WO2025059173A1

WO2025059173A1 - Adipogenic cell culture media

Info

Publication number: WO2025059173A1
Application number: PCT/US2024/046186
Authority: WO
Inventors: Seyedvahid HOSSEINI
Original assignee: Hatchless Inc
Current assignee: Hatchless Inc
Priority date: 2023-09-11
Filing date: 2024-09-11
Publication date: 2025-03-20
Anticipated expiration: 2026-03-11

Abstract

Provided are media compositions for culturing of cells, biomass and meat products using a cell culture medium, wherein the medium comprises egg yolks or a derivative thereof. The disclosed media can support adipogenesis without the requirement for frequent media changes or addition of additional ingredients. Also, provided are methods of producing meat product and other cell products by supporting in vitro or ex vivo growth and proliferation of cells in a medium comprising egg-yolk ingredients.

Description

ADIPOGENIC CELL CULTURE MEDIA

BACKGROUND

1. Field

[0001] The present invention relates to compositions and methods for making cultured cell and meat products using an egg-based media. Use of the disclosed compositions in the field of cellular agriculture and other fields is provided.

2. Background

[0002] Currently, factory farming is the primary way for production of most animal products all around the world, but especially in the United States of America. However, factory farming is rife with ethical and environmental issues, especially since global demands have increased dramatically. In the recent years, the meat industry in particular has been moving away from factory farming and towards products that mimic animal products, for instance plant-based meat. However, it is hard to achieve the texture, flavor and nutritional benefits of meat from such products.

[0003] Cultured meat, or cell-based meat, is meat that is produced using in vitro cell culture or bioreactors, instead of being harvested from live animals. Such meat may include, for example, chicken, beef, pork, or fish. Cultured meat produced from cell cultures provides a viable alternative to address these issues without having to turn to a completely vegetarian or vegan diet. Assessments have shown that cultured meat has the capacity to significantly reduce land and water use, emit fewer greenhouse gases, and reduce agriculture-related pollution and eutrophication. It also provides a more ethical way of producing food, avoiding animal confinement, mistreatment of animals and animal slaughter. Cultured meat can be obtained from animal-based components, but with the advantage of sustainably increased yields, similarity of underlying structure and nutrition to factory farmed animals. In addition, cultured animal cells can also be used for production of other animal products like leather, horns, or feathers. Industrialized animal cell culture has also proven to be useful for medical purposes, for example for production of recombinant proteins, tissues, organs and organoids.

[0004] Animal cell culture and meat cultivation technologies have the potential to revolutionize agriculture. However, it is still difficult and expensive to culture cells for applications such as cultured meat, and thus improvements are needed. Cellular agriculture in bioreactors currently use media compositions that are not inherently capable of inducing cell differentiation into adipocytes and myoblast simultaneously. To promote differentiation, specialized media are used

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SUBSTITUTE SHEET (RULE 26) during different stages of growth increasing media costs and time. Additionally, signaling molecules, drugs and fatty acids may be added to induce cell differentiation. This further drives the costs and may have safety concerns.

[0005] There is an unmet need in the market for an integrated media that does not require frequent changes or additions in order to make a robust product.

SUMMARY OF THE INVENTION

[0006] In some aspects, the current disclosure encompasses an adipogenic medium, comprising a base medium and an egg yolk, or a derivative thereof comprising at least one protein, and at least one lipid from an egg yolk. In an aspect, the adipogenic medium comprises an egg yolk. In an aspect, the adipogenic medium comprises at least one protein or a derivative thereof, naturally present in an egg yolk, for example apovitellenin l-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or a recombinant variant or derivative thereof, or any combination thereof. In an aspect the adipogenic medium comprises at least one lipid or a derivative thereof, naturally present in egg yolk, non-limiting examples of which include a triglyceride, a phosphotidylcholine, phosphotidylethanolamine, lysophosphatidylcholine, cholesterol, lanosterol, desmosterol, 57-cholestenol, cholestanol, 57- and 58-methostenol, 4a-methyl-58,24-cholestenol and its 57-isomer, 4,4a-dimethyl-57, 24-cholestenol, dihydrolanostenol, |3-sitosterol, ergosterol, sphingomyelin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In an aspect, the egg yolk or the derivative thereof is from an oviparous animal. In an aspect, the egg yolk or the derivative thereof is derived from a fish, amphibian, reptile or avian egg. In an aspect, the egg yolk or the derivative thereof is derived from an unfertilized egg. In an aspect, the egg yolk or the derivative thereof is derived from a fertilized egg. In an aspect, the egg yolk or the derivative thereof is derived from the fertilized egg in any one of first, second or third trimester. In an aspect, the egg yolk or the derivative thereof is derived from a fertilized egg in first trimester. In an aspect, the egg is an avian egg comprising a chicken egg, GF chicken egg, ostrich egg, quail egg, or partridge egg. In an aspect, the egg yolk derivative is derived from a recombinant source, selected from a fungus, a bacterium or an alga.

[0007] In an aspect, the adipogenic medium may comprise at least one additional egg ingredient for example a growth factor, vitamins, minerals, choline, carotenoids, lecithins, or a combination thereof.

[0008] In an aspect, the adipogenic medium comprises a basal medium. In an aspect, the basal medium is selected from DMEM, DMEM F12, minimum essential medium, RM1 1640, serum free

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SUBSTITUTE SHEET (RULE 26) medium, bench stable medium, human plasma like medium, primary culture medium, supplemented culture medium.

[0009] In an aspect, the medium may comprise growth promoting ingredient. In an aspect, the additional ingredients may include one or more of growth factors, amino acids, vitamins, minerals, edible surfactants, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fats, fibers, buffering agents or any combinations thereof. Non-limiting examples of growth factors include FGF, VEGF, PGF, TGF, BDNF, GDNF, Shh, BMP-2, BMP-4, EGF, Noggin, R-Spondin 1 , Wnt-3a, FGF-10, FGF-2.FGF-7, Activin A, IGF1 , NRG1, Jagged 1 , HGF, IL-2, IL-4, IL-6, IL-3, IL- 7, Flt3-ligand, PDGF, or TGF-[31 or any combination thereof.

[0010] In an aspect, the adipogenic medium as disclosed herein may comprise about 0.5%- 20% (w/w) of egg yolk. In an aspect, the adipogenic medium comprises at least about 5% (w/w) of egg yolk.

[0011] In an aspect, the medium supports differentiation of adipocytes from a precursor cell. In an aspect, the precursor cell is a stem cell comprising an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell, a fibroblast, a myoblast, or a preadipocyte.

[0012] In an aspect, the adipogenic medium does not require addition of signaling molecules, drugs or fatty acids to the medium to induce adipogenesis. In an aspect, the medium is configured for use in a bioreactor. In an aspect, the media is configured for culturing non-human cells to produce a cultured meat product. In an aspect the media is configured for producing animal fat rich products using a bioreactor. In an aspect, the medium is configured for use in a bioreactor for in vitro cell culture. In an aspect, the adipogenic medium is configured for culturing non-human cells to produce a cultured meat product. In an aspect, the cell culture does not require a media exchange for at least 5-10 days, to maintain a 75%-100% cell proliferation rate. In an aspect, the cell culture does not require a media exchange for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days.

[0013] In an aspect, the adipogenic medium is stable at 37 °C for at least 10-20 days. In an aspect, the medium is stable at 37 °C for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days. In an aspect, the in vitro cell culture has reduced amounts of ammonia compared to a medium not comprising egg yolk after 5-10 days. In an

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SUBSTITUTE SHEET (RULE 26) aspect, the in vitro cell culture has reduced amounts of lactic acid compared to a medium not comprising egg yolk after 5-10 days. In an aspect, the in vitro cell culture maintains a pH of 7-8 for at least about 5-10 days.

[0014] In an aspect, the addition of egg yolk to basal media as disclosed herein enhances the buffering capacity of culture media against pH change derived by atmospheric change of carbon dioxide concentration. In an aspect, the addition of egg yolk can result in concentration of atmospheric carbon dioxide in incubator to decrease by at least 20% as compared to non-yolk supplemented media. In an aspect the medium maintains at least about 75% cell potency after 5- 10 days of use at 37 °C.

[0015] In an aspect, the current disclosure also encompasses a method of differentiating a precursor cell into an adipocyte, comprising culturing the precursor cell in a medium comprising an egg yolk, or a derivative thereof comprising at least one protein, and at least one lipid from an egg yolk, for a time sufficient for the precursor to differentiate into the adipocyte. In an aspect, the precursor cell is a non-human animal cell. In an aspect, the non-human animal cell is a stem cell, a fibroblast, a myoblast, or a preadipocyte. In an aspect, the non-human animal cell is a stem cell selected from an embryonic stem cell, bone marrow derived stem cell, adipose derived stem cell, induced pluripotent stem cell. In an aspect, the non-human cell is a fish, avian, porcine, bovine, ovine, hoofed ruminant mammal, rabbit cell or a derivative thereof. In an aspect, the methods as disclosed herein may be for use in production of a meat product, or a cell product. In an aspect, the precursor cell is a cell derived from a human cell line, for production of a cell product. In an aspect, the cell product is for research, therapeutic, biomedical or industrial goods production. In an aspect of the method as disclosed herein, the culturing of cell is done in a bioreactor or a cell culture system.

[0016] In an aspect of the method as disclosed herein, the egg yolk or the derivative thereof is derived from the egg of an oviparous animal. In an aspect, the egg yolk or the derivative thereof is derived from a fertilized egg. In an aspect, the egg yolk or the derivative thereof is derived from the first, second or third trimester of a fertilized egg. In an aspect, the egg yolk or the derivative thereof is derived from an unfertilized egg. In an aspect, the egg yolk derivative is derived from a recombinant source, selected from a fungus, a bacterium or an alga.

[0017] In an aspect of the method as disclosed herein, the medium comprises an egg yolk. In an aspect, the medium comprises 0.5%-20% egg yolk. In an aspect, the medium comprises 5% egg yolk. In an aspect, the medium further comprises an egg white. In an aspect, the medium comprises 0.1%-20% of egg white. In an aspect, the medium comprises a basal medium, DMEM,

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SUBSTITUTE SHEET (RULE 26) DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench stable medium, human plasma like medium, primary culture medium, supplemented culture medium. In an aspect, the medium further comprises at least one additional egg ingredient. In an aspect, the additional ingredient is a growth factor, vitamins, minerals, choline, carotenoids, lecithins, or a combination thereof. In an aspect of the disclosed method, the medium does not require addition of signaling molecules, drugs or fatty acids to the medium to induce adipogenesis. In an aspect, the adipogenic medium further comprises one or more of amino acids, vitamins, polysaccharides, sugars, minerals, edible surfactants, antibiotics, or growth factors. In an aspect, the growth factor may comprise fibroblast growth factor (FGF), VEGF, PGF, TGF, BDNF, GDNF, Shh, BMP-2, BMP-4, EGF, Noggin, R-Spondin 1 , Wnt-3a, FGF-10, FGF-2,FGF-7, Activin A, IGF1 , NRG1 , Jagged 1 , HGF, IL-2, IL-4, IL-6, IL-3, IL-7, Flt3-ligand, PDGF, or TGF-J31 or any combination thereof.

[0018] In an aspect, the method as disclosed herein is for the production of a cell, organ, tissue, organoid, or whole animal.

[0019] In an aspect, the method uses least 20% - at least 70% less media.

[0020] In an aspect, the current disclosure also encompasses a system for culturing cells, the system comprising: a bioreactor or a cell culture unit, a disclosed cell culture medium, and a precursor cell or cell culture, wherein the cell culture medium does not require addition of signaling molecules, drugs or fatty acids to induce adipogenesis. In an aspect, the system is adapted for production of a cultured meat product. In an aspect, the system is adapted for production of cells, tissue, organoids, or organs. In an aspect, the cultured meat product comprises no added adipocytes or fat.

[0021] In an aspect, the current disclosure also encompasses a media for in vitro cell culture comprising egg yolk or a derivative thereof, wherein the in vitro cell culture does not require a media exchange for at least 5-10 days, to maintain a 75%-100% cell proliferation rate. In an aspect, the cell culture does not require a media exchange for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. In an aspect, the medium is stable at 37 °C for at least 10-20 days. In an aspect, the medium is stable at 37 °C for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days. In an aspect, the in vitro cell culture has reduced amounts of ammonia compared to a medium not comprising egg yolk after 5-10 days. In an aspect, the in vitro cell culture has reduced amounts

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SUBSTITUTE SHEET (RULE 26) of lactic acid compared to a medium not comprising egg yolk after 5-10 days. In an aspect, the in vitro cell culture maintains a pH of 7-8 for at least about 5-10 days. In an aspect, the addition of egg yolk to basal media enhances the buffering capacity of culture media against pH change derived by atmospheric change of carbon dioxide concentration. In an aspect, the addition of egg yolk reduces the concentration of atmospheric carbon dioxide in the bioreactor by at least about 20% as compared to non-yolk supplemented media.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows adherent chicken embryonic myofibroblasts cultivated in an egg-based medium using isolated chicken cells.

[0023] FIG. 2 shows proliferation of chicken embryonic myofibroblasts at different concentration of egg yolk.

[0024] FIG. 3A provides an overview of the production process.

[0025] FIG. 3B is a schematic of the process of meat production using Hatchless media in comparison with the conventional broiler cycle.

[0026] FIG. 4 is a graph showing the relative proliferation potency of control (FBS) or egg yolkbased (EY-based) media after 7 days of storage at 37°C.

[0027] FIG. 5 provides data showing the reduction in pH over days of culture in FBS or egg yolk-based media.

[0028] FIG. 6 Florescence and phase contrast images of fat cells grown in egg yolk media. Fat droplet in cytosol of cells were stained red using Nile Red.

[0029] FIG. 7A provides a phase contrast image of field of grown chicken muscle fibers developed in egg-yolk culture media.

[0030] FIG. 7B provides a phase contrast image showing spontaneous formation of muscle fiber (shown by asterisk) and fat cell (shown by arrows) simultaneously in egg yolk culture media.

[0031] The drawing figures do not limit the present inventive concept to the specific aspects disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain aspects of the present inventive concept.

DETAILED DESCRIPTION

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SUBSTITUTE SHEET (RULE 26) [0032] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense. The scope of the present disclosure is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0033] The present disclosure is based, in part, on the observation that adipogenic yolk-based media compositions egg-yolks can support and enhance growth of cells derived from an animal. Additionally, this yolk-based medium is sufficient for supporting differentiation and proliferation of a variety of cell types such as muscle precursor cells, and fibroblasts. Yolk based media also aid in differentiation of muscle stem cells (myoblasts) to myofiber, the main component of muscle or meat and simultaneously induce the differentiation of fibroblasts to fat cells or adipocyte. This induction was observed at a wide range of yolk concentration in basal media. These adipogenic yolk-based media compositions are demonstrated here to be cheaper and have tangible and intangible benefits over use of conventional media to produce cell products.

[0034] In some aspects, the current disclosure stems from the premise that egg yolks, or components thereof, provide extremely nutritive media that can support growth and differentiation of cells in suspension and adherent cultures. Eggs are relatively inexpensive, can be sustainably sourced and are easily available. They provide a natural and arguably the best possible combination of nutrients to mimic in vivo cell growth.

[0035] Entities working in the field of cellular agriculture use commercially available media options for growth and proliferation of cells. However, cells proliferated using these conventional methods do not show robust differentiation into adipocytes and require frequent media changes and use of additional signaling molecules, drugs and fatty acids to induce the differentiation of cells to fat cells or adipocyte. This process, although doable, needs extended cell culture time and the use of multiple specialized culture media at different stages, that makes the process expensive. At the same time, usage of added drugs and chemicals may result in harmful effects on consumer and require safety considerations. Other mainstream companies in this space are focused on developing recombinant sources from microorganisms that can be used in meat substitute products. However, these methods and trends result in products which lack one or more attributes of natural meat, for example taste, texture, mouth feel, smell or color. The use of media compositions disclosed herein ensures development of animal like fat tissues making the

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SUBSTITUTE SHEET (RULE 26) cultivated products match natural meat in their nutrient make-up, taste, texture, mouth feel, smell and color. Additionally, the yolk-based media disclosed herein may potentially reduce culture media use by at least 20%-70% and bring down the cost of production by as much as -80%.

[0036] Thus, in some aspects the current disclosure provides media compositions combining egg yolks or components thereof and optionally additional egg-based ingredients to provide nutrients for cell growth. The disclosed media can be used in industrial size reactors for cellular agriculture. The technology disclosed herein has the potential to greatly enhance yields and quality of cell-based meat and animal products at a low price. These egg-based systems can effectively decrease resource intensity and increase environmental sustainability of production of meat and other animal products, compared to current industrial animal farming, which is associated with issues of greenhouse gas emission, land usage, deforestation, biodiversity, antibiotic resistance, and animal welfare. In particular, with respect to poultry, the methods disclosed herein can completely do away with the conventional broiler cycle and one-day chicks while providing at least about 20 times higher yields of chicken meat per egg laying hen per year.

[0037] In some aspects, the current disclosure encompasses a cell culture media formulation comprising ingredients sourced from eggs, for use in cellular agriculture and other cell productbased applications.

I. Media formulations

[0038] In some aspects, the current disclosure encompasses a growth medium for production of cultured cells, comprising a base medium and egg yolk or a derivative thereof, wherein the derivative comprises at least one protein and at least one lipid found in an egg yolk.

[0039] As used herein the term “basal medium” or “base medium” is used interchangeably to mean an unsupplemented medium that promotes growth of multiple cell types. In general, basal media comprise amino acids, vitamins, a carbon source, and ions (calcium, magnesium, potassium, sodium, and phosphate) essential for cell survival and growth. In some aspects, the base media for use in the current disclosure may be any media suitable for cell growth. In some aspects, a basal medium for use herein can be a commercially available basal medium, nonlimiting examples of which include cell culture media that may be purchased from commercial vendors (e.g., Gibco, Sartorius, etc.), or synthesized, include SAFC Excell media, BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 (Gibco) and Ham-F10 (Gibco), IMDM (Iscove's Modified Dulbecco's medium), MacCoy's 5A medium,

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SUBSTITUTE SHEET (RULE 26) RPMI 1640, and GTM3. In some aspects, the media disclosed herein can be combined with additional cell culture components. In some aspects, the basal medium can be a defined culture medium with known quantities of each medium ingredient. In some aspects, the medium can be an undefined medium concocted in-house.

[0040] In an aspect, the current disclosure encompasses an adipogenic growth medium comprising an egg yolk or a derivative thereof in a base medium. In some aspects, the egg yolk or a derivative thereof may be sourced naturally from an egg or produced by synthetic or recombinant means. In an aspect, the medium comprises at least one protein and at least one lipid derived from an egg yolk. As used herein the term derived encompasses one or more components either obtained from an egg or components naturally found in an egg but sourced from a recombinant or synthetic source. The terms “derivative,” “variant,” and “fragment,” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function. Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide. Said polypeptides can be sourced from a synthetic, recombinant or natural source. The term “derivative” when used with respect to lipid encompasses a derivative of any lipid that is naturally present in an egg. As used herein, the term lipid encompasses a diverse group of organic molecules that include triglycerides (fats and oils), phospholipids, steroids, and waxes found in eggs. In an aspect, the term lipids also encompass related organic molecules like surfactants. Said lipids for use in the current disclosure may be sourced naturally, or derived from a recombinant source, for example a bacterial or fungal source or synthesized commercially. In an aspect, the current disclosure also encompasses modifications of said lipids and/or surfactants.

[0041] In some aspects, the media as disclosed herein may comprise at least one, or at least two, at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or more egg proteins or variants or derivatives thereof. In some aspects, the media of the current disclosure may comprise at least two, at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 or more of an ovalbumin, ovotransferrin, ovomucoid, ovoglobulin G2, ovoglobulin G3, ovomucin ovoinhibitor, ovoglycoprotein, ovomacroglobulin, avidin, cystatin, lysozyme, apovitellenin l-VI, a lipovitellin, a livetin, phosvitin, biotin binding protein or variants or derivatives thereof, or any combination thereof. In some

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SUBSTITUTE SHEET (RULE 26) aspects, the media may comprise whole eggs or egg yolk. In some aspects, the medium as disclosed herein may comprise about 2% to about 20% of egg yolk by weight. In some aspects, the medium may comprise about 2%, or about 4%, or about 6%, or about 8%, or about 10%, or about 12%, or about 14%, or about 16%, or about 18%, or about 20% of egg yolk by weight. In some aspects, the medium as disclosed herein may further comprise about 2% to about 20% of egg white by weight. In some aspects, the medium may comprise about 2%, or about 4%, or about 6%, or about 8%, or about 10%, or about 12%, or about 14%, or about 16%, or about 18%, or about 20% egg white.

[0042] In some aspects, the media as disclosed herein may comprise at least one, or at least two, at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or more lipids derived from egg yolks. Non-limiting examples of lipids and derivatives thereof suitable for use in a adipogenic medium as disclosed herein include triglyceride, a phosphotidylcholine, phosphotidylethanolamine, lysophosphatidylcholine, cholesterol, lanosterol, desmosterol, 57-cholestenol, cholestanol, 57- and 58-methostenol, 4a-methyl-58, 24-cholestenol and its 57-isomer, 4,4a-dimethyl-57, 24-cholestenol, dihydrolanostenol, [3-sitosterol, ergosterol, sphingomyelin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0043] In some aspects, the media compositions disclosed herein may comprise additional ingredient derived from eggs. Non-limiting examples include growth factors, vitamins, minerals, choline, carotenoids, lecithins, or a combination thereof. Non-limiting examples of vitamins naturally occurring in eggs include vitamin B12, riboflavin (vitamin B2), pantothenic acid (vitamin B5), and vitamin D. They also contain small amounts of other B vitamins, such as folate and niacin. Non-limiting examples of minerals present in eggs include phosphorus, selenium, iron and trace minerals like zinc and copper. Non-limiting examples of carotenoids present in eggs include lutein and zeaxanthin. Examples of growth factors present in eggs include insulin growth factor 1 (IGF-1).

[0044] In some aspects, egg yolk or derivatives thereof including egg proteins and lipids and/or other egg-based ingredients as disclosed herein are usually found in eggs or are a variant or derivative of a molecule usually found in eggs. In some aspects, the molecule may be a natural molecule, an isolated molecule sourced from natural or recombinant source, a recombinant molecule, or a synthetic molecule. In some aspects, the molecule may naturally occur in eggs from any oviparous animal including but not limited to fish, amphibians, reptiles or birds. In some aspects, the egg is a bird egg. Non limiting examples of birds from which the egg molecule may be derived include chicken, duck, emu, goose, guinea fowl, gull, ostrich, pheasant, pigeon, quail,

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SUBSTITUTE SHEET (RULE 26) and turkey. In some aspects, the egg is a fish egg. Non-limiting examples include eggs from fishes that are used as common sources of tobiko, masago, ikura, or caviar for example salmon, paddlefish, bowfin, whitefish, trout, capelin or flying fish, bottarga, lumpfish etc. In some aspects, the egg maybe a reptile egg for example a crocodile, alligator, snake, or lizard egg. In some aspects, the molecule may be isolated from a recombinant source, for example a recombinant animal, bacteria, fungi or algae. In some aspects, the molecule may be synthesized ex vivo using biological or chemical synthesis techniques. Biological techniques may include cell and tissue culture or in vitro translation.

[0045] In some aspects, the one or more egg-based molecules like egg proteins, lipids, other egg ingredients, or egg yolk, egg white as disclosed herein may be isolated from the egg of any oviparous animal, recombinant sources, synthetic source or combinations thereof. In some aspects, the one or more egg-based molecules, egg yolk, egg white or whole egg as disclosed herein may be isolated from a fertilized or an unfertilized egg. In some aspects, the one or more egg-based molecules may be isolated from a fertilized egg. In some aspects, the one or more egg-based molecules may be isolated from a fertilized egg in the first trimester.

[0046] In some aspects, the media disclosed herein may further comprise additional structural or functional ingredients. Non-limiting examples of additional ingredients include growth factors, amino acids, vitamins, minerals, edible surfactants, buffering agents, antibiotics, fibers, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, poly(a-hydroxyacids), polylactic or polyglycolic acids, poly-lactide poly-glycolide copolymers, poly-lactide polyethylene glycol (PEG) copolymers, polyesters, poly(E-caprolactone), poly(3-hydroxy-butyrate), poly(s-caproic acid), poly(p- dioxanone), polypropylene fumarate), poly(ortho esters), polyol/diketene acetal addition polymers, polyanhydrides, poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxyhexane) (PCPP), poly[bis(p-carboxyphenoxy)methane] (PCPM), copolymers of SA, CPP and CPM poly (amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazene], poly[(organo)phosph- azenes]polymers, polyphosphates, polyethylene glycol polypropylene block co-polymers, copolymers prepared from the monomers of these polymers, random blends of these polymers, or mixtures or combinations thereof. In some aspects, the media formulation disclosed herein may comprise at least one additional growth factor. Non-limiting examples of growth factors include is FGF, VEGF, PGF, TGF, BDNF, GDNF, Shh, BMP-2, BMP-4, EGF, Noggin, R-Spondin 1 , Wnt- 3a, FGF-10, FGF-2.FGF-7, Activin A, IGF1 , NRG1, Jagged 1, HGF, IL-2, IL-4, IL-6, IL-3, IL-7,

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SUBSTITUTE SHEET (RULE 26) Flt3-ligand, PDGF, or TGF- 1 or any combination thereof. Non-limiting examples of vitamins that can be incorporated into the disclosed compositions include vitamin D, A, or different vitamin B complexes, thiamine, riboflavin, folic acid, D-Ca pantothenate.

[0047] As another non-limiting example, the medium may comprise edible fibers. In some aspects, the fibers provide structural matrices for adherent cells to grown on. The fibers may be isotropic or anisotropic. A variety of techniques may be used to form fibers from proteins, including electrospinning, wet-spinning, extrusion spinning, direct spinning, dry spinning, gel spinning or semi-melt spinning, or the like. The fibers may also be formed, woven, knitted, etc. to form woven mats, nonwoven fabrics, or other suitable structures.

[0048] In some aspects, the medium may comprise edible microcarriers or scaffold that provide structural support for growth of adherent cells. In some aspects these microcarriers or scaffolds are edible. The microcarriers or scaffold may have any shape or size. In some cases, more than one type of microcarrier may be present, e.g., some of which may have various materials, shapes, sizes, etc., such as are described herein. For example, in some aspects, the microcarrier may be planar, spherical, triangular, tubular, amorphous, spongy, porous or smooth or any combination thereof. In some aspects, at least some of the microcarriers may be substantially spherical or exhibit spherical symmetry, although in other aspects, at least some of the microcarriers may be non-spherically symmetric or may be anisotropic. In addition, in certain cases, at least some of the microcarriers may have a plurality of micropatterns for example grooves, e.g., as discussed herein. In some cases, however, some or all of the microcarriers may not necessarily be spherical. For example, at least some of the microcarriers or scaffold may have shapes such as cubical, rectangular solid, tetrahedral, octahedral, irregular, etc. In some aspects, these microcarriers or scaffold may also comprise egg-based ingredients. In some cases, at least some of the microcarriers have a shape that is substantially planar. For instance, the microcarrier may have a generally rectangular shape where the smallest dimension of the rectangular solid is substantially smaller than either of the other two dimensions, for example, by a factor of at least 3, at least 5, or at least 10, etc. In one set of aspects, at least some of the microcarriers or scaffolds may have one or more micropatterns, including but not limited to grooves, holes, compartments, or craters. Without wishing to be bound by any theory, it is believed that grooves may promote cellular alignment during growth on the microcarriers or other types of scaffolds. Thus, for example, myoblasts seeded within grooves on microcarriers may be induced to grow together to form substantially aligned myotubes, e.g., that are substantially parallel to each other. This can result in muscle fibers grown on the microcarriers. As this is a

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SUBSTITUTE SHEET (RULE 26) biological system, those of ordinary skill in the art would understand that the myotubes will not necessarily grow to be perfectly parallel to a high degree of mathematical precision. Nonetheless, the myotubes may still be readily identified as having substantially parallel myotubes within the cultured animal-derived product, for example, as opposed to myotubes grown on spherical particles not containing grooves, where the myotubes are formed randomly from the myoblasts. For instance, the myotubes may exhibit a strong preference to the direction of the grooves, e.g., having an average directionality that varies by less than 20°, less than 15°, less than 10°, or less than 5° relative to the direction of the grooves. Thus, in some aspects, the grooves may be positioned or sized within the microcarriers or other scaffolds to allow the myoblasts to be directional or aligned, e.g., to allow them to fuse together to become myotubes. One or more grooves may be present. For instance, a microcarrier or other scaffold may have at least 2, at least 3, at least 4, at least 5, at least 7, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 70, or at least 100 or more grooves defined therein. If more than one type of microcarrier or scaffold is present, they may independently have the same or different numbers of grooves. In some cases, the average number of grooves present within the microcarriers may have the ranges described here. The grooves may be positioned in any orientation on the microcarriers. For instance, the grooves may be substantially parallel to each other, e.g., to promote the formation of substantially aligned myotubes.

[0049] In some aspects, the media as disclosed herein may be formulated as liquids, for example ready-to-use media, media concentrates, solids, for example lyophilized media, powders, frozen culture media. In some aspects, all the components of the media may be packaged in a single solution, premixed for use. In some aspects, one or more components of the media may be separately packaged to be added before or during use. In some aspects, the media is formulated for use in laboratory scale culture systems. In some aspects, the media is formulated for use in industrial scale culture systems comprising for example at least a bioreactor. In some aspects, the media is formulated for culturing non-human animal cells for production of cultured meat products. In an exemplary set-up, the media is formulated to culture a cell derived from an avian egg or an avian egg cell line to produce an ex vivo or in vitro generated avian meat product, thus completely forgoing the need for conventional broiler cycle. In some aspects, the media as disclosed herein is formulated to support culture of a non-human animal cell to produce animal products, non-limiting examples of which include tusk, leather, feather, skin or bone. In some aspects, the media is formulated for supporting growth of non-human animal cells or cell lines or human cells or cell lines to generate tissues, organoids, or organs for veterinary or medical purposes. In some aspects, the media is configured to support culture of non-human animal cells

13

SUBSTITUTE SHEET (RULE 26) or human cell lines for production of a recombinant protein, an antigen, a vaccine, a therapeutic polypeptide, an antibody, Fc fusion proteins, hormones, interleukins, enzymes, or anticoagulants. As such the composition of the media may vary based on the application as provided herein.

[0050] In an aspect, the current disclosure also encompasses a media for in vitro cell culture comprising egg yolk or a derivative thereof, wherein the in vitro cell culture does not require a media exchange for at least 5-10 days, to maintain a 75%-100% cell proliferation rate. In an aspect, the cell culture does not require a media exchange for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. In an aspect, the in vitro cell culture has reduced amounts of ammonia compared to a medium not comprising egg yolk after 5-10 days. In an aspect, the in vitro cell culture has reduced amounts of lactic acid compared to a medium not comprising egg yolk after 5-10 days. In an aspect, the in vitro cell culture maintains a pH of 7-8 for at least about 5-10 days. Thus, in some aspects, the media as disclosed herein maintains a pH of about 7, or about 7.5, or about 8 and non-toxic levels of ammonia and/or lactic acid during cell culture for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. In an aspect, the addition of egg yolk to basal media enhances the buffering capacity of culture media against pH change derived by atmospheric change of carbon dioxide concentration. In an aspect, the addition of egg yolk or derivatives thereof reduces the concentration of atmospheric carbon dioxide in the bioreactor by at least about 20% as compared to non-yolk supplemented media.

[0051] In an aspect, the adipogenic or cell culture medium as disclosed herein is stable at 37 °C for at least 10-20 days. In an aspect, the medium is stable at 37 °C for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days.

II. Methods

[0052] In some aspects, the current disclosure also encompasses methods of differentiating a precursor cell into an adipocyte, comprising culturing the precursor cell in a medium comprising an egg yolk, or a derivative thereof wherein the derivative comprises at least one protein, and at least one lipid found in egg yolks, for a time sufficient for the precursor to differentiate into the adipocyte.

[0053] In some aspects, the current disclosure encompasses a method of making a cultured meat product, the method comprising, obtaining a non-human animal cell and propagating the

14

SUBSTITUTE SHEET (RULE 26) non-human animal cell in a medium comprising the composition as disclosed herein. In some aspects, the disclosed method is for use in the production of cultured meat and other animal products. For example, in some aspects, the method can comprise the first step of obtaining a non-human animal cell. As used herein, the term “non-human cell” encompasses any cell from a non-human source or a part thereof (tissue, organ, system or embryo). As used herein, the term “derivative” of a non-human cells encompasses isolated cells, cell lines, or recombinant cell animal lines. In some aspects, the cell is an embryonic cell. In some aspects, the cell is a somatic cell. In some aspects, the cell is a differentiated cell. In some aspects, the cell is an adherent cell, for example a fibroblast, a myoblast, an adipocyte, an endothelial cell, or a mesodermal cell. In some aspects, the cell is a stem cell for example an embryonic stem cell, bone marrow derived stem cell, adipose derived stem cell, or induced pluripotent stem cell. In some aspects, the cell is a vertebrate cell. In some aspects, the non-human animal cell may be from any animal cell, for example a fish, avian, porcine, ovine, bovine, hoofed ruminant mammal, or rabbit cell or a derivative or a recombinant cell line thereof. In some aspects, the non-human cell is an invertebrate cell. Non-limiting examples of invertebrate cells suitable for the current disclosure include cells derived from oysters, mussels, clams, scallop, jelly fishes, squids, prawns, octopus, sea cucumbers, sea squirts or recombinant cell lines thereof. In an exemplary aspect, the cell is an avian cell derived from an egg.

[0054] In some exemplary aspects, the non-human animal cell may be seeded in a laboratory scale culture system, a small-scale bioreactor, or an industrial-scale system comprising at least a bioreactor. In some aspects, any suitable bioreactor may be used in this application, including but not restricted to continuous stirred tank bioreactors, bubble column bioreactors, airlift bioreactors, fluidized bed bioreactors, packed bed bioreactors and photo-bioreactors. In some aspects, the method disclosed herein may also be used at laboratory scale for cell propagation in petri dishes, culture plates, or small-scale bioreactors. In some exemplary aspects, the non- human animal cells may be seeded in a bioreactor or other adipogenic yolk-based media compositions such as are described herein, to produce a cultured meat product. In some aspects, the bioreactor may support a suspension or an adherent cell culture or both. In some aspects, the cells may be seeded onto a microcarrier or scaffold and then cultured in a medium as disclosed herein. In some aspects, the meat product may be formed using an adherent cell culture platform. As a non-limiting example, a cultured meat product may be grown by seeding myoblasts onto microcarriers or scaffolds, then grown within a bioreactor comprising a medium as disclosed herein to form the cultured meat product.

15

SUBSTITUTE SHEET (RULE 26) [0055] In some cases, the cultured meat product thus formed can be used without any additional processing. The cultured meat product may not require subsequent separation or processing steps to convert the cultured cells into a product ready to be cooked or otherwise used as meat. However, it should be understood that in other aspects, additional steps may be used to convert the cells grown within the bioreactor into a cultured meat product, or other cultured animal-derived product. In further aspects, the cultured meat has substantially the same composition with respect to percent proteins, fat, carbohydrates and the like as beef, veal, pork, chicken, or fish. In some aspects, the cultured meat comprises a plurality of layers, wherein each layer comprises non-human myocytes and non-human endothelial cells. In various aspects, the cultured meat disclosed herein comprises a plurality of layers, wherein each layer comprises myocytes, and may include one or more of endothelial cells, adipose cells, and/or fibroblasts, wherein the cells are derived from sources including, but not limited to, mammals, birds, reptiles, fish, crustaceans, mollusks, and cephalopods, or combinations thereof. In some aspects, the myocytes are aligned relative to each other. In further aspects, the myocytes are aligned relative to a layer of the meat. In various aspects, the cultured meat disclosed herein further comprises one or more substances that enhance the nutritional value of the meat, the culinary appeal of the meat, or the growth characteristics of the non-human cells. In some aspects, the cultured meat further comprises one or more nutritional supplements. In further aspects, the nutritional supplements are selected from: vitamins, minerals, fiber, fatty acids, and amino acids. In some aspects, the cultured meat further comprises one or more flavorants and/or colorants. In some aspects, the cultured meat further comprises one or more of: matrix proteins, proteoglycans, antioxidants, , and growth factors. In some aspects, the cultured meat is suitable for human consumption. In other aspects, the cultured meat is suitable for non-human animal consumption. In still other aspects, the cultured meat is suitable for both human and non-human animal consumption. In some aspects, the cultured meat product made using the method herein may further comprises one or more of an emulsifier, surfactant, plasticizer, thickener, salt, sugar, coloring agent, binding agent, stabilizing agent, flavor enhancer, flavoring agent, fragrance enhancer, nutritional supplement (e.g. vitamin, mineral, antioxidants), essential oil, pH regulator, preservative, dietary fiber, or any combination thereof. In some aspects, the cultured meat product as disclosed herein does not contain any added plant-based proteins. In some aspects, the cultured meat product as disclosed herein contains less that about 5% of plant-based proteins. In some aspects, In some aspects, the cultured meat product as disclosed herein contains non more that about 5% or about 4%, or about 3%, or about 2%, or about 1% plant-based proteins.

[0056] In some aspects, the process of making cultured meat as disclosed herein provides

16

SUBSTITUTE SHEET (RULE 26) multiple benefits over currently available methods (see Table 1).

Table 1 :

[0057] In some aspects, the disclosed method provides meat at a faster rate, higher efficiency, high scalability and lower costs. In some aspects, the disclosed method does away with the requirement for genetically modified products, artificial growth factors and ensure almost 100% animal protein that can be labeled vegan, organic, free-range, cage free or grass-fed based on the meat (see FIG. 3B). In some aspects, using the disclosed method of producing meat from cells, may yield at least about 5 times - to at least about 50 times as much meat per year than using conventional methods. In some aspects, the method disclosed herein can produce at least about 5 times, or at least about 10 times, at least about 15 times, or at least about 20 times, at least about 25 times, or at least about 30 times, or at least about 35 times, or at least about 40 times, or at least about 45 times, or at least about 50 times as much meat as obtained from a single animal. In some aspects, the method disclosed herein does not require the use of harmful chemicals and antibiotics for meat production. In some aspects, the disclosed methods reduce labor costs, housing costs, water consumption, medicine costs, vaccination costs, slaughtering and harvesting costs. In some aspects the current method can reduce the cost of meat production by at least about 5 times, or at least about 10 times, at least about 15 times, or at least about 20 times, at least about 25 times, or at least about 30 times, or at least about 35 times, or at least about 40 times, or at least about 45 times, or at least about 50 times, or at least about 55 times, or at least about 60 times, or at least about 65 times, or at least about 70 times, or at least about 75 times, or at least about 80 times, at least about 90 times, or at least about 95 times, or at least about 100 times or more. In some aspects, the method results in at least about 5-20 times faster production times for the same amount of meat. In some aspects the method results in at least about 5 times, or at least about 10 times, at least about 15 times, or at least about 20 times or more faster production times for the same amount of meat.

17

SUBSTITUTE SHEET (RULE 26) [0058] In an exemplary aspect, the current disclosure also encompasses a method of producing avian meat, the method comprising extracting at least one cell from an avian egg; and incubating the at least one cell extracted from the egg in the presence of at least one egg derived protein in a bioreactor or cell culture system wherein the method does not require conventional broiler cycle to produce the avian meat product. In some aspects, the current disclosure also encompasses a method of producing avian meat from an egg, the method comprising: extracting at least one cell from an avian egg; and incubating the at least one cell in a medium as disclosed herein in a bioreactor or cell culture system; wherein the method does not require conventional broiler cycle to produce the avian meat product. Conventional methods of producing avian meat, for example chicken meat use traditional methods of poultry farming using the conventional broiler cycle. As used herein, a broiler is any chicken that is bred and raised for the specific purpose of producing meat. A broiler cycle takes an average of 7 weeks, from hatching of chickens to growing to a final weight. These conventional methods are rife with production problems, humanitarian concerns and environmental issues. In some aspects, the exemplary method as disclosed herein does not require the conventional broiler technology. In a non-limiting aspect, the cell in the current disclosure is obtained from an egg or an avian cell line and cultured in a medium as disclosed herein, comprising at least an egg-based ingredient. In the case of cells derived from an avian egg cell line, the method does not require hatching of an egg. Since the culturing is done in a bioreactor, adequate nutrients can be provided to produce greater biomass than from a single chicken. This biomass can be used directly or further processed to form edible products. In some aspects, using the disclosed method of producing avian meat from cells, an egg laying hen may yield at least about 5 times - to at least about 50 times as much meat per year than using convention broiler methods. In some aspects, the method disclosed herein can produce at least about 5 times, or at least about 10 times, at least about 15 times, or at least about 20 times, at least about 25 times, or at least about 30 times, or at least about 35 times, or at least about 40 times, or at least about 45 times, or at least about 50 times as much meat as obtained from one egg laying hen per year. In some aspects, the method disclosed herein does not require the hatching of an egg. In some aspects, the method disclosed herein does not require the use of one-day old chicks. In some aspects, the method disclosed herein does not require the use of harmful chemicals and antibiotics for meat production. In some aspects, the disclosed methods reduces labor costs, housing costs, water consumption, medicine costs, vaccination costs, slaughtering and harvesting costs. In some aspects the current method can reduce the cost of chicken meat production by at least about 5 times, or at least about 10 times, at least about 15 times, or at least about 20 times, at least about 25 times, or at least about 30 times, or at least

18

SUBSTITUTE SHEET (RULE 26) about 35 times, or at least about 40 times, or at least about 45 times, or at least about 50 times, or at least about 55 times, or at least about 60 times, or at least about 65 times, or at least about 70 times, or at least about 75 times, or at least about 80 times, at least about 90 times, or at least about 95 times, or at least about 100 times or more. In some aspects, the method results in at least about 5-20 times faster production times for the same amount of avian meat. In some aspects the method results in at least about 5 times, or at least about 10 times, at least about 15 times, or at least about 20 times or more faster production times for the same amount of avian meat.

[0059] In some aspects, the meat products prepared using the methods disclosed herein require no additional fats to be added to match the nutrition, taste, texture or mouth feel of naturally obtained meat. In an aspect, the methods as disclosed herein allows for production of meat products with minimal media changes, while still comprising lipids. In an aspect, the methods disclosed herein do not require additional media components that induce adipogenesis making the product healthier and with less processing requirements. This not only reduces the cost of production but also saves time. In an aspect, the method disclosed herein, does not require a media exchange for at least 5-10 days, to maintain a 75%-100% cell proliferation rate. In an aspect, the cell culture does not require a media exchange for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days. Thus, in some aspects, the method disclosed herein may result in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least reduction of culture media uses due to less frequent to no media change requirements. Culture media is significant driver of cost in cellular agriculture and accounts for ~80% of cost of production. The reduced amounts of media use could similarly reflect on the reduction in the total cost of production of cell culture products, particularly meat products. In an aspect, the cell culture grown by the method disclosed herein has reduced amounts of ammonia compared to a medium not comprising egg yolk after 5-10 days. In an aspect, the cell culture has reduced amounts of lactic acid compared to a cell culture method wherein the medium does not comprise, egg yolk after 5-10 days. In an aspect, the in vitro cell culture maintains a pH of 7-8 for at least about 5-10 days. In an aspect, the addition of egg yolk to basal media enhances the buffering capacity of culture media against pH change derived by atmospheric change of carbon dioxide concentration. In an aspect, the addition of egg yolk or derivatives thereof reduces the concentration of atmospheric carbon dioxide in the bioreactor by at least about 20% as compared to non-yolk supplemented media.

[0060] In some aspects, the current disclosure also encompasses the use of the disclosed

19

SUBSTITUTE SHEET (RULE 26) medium and methods, for the production of other animal products for instance structural animal products. Non-limiting examples of structural animal products include leather, tusks, feathers, or horns. In some aspects, structural animal product made using the method herein may further comprises additional ingredients, non-limiting examples of which include a plasticizer, polymer, thickener, gelatin, collagen, fibers or any combination thereof.

[0061] In some aspects, the current disclosure also encompasses the use of the disclosed media for production of cells, organs, tissues, or organoids for use in medical or veterinary purposes in a subject in need thereof. In some aspects, the subject is a vertebrate. In some aspects, the subject is a bird. In some aspects, the subject is a mammal. In some aspects, the subject is an ovine, a bovine, a feline, a porcine, an equine, or a primate. In some aspects, the subject is a human.

[0062] In some aspects, the current disclosure also encompasses the use of the disclosed medium for production of cell products including but not limited to proteins, glycoproteins, polysaccharides, lipids, biomass, or cells. In some aspects, the cell product may be derived from a human cell line. In some aspects, the cell product may be derived from a non-human animal cell, for example a mammalian, vertebrate or an invertebrate cell. In some aspects, the cell product is a protein. In some aspect the cell product is a recombinant protein. Non limiting examples of proteins that can be obtained using the methods disclosed herein include antibodies, antigens, vaccines, a therapeutic polypeptide, Fc fusion proteins, hormones, interleukins, enzymes, or anticoagulants. Thus, in some aspects, the current disclosure encompasses a method of making a recombinant protein, the method comprising, obtaining a cultured cell; propagating the cultured cell in a medium comprising the composition as disclosed herein, and harvesting the recombinant protein.

[0063] In some exemplary aspects of the method disclosed herein, cells can be seeded onto a microcarriers or scaffolds, and grown in vitro in a bioreactor or cell culture system comprising the medium as disclosed herein and using any method known in the art. In some aspects, cells may be confined on the cultured surface or material of the scaffold having a micro-nanotopography as contact guidance, or by applying mechanical forces generated either by the contractile activity of the cells or by an external strain. In some aspects, the bioreactor or cell culture system comprises a cell culture medium as disclosed herein and one or more of the microcarrier or scaffold or a combination thereof for supporting growth of adherent cells. In some aspects, the scaffold or microcarrier can be edible. In some aspects, the medium as disclosed herein comprises at least a basal medium and egg yolk or a derivative there of. In some aspects, the basal medium may

20

SUBSTITUTE SHEET (RULE 26) be any medium known in the art. Non-limiting exemplary cell culture media that may be purchased from commercial vendors (e.g., Gibco, Sartorius, etc.), or synthesized, include SAFC Excell media, BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eagle medium), DMEM- HamF12, Ham-F12 (Gibco) and Ham-F10 (Gibco), IMDM (Iscove's Modified Dulbecco's medium), MacCoy's 5A medium, RPMI 1640, and GTM3. In some aspects, the medium may comprise additional cell culture components including amino acids, growth factors, proteins, fats, vitamins, sugars, polysaccharides, minerals, edible surfactants, antibiotics, buffering agents, platelet rich plasma (PRP), platelet poor plasma (referred to as plasma), a platelet concentrate, a lysate of red blood cells, a platelet lysate (PL), cytokines, fibers, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, sugars, alginates, collagen, gelatin, polymers or any combination thereof.

[0064] In some aspects, the current disclosure encompasses cultured meat and cell products obtained from the use of the media compositions and methods disclosed herein. In some aspects, the methods disclosed herein provide at least about 1.5 to 50 times greater yield of propagated cells as compared to the yield of propagated cells in a medium without the use of the media compositions disclosed herein. In some aspects, the methods provides at least about 1.5 times, or at least about 2 times, or at least about 2.5 times, or at least about 3 times, or at least about 3.5 times, or at least about 4 times, or at least about 4.5 times, or at least about 5 times, or at least about 5.5 times, or at least about 6 times, or at least about 6.5 times, or at least about 7 times, or at least about 7.5 times, or at least about 8 times, at least about 8.5 times, or at least about 9 times, or at least about 9.5 times, or at least about 10 times improvement in the yield of cells, biomass, cell products than when the cells are grown without the use of the media compositions disclosed herein. In some aspects, the cell culture methods disclosed herein provides better cell growth rates than a cell culture method that does not use the compositions disclosed herein. In some aspects, the cell culture or cultured meat production methods disclosed herein provide at least about 1.5 to 10 times greater cell growth rate of as compared to the cell growth rate of propagated cells in a medium without the use of the compositions disclosed herein. In some aspects, the method provides at least about 1.5 times, or at least about 2 times, or at least about 2.5 times, or at least about 3 times, or at least about 3.5 times, or at least about 4 times, or at least about 4.5 times, or at least about 5 times, or at least about 5.5 times, or at least about 6 times, or at least about 6.5 times, or at least about 7 times, or at least about 7.5 times, or at least about 8 times, at least about 8.5 times, or at least about 9 times, or at least about 9.5 times, or at least about 10 times improvement in the cell growth rate.

21

SUBSTITUTE SHEET (RULE 26) III. Terminology

[0065] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0066] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and/or the like in the description mean that the feature and/or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and/or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and/or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and/or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0067] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%,

22

SUBSTITUTE SHEET (RULE 26) such as less than or equal to ± 2%, such as less than or equal to ± 1 %, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1 %, such as less than or equal to ± 0.05%.

[0068] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0069] Lastly, the terms “or” and “and/or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and/or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

EXAMPLES

[0070] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

EXAMPLE 1 : Chicken embryonic myofibroblast isolation

[0071] Fertile chicken eggs were placed in an incubator at 100° F at a relative humidity of 40- 70%. Chicken embryos at second trimester (day 7-14 after incubation) were harvested and the embryo legs were dissected after decapitation of embryo. Thereafter, the tissue was minced thoroughly using sterile tweezer/scissors. 5-6 g of the muscle tissue was transferred into a clean 50 mL tube containing 20 mL of the Isolation medium (Tube 1). Tube 1 was incubated in a water bath at 37° C for 1.5 hours with vigorous shaking every 10 min. The tube was centrifuged at 200 x g for 10 min and the supernatant transferred to another clean tube (Tube 2). 20 mL of prewarmed Trypsin-EDTA solution was added to Tube 1 and the tube was incubated in a water bath for 20 min. In parallel, Tube 2 was centrifuged at 300 x g for 5 min. Supernatant was discarded and the cell pellet resuspended in 10 mL Isolation medium. Upon completion of the trypsinization, Tube 1 was centrifuged at 300 x g for 5 min. The supernatant was discarded, and 5 mL culture medium was added. Tube 1 was centrifuged at 200 x g for 5 min. The top and middle phase of

23

SUBSTITUTE SHEET (RULE 26) Tube 1 was transferred to Tube 2. The tube 2 was centrifuged at 500 x g for 10 min. The supernatant was discarded, and the cell pellet was resuspended in 10 mL culture medium. Thereafter the suspension was transferred into cell culture flask and incubated overnight at 37° C under 5% CO2. The myofibroblasts attach to the flask while other cells and tissue debris remain in the suspension. The myofibroblasts were stored in the culture medium and media exchanged every other day to reach to the confluency as provided in FIG. 1 .

EXAMPLE 2: Effect of egg yolk concentration on cell proliferation

[0072] Chicken embryonic myofibroblasts were trypsinized and detached from culture flask and suspended in a serum free culture medium. Several cell-culture dishes were coated with gelatin and the cell suspension was added at initial density of 50000 cells per dish. The cells were incubated at different concentrations of egg yolk in basal media (DM EM high glucose, supplemented with L-glutamine, HEPES buffer, penicillin, and streptomycin) at 38° C and 95% humidity for 4 days. The cells were then detached from the tissue culture flask and counted using the hemocytometer. Basal media without yolk was used as control. The non-fertile egg yolk were obtained from local grocery stores and added to basal media at different concentration. The results showed that supplementation with egg yolk enhanced cell proliferation as compared to basal media (see FIG. 2). Also, 5-7.5% concentration of yolk was found optimal in this setup of experiment for cellular proliferation.

EXAMPLE 3: Production scale-up

[0073] Basal medium containing 5-7.5% concentration of yolk and further supplemented with vitamins, minerals and amino acids (see FIG. 3A) is provided in a bioreactor along with myofibroblasts, adipocytes or both and the resulting culture is processed to produce products. The production method provides several benefits to preexisting culture methods as shown in FIG. 3B.

[0074] Based on the results from these experiments, a media composition comprising 5-7.5% concentration of egg yolk in basal media along with other supplements is formulated. The formulated media is called Hatchless and provides significant yield and cost advantage over commercially available media.

Example 4: Egg yolk-based media have longer shelf life at room temperature.

[0073] Preliminary experiments were conducted with egg yolk-based media disclosed herein to check for stability under culture conditions. Embryonic chicken fibroblasts were cultured in freshly made FBS-based media as well as egg-yolk containing media. Both FBS and egg-yolk

24

SUBSTITUTE SHEET (RULE 26) was used at 10% v/v concentration. The initial cell density was set at 10⁵ cells/ml, and the cells were counted 4 days after and recorded.

[0074] At the same time, freshly prepared media were kept at 37 °C for 7 days and the same experiment was repeated, and the cell count taken after 4 days of culture were compared to prior results and presented as relative proliferation potency percentage. The results show that the proliferation potency of FBS-based media was significantly dropped after preservation in 37 °C while egg-yolk based media was preserve it potency (see FIG. 4 ).

Example 5: Egg yolk-based media require fewer media exchange during cell culture

[0075] Commercially established protocols use serum-based and serum-free cell culture media, and recommend media exchange every other day to support proper cell growth. When media is not changed frequently, cell growth can be hampered and lead to cell loss. Media exchange is practiced for two reasons. Many growth factors and some nutrients such as glutamine in media are not stable at 37° C and degrade over time thus reducing the growth capacity of cell culture media. Glutamine is most stable when stored at 4 °C and at neutral pH. Loss of glutamine accelerates at temperatures above 4 °C, such as room temperature or above. Secondly, the accumulation of cell waste such as ammonia and lactic acid create cell toxic environment that may lead the cell death. T oxic waste typically increases with stressful environment and metabolic activity of cells triggered by lack of proper nutrient and or growth factors bioavailability.

[0076] Embryonic chicken fibroblasts were cultured in freshly made egg-yolk media in tissue culture flask for 7 days up to the full confluency. The pH of media was measured every other day. No significant change of pH was observed during cell culture period. In comparative example, FBS-based media showed pH reduction at day 5 of culture toward the acidic condition (below 6.5) which is known can damage cells and their proliferation (see FIG. 5).

[0077] Additionally, it was discovered that egg-based media are highly stable at 37 °C with no significant reduction in cellular growth potency over the duration of 7 days. While, nutrient rich basal media was used in these experiments, that substitute glutamine with more stable dipeptide such glutamine-alanine, it was observed that chicken cells grew well in such media during 7 days of continuous culture without media exchange until confluency. Egg yolk-based media surprisingly helped to stabilize media and cells to produce lesser cellular waste such as ammonia and lactic acid as seen from the current analysis. For example, pH drop in the culture was less as compared to standard FBS-based media. It might be that egg yolk has some additional substances that

25

SUBSTITUTE SHEET (RULE 26) activate different metabolic pathways, resulting in lesser cellular waste production such as ammonia or lactic acid, thus providing a stable environment for cellular growth.

EXAMPLE 6: Detection of fat droplets in chicken fibroblast cultures grown in egg yolkbased media

[0078] Most cell culture media require the addition of additional growth promoting factors and media changes to induce adipocyte formation. To test if egg yolk-based media is capable of inducing fibroblast differentiation to adipocytes without the need for additional factors, chicken fibroblasts were isolated from chicken embryo at day 8 of incubation of fertile eggs using the following method. Chicken legs were dissected from an embryo and minced thoroughly using sterile tweezer/scissors. For every required T175 flasks of isolated cells, the tissue was transferred into a clean 50mL tube with collagenase I. The tube was incubated in a water bath at 37° C for 1.5 hours and shaken vigorously every 10 min. Next trypsin was similarly used for digestion of the tissue.

[0079] Upon the completion of the trypsinization, the tube was centrifuged at 600 x g for 8 min. Around three quarter of supernatant was carefully discarded with a pipet-boy (no suction) and 5mL DMEM/F12 and P/S at 3% was added. The contents were mixed thoroughly. The tube was centrifuged at 200 x g for 5 min. The supernatant was discarded, and the cell pellet resuspended in 26 mL isolation medium. The suspension was transferred into 2 T-175 flasks and incubated overnight at 37°C under 5% CO2. The fibroblasts attach to the flask while the satellite muscle precursor cells remain predominantly in the suspension.

[0080] After isolation the fibroblasts, the cells were exposed to 5% yolk-based DMEM/F12 medium. While the cells were proliferating, the formation of fat droplet in the cytoplasm of cells was noticed. To confirm the presence of adipocyte, the cells were stained with lipophilic dye, red Nile. Microscopic images showed the formation of fat cells (see FIG. 6).

Example 7: Spontaneous formation of muscle fiber and fat cells in cultures

[0081] Fibroblasts and myoblasts were cocultured in a cell culture flask and the cells were allowed to proliferate up to confluency for 5 days in yolk-based culture media (5% yolk and 95% DMEM/F12). Thereafter the cells were visually examined for the formation of myofibers and fat cell. Formation of long fiber and fat cells were observed, thus confirming the co-stimulation of fat cells and muscle fiber (see FIG. 7A and 7B).

26

SUBSTITUTE SHEET (RULE 26)

Claims

1. An adipogenic medium, comprising: a) a base medium; b) an egg yolk, or a derivative thereof comprising at least one protein, and at least one lipid from an egg yolk.

2. The adipogenic medium of claim 1 , comprising an egg yolk.

3. The adipogenic medium of claim 1, wherein the at least one protein is selected from a group consisting of apovitellenin l-VI , a lipovitellin, a livetin, phosvitin, biotin binding protein or a recombinant variant or derivative thereof, or any combination thereof.

4. The adipogenic medium of claim 1 , wherein the at least one lipid is selected from a triglyceride, a phosphotidylcholine, phosphotidylethanolamine, lysophosphatidylcholine, cholesterol, lanosterol, desmosterol, 57-cholestenol, cholestanol, 07- and 08- methostenol, 4a-methyl-08,24-cholestenol and its 07-isomer, 4,4a-dimethyl-07, 24- cholestenol, dihydrolanostenol, [3-sitosterol, ergosterol, sphingomyelin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

5. The adipogenic medium of any one of claims 1-4, wherein the egg yolk or the derivative thereof is from an oviparous animal.

6. The adipogenic medium of claim 5, wherein the egg yolk or the derivative thereof is derived from a fish, amphibian, reptile or avian egg.

7. The adipogenic medium of any one of claims 5 or 6, wherein the egg yolk or the derivative thereof is derived from an unfertilized egg.

8. The adipogenic medium of any one of claims 5 or 6, wherein the egg yolk or the derivative thereof is derived from a fertilized egg.

9. The adipogenic medium of claim 8, wherein the egg yolk or the derivative thereof is derived from the fertilized egg in any one of first, second or third trimester.

10. The adipogenic medium of claim 9, wherein the egg yolk or the derivative thereof is derived from a fertilized egg in first trimester.

27

SUBSTITUTE SHEET (RULE 26)

11. The adipogenic medium of any of claims 1-10, wherein the egg is an avian egg comprising a chicken egg, GF chicken egg, ostrich egg, quail egg, or partridge egg.

12. The adipogenic medium of any one of claims 1-10 wherein the egg yolk derivative is derived from a recombinant source, selected from a fungus, a bacterium or an alga.

13. The adipogenic medium of any one of claims 1-12, comprising at least one additional egg ingredient.

14. The adipogenic medium of claim 13, wherein the egg ingredient is selected from a growth factor, vitamins, minerals, choline, carotenoids, lecithin, or a combination thereof.

15. The adipogenic medium of any one of claims 1-16, comprising additional ingredients to promote cell culture.

16. The adipogenic medium of any one of claims 1-14, comprising a basal medium.

17. The adipogenic medium of claim 16, wherein the basal medium is selected from DMEM, DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench stable medium, human plasma like medium, primary culture medium, supplemented culture medium.

18. The adipogenic medium of claim 17, wherein the additional ingredients are selected from a group consisting of one or more of growth factors, amino acids, vitamins, minerals, edible surfactants, antibiotics, pectin, alginate, agarose, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides, alginates, collagen, gelatin, poly(amino acids), peptides, polypeptides, fats, fibers, buffering agents or any combinations thereof.

19. The adipogenic medium of claims 18, wherein the growth factor is FGF, VEGF, PGF, TGF, BDNF, GDNF, Shh, BMP-2, BMP-4, EGF, Noggin, R-Spondin 1 , Wnt-3a, FGF-10, FGF- 2.FGF-7, Activin A, IGF1 , NRG1 , Jagged 1 , HGF, IL-2, IL-4, IL-6, IL-3, IL-7, Flt3-ligand, PDGF, or TGF-pi or any combination thereof.

20. The adipogenic medium of any one of claims 1-19, comprising about 0.5%-20% (w/w) of egg yolk.

21. The adipogenic medium of claim 20, comprising at least about 5% (w/w) of egg yolk.

28

SUBSTITUTE SHEET (RULE 26)

22. The adipogenic medium of any one of claims 1-21, wherein the medium supports differentiation of adipocytes from a precursor cell.

23. The adipogenic medium of claim 22, wherein the precursor cell is a stem cell comprising an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell, a fibroblast, a myoblast, or a preadipocyte.

24. The adipogenic medium of any one of claim 1-23, wherein the medium does not require addition of signaling molecules, drugs or fatty acids to the medium to induce adipogenesis.

25. The adipogenic medium of any one of claims 1-24, wherein the medium is configured for use in a bioreactor for in vitro cell culture.

26. The adipogenic medium of claim 25, configured for culturing non-human cells to produce a cultured meat product.

27. The adipogenic medium of claim 26, configured for producing animal fat rich products.

28. The adipogenic media of claim 25, wherein cell culture does not require a media exchange for at least 5-10 days, to maintain a 75%-100% cell proliferation rate.

29. The adipogenic medium of claim 28, wherein the cell culture does not require a media exchange for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days.

30. The adipogenic medium of claim 29, wherein the medium is stable at 37 °C for at least 1 fl- 20 days.

31. The adipogenic medium of claim 30, wherein the medium is stable at 37 °C for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days, or about 11 days, or about 12 days, or about 13 days, or about 14 days, or about 15 days, or about 16 days, or about 17 days, or about 18 days, or about 19 days, or about 20 days.

32. The adipogenic medium of any one of claims 25-31 , wherein the in vitro cell culture has reduced amounts of ammonia compared to a medium not comprising egg yolk after 5-10 days.

29

SUBSTITUTE SHEET (RULE 26)

33. The adipogenic medium of any one of claims 25-32, wherein the in vitro cell culture has reduced amounts of lactic acid/lactate compared to a medium not comprising egg yolk after 5-10 days.

34. The adipogenic medium of any one of claims 25-33, wherein the in vitro cell culture maintains a pH of 7-8 for at least about 5-10 days.

35. The adipogenic medium of any one of claims 25-34, wherein the addition of egg yolk to basal media enhances the buffering capacity of culture media against pH change derived by atmospheric change of carbon dioxide concentration.

36. The adipogenic medium of any one of claims 25-35, wherein the addition of egg yolk reduces the atmospheric carbon dioxide in a bioreactor by at least 20% as compared to non-yolk supplemented media.

37. The adipogenic medium of any one of claim 25-36, wherein the medium maintains at least about 75% cell potency after 5-10 days of use at 37 °C.

38. A method of differentiating a precursor cell into an adipocyte, comprising culturing the precursor cell in a medium comprising an egg yolk, or a derivative thereof comprising at least one protein, and at least one lipid from an egg yolk for a time sufficient for the precursor to differentiate into the adipocyte.

39. The method of claim 28, wherein the precursor cell is a non-human animal cell.

40. The method of claim 29, wherein the non-human animal cell is a stem cell, a fibroblast, a myoblast, or a preadipocyte.

41. The method of claim 30, wherein the non-human animal cell is a stem cell selected from an embryonic stem cell, bone marrow derived stem cell, adipose derived stem cell, induced pluripotent stem cell.

42. The method of any one of claim 29-31, wherein the non-human cell is a fish, avian, porcine, bovine, ovine, hoofed ruminant mammal, rabbit cell or a derivative thereof.

43. The method of any one of claims 28-32, for use in production of a meat product.

44. The method of any one of claims 28-32 for use in production of a cell product.

30

SUBSTITUTE SHEET (RULE 26)

45. The method of claim 28, wherein the precursor cell is a cell derived from a human cell line, for production of a cell product.

46. The method of claim 34 or 35, wherein the cell product is for research, therapeutic, biomedical or industrial goods production.

47. The method of any one of claims 28-36, wherein the culturing of cell is done in a bioreactor or a cell culture system.

48. The method of claim 28, wherein the egg yolk or the derivative thereof is derived from the egg of an oviparous animal.

49. The method of claim 38, wherein the egg yolk or the derivative thereof is derived from a fertilized egg.

50. The method of claim 39, wherein the egg yolk or the derivative thereof is derived from the first, second or third trimester of a fertilized egg.

51. The method of claim 39, wherein the egg yolk or the derivative thereof is derived from an unfertilized egg.

52. The method of claim 31, wherein the egg yolk derivative is derived from a recombinant source, selected from a fungus, a bacterium or an alga.

53. The method of claim 28, wherein the medium comprises an egg yolk.

54. The method of claim 43, wherein the medium comprises 0.5%-20% egg yolk.

55. The method of claim 44, wherein the medium comprises 5% egg yolk.

56. The method of any one of claims 28-45, wherein the medium further comprises an egg white.

57. The method of any one of claims 28-46, wherein the medium comprises 0.1%-20% of egg white.

58. The method of any one of claims 28-47, wherein medium comprises a basal medium, DMEM, DMEM F12, minimum essential medium, RMI 1640, serum free medium, bench

31

SUBSTITUTE SHEET (RULE 26) stable medium, human plasma like medium, primary culture medium, supplemented culture medium.

59. The method of any one of claims 28-48, wherein the medium further comprises at least one additional egg ingredient.

60. The method of claim 49, wherein the additional ingredient is growth factor, vitamins, minerals, choline, carotenoids, lecithins, or a combination thereof.

61. The method of any one of claims 28-50, wherein the medium does not require addition of signaling molecules, drugs or fatty acids to the medium to induce adipogenesis.

62. The method of any one of claims 28-51 , wherein the adipogenic medium further comprises one or more of amino acids, vitamins, polysaccharides, sugars, minerals, edible surfactants, antibiotics, or growth factors.

63. The method of claim 52, wherein the growth factor is selected from fibroblast growth factor (FGF), VEGF, PGF, TGF, BDNF, GDNF, Shh, BMP-2, BMP-4, EGF, Noggin, R-Spondin 1 , Wnt-3a, FGF-10, FGF-2.FGF-7, Activin A, IGF1 , NRG1 , Jagged 1 , HGF, IL-2, IL-4, IL- 6, IL-3, IL-7, Flt3-ligand, PDGF, or TGF-[31 or any combination thereof.

64. The method of any one of claims 28-53, wherein the method is for production of a cell, organ, tissue, organoid, or whole animal.

65. The method of any one of claims 28-54, wherein the method uses least 20%, or at least 50%, or at least 70% less media.

66. A system for culturing cells, the system comprising: a. a bioreactor or a cell culture unit; b. a cell culture medium of any one of claims 1-27; c. a precursor cell or cell culture; wherein the cell culture medium does not require addition of signaling molecules, drugs or fatty acids to induce adipogenesis.

67. The system of claim 66, wherein the system is adapted for production of a cultured meat product.

32

SUBSTITUTE SHEET (RULE 26)

68. The system of claim 66, wherein the system is adapted for production of cells, tissue, organoids, or organs.

69. The system of claim 67, wherein the cultured meat product comprises no added adipocytes or fat.

70. A media for in vitro cell culture comprising egg yolk, wherein the in vitro cell culture does not require a media exchange for at least 5-10 days, to maintain a 75%-100% cell proliferation rate.

71. The media of claim 70, wherein the cell culture does not require a media exchange for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days.

72. The media of claim 71 , wherein the medium is stable at 37 °C for at least 10-20 days.

73. The media of claim 72, wherein the medium is stable at 37 °C for at least about 5 days, or about 6 days, or about 7 days, or about 8 days, or about 9 days, or about 10 days up to 20 days.

74. The media of any one of claims 70-73, wherein the in vitro cell culture has reduced amounts of ammonia compared to a medium not comprising egg yolk after 5-10 days.

75. The media of any one of claim 70-74, wherein the in vitro cell culture has reduced amounts of lactic acid compared to a medium not comprising egg yolk after 5-10 days.

76. The media of any one of claims 70-75, wherein the in vitro cell culture maintains a pH of 7-8 for at least about 5-10 days.

77. The media of any one of claims 70-76, wherein the addition of egg yolk to basal media enhance the buffering capacity of culture media against pH change derived by atmospheric change of carbon dioxide concentration.

78. The media of any one of claims 70-77, wherein the addition of egg yolk reduces the concentration of atmospheric carbon dioxide in the bioreactor by at least about 20% as compared to non-yolk supplemented media.

33

SUBSTITUTE SHEET (RULE 26)

9. The media of any one of claim 70-78, wherein the medium maintains at least about 75% cell potency after 5-10 days of use at 37 °C.

34

SUBSTITUTE SHEET (RULE 26)