using the cellular scaffolds described herein comprises at least 70% cultured cells or differentiated tissue by composition. The composition percentage refers to the proportion of the total mass of the cultivated meat product that consists of cells and tissues grown on the scaffold during the cell culture process, as opposed to the scaffold material itself or other non-cellular components. A cultivated meattissue comprising at least 70% cultured cells or differentiated tissue provides a product with cellular content comparable to conventional meat products harvested from animals, delivering the protein content, texture, and organoleptic properties expected by consumers of meat products.

[0212] The high cellular content of the cultivated meat tissue results from the three-dimensional porous structure of the cellular scaffold, which provides sufficient surface area for cell attachment while maintaining mass transfer of nutrients and oxygen throughout the scaffold volume. The gas expansion process described previously creates interconnected porosity that supports cell infiltration beyond the scaffold surface, enabling cells to populate the interior regions of the scaffold and achieve high volumetric cell densities. The combination of high surface area for cell attachment and efficient mass transfer throughout the scaffold volume enables the production of cultivated meat tissue with cellular content exceeding 70% of the total product composition.

[0213] In an embodiment, the cultivated meat tissue comprises at least 90% cultured cells or differentiated tissue by composition. A composition of at least 90% cultured cells or differentiated tissue provides a cultivated meat product with minimal scaffold content, where the scaffold serves primarily as a structural template during cell culture and contributes minimally to the final product mass. The high cellular content in this embodiment results from extended culture periods that allow cells to proliferate extensively on the scaffold surfaces and within the porous scaffold structure, combined with scaffold materials that are thin or low-density relative to the cellular mass accumulated during culture.

[0214] In an embodiment, the cultivated meat tissue comprises at least 5% cultured cells or differentiated tissue by composition. A composition of at least 5% cultured cells or differentiated tissue provides a cultivated meat product where the scaffold material constitutes a substantial portion of the final product mass. This embodiment is suitable for applications where the scaffold contributes desired textural, nutritional, or flavor properties to the cultivated meat product, and where the cellular component provides protein content and meat-like characteristics while the scaffold provides bulk and structure.

[0215] In an embodiment, the cultivated meat tissue comprises at least 10% cultured cells or differentiated tissue by composition. A composition of at least 10% cultured cells or differentiated tissue provides a cultivated meat product with increased cellular content relative to the 5% embodiment while maintaining a substantial scaffold contribution to the final product. This embodiment balances cellular protein content with scaffold-derived properties in the cultivated meat product.

[0216] In an embodiment, the cultivated meat tissue comprises at least 20% cultured cells or differentiated tissue by composition. A composition of at least 20% cultured cells or differentiated tissue provides a cultivated meat product where the cellular component constitutes a more substantial portion of the final product mass. This embodiment provides increased protein content from cultured cells while retaining scaffold-derived structural and textural properties.

[0217] In an embodiment the cultivated meat tissue comprises at least 30% cultured cells or differentiated tissue by composition. A composition of at least 30% cultured cells or differentiated tissue provides a cultivated meat product with cellular content approaching the levels found in conventional meat products. This embodiment provides substantial protein content from cultured cells and differentiated tissue while the scaffold material contributes structural support and additional properties to the final product.

[0218] In an embodiment, the cultivated meat tissue comprises at least 40% cultured cells or differentiated tissue by composition. In an embodiment, the cultivated meat tissue comprises at least 50% cultured cells or differentiated tissue by composition. In an embodiment, the cultivated meat tissue comprises at least 60% cultured cells or differentiated tissue by composition. In an embodiment, the cultivated meat tissue comprises at least 80% cultured cells or differentiated tissue by composition.

[0219] In an embodiment, the cultivated meat tissue comprises 5% to 30% cultured cells or differentiated tissue by composition. In an embodiment, the cultivated meat tissue comprises 30% to 70% cultured cells or differentiated tissue by composition. In anembodiment, the cultivated meat tissue comprises 70% to 90% cultured cells or differentiated tissue by composition.

[0220] The percentage of cultured cells or differentiated tissue in the cultivated meat composition is controlled through selection of scaffold parameters, cell seeding density, culture duration, and culture conditions. Scaffolds with higher surface area to mass ratios support greater cellular accumulation relative to scaffold mass, resulting in higher percentages of cultured cells in the final product. Extended culture durations allow cells to proliferate and accumulate additional cellular mass on the scaffold. Culture conditions that promote cell proliferation and differentiation increase the cellular content of the cultivated meat tissue.

[0221] The scaffold density ranges between 1 x 1010cells / mL and 1 x 106cells / mL. Scaffold density refers to the volumetric cell density achieved within the scaffold structure during cell culture, expressed as the number of cells per milliliter of scaffold volume. The scaffold density' range encompasses both high-density cellular scaffolds (HDCS) that support cell densities approaching IxlO10cells / mL and medium-density cellular scaffolds (MDCS) that support cell densities of approximately IxlO6cells / mL, as defined previously.

[0222] In an embodiment, the scaffold density is 1x10 cells / mL. A scaffold density of 1x10 cells / mL provides a balance between high volumetric productivity and manageable nutrient and oxygen demands during cell culture. This scaffold density supports efficient cultivated meat production while maintaining culture conditions that are compatible with standard bioreactor configurations and media exchange protocols. The 1x10 cells / mL density provides sufficient cellular content for cultivated meat products with desirable protein content and texture while avoiding the challenges associated with extremely high cell densities, including increased nutrient consumption, waste accumulation, and mass transfer limitations.

[0223] In an embodiment, the scaffold density is 1 xlO6to IxlO7cells / mL. In an embodiment, the scaffold density is 1 x 107 to 1 x 108 cells / mL. In an embodiment, the scaffolddensity is IMO8to l*109cells / mL. In an embodiment, the scaffold density is l*109to 1 xlO10cells / mL.

[0224] The scaffold density achieved during cell culture depends on the scaffold architecture, cell t pe, culture conditions, and culture duration. Scaffolds with higher porosity and interconnected pore networks support higher cell densities by providing greater surface area for cell attachment and improved mass transfer of nutrients and oxygen to cells throughout the scaffold volume. Cell types with higher proliferation rates achieve higher scaffold densities within a given culture period. Culture conditions that optimize nutrient availability, oxygen delivery, and waste removal support higher cell densities. Extended culture durations allow cells to proliferate and fill available attachment surfaces within the scaffold structure.

[0225] The scaffold density is monitored during cell culture to track the progress of tissue formation and to determine the appropriate harvest time for the cultivated meat product. Monitoring methods include sampling of the culture media to measure metabolic indicators such as glucose consumption and lactate production, which correlate with cell number and metabolic activity. Direct cell counting methods, including enzymatic dissociation of cells from scaffold samples followed by cell counting using hemocytometers or automated cell counters, provide quantitative measurements of scaffold density at specific time points during culture.Bioreactor Process Parameters and Harvest

[0226] Bioreactor parameters are altered over time during the cellular proliferation phase to support cell growth and tissue development on the cellular scaffold. The temporal adjustment of bioreactor parameters accommodates the changing requirements of the cell culture as cell density increases and tissue formation progresses. The bioreactor parameters that are altered over time include temperature, mixing, and pH.

[0227] Temperature is altered over time during the cellular proliferation phase to optimize cell proliferation and differentiation at different stages of the culture process. In an embodiment, the temperature is maintained at 37 degrees Celsius during initial cell attachment and early proliferation phases for mammalian cells, then adjusted to different temperatures during later culture phases to promote specific cellular behaviors. In an embodiment, the temperature is reduced slightly during differentiation phases to slow proliferation and promote myogenic differentiation of myoblasts into myotubes. In an embodiment, the temperature is increased transiently to induce heat shock responses that promote protein expression or cellular stress responses that enhance tissue maturation. The temperature adjustments are implemented through the bioreactor temperature control system, which regulates heating and cooling elements to maintain the target temperature at each phase of the culture process.

[0228] Mixing is altered over time during the cellular proliferation phase to accommodate changes in cell density and tissue structure as the culture progresses. In an embodiment, mixing intensity is reduced during initial cell attachment phases to minimize shear stress on cells and promote adhesion to the scaffold surfaces. In an embodiment, mixing intensity is increased during proliferation phases to enhance mass transfer of nutrients and oxygen to the growing cell population. In an embodiment, mixing patterns are altered to provide intermittent agitation with rest periods that allow cells to establish cell-cell contacts and tissue organization. The mixing parameters include impeller rotation speed for stirred tank bioreactors, rocking frequency and angle for wave bag bioreactors, and perfusion flow rate for packed bed bioreactors. The mixing adjustments are implemented through the bioreactor control system, which regulates the agitation mechanism according to programmed profiles or in response to measured culture parameters.

[0229] pH is altered over time during the cellular proliferation phase to maintain optimal conditions for cell viability and function as metabolic activity changes with increasing cell density. In an embodiment, the pH setpoint is adjusted during different culture phases to accommodate the metabolic requirements of proliferating versus differentiating cells. In anembodiment, the pH is maintained at 7.4 during proliferation phases and adjusted to slightly lower or higher values during differentiation phases to promote specific cellular responses. The pH is controlled through the addition of acid or base solutions to the culture media, through adjustment of carbon dioxide concentration in the headspace gas, or through media exchange that replaces spent media with fresh media at the target pH. The pH adjustments are implemented through the bioreactor pH control system, which monitors pH using in-line sensors and regulates acid / base addition or carbon dioxide sparging to maintain the target pH at each phase of the culture process.

[0230] The bioreactor parameters change in response to cellular behavior during the culture process. In an embodiment, the bioreactor control system monitors indicators of cellular activity such as dissolved oxygen consumption rate, glucose consumption rate, lactate production rate, and pH drift, and adjusts bioreactor parameters in response to measured changes in these indicators. In an embodiment, increased oxygen consumption rate triggers increased aeration or oxygen supplementation to maintain dissolved oxygen levels. In an embodiment, increased lactate production triggers increased media exchange rate to remove accumulated lactate and replenish depleted nutrients. The responsive adjustment of bioreactor parameters maintains optimal culture conditions as the cell population grows and metabolic demands increase.

[0231] The bioreactor parameters change according to a predefined process during the culture process. In an embodiment, the bioreactor control system implements a programmed sequence of parameter changes at specified time points during the culture process, independent of measured cellular behavior. In an embodiment, the predefined process specifies temperature, mixing, and pH setpoints for each day of the culture period, with transitions between setpoints occurring at programmed times. In an embodiment, the predefined process specifies gradual ramps in parameter values over specified time periods to provide smooth transitions between culture phases. The predefined process is developed based on prior characterization of the cell culture system and is optimized to achieve desired tissue formation outcomes.

[0232] A harvest phase transfers the bioprocess product from a bioreactor cultivation space into a food process space. The harvest phase marks the transition from cell culture conditions to food processing conditions, and bioreactor parameters are adjusted during the harvest phase to prepare the cultivated meat tissue for downstream food processing and consumption.

[0233] During the harvest phase, bioreactor temperature is reduced to preserve the cultivated meat tissue. Temperature reduction slows metabolic activity and enzymatic reactions within the tissue, preserving the cellular components and preventing degradation of proteins, lipids, and other tissue constituents. In an embodiment, the bioreactor temperature is reduced from the culture temperature of 37 degrees Celsius to a refrigerated temperature between 0 and 4 degrees Celsius during the harvest phase. In an embodiment, the temperature reduction is implemented gradually over a period of hours to avoid thermal shock to the tissue. In an embodiment, the temperature reduction is implemented rapidly to minimize the time during which the tissue is at intermediate temperatures where enzymatic activity remains elevated. The reduced temperature is maintained during transfer of the cultivated meat tissue from the bioreactor to downstream food processing equipment.

[0234] During the harvest phase, bioreactor temperature is increased to inactivate enzymatic activity within the cultivated meat tissue. Temperature increase denatures enzymes that w ould otherwise degrade tissue components during storage and processing. In an embodiment, the bioreactor temperature is increased from the culture temperature of 37 degrees Celsius to an elevated temperature between 50 and 70 degrees Celsius during the harvest phase to inactivate proteases, lipases, and other degradative enzymes. In an embodiment, the temperature increase is maintained for a duration sufficient to achieve enzyme inactivation throughout the tissue thickness. In an embodiment, the elevated temperature treatment also provides partial cooking of the cultivated meat tissue, reducing subsequent cooking time required during food preparation. The selection between temperature reduction and temperature increase during the harvest phase depends on theintended downstream processing and the desired characteristics of the final cultivated meat product.

[0235] During the harvest phase, gas flows and mixtures are changed to reduce oxygen content in the bioreactor environment. Reducing oxygen content minimizes oxidative reactions that degrade lipids, proteins, and other tissue components during the harvest and transfer process. In an embodiment, the oxygen concentration in the bioreactor headspace is reduced by displacing air with nitrogen gas or other inert gases. In an embodiment, the sparging gas is switched from air or oxygen-enriched air to pure nitrogen or a nitrogencarbon dioxide mixture that maintains pH without providing oxygen. In an embodiment, the bioreactor headspace is purged with nitrogen to remove residual oxygen before opening the bioreactor for tissue harvest. The reduced oxygen environment is maintained during transfer of the cultivated meat tissue to downstream processing equipment that is also maintained under reduced oxygen conditions. The reduction of oxygen content during the harvest phase extends the shelflife of the cultivated meat tissue and preserves the color, flavor, and nutritional quality of the final product.Cell Culture Results and Scaffold Performance

[0236] In an embodiment, the surface morphology is a dense network of intertwined nanofibers with cells adhered and spread across the fibrous matrix. The fibrous architecture creates an interconnected porous structure that supports cellular attachment and proliferation.

[0237] In an embodiment, the cells exhibit spread morphology on the nanofiber surfaces. The spread cell morphology enables the scaffold surface to provide appropriate attachment sites and mechanical properties for cell spreading, which is a prerequisite for subsequent cell proliferation and differentiation.

[0238] In an embodiment, the scaffold has an internal three-dimensional porous architecture achieved through a gas expansion process. The cross-section has a layered, openstructure with visible cellular material integrated within the scaffold matrix. The presence of a layered, open structure enables cell infiltration throughout the scaffold thickness. Cell infiltration beyond the scaffold surface is achieved through interconnected porosity’ created by the gas expansion process, which provides channels for cell migration into the scaffold interior.

[0239] In an embodiment, the cell distribution when the three-dimensional porous structure is formed by gas expansion enables mass transfer of nutrients and oxygen to cells throughout the scaffold volume. Cells located within the interior regions of the scaffold receive sufficient nutrients and oxygen to maintain viability and support proliferation. The mass transfer through the interconnected pore network addresses the limitation of conventional porous scaffolds that do not permit cell infiltration beyond several hundred micrometers due to diffusion constraints.

[0240] In an embodiment, the dynamic culture conditions under which the cells are cultured on the scaffold include perfusion of cell culture media through the scaffold structure or agitation of the scaffold within the culture media. Dynamic culture conditions enhance mass transfer compared to static culture conditions, delivering nutrients and oxygen to cells throughout the scaffold volume while removing metabolic waste products.

[0241] In an embodiment, the combination of nanofibrous texture at the cellular scale, promotes cell adhesion and spreading, with macroscopic porosity that enables cell infiltration and mass transfer, provides a scaffold architecture that supports the production of thick cultivated meat tissue with high cellular content.Integration of Scaffold System Components

[0242] The cellular scaffold system for cultivated meat production integrates multiple components that function together to achieve high-density cell culture and the production of thick cultivated meat tissue. The nanofiber scaffold, gas expansion process, freeze-dryingstabilization, bioreactor systems, and cell culture methods interact synergistically to create cultivated meat products with organoleptic qualities closer to conventional whole-cut meat harvested from animals.

[0243] The nanofiber scaffold provides the structural foundation for the cellular scaffold system. Nanofibers formed by electrospinning, electrospray, or extrusion from zein, cellulose acetate, or combinations thereof create two-dimensional mats with nanoscale surface texture that promotes cell adhesion and spreading. The nanofiber diameter and surface chemistry’ provide attachment sites for cells, while the mechanical stiffness of the nanofiber network supports myogenic differentiation of myoblasts into myotubes. The nanofiber scaffold serves as the starting material for subsequent processing steps that transform the two-dimensional mat into a three-dimensional porous structure suitable for high-density cell culture.

[0244] The gas expansion process converts the two-dimensional nanofiber mat into a three-dimensional porous scaffold with volumetric expansion and interconnected porosity. Gas generation within the fibrous network, whether through reaction of acid with sodium bicarbonate to produce carbon dioxide or through reaction of sodium borohydride with water to produce hydrogen gas, creates bubbles that separate adjacent nanofibers and expand the mat thickness. The gas expansion process creates channels and pores throughout the scaffold structure that enable cell infiltration beyond the scaffold surface and into the interior regions of the scaffold. The interconnected pore network provides pathways for mass transfer of nutrients, oxygen, and growth factors to cells throughout the scaffold volume while facilitating removal of metabolic waste products from the cellular environment.

[0245] Freeze-drying stabilization preserves the expanded porous architecture achieved during gas expansion. The freeze-drying process removes water from the expanded scaffold through sublimation, locking the volumetric expansion and interconnected porosity into a stable, dry scaffold structure. The stabilized scaffold retains the three-dimensional configuration upon subsequent handling, storage, and rehydration. Freeze-drying provides a method for preserving the scaffold architecture without collapse of the porous structure that would occur if water were removed through evaporation from the liquid phase. The stabilizedscaffold is stored in dry conditions until use, then rehydrated prior to introduction into the bioreactor for cell culture.

[0246] The bioreactor system provides the controlled environment for cell culture on the cellular scaffold. The bioreactor maintains temperature, pH, dissolved oxygen, and nutrient concentrations at levels that support cell attachment, proliferation, and differentiation. The bioreactor configuration is selected based on the scaffold format, with stirred tank, packed bed, fluidized bed, airlift, rotating wall, spinning basket, rotary wheel, hollow fiber, shake flask, and rocking motion wave bag bioreactors providing options for different scaffold sizes and production scales. The bioreactor agitation or perfusion system circulates cell culture media through and around the scaffold, delivering nutrients and oxygen to cells on the scaffold surfaces and within the porous scaffold interior.

[0247] The cell culture methods establish the conditions for cell attachment, proliferation, and differentiation on the cellular scaffold within the bioreactor. Cells are seeded onto the scaffold and cultured through attachment, proliferation, and maturation phases with temporal adjustments to media composition and bioreactor parameters. The cell culture methods support the grow th of myoblasts, adipocytes, fibroblasts, and other cell types relevant to cultivated meat production. The differentiation of myoblasts into myotubes on the scaffold produces muscle tissue with myosin heavy chain expression that contributes to the protein content and texture of the cultivated meat product.

[0248] The integration of these components enables the production of cultivated meat tissue with high cellular content. The three-dimensional porous architecture created by gas expansion provides surface area for cell attachment that exceeds the surface area of the original two-dimensional nanofiber mat. The increased surface area supports attachment of greater numbers of cells per unit scaffold mass, increasing the ratio of cellular content to scaffold content in the final cultivated meat product. The interconnected porosity enables cells to populate the interior regions of the scaffold, achieving volumetric cell densities that approach the cell densities found in native muscle tissue.

[0249] The mass transfer of nutrients and oxygen throughout the scaffold volume is achieved through the interconnected pore network created by gas expansion. Nutrients and oxygen from the cell culture media diffuse through the porous channels to reach cells located within the scaffold interior. The pore dimensions and interconnectivity are sufficient to support mass transfer over distances of millimeters to centimeters, enabling the production of thick cultivated meat tissue that exceeds the thickness limitations of conventional porous scaffolds. Conventional porous scaffolds without the interconnected porosity achieved through gas expansion do not permit cell infiltration beyond several hundred micrometers due to diffusion constraints, limiting the thickness of cultivated meat tissue that is produced using such scaffolds.

[0250] The bioreactor perfusion or agitation enhances mass transfer beyond what is achieved through diffusion alone. Convective transport of nutrients and oxygen through the scaffold pore network, driven by fluid flow from bioreactor agitation or perfusion, delivers nutrients and oxygen to cells at rates that support the metabolic demands of high-density cell populations. The combination of diffusive and convective mass transfer through the three-dimensional porous scaffold architecture supports cell densities ranging from P IO6cells / mL to 1 xlO10cells / mL, depending on the scaffold configuration and culture conditions.

[0251] The production of thick cultivated meat tissue with organoleptic qualities closer to conventional whole-cut meat results from the integrated scaffold system. The three-dimensional porous scaffold provides a structural template that guides tissue formation in three dimensions, producing cultivated meat tissue with thickness and structure that mimics the architecture of native muscle tissue. The nanofibrous texture of the scaffold surfaces promotes cell alignment and organization that contributes to the texture of the cultivated meat product. The mechanical properties of the scaffold support myogenic differentiation, resulting in the formation of myotubes that express muscle-specific proteins contributing to the taste and texture profile of the cultivated meat.

[0252] The edible and animal-component free nature of the scaffold allows the scaffold to remain incorporated within the final cultivated meat product. The scaffold materials,including zein and cellulose acetate, are food-grade compounds that contribute to or do not detract from the organoleptic qualities of the finished product. The scaffold provides structural support and texture to the cultivated meat product while the cultured cells and differentiated tissue provide the protein content, flavor compounds, and nutritional value associated with conventional meat. The integration of scaffold and cellular components produces a cultivated meat product that is consumed as a whole, without requiring separation of the scaffold from the cellular tissue prior to consumption.

[0253] The scaffold system supports the production of whole-cut cultivated meat products with dimensions similar to conventional meat cuts. In an embodiment, the scaffold system produces cultivated meat tissue sized approximately 200 mm length x 200 mm width x 20 mm thickness, equivalent to a Top Sirloin Steak cut weighing approximately 225 grams. The monolithic scaffold configuration supports cultivation of cells on a single large scaffold with dimensions matching the target product dimensions. The three-dimensional porous architecture enables mass transfer throughout the scaffold volume, supporting cell viability’ and tissue formation across the full thickness of the scaffold. The resulting cultivated meat product provides the size, structure, and organoleptic qualities expected by consumers of whole-cut meat products.

[0254] In an embodiment, the scaffold system produces cultivated meat products by combining multiple scaffold pieces and microcarriers with cultivated meat tissue grown on the scaffolds and microcarriers. The combination of multiple scaffold pieces outside of the bioreactor enables the production of cultivated meat products of various sizes ranging from 5 grams to 350 kilograms. Adhesives and binders including starch, pectin, transglutaminase, and methylcellulose join the scaffold pieces together to form cohesive cultivated meat products. The combination approach provides manufacturing flexibility for producing cultivated meat products of arbitrary size and shape while utilizing the high-density cell culture capabilities of the integrated scaffold system.

[0255] The integrated scaffold system achieves volumetric productivity that exceeds the productivity of conventional cell culture approaches for cultivated meat production. The highsurface area to scaffold mass ratio of the three-dimensional porous scaffold, combined with the mass transfer capabilities that support high cell densities throughout the scaffold volume, enables efficient utilization of bioreactor volume for cultivated meat production. The scaffold system produces cultivated meat tissue with cellular content comprising at least 70% of the total product composition, providing products with protein content and organoleptic qualities comparable to conventional meat products. The integration of nanofiber scaffold, gas expansion, freeze-drying stabilization, bioreactor systems, and cell culture methods provides a complete system for producing cultivated meat products that meet consumer expectations for taste, texture, and nutritional value.

[0256] Provided herein as Embodiment 1 is a cellular scaffold comprising:

[0257] at least one fiber arranged to provide cellular attachment, thereby forming the cellular scaffold having a first surface and a second surface.

[0258] Provided herein as Embodiment 2 is the cellular scaffold of Embodiment 1, wherein the scaffold is a high-density cellular scaffold.

[0259] Provided herein as Embodiment 3 is the cellular scaffold of Embodiment 1 or 2, wherein the scaffold is edible.

[0260] Provided herein as Embodiment 4 is the cellular scaffold of any one of Embodiments 1 to 3. wherein the scaffold is animal-component free.

[0261] Provided herein as Embodiment 5 is the cellular scaffold of any one of Embodiments 1 to 4, wherein the at least one fiber is arranged to form multiple layers relative to an Y axis.

[0262] Provided herein as Embodiment 6 is the cellular scaffold of any one of Embodiments 1 to 5, wherein the cellular scaffold comprises a structural construct providing for combining with a second cellular scaffold.

[0263] Provided herein as Embodiment 7 is the cellular scaffold of any one of Embodiments 1 to 6, wherein the cellular scaffold allows for cellular adhesion in a serum-free growth medium.

[0264] Provided herein as Embodiment 8 is the cellular scaffold of any one of Embodiments 1 to 7, wherein the cellular scaffold is a monolithic structure.

[0265] Provided herein as Embodiment 9 is the cellular scaffold of any one of Embodiments 1 to 8, wherein the cellular scaffold is at least 300 um in any one of an X, Y, or Z dimension.

[0266] Provided herein as Embodiment 10 is the cellular scaffold of any one of Embodiments 1 to 8, wherein the cellular scaffold is at least 300 um in at least two of the X, Y, or Z dimension.

[0267] Provided herein as Embodiment 11 is the cellular scaffold of any one of Embodiments 1 to 8. wherein the cellular scaffold is at least 300 um in each one of the X, Y. and Z dimension.

[0268] Provided herein as Embodiment 12 is the cellular scaffold of any one of Embodiments 1 to 8, wherein the cellular scaffold is less than 300 um in any one of an X, Y, or Z dimension.

[0269] Provided herein as Embodiment 13 is the cellular scaffold of any one of Embodiments 1 to 8, wherein the cellular scaffold is less than 300 um in at least two of the X, Y, or Z dimension.

[0270] Provided herein as Embodiment 14 is the cellular scaffold of any one of Embodiments 1 to 8, wherein the cellular scaffold is less than 300 um in each one of the X, Y, and Z dimension.

[0271] Provided herein as Embodiment 15 is the cellular scaffold of any one of Embodiments 1 to 14, wherein the fiber comprises at least 80% of the total weight of the cellular scaffold.

[0272] Provided herein as Embodiment 1 is the cellular scaffold of any one of Embodiments 1 to 14, wherein the fiber comprises at least 90% of the total weight of the cellular scaffold.

[0273] Provided herein as Embodiment 17 is the cellular scaffold of any one of Embodiments 1 to 14, wherein the fiber comprises at least 95% of the total weight of the cellular scaffold.

[0274] Provided herein as Embodiment 18 is the cellular scaffold of any one of Embodiments 1 to 17, wherein the porosity of the cellular scaffold is at least X porosity' units (or X% porosity).

[0275] Provided herein as Embodiment 19 is the cellular scaffold of any one of Embodiments 1 to 18, wherein the cellular scaffold further comprises flavoring or additives.

[0276] Provided herein as Embodiment 20 is the cellular scaffold of any one of Embodiments 1 to 19, wherein the cellular scaffold comprises an ingredient selected from a group comprising, Alginate, Soy protein, Pea protein, Zein, Pumpkin seed protein, Cottonseed Feedstock, Hemp seed protein, Mung bean protein. Fava bean protein. Kidney beans, Chia, Quinoa, Canola Protein, Garbanzo, Recombinant peptides, recombinant proteins, Chitin, Chitosan, Cellulose, Probiotics, Prebiotics, Plant extracts, plant oils, Dietary supplements. Oleogels, vegan beef extracts, vegan chicken extracts, Encapsulated fats.Vitamins, minerals, Antioxidants, Aromatics, Tapioca, Rice, Microbial polysaccharides, Curdlan, Gellan, Konjac and any combination thereof.

[0277] Provided herein as Embodiment 21 is the cellular scaffold of Embodiment 20, wherein the ingredient is an isolate, a concentrate, or functionalized.

[0278] Provided herein as Embodiment 22 is a cellular scaffold system comprising:

[0279] a first cellular scaffold in accordance with any one of Embodiments 1 to 21 and a second cell scaffold in accordance with any one of Embodiments 1 to 21. wherein the first cellular scaffold and the second cellular scaffold comprise a structural construct providing for cell transfer between the first and second cellular scaffold.

[0280] Provided herein as Embodiment 23 is the cellular scaffold system of any one of Embodiments 1 to 22, wherein the system further comprises a bioreactor.

[0281] Provided herein as Embodiment 24 is the cellular scaffold system of any one of Embodiments 1 to 23, wherein the system is sterilized.

[0282] Provided herein as Embodiment 25 is a method of making a cellular scaffold comprising the steps of:

[0283] 1. preparation of at least one fiber and

[0284] 2. forming the at least one fiber into a sheet, thereby forming a cellular scaffold.

[0285] Provided herein as Embodiment 26 is the method of Embodiment 25, wherein the fiber is formed from electrospinning, electrospray or extrusion.

[0286] Provided herein as Embodiment 27 is the method of Embodiment 25 or 26, wherein the at least one fiber comprises a plurality of uniaxially-aligned fibers, random fibers, and / or entangled fibers.

[0287] Provided herein as Embodiment 28 is the method of any one of Embodiments 25 to 27, further comprising exposing the at least one fiber to a foaming agent.

[0288] Provided herein as Embodiment 29 is the method of any one of Embodiments 25 to 28, wherein the foaming agent is selected from a group comprising dry ice (CO2), supercritical CO2, sodium bicarbonate, or gas.

[0289] Provided herein as Embodiment 30 is the method of any one of Embodiments 25 to 29, wherein foaming agent is added to the cellular scaffold in combination with a polymer.

[0290] Provided herein as Embodiment 31 is the method of any one of Embodiments 25 to 30, wherein the polymer comprises hydrophilic polymers.

[0291] Provided herein as Embodiment 32 is the method of any one of Embodiments 25 to 30, wherein polymer comprise hydrophobic polymers.

[0292] Provided herein as Embodiment 33 is the method of any one of Embodiments 25 to 32, wherein the at least one fiber is formed into a sheet.

[0293] Provided herein as Embodiment 34 is the method of Embodiment 33, further comprising the step of combining at least tw o sheets of fibers.

[0294] Provided herein as Embodiment 35 is the method of Embodiment 33 or 34, wherein the at least two sheets are fused together.

[0295] Provided herein as Embodiment 36 is the method of any one of Embodiments 33 to 35, wherein the at least two sheets are bonded together.

[0296] Provided herein as Embodiment 37 is the method of any one of Embodiments 25 to 33, further comprising the step of adding an adhesion factor.

[0297] Provided herein as Embodiment 38 is the method of Embodiment 37, wherein the adhesion factor is added to the at least one fiber composition.

[0298] Provided herein as Embodiment 39 is the method of Embodiment 37 or 38, wherein the adhesion factor is added to the surface of the at least one fiber.

[0299] Provided herein as Embodiment 40 is the method of any one of Embodiments 25 to 39, further combining a hydrogel to the cellular scaffold.

[0300] Provided herein as Embodiment 41 is the method of any one of Embodiments 25 to 40, wherein the cellular scaffold is at least 300 um in any one of an X, Y, or Z dimension.

[0301] Provided herein as Embodiment 42 is the method of any one of Embodiments 25 to 40, wherein the cellular scaffold is at least 300 um in at least two of the X, Y, or Z dimension.

[0302] Provided herein as Embodiment 43 is the method of any one of Embodiments 25 to 40, wherein the cellular scaffold is at least 300 um in each one of the X, Y, and Z dimension.

[0303] Provided herein as Embodiment 44 is the method of any one of Embodiments 25 to 40, wherein the cellular scaffold is less than 300 um in any one of an X, Y, or Z dimension.

[0304] Provided herein as Embodiment 45 is the method of any one of Embodiments 25 to 40, wherein the cellular scaffold is less than 300 um in at least two of the X, Y, or Z dimension.

[0305] Provided herein as Embodiment 46 is the method of any one of Embodiments 25 to 40, wherein the cellular scaffold is less than 300 um in each one of the X, Y, and Z dimension.

[0306] Provided herein as Embodiment 47 is the method of any one of Embodiments 25 to 46, wherein the fiber comprises at least 80% of the total weight of the cellular scaffold.

[0307] Provided herein as Embodiment 48 is the method of any one of Embodiments 25 to 46, wherein the fiber comprises at least 90% of the total weight of the cellular scaffold.

[0308] Provided herein as Embodiment 49 is the method of any one of Embodiments 25 to 46, wherein the fiber comprises at least 95% of the total weight of the cellular scaffold.

[0309] Provided herein as Embodiment 50 is the method of any one of Embodiments 25 to 49, wherein the porosity of the cellular scaffold is at least X porosity units (or X% porosity).

[0310] Provided herein as Embodiment 51 is a method of using a cellular scaffold comprising:

[0311] combining the cellular scaffold of any one of Embodiments 1 to 21 with a bioreactor.

[0312] Provided herein as Embodiment 52 is the method of Embodiment 51, wherein the bioreactor further comprises cell culture media to promote cellular attachment.

[0313] Provided herein as Embodiment 53 is the method of Embodiment 51 or 52, wherein the cellular scaffold is sterile prior to being added to a bioreactor.

[0314] Provided herein as Embodiment 54 is the method of any one of Embodiments 51 to 53, further comprising the step of adding cells into the bioreactor.

[0315] Provided herein as Embodiment 55 is the method of any one of Embodiments 51 to 54, wherein additional media is added to promote cellular proliferation.

[0316] Provided herein as Embodiment 56 is the method of Embodiment 55, wherein the additional media is the same media as the attachment phase.

[0317] Provided herein as Embodiment 57 is the method of Embodiment 55 or 56, wherein the additional media is a different media as the attachment phase.

[0318] Provided herein as Embodiment 58 is the method of any one of Embodiments 51 to 57, further comprising the step of adding a third mediate promote cellular maturation.

[0319] Provided herein as Embodiment 59 is the method of Embodiment 58, further comprising the step of harvesting the cellular scaffold from the bioreactor.

[0320] Provided herein as Embodiment 60 is the method of Embodiment 58 or 59, further comprising the step of processing the harvested cellular scaffold harvesting the cellular scaffold from the bioreactor.

[0321] Provided herein as Embodiment 61 is a cellular scaffold for cultivated meat production, comprising:

[0322] at least one nanofiber arranged to provide cellular attachment, the at least one nanofiber forming a three-dimensional porous structure;

[0323] wherein the three-dimensional porous structure is formed by gas expansion of a two-dimensional nanofiber mat, the gas expansion creating volumetric expansion and increased porosity within the nanofiber structure; and

[0324] wherein the cellular scaffold is edible and animal-component free.

[0325] Provided herein as Embodiment 62 is the cellular scaffold of Embodiment 61, wherein the at least one nanofiber comprises zein.

[0326] Provided herein as Embodiment 63 is the cellular scaffold of Embodiment 61 or 62, wherein the at least one nanofiber further comprises cellulose acetate.

[0327] Provided herein as Embodiment 64 is the cellular scaffold of any one of Embodiments 61 to 63, wherein the gas expansion is produced by reacting an acid with sodium bicarbonate to generate carbon dioxide in situ within the two-dimensional nanofiber mat.

[0328] Provided herein as Embodiment 65 is the cellular scaffold of any one of Embodiments 61 to 64, wherein the acid comprises acetic acid.

[0329] Provided herein as Embodiment 66 is the cellular scaffold of any one of Embodiments 61 to 65, wherein the gas expansion is produced by immersing the two-dimensional nanofiber mat in an aqueous sodium borohydride solution to generate hydrogen gas within the nanofiber mat.

[0330] Provided herein as Embodiment 67 is the cellular scaffold of any one of Embodiments 61 to 66, wherein the three-dimensional porous structure is stabilized by freeze-drying following the gas expansion.

[0331] Provided herein as Embodiment 68 is the cellular scaffold of any one of Embodiments 61 to 67, wherein the at least one nanofiber is formed by a process selected from the group consisting of electrospinning, electrospray, and extrusion.

[0332] Provided herein as Embodiment 69 is the cellular scaffold of any one of Embodiments 61 to 68, wherein the cellular scaffold is a monolithic structure having a dimension of at least 300 micrometers in each of an X, Y, and Z dimension.

[0333] Provided herein as Embodiment 70 is the cellular scaffold of any one of Embodiments 61 to 69, wherein the cellular scaffold is a microcarrier having a dimension of less than 300 micrometers in each of an X, Y, and Z dimension.

[0334] Provided herein as Embodiment 71 is the cellular scaffold of any one of Embodiments 61 to 70, wherein the at least one nanofiber is crosslinked with a crosslinking agent selected from the group consisting of genipin, transglutaminase, glutaraldehyde, citric acid, tannic acid, succinic anhydride, epigallocatechin, tyrosinase, phosphoryl chloride, sodium trimetaphosphate, sodium tripolyphosphate, plant-derived proanthocyanidins, plant-derived epigallocatechin gallate, and mixtures thereof.

[0335] Provided herein as Embodiment 72 is a method of making a cellular scaffold for cultivated meat production, comprising:

[0336] forming at least one nanofiber into a two-dimensional mat by electrospinning, electrospray, or extrusion;

[0337] immersing the two-dimensional mat in an effervescent system to generate gas within the mat, thereby inducing volumetric expansion and creating a three-dimensional porous scaffold; and

[0338] stabilizing the three-dimensional porous scaffold by freeze-drying;

[0339] wherein the cellular scaffold is edible and animal-component free.

[0340] Provided herein as Embodiment 73 is the method of Embodiment 72, wherein the at least one nanofiber comprises zein.

[0341] Provided herein as Embodiment 74 is the method of Embodiment 72 or 73, wherein the at least one nanofiber further comprises cellulose acetate.

[0342] Provided herein as Embodiment 75 is the method of any one of Embodiments 72 to 74, wherein the effervescent system comprises an aqueous solution of acetic acid reacted with sodium bicarbonate to generate carbon dioxide in situ within the two-dimensional mat.

[0343] Provided herein as Embodiment 76 is the method of any one of Embodiments 72 to 75, further comprising rinsing the three-dimensional porous scaffold with deionized water prior to freeze-drying.

[0344] Provided herein as Embodiment 77 is a method of culturing cells for cultivated meat production, comprising:

[0345] providing a cellular scaffold comprising at least one nanofiber arranged in a three-dimensional porous structure formed by gas expansion, wherein the cellular scaffold is edible and animal -component free;

[0346] introducing the cellular scaffold into a bioreactor containing cell culture media;

[0347] seeding cells onto the cellular scaffold; and

[0348] culturing the cells on the cellular scaffold to produce cultivated meat tissue.

[0349] Provided herein as Embodiment 78 is the method of Embodiment 77, wherein the cells comprise one or more cell types selected from the group consisting of myoblasts, mesangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, and endothelial cells.

[0350] Provided herein as Embodiment 79 is the method of Embodiment 77 or 78, wherein the cell culture media is a serum-free growth medium.

[0351] Provided herein as Embodiment 80 is the method of any one of Embodiments 77 to 79, wherein the cultivated meat tissue comprises at least 70% cultured cells or differentiated tissue by composition.

[0352] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

[0353] In the context of the present description, all publications, patent applications, patents and other references mentioned herein, if not otherwise indicated, are explicitly incorporated by reference herein in their entirety for all purposes as if fully set forth, and shall be considered part of the present disclosure in their entirety.

[0354] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, will control.

[0355] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found inthe marketplace, or to enable others or ordinary7skill in the art to understand the embodiments disclosed herein.

[0356] Except where expressly noted, trademarks are shown in upper case.

[0357] Unless stated otherwise, all percentages, parts, ratios, etc., are by weight.

[0358] When an amount, concentration, or other value or parameter is given as a range, or a list of upper and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper and lower range limits, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range. Further, where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

[0359] Further, unless otherwise explicitly stated to the contrary, when one or multiple ranges or lists of items are provided, this is to be understood as explicitly disclosing any single stated value or item in such range or list, and any combination thereof with any other individual value or item in the same or any other list.

[0360] When the term 'About” or "approximately" is used, it is used to mean a certain effect or result can be obtained within a certain tolerance (e.g., ±10%), and the skilled person knows how to obtain the tolerance. When the term "about" is used in describing a value or an end-point of a range, the disclosure should be understood to include the specific value or endpoint referred to.

[0361] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover anon-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0362] The transitional phrase "consisting of' excludes any element, step, or ingredient not specified in the claim, closing the claim to the inclusion of materials other than thoserecited except for impurities ordinarily associated therewith. When the phrase "consists of appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0363] The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. A “consisting essentially of’ claim occupies a middle ground between closed claims that are written in a “consisting of’ format and fully open claims that are drafted in a “comprising” format. Optional additives as defined herein, at a level that is appropriate for such additives, and minor impurities are not excluded from a composition by the term “consisting essentially of’.

[0364] As used herein, an "embodiment" means that a particular feature, structure or characteristic is included in at least one or more manifestations, examples, or implementations of this invention. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art. Combinations of features of different embodiments are all meant to be within the scope of the invention, without the need for explicitly describing every' possible permutation by example. Thus, any of the claimed embodiments can be used in any combination.

[0365] Further, unless expressly stated to the contrary, “and / or” refers to an inclusive and not to an exclusive. Thus, “and / or” should be understood to mean “either or both” of the elements so conjoined, e g., elements that are conjunctively present in some cases and disjunctively present in other cases. For example, a condition A and / or B, is satisfied by any one of the following: A is true (or present) and B is false (or not present). A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0366] The use of "a" or "an" to describe the various elements and components herein is merely for convenience and to give a general sense of the disclosure. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise. Similarly, the adjective “another,” when used to introduce an element, is intended to mean one or more elements. The terms “including” and “having”are intended to be inclusive such that there may be additional elements other than the listed elements.

[0367] The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

[0368] As used herein, the phrases “selected from the group consisting off’ “chosen from,” and the like include mixtures of the specified materials.

[0369] The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

[0370] Although this invention has been described with a certain degree of particularity, it is to be understood that the present disclosure has been made only by way of illustration and that numerous changes in the details of construction and arrangement of parts may be resorted to without departing from the spirit and the scope of the invention.

[0371] The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.

[0372] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.EXAMPLES

[0373] The examples herein are intended to illustrate certain aspects of the present disclosure to one of ordinary skill in the art. However, the examples are not intended to limit the scope of the present disclosure.

[0374] Example 1: Gas Expansion of Electrospun Zein Nanofiber Mat Using Acetic Acid and Sodium Bicarbonate

[0375] A two-dimensional electrospun zein nanofiber mat was converted into a three-dimensional porous scaffold via controlled gas expansion followed by freeze-drying. The electrospun mat was cut to desired dimensions and briefly immersed in an effervescent aqueous system generated by reacting dilute acetic acid (vinegar) with sodium bicarbonate (approximately 1% w / v), thereby producing carbon dioxide in situ. Gas formation within the fibrous network induced rapid volumetric expansion and increased porosity. As shown in FIG. 1, the expanded scaffold floated at the surface of the liquid, demonstrating the presence of entrapped gas within its porous structure. The expanded scaffold was rinsed with deionized water, frozen at -80 °C. and lyophilized under controlled conditions for at least 24 hours. As shown in FIG. 2, the resulting scaffold exhibited a pale yellow, disc-shaped structure with an irregular, porous texture and demonstrated significant three-dimensional thickness with visible layered striations along its cross-section. The expanded porous architecture enabled mass transfer of nutrients and oxygen throughout the scaffold volume.

[0376] The examples demonstrated that two-dimensional electrospun nanofiber mats were successfully converted into three-dimensional porous scaffolds through controlled gas expansion using either carbon dioxide generated from acetic acid and sodium bicarbonate or hydrogen gas generated from sodium borohydride. The expanded scaffolds exhibited significant volumetric expansion with layered, porous architecture suitable for high-density cell culture applications. Cell culture experiments confirmed that the expanded zein / cellulose acetate scaffolds supported cell adhesion, spreading, and infiltration throughout the scaffold thickness under dynamic culture conditions, demonstrating the suitability of these scaffolds for cultivated meat production.

[0377] Prophetic Example 1: Spontaneous Microtissue Adhesion Using Zein Sheets for Structured Multicellular Constructs

[0378] Below is a prophetic example illustrating the preparation, application, and performance of the cellular scaffolds according to the present invention. This example is provided to demonstrate a potential embodiment and is not based on actual experimental data.

[0379] The expanded zein scaffold is used for spontaneous microtissue adhesion to create structured, multicellular constructs. The scaffold is prepared by gas expansion of electrospun zein nanofiber mats using the acetic acid and sodium bicarbonate effervescent system, followed by freeze-drying to stabilize the three-dimensional porous architecture. The expanded zein sheets serve as the primary scaffold structure for tissue assembly.

[0380] Layers of hydrogel, cell-laden hydrogel, pure cell layers, or microcarriers are incorporated onto the zein scaffold surfaces. The hydrogel layers comprise food-grade materials selected from alginate, pectin, or other plant-derived polysaccharides. Cell-laden hydrogels contain myoblasts or adipocytes suspended within the hydrogel matrix prior to application onto the scaffold surface.

[0381] Additional adhesives or binders are applied to facilitate the assembly process and promote stable multilayered tissue formation. The adhesives and binders include starch, pectin, transglutaminase, and methylcellulose. Transglutaminase catalyzes covalent crosslinks between protein layers, enhancing the structural integrity of the assembled construct. Methylcellulose provides temperature-responsive gelation properties that aid in layer adhesion during the assembly process.

[0382] The assembly process proceeds by alternating layers of expanded zein scaffold sheets with cell-laden hydrogel layers. Each layer is allowed to adhere to the preceding layer before the next layer is applied. The transglutaminase is applied between protein-containing layers to promote enzymatic crosslinking. The assembled multilayered construct is incubated under cell culture conditions to allow cellular integration and tissue maturation.

[0383] The resulting structured multicellular construct is expected to exhibit stable adhesion between layers, with cells distributed throughout the three-dimensional structure. The layered architecture is expected to mimic the organization of native muscle tissue, with distinct regions of muscle cells and adipocytes. The construct is expected to maintain structural integrity during handling and subsequent processing for cultivated meat applications.

[0384] This prophetic example demonstrates the versatility of the inventive approaches to fabricating expanded cellular scaffolds for cultivated meat production. The example highlights specific features and benefits of using the gas-expanded zein scaffolds as a platform for spontaneous microtissue adhesion and multilayered tissue assembly, showcasing the adaptability and effectiveness of the invention for creating structured cultivated meat products with organoleptic qualities closer to conventional whole-cut meat.

[0385] Numerous modifications and variations on the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the accompanying claims, the invention may be practiced otherwise than as specifically described herein.

Claims

CLAIMS1. A cellular scaffold for cultivated meat production, comprising:at least one nanofiber arranged to provide cellular attachment, the at least one nanofiber forming a three-dimensional porous structure;wherein the three-dimensional porous structure is formed by gas expansion of a two-dimensional nanofiber mat, the gas expansion creating volumetric expansion and increased porosity within the nanofiber structure; andwherein the cellular scaffold is edible and animal-component free.

2. The cellular scaffold of claim 1, wherein the at least one nanofiber comprises zein.

3. The cellular scaffold of claim 2, wherein the at least one nanofiber further comprises cellulose acetate.

4. The cellular scaffold of claim 1, wherein the gas expansion is produced by reacting an acid with sodium bicarbonate to generate carbon dioxide in situ within the two-dimensional nanofiber mat.

5. The cellular scaffold of claim 4, wherein the acid comprises acetic acid.

6. The cellular scaffold of claim 1, wherein the gas expansion is produced by immersing the two-dimensional nanofiber mat in an aqueous sodium borohydride solution to generate hydrogen gas within the nanofiber mat.

7. The cellular scaffold of claim 1, wherein the three-dimensional porous structure is stabilized by freeze-drying following the gas expansion.

8. The cellular scaffold of claim 1, wherein the at least one nanofiber is formed by a process selected from the group consisting of electrospinning, electrospray, and extrusion.

9. The cellular scaffold of claim 1, wherein the cellular scaffold is a monolithic structure having a dimension of at least 300 micrometers in each of an X, Y, and Z dimension.

10. The cellular scaffold of claim 1, wherein the cellular scaffold is a microcarrier having a dimension of less than 300 micrometers in each of an X, Y, and Z dimension.

11. The cellular scaffold of claim 1, wherein the at least one nanofiber is crosslinked with a crosslinking agent selected from the group consisting of genipin, transglutaminase, glutaraldehyde, citric acid, tannic acid, succinic anhydride, epigallocatechin, tyrosinase, phosphoryl chloride, sodium trimetaphosphate, sodium tripolyphosphate, plant-derived proanthocyanidins, plant-derived epigallocatechin gallate, and mixtures thereof.

12. A method of making a cellular scaffold for cultivated meat production, comprising: forming at least one nanofiber into a two-dimensional mat by electrospinning, electrospray, or extrusion;immersing the two-dimensional mat in an effervescent system to generate gas within the mat, thereby inducing volumetric expansion and creating a three-dimensional porous scaffold; and stabilizing the three-dimensional porous scaffold by freeze-drying;wherein the cellular scaffold is edible and animal-component free.

13. The method of claim 12, wherein the at least one nanofiber comprises zein.

14. The method of claim 13, wherein the at least one nanofiber further comprises cellulose acetate.

15. The method of claim 12, wherein the effervescent system comprises an aqueous solution of acetic acid reacted with sodium bicarbonate to generate carbon dioxide in situ within the two-dimensional mat.

16. The method of claim 12, further comprising rinsing the three-dimensional porous scaffold with deionized water prior to freeze-drying.

17. A method of culturing cells for cultivated meat production, comprising: providing a cellular scaffold comprising at least one nanofiber arranged in a three-dimensional porous structure formed by gas expansion, wherein the cellular scaffold is edible and animal-component free;introducing the cellular scaffold into a bioreactor containing cell culture media; seeding cells onto the cellular scaffold; andculturing the cells on the cellular scaffold to produce cultivated meat tissue.

18. The method of claim 17, wherein the cells comprise one or more cell types selected from the group consisting of myoblasts, mesangioblasts, myofibroblasts, mesenchymal stem cells, hepatocytes, fibroblasts, pericytes, adipocytes, epithelial cells, chondrocytes, osteoblasts, osteoclasts, pluripotent cells, somatic stem cells, and endothelial cells.

19. The method of claim 17, wherein the cell culture media is a serum-free growth medium.

20. The method of claim 19, wherein the cultivated meat tissue comprises at least 70% cultured cells or differentiated tissue by composition.

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