Production of meat ex vivo

Abstract

To provide a method for producing cultured cells.SOLUTION: A method of generating cultured cells, the method comprising culturing a population of self-renewing cells to grow in suspension culture in a bioreactor without the use of an extracellular scaffold or matrix, wherein the population of self-renewing cells comprises fibroblasts that do not require attachment to an extracellular scaffold or matrix for growth, and seeding a plant-based scaffold with the self-renewing cells.SELECTED DRAWING: Figure 21

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 516,575, filed June 7, 2017, and U.S. Provisional Patent Application No. 62 / 653,332, filed April 5, 2018, both of which are incorporated by reference herein in their entireties. [Background technology]

[0002] Traditional meat production is a resource-intensive process that leaves a large environmental footprint. Farmed animals are raised in agricultural environments that require large amounts of water, feed, land, and other resources. Similarly, fish consumption is vulnerable to many problems, including overfishing, bycatch, and pollution from fishing industries. Summary of the Invention

[0003] Disclosed herein are methods for producing cultured fish meat for human consumption. Some such methods include: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells in a medium comprising a microscaffold; c) inducing differentiation in the population of cells to form at least one of muscle cells and adipocytes; and d) processing the population of cells into fish meat for human consumption. Various embodiments incorporate at least one of the following elements: The fish meat is often sushi. In some instances, the fish meat is surimi. Often, the fish meat is suitable for raw consumption. In certain instances, the fish meat is cooked. The fish meat is typically salmon meat. In certain embodiments, the fish meat is sushi-grade salmon meat. Alternatively, the fish meat is often tuna meat. Often, the fish meat is sushi-grade tuna meat. Optionally, the inducing differentiation in (c) causes the population of cells to form muscle cells and adipocytes. Optionally, differentiation includes transdifferentiation of the cells into different cell types. Fish meat is often composed of at least 50% highly glycolytic and anaerobic muscle fibers. The cell population is often derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. The treating step in (d) typically includes combining the cell population with a second cell population consisting of myocytes or adipocytes. In various embodiments, the cell population is isolated as an embryonic stem cell. Often, the cell population has been modified to induce pluripotency. In certain embodiments, the cell population is isolated as a pluripotent adult stem cell. The culturing step typically includes growing and expanding the cell population in cell culture. The differentiation step often includes exposing the cell population to culture conditions that stimulate differentiation. Often, the differentiation step includes exposing the cell population to at least one growth factor that stimulates differentiation. In certain examples, the culturing step includes growing the cell population on a two-dimensional surface. Alternatively, the culturing step involves growing a population of cells on a three-dimensional scaffold. The culturing step often involves growing a population of cells on a microscaffold in a bioreactor, where the microscaffold allows for cell attachment.Often, the cell population forms a tissue without texture after differentiation. In various embodiments, the culturing step comprises growing the cell population in a medium formulation comprising at least one nutrient. The at least one nutrient typically comprises omega-3 fatty acids. The at least one nutrient often comprises a polyunsaturated fatty acid. In some instances, the at least one nutrient comprises a monounsaturated fatty acid. Often, the cell population is cultured using a serum-free medium formulation. In many instances, the cell population is cultured using a mushroom-based medium formulation.

[0004] In some embodiments, methods for producing cultured fish tissue are disclosed herein, the methods comprising: a) culturing a population of fish preadipocytes and a population of fish satellite cells; b) inducing differentiation in the population of fish preadipocytes to form adipocytes; c) inducing differentiation in the population of fish satellite cells to generate myocytes; d) co-culturing the adipocytes and myocytes; and e) processing the adipocytes and myocytes into fish tissue for human consumption. Various embodiments include at least one of the following elements: Often, the fish tissue comprises fast-twitch muscle fibers. Often, the fish tissue is salmon tissue. In some instances, the fish tissue is tuna tissue. The fish tissue is occasionally trout tissue. In many instances, the fish tissue is surimi. The fish tissue is often sushi. The fish tissue is sometimes prepared for raw human consumption. The fish tissue is often cooked for human consumption. In various embodiments, the adipocytes and myocytes are co-cultured in a media formulation comprising at least one nutrient. At least one nutritional agent typically includes an omega-3 fatty acid. Often, at least one nutritional agent includes a polyunsaturated fatty acid. At least one nutritional agent occasionally includes a monounsaturated fatty acid. Often, a serum-free media formulation is used for cell culture. In one example, a mushroom-based media formulation is used for cell culture.

[0005] In some aspects, disclosed herein are methods for producing cultured fish tissue, the methods comprising: a) culturing a population of fish preadipocytes and a population of fish satellite cells suitable for suspension culture; b) inducing differentiation in the population of fish preadipocytes to form adipocytes; c) inducing differentiation in the population of fish satellite cells to form myocytes; d) co-culturing the adipocytes and myocytes; and e) processing the adipocytes and myocytes into fish tissue for human consumption.

[0006] In some aspects, disclosed herein are human edible compositions comprising fish tissue generated from co-cultured muscle cells and adipocytes.

[0007] In some aspects, disclosed herein are human edible compositions comprising fish tissue generated from preadipocytes and satellite cells.

[0008] In some embodiments, disclosed herein are methods for producing cultured fish meat for human consumption, the methods comprising: a) obtaining a population of preadipocytes and a population of satellite cells; b) adapting the population of preadipocytes and the population of satellite cells to suspension culture; c) inducing differentiation in the population of preadipocytes and the population of satellite cells; d) co-culturing the populations in suspension culture; and e) processing the populations into fish meat for human consumption. In some cases, differentiation comprises transdifferentiation of the cells into a different cell type. Various embodiments include at least one of the following elements: Often, the fish meat is sushi. Often, the fish meat is surimi. In certain instances, the fish meat is suitable for raw consumption. Often, the fish meat is cooked. In various embodiments, the fish meat is salmon meat. In certain instances, the fish meat is sushi-grade salmon meat. The fish meat is tuna meat. Often, the fish meat is sushi-grade tuna meat. Occasionally, the fish meat is trout meat. In many instances, fish meat is composed of at least 50% highly glycolytic and anaerobic muscle fibers. Populations of preadipocytes are typically derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. Populations of satellite cells are often derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. The co-culturing step typically involves growing and expanding the population in cell culture. In certain instances, the differentiation step involves exposing the population of preadipocytes to at least one growth factor that stimulates differentiation into adipocytes. Often, the differentiation step involves exposing the population of satellite cells to at least one growth factor that stimulates differentiation into myocytes. The culturing step often involves growing the population of cells in a bioreactor. In many instances, myocytes and adipocytes form textureless tissue after differentiation. Myocytes and adipocytes are often cultured in a media formulation that includes at least one nutrient. At least one nutrient typically comprises an omega-3 fatty acid. In many instances, at least one nutrient comprises a polyunsaturated fatty acid. Occasionally, at least one nutrient comprises a monounsaturated fatty acid. Often, serum-free media formulations are used for cell culture. In some instances, mushroom-based media formulations are used for cell culture.

[0009] In some aspects, disclosed herein is a processed fish product suitable for human consumption comprising fish paste produced from cultured muscle cells and adipocytes.

[0010] In some aspects, disclosed herein is a synthetic food suitable for human consumption comprising fish meat derived from cultured satellite cells and preadipocytes.

[0011] In some aspects, disclosed herein is a processed fish product suitable for human consumption that includes fish meat produced from muscle cells and adipocytes grown in suspension culture.

[0012] In some embodiments, disclosed herein are methods for producing cultured fish meat for human consumption, the methods comprising: a) obtaining a population of fish preadipocytes capable of growth in suspension culture; b) obtaining a population of fish satellite cells capable of growth in suspension culture; c) inducing differentiation in the population of fish preadipocytes and the population of fish satellite cells to form adipocytes and myocytes; d) co-culturing the adipocytes and myocytes in a suspension culture comprising at least one nutrient; and d) processing the population of cells into fish meat for human consumption. Various embodiments include at least one of the following elements: Often, the fish meat is sushi. Often, the fish meat is surimi (surimi) that is used for sushi. In many instances, the fish meat is suitable for raw consumption. The fish meat is occasionally cooked. The fish meat is often salmon meat. In particular instances, the fish meat is sushi-grade salmon meat. Often, the fish meat is tuna meat. The fish meat is sushi-grade tuna meat. In various embodiments, fish meat is composed of at least 50% highly glycolytic and anaerobic muscle fibers. Typically, the cell population is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. Often, the step (c) of inducing differentiation comprises exposing the population of preadipocytes and the population of satellite cells to culture conditions that stimulate differentiation. The step (c) of inducing differentiation typically comprises exposing the population of preadipocytes to at least one growth factor that stimulates differentiation. In certain examples, the step (c) of inducing differentiation comprises exposing the population of satellite cells to at least one growth factor that stimulates differentiation. Adipocytes and myocytes typically form tissues without texture. Often, at least one nutritional agent comprises an omega-3 fatty acid. Often, at least one nutritional agent comprises a polyunsaturated fatty acid. Often, at least one nutritional agent comprises a monounsaturated fatty acid. Often, a serum-free medium formulation is used for cell culture. In some instances, the mushroom-based medium formulation is used for cell culture. In some instances, the population of cells is transdifferentiated into at least one cell type. In some instances, the population of cells is transdifferentiated into at least one of hepatocytes, myocytes, and adipocytes.

[0013] In some embodiments, methods for producing cultured tissue for human consumption are disclosed herein, the methods comprising: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation in the population of self-renewing cells to form the cultured tissue; and d) processing the cultured tissue for human consumption. Various embodiments include at least one of the following elements. Optionally, obtaining the population of self-renewing cells comprises transitioning the population of cells from a two-dimensional adherent culture to a three-dimensional culture in a bioreactor. Often, the population of self-renewing cells comprises immortalized differentiated cells. Inducing differentiation in the population of self-renewing cells often comprises inducing transdifferentiation of cells in the population into myocytes, adipocytes, or a combination thereof. Optionally, the population of cells is transdifferentiated into at least one cell type. Optionally, the population of cells is transdifferentiated into at least one of hepatocytes, myocytes, and adipocytes. In some examples, culturing comprises seeding the population of self-renewing cells on a three-dimensional microscaffold. In some cases, the three-dimensional microscaffold promotes cell proliferation, adhesion, differentiation, or a combination thereof. In various embodiments, the three-dimensional microscaffold is conjugated to at least one factor that promotes cell growth, adhesion, differentiation, or a combination thereof. Often, the microscaffold comprises at least one of hydrogel, chitosan, polyethylene terephthalate, collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin, fibronectin, cellulose, hemicellulose, pectin, lignin, alginate, glucomannan, polycaprolactone (PCL), textured vegetable protein (TVP), textured soy protein (TSP), and acrylate. In some examples, the population of self-renewing cells comprises at least one cell modified to undergo induced differentiation.In some examples, at least one cell is modified to incorporate: a) a first genetic construct comprising an open reading frame (ORF) of at least one pluripotency gene; and b) a second genetic construct comprising an open reading frame (ORF) of a regulatory factor configured to inactivate at least one pluripotency gene. Often, the population of self-renewing cells comprises at least one cell that has undergone at least 50 cell divisions in culture. In some instances, the regulatory factor is a recombinase, and the open reading frame (ORF) of the at least one pluripotency gene is flanked by recombination sequences recognized by the recombinase, such that expression of the recombinase catalyzes excision of the open reading frame (ORF) of the at least one pluripotency gene. In some instances, the second genetic construct comprises an ORF of at least one hepatocyte differentiation factor selected from hepatocyte nuclear factor 1 alpha (HNF1A), forkhead box A2 (FOXA2), and hepatocyte nuclear factor 4 alpha (HNF4A). In various embodiments, the second genetic construct comprises at least one myogenic factor selected from myogenin (MyoG), myogenic differentiation 1 (MyoD), myogenic factor 6 (MRF4), and myogenic factor 5 (MYF5). The second genetic construct often comprises at least one adipogenic factor selected from fatty acid binding protein 4 (FABP4), insulin-responsive glucose transporter type 4 (GLUT4), adiponectin, C1Q and collagen domain-containing (ADIPOQ), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), perilipin 1 (PLIN1), leptin (LEP), and lipoprotein lipase (LPL). Often, the second genetic construct further comprises: a) an open reading frame (ORF) of at least one differentiation gene; and b) an inducible promoter controlling expression of i) the open reading frame (ORF) of the at least one differentiation gene; and ii) the open reading frame (ORF) of the regulatory factor. In some instances, inducing differentiation comprises exposing the at least one cell to an inducer to induce expression of the ORF of the at least one cell lineage gene and the ORF of the regulatory factor.The method typically includes removing the inducer after treating the population of self-renewing cells with the inducer in step d) and before processing for human consumption. Inducing differentiation may include generating myotubes within the population of self-renewing cells. In many instances, inducing differentiation further includes generating adipocytes within the population of self-renewing cells. Often, the population of self-renewing cells includes pluripotent cells induced to differentiate into myocytes and adipocytes during step c). The pluripotent cells often include a first subpopulation of muscle satellite cells and a second subpopulation of preadipocytes. In some instances, inducing differentiation includes generating hepatocytes within the population of self-renewing cells. In some embodiments, the population of self-renewing cells is derived from a bird selected from duck, goose, chicken, and turkey. The method often includes inducing steatosis within at least one hepatocyte. In certain instances, the population of self-renewing cells includes at least one cell modified to express at least one gene to enhance steatosis upon treatment with the inducer. Often, at least one cell is stably transformed with a construct containing an open reading frame (ORF) encoding ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, or KLF6. 433. In various embodiments, inducing steatosis includes incubating the hepatocytes in a medium containing at least one nutrient. The at least one nutrient often includes a polyunsaturated fatty acid, a monounsaturated fatty acid, or a combination thereof. Optionally, the at least one nutrient includes palmitic acid, oleic acid, docosahexaenoic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, or a combination thereof. In some embodiments, the cultured tissue includes muscle cells from octopus, squid, or cuttlefish. Often, the cultured tissue includes fish muscle tissue. The population of self-renewing cells can be derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. In many cases, fish muscle tissue is combined with separately cultured fish adipose tissue during step d). In some instances, the population of cells is cultured using a serum-free media formulation.The serum-free media formulation, in some embodiments, includes mushroom extract or soy hydrolysate.

[0014] Disclosed herein are methods for producing cultured meat for human consumption. Some such methods include: a) obtaining a population of self-renewing cells capable of growth in suspension culture; b) culturing the population of self-renewing cells in suspension; c) inducing differentiation in the population of cells to form at least one of muscle cells and adipocytes; and d) processing the population of cells into meat for human consumption. Various aspects incorporate at least one of the following elements. Often, the meat is fish meat. The fish meat is typically sushi. In some embodiments, the fish meat is surimi. Often, the fish meat is suitable for raw consumption. In certain instances, the fish meat is cooked. In certain instances, the fish meat is salmon meat. In certain aspects, the fish meat is sushi-grade salmon meat. In some instances, the fish meat is tuna meat. Often, the fish meat is sushi-grade tuna meat. Often, the inducing differentiation in (c) causes the population of cells to form muscle cells and adipocytes. Fish meat is typically composed of at least 50% highly glycolytic and anaerobic muscle fibers. The population of cells is typically derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. The treating step in (d) often includes combining the population of cells with a second population of cells composed of muscle cells or adipocytes. In some embodiments, the population of cells is isolated as embryonic stem cells. Often, the population of cells has been modified to induce pluripotency. A population of cells is isolated as pluripotent adult stem cells. Often, the population of self-renewing cells is immortalized cells. The culturing step typically includes growing and expanding the population of cells in cell culture. Often, inducing differentiation includes exposing the population of cells to culture conditions that stimulate differentiation. In some cases, differentiation includes transdifferentiation of the cells into a different cell type. Often, inducing differentiation includes, for example, exposing the population of cells to at least one growth factor that stimulates differentiation. The culturing step often involves growing a population of cells on a two-dimensional surface. Some populations of cells form textureless tissue after differentiation. In some embodiments, the culturing step involves growing the population of cells in a media formulation comprising at least one nutrient. Often, the at least one nutrient comprises omega-3 fatty acids.In other cases, at least one nutrient comprises a polyunsaturated fatty acid. Occasionally, at least one nutrient comprises a monounsaturated fatty acid. Often, the population of cells is cultured using a serum-free media formulation. In many instances, the population of cells is cultured using a mushroom-based media formulation.

[0015] Disclosed herein are methods for producing cultured cells with high lipid accumulation for human consumption. Some methods include: a) culturing a population of cells; b) inducing differentiation in the population of cells; c) inducing high lipid accumulation in the population of cells; and d) processing the population of cells for human consumption. Various embodiments incorporate at least one of the following elements. In some instances, the differentiated population of cells includes hepatocytes. Often, the processing step includes using the population of cells as an ingredient in foie gras. The population of cells is optionally derived from duck or goose. The population of cells is often derived from at least one of poultry and livestock. In some instances, inducing high lipid accumulation includes inducing steatosis. In some embodiments, high lipid accumulation is characterized by excessive accumulation of cytoplasmic lipid droplets. Inducing high lipid accumulation often includes exposing the population of cells to an exogenous compound that modulates at least one lipid metabolic pathway. In some instances, inducing high lipid accumulation includes exposing the population of cells to at least one of a toxin and a high lipid concentration. Inducing high fat accumulation often involves modulating at least one lipid metabolic pathway to enhance lipid retention in the population of cells. In some examples, inducing high fat accumulation involves altering at least one gene in the population of cells to modulate lipid metabolism. Often, the differentiated population of cells includes liver, heart, kidney, stomach, intestine, lung, diaphragm, esophagus, thymus, pancreas, or tongue cells. In various aspects, processing the population of cells for human consumption includes combining the population of cells with cells having low lipid accumulation. The population of cells is often isolated as embryonic stem cells. In some cases, the population of cells has been modified to induce pluripotency. In some examples, the population of cells is isolated as pluripotent adult stem cells. Culturing typically involves growing and expanding the population of cells in cell culture. In some aspects, inducing differentiation involves exposing the population of cells to culture conditions that stimulate differentiation. In some embodiments, inducing differentiation involves exposing the population of cells to at least one growth factor that stimulates differentiation. In some cases, differentiation involves transdifferentiation of cells into a different cell type.The culturing step often involves growing a population of cells on a two-dimensional surface. Often, the culturing step involves growing a population of cells on a three-dimensional scaffold. In certain instances, the culturing step involves growing a population of cells on a microscaffold in a bioreactor, where the microscaffold allows for cell attachment. In some embodiments, the population of cells does not require an adhesive substrate for survival and proliferation. Often, the population of cells is suitable for suspension culture. The population of cells often forms non-textured tissue after differentiation. In some embodiments, the population of cells forms non-muscle tissue after differentiation. In various instances, the culturing step involves growing the population of cells in a media formulation including at least one nutrient agent. In some embodiments, the at least one nutrient agent includes an omega-3 fatty acid. The at least one nutrient agent frequently includes at least one polyunsaturated fatty acid. Often, the population of cells is cultured using a serum-free media formulation. In many instances, the population of cells is cultured using a mushroom-based media formulation.

[0016] Disclosed herein are methods for producing a non-textured cultured tissue having a high lipid content. Some such methods include: a) obtaining a population of differentiated cells capable of self-renewal; b) culturing the population of differentiated cells; c) manipulating at least one lipid metabolic pathway to induce adiposity in the population of differentiated cells such that the cells accumulate a high lipid content; and d) processing the population of differentiated cells into a non-textured tissue. Various embodiments incorporate at least one of the following elements: In some cases, obtaining a population of differentiated cells capable of self-renewal includes transforming the differentiated cells into immortalized cells. Often, obtaining a population of differentiated cells capable of self-renewal includes culturing the differentiated cells until spontaneous mutations occur in the immortalized cells. In some cases, differentiation includes transdifferentiation of the cells into a different cell type. Often, the population of differentiated cells includes fibroblasts. In some examples, the population of differentiated cells is transdifferentiated into myocytes, adipocytes, or a combination thereof. In some embodiments, the population of differentiated cells is derived from a fish, such as salmon or trout. In some examples, the population of differentiated cells includes hepatocytes. In some embodiments, the processing step includes using the population of differentiated cells as an ingredient in foie gras. In some embodiments, the population of differentiated cells is derived from duck or goose. The population of differentiated cells is often derived from at least one of poultry and livestock. Typically, steatosis is characterized by excessive accumulation of cytoplasmic lipid droplets. In some embodiments, manipulating at least one lipid metabolic pathway includes exposing the population of cells to an exogenous compound. In some embodiments, manipulating at least one lipid metabolic pathway includes exposing the population of differentiated cells to at least one of a toxin and a high lipid concentration. Alternatively, or in combination, manipulating at least one lipid metabolic pathway includes modifying at least one gene in the population of cells to regulate lipid metabolism. In many cases, the population of differentiated cells includes liver, heart, kidney, stomach, intestine, lung, diaphragm, esophagus, thymus, pancreas, or tongue cells. In some embodiments, the processing step includes mixing the population of differentiated cells with cells having low lipid accumulation. In many embodiments, the step of culturing includes growing and expanding a population of cells in cell culture.The culturing step often includes growing a population of cells on a two-dimensional surface. In some embodiments, the culturing step includes growing a population of cells on a three-dimensional scaffold. Optionally, the culturing step includes growing a population of cells on a microscaffold in a bioreactor, where the microscaffold allows for cell attachment. The population of cells, in some instances, does not require an adhesive substrate for survival and proliferation. Often, the population of cells is suitable for suspension culture. Often, the population of differentiated cells forms tissue without texture. In some instances, the population of cells forms non-muscle tissue. In many embodiments, the culturing step includes growing the population of cells in a media formulation comprising at least one nutrient agent. In some instances, the at least one nutrient agent comprises an omega-3 fatty acid. Often, the at least one nutrient agent comprises a polyunsaturated fatty acid. Often, the at least one nutrient agent comprises a monounsaturated fatty acid. Often, the population of cells is cultured using a serum-free media formulation. In many instances, the population of cells is cultured using a mushroom-based media formulation.

[0017] Disclosed herein are methods for producing cultured non-muscle tissue for human consumption. Some such methods include: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation in the population of cells to form non-muscle tissue; and d) processing the cultured non-muscle tissue for human consumption. Various embodiments incorporate at least one of the following elements. In some cases, differentiation includes transdifferentiation of cells into a different cell type.

[0018] In some embodiments, disclosed herein are methods for producing cultured tissue for human consumption, the methods comprising: obtaining a population of self-renewing cells; adapting the population of self-renewing cells to a suspension culture; culturing the population of self-renewing cells; inducing differentiation in the population of cells to form the cultured tissue; and processing the cultured tissue for human consumption. In some instances, differentiation comprises transdifferentiation of the cells into a different cell type.

[0019] Disclosed herein are methods for producing cultured, textureless muscle tissue for human consumption. Some such methods include: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation in the population of cells to form textureless muscle tissue; and d) processing the cultured textureless muscle tissue for human consumption. Various embodiments incorporate at least one of the following elements. Optionally, the textureless muscle tissue is octopus, squid, or cuttlefish muscle. Often, the textureless muscle tissue is fish muscle tissue. In some instances, fish muscle tissue contains highly glycolytic and anaerobic muscle fibers. Highly glycolytic and anaerobic muscle fibers often comprise up to 80% of the fish muscle tissue. Optionally, the population of cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. In various embodiments, the textureless muscle tissue is combined with adipose tissue. Often, muscle tissue and adipose tissue are combined to make surimi products. In some cases, the fish muscle and adipose tissue are sushi-quality. The population of cells, in certain embodiments, is isolated as pluripotent adult stem cells. In certain aspects, the population of cells has been modified to induce pluripotency. Often, the population of cells is isolated as pluripotent adult stem cells. The culturing step, in various instances, includes growing and expanding the population of cells in cell culture. Often, inducing differentiation includes exposing the population of cells to culture conditions that stimulate differentiation. In some cases, differentiation includes transdifferentiation of the cells into a different cell type. Often, inducing differentiation includes exposing the population of cells to at least one growth factor that stimulates differentiation. In various embodiments, the culturing step includes growing the population of cells on a two-dimensional surface. Often, the culturing step includes growing the population of cells on a three-dimensional scaffold. In some instances, the culturing step includes growing the population of cells on a microscaffold in a bioreactor, where the microscaffold allows for cell attachment. In some scenarios, the population of cells does not require an adhesive substrate for survival and proliferation. Often, the population of cells is suitable for suspension culture. In some embodiments, the population of cells forms a non-textured tissue after differentiation.The population of cells often forms muscle tissue without texture after differentiation. In some cases, the culturing step includes growing the population of cells in a media formulation including at least one nutrient. In some examples, the at least one nutrient includes an omega-3 fatty acid. Typically, the at least one nutrient includes a polyunsaturated fatty acid. Often, the at least one nutrient includes a monounsaturated fatty acid. Often, the population of cells is cultured using a serum-free media formulation. In many examples, the population of cells is cultured using a mushroom-based media formulation.

[0020] Disclosed herein are methods for preparing foie gras, including cultured avian liver tissue. Some such methods include: a) obtaining a population of self-renewing avian-derived cells; b) differentiating the population of avian-derived cells into hepatocytes; and c) inducing steatosis in the hepatocytes to produce cultured avian liver tissue having a high lipid content; and d) preparing the cultured avian liver tissue as foie gras. Various embodiments incorporate at least one of the following elements: Often, the cells are duck cells. In some embodiments, the cells are goose cells. In some cases, differentiation includes transdifferentiation of the cells into a different cell type.

[0021] Disclosed herein is a culinary foie gras composition comprising tissue culture hepatocytes having a high lipid content and processed for human consumption. Various embodiments incorporate at least one of the following elements: Optionally, the composition is processed into multiple sections. In some instances, each section weighs no more than about 5 ounces. Each section is often individually packaged. Often, the foie gras composition weighs at least about 1.5 pounds and is round, firm, and free of blemishes. In various embodiments, the foie gras composition has a packaging label indicating an A grade rating. In some embodiments, the foie gras composition weighs about 0.75 to about 1.5 pounds. In some instances, the foie gras composition has a packaging label indicating a B grade rating. The foie gras composition optionally weighs less than about 1 pound and has no more than three blemishes. In some instances, the foie gras composition has a packaging label indicating a C grade rating. In some embodiments, the tissue culture hepatocytes are fatty. In many instances, the tissue culture hepatocytes are characterized by excessive accumulation of cytoplasmic lipid droplets. In some embodiments, the high lipid content is achieved by exposure to an exogenous compound that modulates at least one lipid metabolic pathway. The high lipid content is often achieved by exposure to at least one of a toxin and a high lipid concentration. Often, the high lipid content is achieved by modulating at least one lipid metabolic pathway to enhance lipid retention within the cell population. The high lipid content is achieved by modifying at least one gene in tissue culture hepatocytes. Often, the foie gras composition comprises cells with low lipid accumulation. In various embodiments, the tissue-cultured hepatocytes are differentiated from isolated embryonic stem cells. In some cases, the tissue-cultured hepatocytes are differentiated from induced pluripotent stem cells. In some instances, the tissue-cultured hepatocytes are differentiated from isolated pluripotent adult stem cells. In some embodiments, the composition comprises tissue cultured hepatocytes generated by differentiation of a population of self-renewing cells. Often, the differentiation step includes exposing the population of cells to culture conditions that stimulate differentiation. In some cases, differentiation includes transdifferentiation of the cells into a different cell type. In some embodiments, the step of differentiating comprises exposing the population of cells to at least one growth factor that stimulates differentiation.Often, tissue culture hepatocytes are grown on two-dimensional surfaces. In some instances, tissue culture hepatocytes are grown on three-dimensional scaffolds. In various embodiments, tissue culture hepatocytes are grown on microscaffolds in a bioreactor, where the microscaffolds allow for cell attachment. In certain embodiments, tissue culture hepatocytes do not require an adhesive substrate for survival and proliferation. Often, tissue culture hepatocytes are suitable for suspension culture. Often, tissue culture hepatocytes form non-textured tissue. In various instances, tissue culture hepatocytes form non-textured muscle tissue. Tissue culture hepatocytes are often cultured in a media formulation that includes at least one nutrient. Often, the at least one nutrient includes an omega-3 fatty acid. In certain embodiments, the at least one nutrient includes a polyunsaturated fatty acid. Often, the at least one nutrient includes a monounsaturated fatty acid. Often, tissue culture hepatocytes are cultured using a serum-free media formulation. In many instances, tissue culture hepatocytes are cultured using a mushroom-based media formulation.

[0022] Disclosed herein are compositions comprising cultured organ cells processed into a textureless, non-muscle food product suitable for human consumption. Various embodiments incorporate at least one of the following elements. In some cases, the cultured organ cells comprise hepatocytes. In some embodiments, the cultured organ cells comprise avian cells. Often, the food product is processed into multiple pieces. Often, each piece weighs no more than about 5 ounces. Each piece is typically individually packaged. In many embodiments, the food product is foie gras. The foie gras typically weighs at least about 1.5 pounds, is round, firm, and free of blemishes. In some instances, the foie gras has a packaging label indicating an A grade rating. In various embodiments, the foie gras weighs about 0.75 to about 1.5 pounds. In some embodiments, the foie gras has a packaging label indicating a B grade rating. Often, the foie gras weighs less than about 1 pound and has no more than three blemishes. In various examples, the foie gras has a packaging label indicating a C grade rating. Often, the tissue culture hepatocytes are fatty. In some embodiments, foie gras is characterized by a high lipid content. In some embodiments, the high lipid content is achieved by exposure to an exogenous compound that modulates at least one lipid metabolic pathway. The high lipid content is often achieved by exposure to at least one of a toxin and a high lipid concentration. In some embodiments, the high lipid content is achieved by modulating at least one lipid metabolic pathway to enhance lipid retention within a population of cells. Often, the high lipid content is achieved by genetically modifying at least one tissue culture hepatocyte. In some embodiments, the foie gras composition further comprises cells with low lipid accumulation. In some instances, the cultured organ cells are grown on a two-dimensional surface. Often, the cultured organ cells are grown on a three-dimensional scaffold. The cultured organ cells are grown on a microscaffold within a bioreactor, where the microscaffold allows for cell attachment in various embodiments. The cultured organ cells often do not require an adhesive substrate for survival and proliferation. Often, tissue culture hepatocytes are suitable for suspension growth. In various embodiments, the cultured organ cells form tissue without texture. Often, the cultured organ cells form non-muscle tissue. In various cases, the cultured organ cells are cultured in a media formulation that includes at least one nutrient.Often, at least one nutritional agent comprises an omega-3 fatty acid. In many instances, the at least one nutritional agent comprises a polyunsaturated fatty acid. Often, the at least one nutritional agent comprises a monounsaturated fatty acid. Often, the cultured organ cells are cultured using a serum-free media formulation. In many instances, the cultured organ cells are cultured using a mushroom-based media formulation.

[0023] Disclosed herein is a foie gras composition for human consumption, comprising cultured fatty avian liver cells and seasonings, optionally including at least one of salt, pepper, and sugar.

[0024] Disclosed herein is a foie gras composition comprising cultured liver cells having a high lipid content and liver cells having a low lipid content. Various embodiments incorporate at least one of the following elements: In some cases, the cultured liver cells having a high lipid content and the liver cells having a low lipid content are mixed together. In some examples, the foie gras composition is suitable as an ingredient for preparing one of a mousse, a parfait, and a pâté. Typically, the liver cells having a low lipid content are cultured cells. In some embodiments, the liver cells having a low lipid content are uncultured cells.

[0025] Disclosed herein is a human edible composition comprising avian liver cells grown in cell culture and processed for human consumption.

[0026] Disclosed herein is a packaged foie gras composition, the package comprising cultured liver cells and a package having a label indicating that the foie gras composition has not been produced by force-feeding.

[0027] Disclosed herein is a packaged foie gras composition, the package comprising cultured liver cells and a package having a label indicating that the foie gras was produced in a pathogen-free environment. In some examples, the label indicates that the composition was produced without exposure to avian influenza viruses.

[0028] Disclosed herein is a packaged composition for human consumption, the composition comprising cultured cells processed into a food product and a label indicating that the composition was produced without exposure to a toxin, in some cases the toxin is one of a pesticide, a herbicide, and a fungicide.

[0029] Disclosed herein are methods for producing cultured cells for human consumption without the use of antibiotics. Some such methods include: a) culturing a population of cells without the use of antibiotics; b) inducing differentiation in the population of cells; c) inducing lipid accumulation in the population of cells; and d) processing the population of cells for human consumption. In some cases, differentiation includes transdifferentiation of the cells into a different cell type.

[0030] Disclosed herein are methods for producing cultured cells for human consumption without exposure to pathogens. Some such methods include: a) culturing a population of cells in a pathogen-free culture environment; b) inducing differentiation in the population of cells; c) inducing lipid accumulation in the population of cells; and d) processing the population of cells for human consumption. In some cases, differentiation includes transdifferentiation of the cells into a different cell type.

[0031] Disclosed herein are methods for producing cultured cells for human consumption without exposure to toxins. Some such methods include: a) culturing a population of cells in a toxin-free culture environment; b) inducing differentiation in the population of cells; c) inducing lipid accumulation in the population of cells; and d) processing the population of cells for human consumption. In some cases, differentiation includes transdifferentiation of the cells into a different cell type.

[0032] Disclosed herein are methods for producing cultured, untextured tissue with a high lipid content and no vasculature. Some such methods include: a) culturing a population of cells; b) inducing differentiation within the population of cells; c) manipulating lipid metabolic pathways to induce adiposity in the population of differentiated cells such that the cells accumulate a high lipid content; and d) processing the population of differentiated cells into a untextured tissue with no vasculature. In some cases, differentiation includes transdifferentiation of the cells into a different cell type.

[0033] Disclosed herein are methods for producing cultured tissue with high nutrient content for human consumption. Some such methods include: a) culturing a population of cells in a culture medium with at least one nutrient agent; b) manipulating lipid metabolic pathways to induce adiposity in the population of differentiated cells such that the cells accumulate a high lipid content; and c) processing the population of differentiated cells into avascular, textureless tissue for human consumption. Various embodiments incorporate at least one of the following elements: In some instances, the at least one nutrient agent comprises an omega-3 fatty acid. Often, the at least one nutrient agent comprises a polyunsaturated fatty acid. Often, the at least one nutrient agent comprises a monounsaturated fatty acid.

[0034] Disclosed herein are methods for producing cultured organ tissue for human consumption. Some such methods include: a) culturing a population of self-renewing cells; b) inducing differentiation in the population of cells to generate organ tissue; and c) processing the organ tissue for human consumption. Various embodiments incorporate at least one of the following elements. In some cases, the organ cells are liver, heart, kidney, stomach, intestine, lung, diaphragm, esophagus, thymus, pancreas, or tongue tissue. In some cases, differentiation includes transdifferentiation of cells into a different cell type. In various embodiments, the organ tissue is liver tissue. Often, the processing step includes combining the organ tissue with additional cellular tissue. The additional cellular tissue, in many instances, includes non-fatty liver cells.

[0035] Disclosed herein are methods for producing cultured fish tissue with enhanced nutrient content for human consumption. Some such methods include: a) culturing a population of fish muscle cells in a culture medium with at least one nutrient; B) expanding the population of muscle cells; and c) processing the population of muscle cells into fish tissue for human consumption. Various aspects incorporate at least one of the following elements: In some cases, the fish tissue comprises fast-twitch muscle fibers. In some embodiments, the method further includes combining the population of muscle cells with a population of fat cells. The fish muscle cells are often salmon muscle cells. The fish muscle cells are often tuna muscle cells. In some cases, the fish muscle cells are trout muscle cells.

[0036] Disclosed herein are human edible compositions comprising fish tissue produced from cultured muscle cells and adipocytes according to any of the methods described herein.

[0037] Disclosed herein is a bioreactor system for producing cultured tissue suitable for human consumption, the system comprising: a) a reactor chamber including a plurality of microscaffolds that provide an adhesive surface for cell attachment; b) a population of self-renewing cells cultivated within the bioreactor; c) a first source providing at least one maintenance medium containing ingredients for maintaining the population of self-renewing cells without spontaneously differentiating; and d) a second source providing at least one differentiation medium containing ingredients for differentiating the population of self-renewing cells toward a specific lineage; wherein the reactor chamber receives the maintenance medium from the first source for cultivating the population of cells and the differentiation medium from the second source for differentiating the population of cells, and wherein the population of cells produced in a single batch comprises a cultured tissue suitable for human consumption and having a dry weight of at least 1 kg. Various embodiments incorporate at least one of the following elements. Optionally, the system further comprises at least one sensor for monitoring the reactor chamber. In certain embodiments, the at least one sensor is a biosensor, a chemical sensor, or an optical sensor. Often, the at least one sensor is configured to monitor at least one of pH, temperature, oxygen, carbon dioxide, glucose, lactate, ammonia, hypoxanthine, amino acids, dopamine, and lipids. Often, the system further includes at least one additional reactor chamber. A single batch often has a dry weight of at least 5 kg. Often, the bioreactor system further includes a plurality of microscaffolds. Alternatively, the bioreactor system further includes at least one 3D scaffold. The bioreactor system frequently includes a third source providing at least one lipid-based medium containing components for inducing adiposity or lipid accumulation in the population of cells. In various cases, the population of cells is cultured in a medium containing at least one nutrient. Often, the population of cells is cultured using a serum-free medium formulation. In many instances, the population of cells is cultured using a mushroom-based medium formulation.

[0038] In some embodiments, disclosed herein is a cultured food product for human consumption, the cultured food product comprising tissue produced according to any one of the aforementioned methods. Often, the cultured food product includes packaging with a label indicating that the cultured tissue was produced in a pathogen-free environment, a toxin-free environment, an environment without force-feeding to animals, or any combination thereof. In some examples, the cultured tissue is processed into multiple sections and packaged to form the cultured food product. [Brief explanation of the drawings]

[0039] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Figure 1] 1 shows a flow chart of a process for producing cultured cells with lipid accumulation for human consumption. [Figure 2] A flowchart of the process for producing cultured meat containing muscle cells and fat cells for human consumption. [Figure 3] 1 shows a schematic of an exemplary process for culturing meat. [Figure 4A] 1 shows a schematic illustrating an exemplary method for generating fatty liver cells to produce cultured foie gras. [Figure 4B] 1 shows a schematic illustrating a method for producing cultured fish tissue for human consumption. [Figure 5A] 1 shows isolated trout muscle satellite cells. [Figure 5B] 1 shows the expression of genetic markers in isolated trout muscle satellite cells. [Figure 5C] This shows mature myotubes formed by differentiating muscle satellite cells. [Figure 5D] Shows sheets of myotubes after differentiation from muscle satellite cells. [Figure 6A]Co-culture of salmon muscle satellite cells (arrowheads) and salmon preadipocytes (arrows) is shown. As shown in Figure 6B, preadipocytes can differentiate into adipocytes, and muscle satellite cells can differentiate into myocytes (arrowheads). [Figure 6B] Co-culture of salmon muscle satellite cells (arrowheads) and salmon preadipocytes (arrows) is shown. As shown in Figure 6B, preadipocytes can differentiate into adipocytes, and muscle satellite cells can differentiate into myocytes (arrowheads). [Figure 7A] 1 shows salmon fibroblasts induced to form spheroids grown in a bioreactor. [Figure 7B] Confirmation of spheroid viability after returning spheroids to 2D culture conditions and observing that fibroblasts migrated to the periphery and formed colonies is shown. [Figure 8] We demonstrate successful cell culture of bath muscle satellite cells. [Figure 9] Shown are spheroids formed from duck hepatocytes growing in spinner flasks and hanging drops, where spheroids can be transferred for 3D suspension culture. [Figure 10A] 1 shows duck hepatocytes growing in culture after successful differentiation. [Figure 10B] Successful hepatocyte differentiation was confirmed by measuring markers of hepatocyte differentiation. [Figure 11A]

[0023] Figure 1 shows duck self-renewing cells generated by culturing primary fibroblasts and picking colonies of dividing cells. [Figure 11B] 1 shows trout self-renewing cells generated by culturing primary fibroblasts and picking colonies of dividing cells. [Figure 12] 1 shows an exemplary embodiment of a genetic construct that can be introduced into cells to cause inducible differentiation into hepatocytes. [Figure 13] 1 shows an exemplary embodiment of a construct that can be introduced into a cell to allow for inducible expression of one or more genes that predispose the cell to steatosis. [Figure 14]1 shows an exemplary embodiment of a DNA construct system that can be introduced into cells to enable a proliferation / differentiation switch from a pluripotent to a differentiated phenotype. [Figure 15] 1 shows an exemplary construct that can be introduced into a cell to confer an inducible "off switch." [Figure 16A] Figure 1 shows successful induction of steatosis in duck hepatocytes after incubation with linoleic acid. [Figure 16B] 1 shows a dose-response curve correlating the percentage of fatty liver cells with the concentration of linoleic acid. [Figure 17] 1 shows hepatocyte populations when cultured in media with decreasing concentrations of fetal bovine serum (FBS) in the presence of soy hydrolysate. [Figure 18] 1 shows duck fibroblasts successfully grown in 10% shiitake mushroom extract after serial reduction of fetal bovine serum from the cell culture medium. [Figure 19A] Shown are duck fibroblasts grown in serum-free medium without additional serum-free medium; [Figure 19B] Control cultures grown in DMEM supplemented with 10% fetal bovine serum are shown. [Figure 20] 1 shows a schematic diagram of a bioreactor system for culturing cells for human consumption. [Figure 21] 1 shows another schematic diagram of a bioreactor system used as part of a meat production process. [Figure 22A] Shown are embryoid bodies generated using the hanging drop method (left panel) and an exemplary bioreactor into which embryoid bodies are transferred for growth in 3D culture. [Figure 22B] Another exemplary bioreactor (left panel) and cells from spheroids growing in 3D culture (right panel) are shown. [Figure 23A] 1 shows trout myotubes successfully differentiated from muscle satellite cells attached to glucomannan microscaffolds. [Figure 23B]A negative control of undifferentiated muscle satellite cells from the same preparation grown under identical cell culture conditions is shown. [Figure 24A] Duck fibroblasts (arrowheads) successfully grown on glucomannan microscaffolds (arrows) are shown. [Figure 24B] A representative glucomannan microscaffold is shown. [Figure 25] 1 shows images captured from a video of duck muscle tissue demonstrating spontaneous contractions. [Figure 26] Showing duck liver pate and foie gras butter made using duck fatty liver cells. [Figure 27A] 1 shows salmon pate and duck pate prototypes made according to the methods described herein. [Figure 27B] 1 shows salmon pate and duck pate prototypes made according to the methods described herein. [Figure 28A] 1 shows an exemplary embodiment of a method for Cre delivery for the purpose of activating / silencing specific genes. [Figure 28B] B shows another method of using Cre to induce a “switch” between activated gene sets associated with carniogenesis (e.g., proliferation and differentiation). DETAILED DESCRIPTION OF THE INVENTION

[0040] Disclosed herein are systems and methods for producing food using cellular agriculture. Cell-cultured foods offer many benefits that eliminate or significantly reduce the negative impacts caused by traditional food production. These benefits are particularly felt in the area of ​​meat production, which is typically produced using intensive livestock production or through fishing and aquaculture. Instead of raising or capturing live animals or fish from which meat is harvested, cells with self-renewal capabilities are isolated or created and grown in cell culture. In some cases, cells such as embryonic stem cells and pluripotent progenitor cells are naturally capable of self-renewal. Alternatively, or in combination, cells are engineered to acquire the ability to self-renew. These cells are cultured and expanded to desired quantities. Often, cells are cultured in a scalable manner, for example, using bioreactors that enable large-scale production. Various media formulations are optionally used to maintain the capacity for self-renewal, such as during expansion of the cell population, or to drive cells down specific differentiation pathways to generate desired cell types. For example, in some instances, cultured cells are induced to differentiate into muscle cells, adipocytes, or organ cells. In some cases, differentiation involves transdifferentiation of cells into different cell types. For example, immortalized fibroblasts can be expanded and then transdifferentiated into muscle cells, adipocytes, hepatocytes, and / or other desired cell types. Often, media formulations are modified from traditional media so that they do not require fetal bovine serum or serum substitutes and remain untested for human consumption. Media formulations may include plant-derived, low-serum or serum-free formulations that reduce or eliminate the use of animal components, such as fetal bovine serum. Examples of plant-based formulations include soy-based and plant hydrolysate-based media formulations. Media formulations often include at least one mushroom-based ingredient. In some cases, at least one mushroom-derived extract replaces fetal bovine serum in the media formulation. Some media formulations include at least one ingredient to enhance the nutrient content of cultured cells.Instead, co-culture systems are used to provide conditioned medium systems, which increase efficiency by eliminating the need for recombinant protein production and allowing for medium reuse. In addition to alternative media formulations, 3D scaffolds and tissue engineering platforms are often used to facilitate large-scale growth. Often, scalable bioreactors provide the requisite growth required for mass production. In some instances, 3D scaffolds are used to provide structural support and guide the growth of cultured cells into desired structures and / or tissues that resemble equivalent food products using traditional methods. Alternatively, or in combination, microscaffolds enable the growth of adherent cells in suspension culture, such as in bioreactors. These microscaffolds can be engineered to enhance stem cells, direct cell differentiation into relevant lineages, and adjust the flavor, texture, and tensile modulus of the final meat product. Some adherent cells are modified to grow in suspension culture without the need for an adhesive surface. Some foods are produced using homogenous populations of cells, such as liver cells to make foie gras. Alternatively, some foods are produced using heterogeneous populations of cells, such as a combination of muscle and fat cells. In some cases, a population of cells is differentiated into multiple cell types to generate a heterogeneous population of differentiated cells. Alternatively, independent cell populations are differentiated into distinct cell types and then combined. This method using heterogeneous cell populations allows for the production of specific tissues, such as salmon, which is composed of a combination of muscle and fat cells. Often, cultured cells are modified to produce a desired cell or tissue phenotype. Cultured cells can be modified with one or more genetic constructs to confer a desired phenotype, such as a state of self-renewal, differentiation into a cell or tissue type, or a tendency toward steatosis. Cultured cells can also be modified through adjustments to the culture environment. For example, liver cells are optionally cultured in a lipid-rich medium to induce steatosis through excessive lipid uptake and storage within the cytoplasm. Fatty liver cells are often harvested and processed into foie gras or foie gras foods.The harvested cells are typically processed to provide a desired firmness and / or texture, and in some cases, the harvested cells are processed to achieve special flavors, textures, and other characteristics indistinguishable from the high-quality meat they are intended to reproduce.

[0041] The systems and methods for producing cell-cultured foods disclosed herein offer many advantages. Cultured meat is not exposed to pathogens, such as avian flu or various bacterial strains, during production. Similarly, the systems and methods disclosed herein can provide antibiotic-free meat production. This has the advantage of avoiding inadvertent human exposure to antibiotics while also avoiding the increased risk of bacteria developing antibiotic resistance. Additionally, cultured food production does not require feed grains and avoids the production of animal waste, which often contains fecal coliform bacteria, ammonia, and phosphorus. For example, vast amounts of land are allocated to growing feed grains for livestock, which involves extensive use of fertilizers, pesticides, and herbicides. In contrast, cultured foods can be produced with a relatively small environmental footprint.

[0042] Textureless tissues, such as foie gras, and certain fish meats, such as salmon, can be produced using various systems and methods described herein. Some methods allow for the production of cultured textureless tissues boasting high lipid content, such as fatty liver cells, which are useful for making foie gras. Such methods often include: a) obtaining a population of differentiated cells capable of self-renewal; b) culturing the population of differentiated cells; c) manipulating at least one lipid metabolic pathway to induce adiposity in the population of differentiated cells, such that the cells accumulate a high lipid content; and d) processing the population of differentiated cells into textureless tissue. In some cases, differentiated cells capable of self-renewal are obtained via transdifferentiation (e.g., direct cell reprogramming). Often, the methods described herein produce cultured organ tissue for human consumption. Such methods include: a) culturing a population of cells capable of self-renewal; b) inducing differentiation in the population of cells to generate organ tissue; and c) processing the organ tissue for human consumption.

[0043] FIG. 1 illustrates one embodiment of a process for culturing cells for human consumption. In this example, a population of self-renewing cells is obtained (101). As described herein, the self-renewing cells are often embryonic stem cells, induced pluripotent stem cells, embryonic germ cells, immortalized differentiated cells, or nascent adult stem cells. The population of cells is cultured (102) and typically expanded to a desired population size. Differentiation is then induced in the population (103). In some cases, differentiation involves transdifferentiation of the cells into a different cell type. In this example, cells in the population differentiate into hepatocytes. Often, lipid accumulation is induced in the population of cells, including the differentiated hepatocytes (104). Finally, the population of cells is processed for human consumption (105). For example, hepatocytes are often processed into foie gras or foie gras food products.

[0044] Figure 2 illustrates one embodiment of a process for culturing muscle tissue for human consumption. In this example, a first and second population of self-renewing cells are obtained (201)(204). The second population of cells is cultured (202)(205) and typically expanded to a desired population size. Differentiation is then induced in the two populations (203)(206). In this case, differentiation into muscle cells is induced in the first population of cells (203). In some cases, differentiation includes transdifferentiation of the cells into a different cell type. Differentiation into adipocytes is induced in the second population of cells (206). Finally, the two populations of cells are processed for human consumption (207). In this case, the first and second populations are combined and processed into meat containing both muscle and adipocytes for human consumption.

[0045] An overview of an exemplary process for preparing cultured meat for human consumption is shown in Figure 3. First, stem cells are identified, isolated, and characterized. These cells are initially grown in two-dimensional culture, such as on a feeder cell layer. The cells are eventually transferred to suspension culture in a bioreactor, which allows for large-scale cell growth. After transfer to suspension culture, the cells differentiate into muscle cells. In some cases, differentiation involves transdifferentiation of cells into different cell types (e.g., from immortalized fibroblasts to muscle and / or adipocytes). The meat is then harvested and finally prepared and cooked. Various approaches can be used to obtain cell lines suitable for preparing cultured foods (Figures 4A-4B).

[0046] Disclosed herein in one aspect is a method for producing synthetic foods containing fish-derived tissues. In some embodiments, fish muscle cells and adipocytes are utilized in the development of fish-related foods based on their intrinsic regenerative capacity during early development. In an exemplary embodiment of this process, trout preadipocytes and muscle satellite cells (capable of differentiating into muscle cells) were isolated, cultured, and characterized. As shown in Figures 5A-5D, trout muscle satellite cells were isolated and subsequently characterized. Insets, where present, magnify image details; scale bars equal 10 μm in all micrographs unless otherwise specified. A substantially pure population of fish muscle satellite cells was successfully isolated, as shown in Figure 5A, with satellite cells accounting for approximately 80% of the isolated cells. RT-PCR analysis of these isolated cells revealed expression of the transcriptional markers Mstn1a and Myf5, markers of pluripotency (Figure 5B). Culture conditions were then optimized for these cells. Using this culture protocol, muscle satellite cells (arrowheads) were successfully differentiated into mature, differentiated muscle cells (arrows) (Figure 5C). The resulting sheets of trout myotubes differentiated from muscle satellite cells are shown in Figure 5D (scale bar is 100 μm). In some embodiments, muscle satellite cells can be co-cultured with preadipocytes. In an illustrative embodiment, salmon muscle satellite cells (arrowheads) were co-cultured with salmon preadipocytes (arrows) to produce a food product containing both muscle and fat cells or tissues, as shown in Figure 6A (scale bar is 100 μm). As shown in Figure 6B, the preadipocytes were differentiated into adipocytes, and the muscle satellite cells were differentiated into myocytes (arrowheads) (scale bar is 100 μm).

[0047] In some cases, salmon fibroblasts are used to produce food. Salmon fibroblasts can be induced to form spheroids for growth in bioreactors (Figure 7A) (scale bar is 100 µm). The viability of these spheroids is confirmed by returning them to two-dimensional culture conditions and observing that the fibroblasts migrate to the periphery to form colonies (Figure 7B) (scale bar is 100 µm). In some cases, the fibroblasts proliferate and then transdifferentiate into desired cell types, such as myocytes, adipocytes, hepatocytes, or any combination of these.

[0048] The methods disclosed herein are applicable to a variety of aquatic species. For example, cell cultures of bass muscle satellite cells have been successfully cultivated using standard cell culture protocols (Figure 8). In certain embodiments, disclosed herein are meat products comprising cells or tissues from one or more species of aquatic organisms. Often, the aquatic organism is selected from the group consisting of grouper, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, trout, eel, abalone, squid, clams, ark shell, sweetfish, scallop, sea bream, halfbeak, shrimp, flounder, cockle, octopus, or crab. In some cases, the aquatic organism is a species of fish selected from the group consisting of grouper, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, trout, or flounder. In some examples, the aquatic organism can be a round or flat fish. Round fish can include bass, catfish, alpine char, cod, haddock, herring, sardines, tilapia, trout, snapper, salmon, swordfish, and tuna. Flat fish can include flounder, oysterfish, halibut, and turbot. Tuna species include yellowfin tuna, southern bluefin tuna, Atlantic bluefin tuna, albacore tuna, Atlantic bluefin tuna, and bigeye tuna. Salmon species include Atlantic salmon, sockeye salmon, Chinook salmon (also called king salmon), coho salmon, chum salmon, and pink salmon. Trout species include rainbow trout, cutthroat trout, brown trout, red mountain trout, brook trout, and lake trout.

[0049] In some embodiments, methods for producing synthetic foods containing tissue derived from birds are disclosed herein. In some embodiments, the synthetic foods contain avian hepatocytes and / or liver tissue derived from ducks or geese. In some embodiments, the synthetic foods contain fatty liver tissue. In some embodiments, these methods utilize self-renewing cells (e.g., pluripotent or multipotent cells) for the development of bird-related foods based on their intrinsic regenerative capacity during early development. Successfully isolated duck embryonic stem cells grown in culture are shown in Figure 9.

[0050] cell line Some systems and methods disclosed herein involve the generation of cell lines capable of self-renewal for the production of cultured foods. In one approach, embryonic stem cells are isolated. Embryonic stem cells are pluripotent stem cells generated from early embryos. Typically, embryonic stem cells are harvested from blastocysts 4-5 days after fertilization. Blastocysts have an inner cell mass, which is removed and placed in culture. These cells, which remain viable in cell culture, are used to establish cell lines capable of self-renewal. For example, Figure 4A illustrates one approach for generating fatty liver cells. In some examples, embryonic stem cells are obtained from avian embryos, such as duck or goose embryos (401). These duck or goose embryonic stem cells are pluripotent stem cells (411) that are optionally used to produce cultured foie gras. Often, avian embryonic stem cells are isolated from blastoderm cells in Eyal-Giladi and Kochav stage 10 (EGK-X) avian embryos. For example, avian embryonic stem cells can be isolated by culturing them on inactivated STO feeder cells in sexual stem cell medium (ESA) with specific growth factors such as bFGF, IGF-1, mSCF, IL-6, OSM, LIF, IL-6, and IL-11, as described in Aubel P., Pain B. Chicken embryonic stem cells: establishment and characterization. Methods Mol. Biol. 2013; 1074:137-150. Successfully isolated duck embryonic stem cells growing in culture are shown in Figure 9. These approaches can also be used to generate fish muscle and / or adipocytes (Figure 4B).

[0051] Once isolated, embryonic stem cells are typically maintained in an undifferentiated state. Published protocols are often modified to maintain embryonic stem cells in an undifferentiated state. Modifications to published protocols can include the use of optimized matrix substrates and media formulations to achieve sustained cell proliferation and maintenance of a dedifferentiated state. In some cases, avian embryonic stem cells are maintained in an undifferentiated state using leukemia inhibitory factor (LIF), a member of the interleukin-6 family of cytokines, as described in Horiuchi et al., Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J. Biol. Chem. 2004;279:24514-24520. Alternatively, avian embryonic stem cells are maintained in an undifferentiated state without the need for LIF in the medium. In some instances, avian embryonic stem cells are maintained in an undifferentiated state in a media formulation containing LIF without the use of other cytokines or feeder cells. Media formulations often contain recombinant LIF. In some cases, recombinant LIF is produced as a fusion protein with an affinity tag for purification. Typically, the fusion protein with an affinity tag for purification uses at least one affinity tag selected from glutathione S-transferase (GST), FLAG tag, S-tag, heavy chain of protein C (HPC), streptavidin-binding peptide, streptavidin tag, histidine affinity tag, polyhistidine tag, polycysteine ​​tag, polyaspartate tag, albumin-binding protein (ABP), calmodulin-binding peptide, cellulose-binding domain, chitin-binding domain, and choline-binding domain. In some examples, the affinity-purified fusion protein is cleaved or digested to remove the affinity tag.

[0052] Isolated embryonic stem cells are typically differentiated into the desired cell type. The desired cell type is usually a fully differentiated cell that will construct a food product or a portion of a food product. Often, the differentiated cells are liver parenchymal cells or hepatocytes (412). Figure 10A shows duck hepatocytes growing in culture after successful differentiation. Differentiation was confirmed using RT-PCR (Figure 10B) to measure markers of hepatocyte differentiation (L-FABP, alpha-fetoprotein, and HNF3b, with beta-actin as a loading control). As shown in Figure 10B, hepatocytes (right lane) exhibit significant expression of hepatocyte differentiation markers compared to a lack of expression in control undifferentiated cells (left lane). Cultured hepatocytes are often used to produce foie gras. In some instances, the differentiated cells are muscle cells or skeletal muscle cells. Differentiated cells are often adipocytes, optionally used in combination with other cell types, such as muscle cells, for the production of cultured meat products containing both fat and muscle tissue. For example, salmon muscle cells and adipocytes are often used to produce sushi-grade salmon meat for human consumption. Often, the differentiated cells are organ cells, such as striated or skeletal muscle cells, smooth muscle cells, cardiac myocytes, spleen cells, thymocytes, endothelial cells, blood cells, gastrointestinal cells, liver cells, kidney cells, pancreatic cells, lung cells, or any combination thereof. Instead, the desired cell type is often an intermediate cell type, such as an adult stem cell or progenitor cell, useful for generating a fully differentiated cell type. Differentiation is often achieved by optimizing standard protocols, such as those described on the website www.abcam.com / protocols / hepatocyte-differentiation-protocol. For example, both embryonic stem cells and induced pluripotent stem cells can be differentiated into hepatocytes by splitting the cells onto Matrigel-coated plates in mTESR medium with the ROCK inhibitor Y27632, treating them with definitive endoderm (DE) medium, followed by hepatic endoderm (HE) medium, immature hepatocyte (IMH) medium, and finally mature hepatocyte (MH) medium. Some media formulations are modified to enhance proliferation, differentiation, or other desired qualities of the cultured cells.

[0053] Several methods result in the generation of induced pluripotent stem cells (402) used to produce the foods disclosed herein. Often, episomal reprogramming strategies are employed to generate induced pluripotent stem cells (iPSCs) from fibroblasts using the episomal reprogramming strategy outlined in Drozd et al., "Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP / EBNA-1 episomal reprogramming system." Stem Cell Research & Therapy. 2015;6:122. For example, in some cases, at least one episomal vector expressing a combination of reprogramming factors, such as Oct3 / 4, Sox2, Klf4, L-Myc, c-Myc, Lin28, Nanog, and Lin4, is transfected into cells derived from adult avian fibroblasts. Additional factors often added include p53 to overcome reprogramming barriers, such as cellular senescence. As an example, (ori-P / EBNA-1)-based episomal vectors persist episomally within reprogrammed cells, allowing for reprogramming. Episomal reprogramming provides an approach that prevents the generation of a "gene footprint," as the episomal approach generates iPSC lines without the integration of reprogramming vectors that results from classical viral reprogramming strategies. Finally, in some cases, differentiation of iPSCs is achieved using optimized standard protocols, such as those described elsewhere herein for embryonic stem cells.

[0054] In some cases, embryonic germ cells are used as a source of self-renewing, pluripotent stem cells (403). For example, embryonic germ cells can differentiate into desired cell types, such as mature hepatocytes, for the purpose of liver tissue generation. Often, embryonic germ cells are isolated using protocols such as those described in Guan et al., Derivation and characteristics of pluripotent embryonic germ cells in duck. Poultry Science. 2010; 89(2): 312-317. For example, stage 28 duck embryo tissue is obtained and then dissociated using trypsin. The dissociated cells are collected by centrifugation and then cultured in suspension in the presence of stem cell stimulating factor (SCF), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (FGF). Embryonic germ cells typically form colonies, which are then replated onto plates with feeder cells. In some instances, the isolated embryonic germ cells are expanded and optionally differentiated into desired cell types for food production as described herein.

[0055] In some cases, differentiated cells are reprogrammed into a desired cell type without generating an intermediate pluripotent cell type (404). This process is often referred to as transdifferentiation, in which a desired cell type is generated from a non-stem cell. Often, this method is performed as described in Simeonov KP and Uppal H, Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetically modified mRNAs. PLOS ONE. 2014; 9(6): e100134. Often, isolated fibroblasts are reprogrammed into hepatocytes or hepatocyte-like cells by culturing the fibroblasts in an optimized liver growth medium while expressing at least one of the following factors: FOXA1, FOXA3, HNF1A, and HNF4A. In some cases, HNF1A and at least two of FOXA1, FOXA3, and HNF4A are expressed in fibroblasts, resulting in their conversion into hepatocytes. Often, the expression or overexpression of any of the aforementioned factors in differentiated cells for reprogramming is achieved by introducing exogenous DNA or RNA into cells using genetic techniques known in the art. Alternatively, isolated fibroblasts are reprogrammed into muscle cells or adipocytes. In some cases, reprogramming involves the transdifferentiation of isolated cells, such as fibroblasts, into different cell types. For example, reprogrammed salmon muscle cells and adipocytes are useful for producing salmon meat for human consumption, such as salmon-grade sushi.

[0056] Often, fully differentiated cells of a desired cell type are immortalized to generate self-renewing cell lines. For example, myocytes, adipocytes, and / or hepatocytes can be immortalized for food production purposes. Often, classically defined immortalization strategies using transformation are applied to differentiated adult cells to generate cell lines with indefinite proliferation capacity (405). In various instances, cell immortalization is achieved by artificial expression of key proteins required for immortality. In some instances, differentiated adult cells are immortalized by expression or overexpression of at least one of SV4040 large T antigen, hTERT, HPV E6 / E7, EBV, MycT58A, RasV12, and p53. In some cases, avian hepatocytes are immortalized to generate self-renewing adult avian hepatocyte cell lines. Such cell lines facilitate the large-scale production of hepatocyte-based foods, such as foie gras. Often, salmon muscle cells and / or adipocytes are immortalized to generate self-renewing adult salmon muscle and adipocyte cell lines. Such immortalized cell lines enable large-scale production of salmon meat, such as sushi-quality salmon. In some cases, differentiated cell lines (e.g., fibroblasts) are immortalized and then transdifferentiated into desired cell types, such as muscle cells, adipocytes, hepatocytes, or any combination thereof, and can be used to produce various types of food products, such as fish meat or avian liver.

[0057] In some cases, immortalized cell lines capable of self-renewal have been generated, in some instances without transformation or direct genetic modification (406). In this approach, cell populations are typically harvested and serially passaged over several weeks until most cells undergo senescence, during which time a small number of spontaneous mutations occur, leading to the generation of cell lines with unlimited replicative potential. In some cases, cell populations are obtained from differentiated cells of an embryo, such as embryonic (differentiated) liver cells. This process can be applied to avian cells to generate immortalized avian liver cells, such as those described in Lee et al., Establishment of an immortal chicken embryo liver-derived cell line. Poultry Science. 2013; 92(6):1604-12. This method eliminates the need for viral integration to generate immortalized cell lines without the use of exogenous genetic material or genetic manipulation. For example, Figure 11A shows duck self-renewing cells generated by culturing primary fibroblasts and harvesting colonies of dividing cells after 6–8 weeks. Figure 11B shows trout self-renewing cells generated by culturing primary fibroblasts and harvesting colonies of dividing cells after 6-8 weeks. The self-renewing cells were then characterized for morphology, growth rate, and proliferation potential (e.g., the number of passages achieved without changes in morphology, growth rate, and genomic instability). In some cases, differentiated cell lines (e.g., fibroblasts) can be immortalized and then transdifferentiated into desired cell types, such as myocytes, adipocytes, hepatocytes, or any combination thereof, and used to produce various types of food products, such as fish meat or avian liver.

[0058] In some cases, nascent adult stem cells capable of self-renewal are isolated. For example, the liver is one of the few organs in adult mammalian and avian organisms that possess regenerative capabilities. The presence of stem cells in adult liver tissue is reviewed in Navarro-Alvarez et al., Hepatic stem cells and liver development. Methods Mol Biol. 2010;640:181-236. Accordingly, in some instances, nascent liver stem cells have been isolated, cultured, and expanded for use in cultivated food production (407). Frequently, fish preadipocytes and satellite cells are isolated and cultured to form cell lines suitable for expansion and differentiation into adipocytes and myocytes, respectively. Differentiated adipocytes and myocytes are usually co-cultured together in specific ratios to produce the desired final composition of adipocytes and myocytes in the resulting food.

[0059] In some cases, liver cells are treated with toxic chemicals to generate cells with enhanced proliferative capacity. (408) For example, such exposure to toxic compounds has been shown to elicit a proliferative response within the liver parenchyma. Accordingly, these liver cells with enhanced proliferative capacity are cultured and expanded, in various instances, for use in the production of cultivated foods.

[0060] In some cases, cells obtained using any of the aforementioned methods are further modified to generate cell lines that do not require an adhesive substrate for growth or survival. This method often involves generating hepatocytes that do not require an extracellular matrix for attachment to survive and proliferate. The advantages of being able to grow cells in suspension culture include the ability to easily and rapidly scale up growth. Suspension culture is often less labor-intensive and / or resource-intensive because it cultivates cells based on volume rather than surface area and allows for cell passaging without a separation step such as trypsinization. Cell lines that do not require an adhesive substrate are often combined with bioreactor cell culture systems to enhance large-scale food production. In some cases, stem cells are suitable for suspension culture, allowing expansion before differentiation into differentiated cell types such as hepatocytes, myocytes, or adipocytes. Alternatively, in some instances, stem cells are differentiated into hepatocytes and then transferred to a 3D suspension culture. In some cases, differentiated cells are transdifferentiated into desired cell types.

[0061] Genetic modification of cell lines Disclosed herein are methods for performing one or more modifications on cells or cell lines. In some cases, the modifications are genetic modifications performed by introducing a nucleic acid or gene construct into the cells or cell line. Cells can be modified to confer the ability to self-renew, differentiate into a desired cell type, obtain a specific cell phenotype (e.g., steatosis), or other desired changes. In some cases, cells are modified through the introduction of exogenous nucleic acids, such as one or more DNA constructs. Introduction of exogenous nucleic acids into cells can be accomplished using a variety of methods, including, but not limited to, transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, or transformation. For example, cells of a specific cell type can be transdifferentiated into a desired cell type.

[0062] Additionally, gene editing systems such as transcription activator-like effector nucleases (TALENS) or CRISPR are available to perform genetic modifications in cells. For example, CRISPR can be customized because its active form consists of an invariant Cas9 protein and a programmable guide RNA (gRNA). The Cas9-gRNA complex searches DNA for a protospacer adjacent motif (PAM) sequence, followed by the formation of an R-loop. Upon formation of a macromolecular complex containing Cas9, gRNA, and target DNA, the Cas9 protein generates two nicks in the target DNA, creating a blunt double-strand break that is repaired predominantly by nonhomologous end joining or template-directed homologous recombination.

[0063] A gene construct can contain promoters and ORFs for one or more genes. The gene construct can be introduced into a cell population, followed by selection for stable cell lines that have integrated the construct. For example, a plasmid containing the desired gene and the neo gene, which confers resistance to G418, can be linearized (e.g., cleaved once with a restriction endonuclease) and transfected into a duck hepatocyte fibroblast cell line, followed by selection with G418 to obtain fibroblasts that have successfully integrated the linearized plasmid vector into their genome. Examples of promoters include the cytomegalovirus (CMV) promoter, the CMV enhancer fused to the chicken β-actin promoter (CAG), the human elongation factor 1-alpha (HEF-1α), the telomerase reverse transcriptase (hTERT) promoter, and the simian virus (Simian Virus 40) promoter. In some cases, promoters with low or absent basal transcription rates are used to minimize or prevent leaky expression. As an example, expression of the recombinases used in the various constructs described herein can cause irreversible changes to the cell (e.g., by stimulating genes involved in maintaining pluripotency). Thus, in some embodiments, the constructs include a promoter that allows for at most one transcription event per mitotic cell cycle. In some cases, the promoter allows for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or even 50 or fewer transcription events (on average) per mitotic cell cycle. In some cases, the promoter allows for less than one transcription event (on average) per mitotic cycle. Often, the promoter does not allow any transcription events per mitotic cycle (e.g., if the average is less than half a transcription event per mitotic cycle).

[0064] Genetic modification allows for the generation of cell lines possessing desirable characteristics. For example, modified cell lines may express genes that maintain the cell line in a state of self-renewal and / or proliferation. A self-renewal state can be a state of proliferation or division while maintaining an undifferentiated state. As an example, induced pluripotent stem cells with self-renewal properties can be generated from differentiated adult cells by expressing one or more of Oct3 / 4, Sox2, Klf4, and c-Myc. In some cases, cells can be differentiated and have unlimited proliferative capacity (e.g., immortalized fibroblasts). Often, modified cell lines respond to inducers that cause a switch from one phenotype to another. The switch can be from a self-renewal state to a differentiated state (e.g., from muscle satellite cells to myocytes or from preadipocytes to adipocytes). The methods disclosed herein can include one or more constructs for inducible adipogenesis. For example, the method may utilize a first construct containing one or more pluripotency genes to promote cell division and / or maintain preadipocytes in an undifferentiated state, and a second construct containing a TRE, one or more adipogenic genes, and a regulatory factor (e.g., Cre recombinase) to inactivate the pluripotency genes. In some cases, the switch involves a change from one differentiated cell type to another (e.g., from adult fibroblasts to hepatocytes). Often, the switch does not involve a change in cell type, but instead involves a change in cell phenotype or characteristics. As an example, the switch can induce hepatocytes to undergo steatosis, become predisposed to steatosis (e.g., become more likely to undergo steatosis under appropriate conditions, such as incubation with fatty acids), or cause enhanced steatosis (e.g., increase lipid accumulation compared to controls). Examples of genes involved in adipogenesis include FABP4, GLUT4, ADIPOQ, AGPAT2, PLIN1, LEP, and LPL. In some examples, the construct comprises FABP4, GLUT4, ADIPOQ, AGPAT2, PLIN1, LEP, LPL, or any combination thereof.The construct can include at least one, at least two, at least three, at least four, at least five, at least six, or all seven ORFs for genes selected from the group consisting of FABP4, GLUT4, ADIPOQ, AGPAT2, PLIN1, LEP, and LPL.

[0065] In some instances, the cells are modified to express (either inducibly or constitutively active) one or more pluripotency genes that promote cell division. In particular instances, the pluripotency genes promote at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, or at least about 1000 cell divisions. In some instances, the number of cell divisions for a given cell line or cell population is monitored for quality control purposes. For example, in some instances, cell lines or populations that exceed a threshold number of cell divisions are not used to produce cultured food products. In some cases, the threshold number of cell divisions is at least about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 20000, about 30000, about 40000, or about 50000 or more cell divisions. In some cases, the threshold number of cell divisions is at most about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000, about 20000, about 30000, about 40000, or about 50000 or more cell divisions.

[0066] Disclosed herein are inducible cells modified using constructs that respond to inducers. These modified cells can be used to produce cultured food products such as fish meat, avian liver tissue, and other foodstuffs. The modified cells can be used to control proliferation, differentiation, cell phenotype (e.g., adiposity / lipid accumulation), or other cellular properties. A typical, non-limiting example of such an inducible system is the Tet-on / off system, which utilizes tetracycline / doxycycline as an inducer. Other inducible systems are also contemplated for carrying out the methods described herein. Examples of non-Tet inducible systems include the coumermycin-inducible expression system, the RheoSwitch® Mammalian-inducible expression system, the estrogen receptor-inducible system, the cumate-inducible system, and the Cre-Lox recombinase system. In some cases, cell lines stably incorporating the inducible systems or constructs described herein are generated. Alternatively, cells can be engineered to transiently express an inducible system or construct described herein (e.g., via transient transfection of at least one construct).

[0067] Tet-on or Tet-off systems typically utilize the tetracycline transactivator protein. The TetO sequence is typically positioned upstream of any ORF whose expression is desired to be controlled using the Tet system. The promoter and TetO sequence can constitute a tetracycline response element (TRE). In some cases, the TRE consists of a TetO sequence and is placed upstream of the promoter and ORF for one or more genes of interest. In the Tet-on system, the transactivator protein has a strong binding affinity for the TetO operator region when not bound by tetracycline (or a derivative such as doxycycline). In the absence of tetracycline, the transactivator protein does not bind to the tetracycline response element (TRE). When tetracycline is added, it binds to the transactivator protein and causes it to bind to the TRE to induce expression of downstream ORFs. In the Tet-off system, the transactivator protein has a strong binding affinity for the TetO operator region only when not bound by tetracycline. In the absence of tetracycline, the transactivator protein binds to the TetO sequence and promotes expression of the downstream ORF. Added tetracycline binds to the transactivator protein, causing a conformational change that results in reduced or lost binding to the TRE, resulting in reduced expression of the downstream ORF.

[0068] Figure 12 shows an exemplary embodiment of a gene construct that can be introduced into cells to provide inducible differentiation into hepatocytes. The construct contains a tetracycline response element (TRE) and ORFs for hepatocyte reprogramming factors HNF1A, FOXA1, and HNF4A. The construct can be stably transformed into target cells, such as pluripotent or multipotent cells. In some cases, the construct can be stably transformed into terminally differentiated cells, such as immortalized fibroblasts (obtained according to the techniques described herein). Expression of the ORF is normally repressed in the absence of tetracycline. Treatment with tetracycline induces expression of the ORF, thereby driving the cells toward differentiation into hepatocytes. Thus, cell lines stably incorporating this construct can be induced to differentiate into hepatocytes via tetracycline / doxycycline treatment. In certain cases, the construct contains at least one of HNF1A, FOXA1, and HNF4A. Sometimes, the construct comprises at least two of HNF1A, FOXA1, and HNF4A. In some examples, the construct comprises HNF1A, FOXA1, and HNF4A. The construct may comprise HNF1A, FOXA1, HNF4A, or any combination thereof. In particular examples, the construct comprises HNF1A and FOXA1; HNF1A and HNF4A; or FOXA1 and HNF4A.

[0069] Figure 13 shows an exemplary embodiment of a construct that can be introduced into cells to enable inducible expression of one or more proteins that cause the cells to become adipose. The construct contains a tetracycline response element (TRE) and ORFs for one or more genes involved in lipid metabolism. The construct can be stably transformed into target cells, such as pluripotent or multipotent cells. In some cases, the construct can be stably transformed into terminally differentiated cells, such as fibroblasts. The TRE represses expression of the ORF but allows the ORF to be transcribed in the presence of tetracycline or doxycycline. Thus, cell lines that stably integrate this construct can be induced to undergo or become adipose via treatment with tetracycline / doxycycline. In some cases, the construct contains an ORF for ZFP423. In some cases, the construct comprises an ORF for ATF4 (activating transcription factor 3) (Kim JY et al., Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis. J Hepatol. 2017 Aug;67(2):349-359). In some cases, the construct comprises an ORF for SREBP-1c (Ferre P et al., Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c. Diabetes Obes Metab. 2010 Oct;12 Suppl 2:83-92). Other genes are contemplated for use in the methods and construct systems described herein, including LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, KLF6, or any combination thereof.In some cases, the construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 genes selected from the group consisting of: ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, and KLF6.

[0070] Exemplary genes utilized in the methods described herein are listed below in Table 1. Expression of genes involved in lipid metabolism can be induced or enhanced to promote or enhance lipid accumulation and / or adiposity in target cells, such as hepatocytes or adipocytes. Hepatocyte reprogramming factors can be used to reprogram cell types into hepatocytes, such as transdifferentiation of fibroblasts into hepatocytes. Similarly, myocyte reprogramming factors can be used to reprogram cell types, such as fibroblasts, into myocytes. Adipocyte reprogramming can be used to reprogram cell types, such as fibroblasts, into adipocytes. Similarly, genes involved in myocyte differentiation and adipogenesis can be used to induce differentiation of muscle satellite cells into myocytes and preadipocytes into adipocytes, respectively. Finally, various genes that can be used to generate induced pluripotent stem cells (iPSCs) are listed. Any one or combination of the genes listed in Table 1 is contemplated for the purposes described herein.

[0071] [Table 1-1]

[0072] [Table 1-2]

[0073] Figure 14 shows an exemplary embodiment of a DNA construct system that can be introduced into cells to enable a proliferation / differentiation switch from a pluripotent phenotype to a differentiated phenotype. This system has a first construct containing a pluripotency cassette that confers constitutive expression of ORFs for pluripotency factors (e.g., Oct. 4, Sox2, Klf4, I-Myc). The pluripotency factors of the first construct are flanked by pLox sites. The system has a second construct containing a differentiation cassette that confers tetracycline-inducible expression of MyoD and Cre recombinase. Addition of an inducer, such as tetracycline or doxycycline, can induce expression of MyoD and Cre recombinase. MyoD expression can help differentiate cells into muscle cells. The Cre recombinase enzyme can catalyze the excision of the pluripotency factors flanked by pLox sites. The inducer can then be removed to stop the induction of MyoD and Cre recombinase expression. An advantage of this system is the small footprint it leaves after excision of the pluripotency factors and removal of the inducer. Other genes can be used to induce myogenesis, including myogenin (MyoG), MRF4, and Myf5. In some cases, MyoD, MyoG, MRF4, Myf5, or any combination thereof, are used to induce myogenesis. Sometimes, the methods and / or construct systems described herein utilize at least one, at least two, at least three, or all four myogenic factors selected from the group consisting of MyoD, MyoG, MRF4, and Myf5.

[0074] Figure 15 shows an exemplary construct that can be introduced into cells to confer an inducible "off switch." This construct contains an ORF of one or more genes of interest and an expression cassette containing a TRE and Cre recombinase flanked by pLox sites. A construct such as that shown in Figure 15 contains a TRE-Cre expression cassette located downstream of the ORF. Alternatively, the construct can have a TRE-Cre expression cassette located upstream of the ORF. The promoter is typically located upstream of the ORF and is separate from the TRE, so that the ORF is expressed independently of Cre recombinase. By adding an inducer, cell lines that stably incorporate this construct can express Cre recombinase to catalyze the excision of the intervening sequence flanked by pLox sites. Thus, the expression cassettes for one or more genes (e.g., those that promote differentiation) and the TRE and Cre recombinase are removed, resulting in footprint-free excision of the gene of interest. Such constructs can be used to induce the transdifferentiation of cells into different cell types.

[0075] Figure 28A shows one embodiment of a synthetic receptor for regulating gene expression (e.g., activating and / or inactivating a target ORF). This system is modeled after the "synthetic Notch receptor" system described by Morsut et al., Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell. 2016 Feb 11; 164(4):780-9. In this system, the receptor is engineered to contain the same extracellular components as endogenous Notch receptors (which signal by cleaving the intracellular domain upon binding a ligand such as protein δ). Accordingly, the intracellular domain can be replaced with an enzyme such as Cre recombinase. When a ligand is added to a cell, the engineered Notch receptor is activated and the intracellular domain is cleaved, thereby releasing Cre, in this case, into the cytoplasm. Cre enters the nucleus by passive diffusion (alternatively, a nuclear localization sequence can be engineered into the protein to facilitate nuclear entry). There, Cre induces recombination of the loxP sites, as described herein (e.g., Figures 12-15). Such constructs can be used to induce transdifferentiation of cells into different cell types.

[0076] The model shown in Figure 28A represents an irreversible switch whereby Cre is released from the cell membrane to the nucleus to induce recombination events. Such events, including the switch from pluripotency maintenance to differentiation, are described herein. Cre delivery can affect various cellular functions or properties, such as proliferation, adhesion, differentiation, migration, and other cellular characteristics. In various embodiments, this switch allows for a transition from a proliferative state to the activation of genes that induce differentiation (to myocytes, adipocytes, etc.). A key advantage of this system is that no gene activation is required for Cre to function; instead, the enzyme is constitutively expressed and localized as a reservoir at the cell surface. Such constructs can be used to induce the transdifferentiation of cells into different cell types.

[0077] Figure 28B shows additional strategies for switching between gene sets (e.g., inactivating a pluripotency gene set and activating a differentiation gene set). These strategies can be implemented using a site-specific recombinase (SSR) system in combination with an inducible gene expression system (e.g., a tetracycline / doxycycline-inducible system). The SSR / inducibility combination is described in Zhang et al. Conditional gene manipulation: Creating a new biological era. J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2012 13(7):511-524. In some cases, this SSR / inducibility system is used as a switch between pluripotency and differentiation to generate ex vivo meat. Such constructs can be used to induce the transdifferentiation of cells into different cell types.

[0078] As shown in Figure 28B, triangles represent lox sequences (black is loxP and white is lox5171, although other sequences may be used for this purpose). When the triangles align, a recombination event (catalyzed by the Cre enzyme) leads to the excision / deletion of the sequence between them. When the triangles (representing defined DNA sequences) face each other, the recombination event causes a stochastic inversion (flipping back and forth) of the intervening sequence. For purposes of this illustration, proliferation is described generally (and can include genes such as cyclin family members, cyclin-dependent kinases, cell cycle inhibitors such as p27kip, TERT, or others). As described in other genetic recombination mechanisms described herein (e.g., Figures 12-15), proliferation can also be replaced with "pluripotency" genes (such as Yamanaka factors for generating iPSCs). Similarly, differentiation can include various genes (such as MyoD for muscle cells). Other genes may be used in the same way to stimulate differentiation into any lineage. Such constructs can be used to induce the transdifferentiation of cells into different cell types.

[0079] Two representative scenarios are shown in Figure 28B. In the top panel (1), when Cre is added, it first induces recombination in either the black pair or the white pair of triangles. As shown in the left scenario, if the first event involves the black triangle (loxP), this induces an inversion of the intervening DNA sequence, placing two white triangles (lox5171) in parallel; this then allows the Cre enzyme to excise the intervening sequence. The result is a switch from proliferation genes to differentiation genes. In the right scenario, Cre first acts on the white triangle (lox5171). This induces an inversion, which then places the black triangle (loxP) in parallel, allowing Cre to excise the intervening sequence. Again, the result is a switch from transcription of proliferation genes to transcription of differentiation genes. Such constructs can be used to induce transdifferentiation of cells into different cell types.

[0080] In the bottom panel (2), a different scheme results in a switch from proliferation to differentiation. The lox sequences are positioned such that an inversion event occurs when Cre first induces recombination between the white triangles (lox5171). The inversion event places two black triangles in parallel (loxP), allowing Cre to excise the proliferation genes and activate differentiation. Inversion occurs in the right panel, when Cre first induces recombination between the black triangles. Such constructs can be used to induce transdifferentiation of cells into different cell types.

[0081] Unlike other systems (which activate one gene program and then require a second step of gene activation), the process described herein in Figure 28B is unique in that it uses a single input (Cre) to induce a complete switch from one set of genes to another with very high efficiency. This approach results in an improved method of producing cultivated foods that can simplify the process and / or reduce required inputs, especially in large-scale production. At large scale, the process disclosed herein represents a significant improvement in process simplification and reduced required inputs.

[0082] The inducible system described herein is not limited to Tet and / or Cre recombinase-based systems. Other embodiments of the system are contemplated that utilize inducible recombinase expression to excise one or more genes of interest. For example, the Flp-FRT system utilizes Flp (flippase) recombinase to excise DNA adjacent to the FRT (flippase recognition target) sequence. Such a system can be used to induce the transdifferentiation of cells into different cell types.

[0083] The combination of induced expression and gene excision to maintain cells in a self-renewing, undifferentiated state can face technical challenges due to promoter leakiness or basal expression levels. For example, promoter leakiness results in partial expression of the recombinase and subsequent excision of the pluripotent stem cell factor before the inducer is added. However, a key advantage of utilizing this system for the purposes of cultured food production described herein is that those cells that experience leakiness lose their self-renewal phenotype and are likely outcompeted by cells that maintain tighter control of recombinase expression. Therefore, when self-renewing (e.g., pluripotent or multipotent) cell cultures are scaled up to produce commercial quantities of cultured food, most or all of the cultured cells in the population must retain their undifferentiated and self-renewing properties, provided that basal expression levels are sufficiently low. Modified cells can be clonally selected to identify cell lines with strong repression of recombinase expression in the absence of an inducer. In some cases, an inducer is added to induce differentiation (and / or other desired properties) just before the cells are harvested to produce meat products for human consumption.

[0084] Induce lipid accumulation or steatosis In some cases, the systems, methods, and compositions disclosed herein provide for the induction of lipid accumulation or steatosis. Lipid accumulation or steatosis is often induced in a population of cells for the purpose of producing cell culture foods with increased lipid content. As used herein, steatosis is a pathological condition characterized by abnormal retention of lipids within cells. Excess lipid accumulates in vesicles that replace the cytoplasm. Macrodroplet steatosis describes cases where vesicles are large enough to replace or distort the nucleus, while microdroplet steatosis lacks this phenotype. For example, Figure 4A illustrates a process involving the generation of fatty hepatocytes (413) through genetic intervention and / or the addition of exogenous compounds (409).

[0085] In some embodiments, lipid accumulation and / or steatosis are induced in a population of cells by genetic manipulation. For example, some methods disclosed herein provide for the preparation of foie gras comprising cultured avian liver tissue. Some such methods include: a) obtaining a population of self-renewing avian-derived cells; b) differentiating the population of avian-derived cells into hepatocytes; c) inducing steatosis in hepatocytes to produce cultured avian liver tissue with a high lipid content; and d) preparing the cultured avian liver tissue as foie gras. In some examples, non-hepatic cells are induced to undergo lipid accumulation. Some methods produce cultured cells with high lipid accumulation for human consumption. One example of such a method includes: a) culturing a population of cells; b) inducing differentiation within the population of cells; c) inducing high lipid accumulation within the population of cells; and d) processing the population of cells for human consumption.

[0086] Steatosis and / or lipid accumulation can be achieved by manipulating lipid metabolism. For example, the gene profile of hepatocytes undergoing steatosis has been previously characterized by Chiappini et al., Exploration of global gene expression in human liver steatosis by high-density oligonucleotide microarray. Lab Invest. 2006 Feb; 86(2):154-65. The intracellular signaling pathways affected in fatty liver cells are involved in lipid metabolism and lead to the accumulation of lipid droplets in the cytoplasm of hepatocytes. In some cases, the systems and methods described herein provide reliable and highly efficient induction of steatosis in a population of cells. Steatosis is often induced in liver cells or a population of liver cells. Sometimes, steatosis is induced by up-regulating or down-regulating genes involved in hepatic lipid metabolism. For example, in some cases, p53 depletion and / or upregulation of p63 (e.g., overexpression of the N-terminal transactivation domain TAp63) induces lipid accumulation, as described in Porteiro et al., Hepatic p63 regulates steatosis via IKK beta / ER stress. Nature Communications. 2017 May;8:15111. Genetic modification of cells, such as hepatocytes, can be performed using various protocols discussed (e.g., inducible expression of genes involved in steatosis). In some instances, steatosis and / or lipid accumulation is induced by manipulating at least one of the lipid metabolic pathways and the ER pathway involved in ER stress. Sometimes, steatosis or lipid accumulation is induced in hepatocytes or liver cells. Alternatively, in other cases, steatosis or lipid accumulation is induced in non-hepatic cells, such as muscle cells or skeletal muscle cells. Sometimes, steatosis is induced in fish muscle cells, such as salmon muscle cells. In some instances, steatosis or lipid accumulation is induced in non-hepatocyte organ cells, such as, for example, kidney cells.Occasionally, steatosis or lipid accumulation is induced in pluripotent cell populations or adult progenitor cell populations that are used as intermediate cell lines for expansion into differentiated cell populations. In these scenarios, steatosis or lipid accumulation is induced early in the developmental process before the population of cells differentiates. In some cases, high lipid accumulation is induced in the cells.

[0087] Conversely, in some instances, steatosis can be induced by disrupting lipid metabolic pathways without the need for genetic manipulation. For example, certain exogenous compounds can induce steatosis in hepatocytes grown in vitro or ex vivo. These exogenous compounds include toxins, such as alcohols, and lipids, such as fatty acids. In some instances, culturing cells in a media formulation with a high concentration of at least one lipid induces steatosis. In various embodiments, cells are cultured in a lipid-rich media formulation to induce steatosis. Cells cultured in lipid-rich media sometimes contain a population of differentiated cells, such as avian hepatocytes or fish muscle cells. In some instances, the cells cultured in lipid-rich media are multipotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. Alternatively, in some instances, the cells cultured in lipid-rich media are differentiated pluripotent stem cells, such as adult progenitor cells. Sometimes, cells are cultured in lipid-rich media having at least one lipid type selected from saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, and trans fatty acids. Examples of lipids include palmitic acid, oleic acid, docosahexaenoic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, and eicosapentaenoic acid. Optionally, the medium is supplemented with linoleic acid, oleic acid, or a combination thereof to induce lipid accumulation or steatosis in a population of cells cultured in the medium. Optionally, the medium is supplemented with a lipid concentration of at least about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM. Sometimes the medium is supplemented with about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM or less of lipid.In various embodiments, the medium is supplemented with lipid at a concentration of at least about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, or about 1,000 μM. In some instances, the medium is supplemented with a lipid at a concentration of at least about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, or about 1,000 μM or less. Often, cell culture media contain a lipid concentration of about 1 mM to about 20 mM or about 1 μM to about 1,000 μM. Cell culture media often contain a lipid concentration of at least about 1 μM. Typically, cell culture media contain a lipid concentration of up to about 20 mM.

[0088] In some cases, the medium is supplemented with at least one medium such as IBMX (methylxanthine), rosiglitazone (thiazolidinedione), elevated glucose concentration, and / or corticosteroids such as dexamethasone. Other examples of thiazolidinediones that can be used to supplement the medium include pioglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, and balaglitazone. In some examples, the medium is supplemented with at least one thiazolidinedione selected from the group consisting of pioglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone, and balaglitazone. In one example, the thiazolidinedione is rosiglitazone. The media supplements described herein can be added to the media at various concentrations, including the full range of concentrations described for lipid concentrations. For example, the media supplements can be added to the media at a concentration of at least about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM. In some cases, the media supplements can be added to the media at a concentration of at least about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 200 μM, about 300 μM, about 400 μM, about 500 μM, about 600 μM, about 700 μM, about 800 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM or less.

[0089] Figure 16A shows the successful induction of steatosis (accumulation of intracellular lipid-containing vesicles; arrowheads) in duck hepatocytes upon incubation with 2 μM linoleic acid (lower panel) compared to untreated controls (upper panel). Figure 16B shows a dose-response curve correlating the percentage of fatty hepatocytes with the concentration of linoleic acid. Similar results were achieved with oleic acid. In some cases, the protocol was enhanced by incubating hepatocytes with IBMX (methylxanthine), rosiglitazone (thiazolidinedione), elevated glucose concentrations, or other fatty acids, and corticosteroids such as dexamethasone.

[0090] In some cases, the cell culture medium comprises a lipid concentration (or other medium supplements described herein) of about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1.0 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or even 20 μM. In some cases, the cell culture medium comprises a lipid concentration of at most about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1.0 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 15 μM, or about 20 μM.In some cases, the cell culture medium contains a lipid concentration of about 1 μM to about 2 μM, about 1 μM to about 3 μM, about 1 μM to about 4 μM, about 1 μM to about 5 μM, about 1 μM to about 6 μM, about 1 μM to about 7 μM, about 1 μM to about 8 μM, about 1 μM to about 9 μM, about 1 μM to about 10 μM, about 1 μM to about 15 μM, about 1 μM to about 20 μM, about 2 μM to about 3 μM, about 2 μM to about 4 μM, about 2 μM to about 5 μM, about 2 μM to about 6 μM, about 2 μM to about 7 μM, about 2 μM to about 8 μM, about 2 μM to about 9 μM, about 2 μM to about 10 μM, about 2 μM to about 15 μM, about 2 μM to about 20 μM, about 3 μM to about 4 μM, about 3 μM to about 5 μM, about 3 μM to about 6 μM, about 3 μM to about 7 μM, about 3 μM to about 8 μM, about 3 μM to about 9 μM, about 3 μM to about 10 μM, about 3 μM to about 15 μM, about 3 μM to about 20 μM, about 4 μM to about 5 μM, about 4 μM to about 6 μM, about 4 μM to about 7 μM, about 4 μM to about 8 μM, about 4 μM to about 9 μM, about 4 μM to about 10 μM, about 4 μM to about 15 μM, about 4 μM to about 20 μM, about 5 μM to about 6 μM, about 5 μM to about 7 μM, about 5 μM to about 8 μM, about 5 μM to about 9 μM, about 5 μM to about 10 μM, about 5 μM to about 15 μM, about 5 μM to about 20 μM, about 6 μM to about 7 μM, about 6 μM to about 8 μM, about 6 μM to about 9 μM, about 6 μM to about 10 μM, about 6 μM to about 15 μM, about 6 μM to about 20 μM, about 7 μM to about 8 μM, about 7 μM to about 9 μM, about 7 μM to about 10 μM, about 7 μM to about 15 μM, about 7 μM to about 20 μM, about 8 μM to about 9 μM, about 8 μM to about 10 μM, about 8 μM to about 15 μM, about 8 μM to about 20 μM, about 9 μM to about 10 μM, about 9 μM to about 15 μM, about 9 μM to about 20 μM, about 10 μM to about 15 μM, about 10 μM to about 20 μM, or about 15 μM to about 20 μM.

[0091] In some cases, the cell culture medium comprises a lipid concentration (or other medium supplements described herein) of at least about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM. In some cases, the cell culture medium comprises a lipid concentration of up to about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM.In some cases, the cell culture medium contains a lipid concentration of about 1 mM to about 2 mM, about 1 mM to about 3 mM, about 1 mM to about 4 mM, about 1 mM to about 5 mM, about 1 mM to about 6 mM, about 1 mM to about 7 mM, about 1 mM to about 8 mM, about 1 mM to about 9 mM, about 1 mM to about 10 mM, about 1 mM to about 15 mM, about 1 mM to about 20 mM, about 2 mM to about 3 mM, about 2 mM to about 4 mM, about 2 mM to about 5 mM, about 2 mM to about 6 mM, about 2 mM to about 7 mM, about 2 mM to about 8 mM, about 2 mM to about 9 mM, about 2 mM to about 10 mM, about 2 mM to about 15 mM, about 2 mM to about 20 mM, about 3 mM to about 4 mM, about 3 mM to about 5 mM, about 3 mM to about 6 mM, about 3 mM to about 7 mM, about 3 mM to about 8 mM, about 3 mM to about 9 mM, about 3 mM to about 10 mM, about 3 mM to about 15 mM, about 3 mM to about 20 mM, about 4 mM to about 5 mM, about 4 mM to about 6 mM, about 4 mM to about 7 mM, about 4 mM to about 8 mM, about 4 mM to about 9 mM, about 4 mM to about 10 mM, about 4 mM to about 15 mM, about 4 mM to about 20 mM, about 5 mM to about 6 mM, about 5 mM to about 7 mM, about 5 mM to about 8 mM, about 5 mM to about 9 mM, about 5 mM to about 10 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 6 mM to about 7 mM, about 6 mM to about 8 mM, about 6 mM to about 9 mM, about 6 mM to about 10 mM, about 6 mM to about 15 mM, about 6 mM to about 20 mM, about 7 mM to about 8 mM, about 7 mM to about 9 mM, about 7 mM to about 10 mM, about 7 mM to about 15 mM, about 7 mM to about 20 mM, about 8 mM to about 9 mM, about 8 mM to about 10 mM, about 8 mM to about 15 mM, about 8 mM to about 20 mM, about 9 mM to about 10 mM, about 9 mM to about 15 mM, about 9 mM to about 20 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0092] In some cases, cells are cultured for a period of time at a high lipid concentration to induce steatosis. The length of time the cells are exposed to the high lipid concentration varies depending on the cell type, the size of the cell population, the age of the cell population, the number of passages, any genetic modifications or manipulations of the cells, the type and composition of the medium, the desired amount of lipid accumulation or steatosis, or any combination thereof. For example, certain cell types take up exogenous lipids in the medium at a slower rate than other cell types and therefore require a longer incubation time in lipid-rich medium to induce the desired amount of steatosis. In various cases, cells are cultured for a period of time in a cell culture medium containing at least one lipid. Sometimes, cells are cultured for at least a period of time in a medium having a high lipid concentration. In many cases, cells are cultured for about 1 day to about 20 days in a medium having a high lipid concentration. Cells are often cultured for at least about 1 day in a medium having a high lipid concentration. Typically, cells are cultured for up to about 20 days in a medium having a high lipid concentration.

[0093] In some examples, the cells are cultured in a medium having a high lipid concentration (or other medium supplement as described herein) for about 1 to about 2 days, about 1 to about 3 days, about 1 to about 4 days, about 1 to about 5 days, about 1 to about 6 days, about 1 to about 7 days, about 1 to about 8 days, about 1 to about 9 days, about 1 to about 10 days, about 1 to about 15 days, about 1 to about 20 days, about 2 to about 3 days, about 2 to about 4 days. , about 2 to about 5 days, about 2 to about 6 days, about 2 to about 7 days, about 2 to about 8 days, about 2 to about 9 days, about 2 to about 10 days, about 2 to about 15 days, about 2 to about 20 days, about 3 to about 4 days, about 3 to about 5 days, about 3 to about 6 days, about 3 to about 7 days, about 3 to about 8 days, about 3 to about 9 days, about 3 to about 10 days, about 3 to about 15 days, about 3 to about 20 days, about 4 to about 5 days, about 4 to About 6 days, about 4 to about 7 days, about 4 to about 8 days, about 4 to about 9 days, about 4 to about 10 days, about 4 to about 15 days, about 4 to about 20 days, about 5 to about 6 days, about 5 to about 7 days, about 5 to about 8 days, about 5 to about 9 days, about 5 to about 10 days, about 5 to about 15 days, about 5 to about 20 days, about 6 to about 7 days, about 6 to about 8 days, about 6 to about 9 days, about 6 to about 10 days, about 6 to about 15 days The cells are cultured for about 6 to about 20 days, about 7 to about 8 days, about 7 to about 9 days, about 7 to about 10 days, about 7 to about 15 days, about 7 to about 20 days, about 8 to about 9 days, about 8 to about 10 days, about 8 to about 15 days, about 8 to about 20 days, about 9 to about 10 days, about 9 to about 15 days, about 9 to about 20 days, about 10 to about 15 days, about 10 to about 20 days, or about 15 to about 20 days.

[0094] <Culture medium preparation> Provided herein are systems and methods that utilize at least one media formulation that enables cultured food production. In some cases, the media formulation does not require the use of serum, such as fetal bovine serum. Sometimes, the media formulation does not require one or more other supplements used in a particular cell culture medium. Cell culture media are typically divided into two categories: serum media and serum-free media. Traditional media formulations often utilize fetal bovine serum and other supplements, which are too costly for large-scale cultured food production. Because serum (e.g., fetal bovine serum) is produced from animals, it is prone to batch-to-batch variation. For example, fetal bovine serum (FBS) is extracted from the blood of fetal calves and is prone to batch-to-batch variation in composition. In addition, the use of serum can create the potential for contamination by viruses, mycoplasma, prions, toxins, and other undesirables present in the animal from which the serum is extracted. Finally, serum is expensive and requires raising livestock, which is counter to part of the goal of providing cultured food. However, the use of serum-free media circumvents these challenges. Serum substitutes or supplements are used in various media formulations for producing cultured foods described herein. The serum substitutes or supplements are derived from non-domestic (e.g., non-fetal bovine) sources. Examples include mammalian cell overexpression systems and transgene expression in yeast, larger fungi (e.g., mushrooms), bacteria, algae, or insect cell (e.g., baculovirus) systems. An exemplary embodiment is a mushroom-based system for producing serum substitutes for serum-free media formulations, as described in Benjaminson et al., "In vitro edible muscle protein production system (mpps): Stage 1, fish." Acta Astronautica (2002): 51(12), 879-889. Sometimes, the systems, methods, and compositions described herein include generating or obtaining at least one cell line suitable for culturing using a mushroom-based media formulation.In some cases, hepatocyte, preadipocyte, or satellite cell lines are adapted or modified to allow for culture in a mushroom-based serum-free media formulation.

[0095] In some cases, media formulations include natural media. Often, media formulations include synthetic media or modifications thereof. Examples of synthetic media include Minimum Essential Medium (MEM), Essential 8 Medium, Basal Eagle's Medium (BME), Ham's F12, Ham's F-10, Fisher's Medium, CMRL-1066 Medium, Crick's Medium, Medium 199, Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640, L-15 Medium, McCoy's Modified 5A Medium, William's Medium E, and Iscove's Modified Dulbecco's Medium (IMDM).

[0096] In some cases, the medium formulation is modified to culture embryonic stem cells, induced pluripotent stem cells, embryonic germ cells, differentiated cells (e.g., hepatocytes or myocytes), immortalized differentiated cells, or nascent liver stem cells. In one example, a medium formulation for culturing an isolated duck stem cell line, such as that described in WO2008129058A1, is used with one or more modifications. For example, in some cases, interleukin 6 and stem cell stimulatory factors are optionally removed from the medium formulation. Sometimes, the medium formulation is modified from WO2008129058A1. Medium formulations typically allow for the proliferation and / or maintenance of stem cell self-renewal capacity without the need for feeder cells. For example, Essential 8 Medium provides the most important components for maintaining pluripotent stem cells in a feeder-free environment. A feeder-free culture environment enhances the large-scale production of cultured foods because it eliminates the need to constantly re-seed a feeder cell layer to grow pluripotent stem cells. Often, multiple medium formulations are used during the culture of a population of cells. In some cases, an initial population of cells capable of self-renewal is cultured using a medium formulation that maintains self-renewal capacity, such as maintaining a population of embryonic stem cells in an undifferentiated state. Differentiation is then sometimes induced in the population of cells capable of self-renewal. For example, embryonic stem cells are induced to differentiate into hepatocytes. This differentiation process sometimes requires a differentiation medium formulation. For example, in some cases, specific differentiation factors are added and / or factors necessary for maintaining self-renewal capacity are removed from the differentiation medium. Furthermore, when differentiation in a population of cells leads to the generation of hepatocytes, there is often an additional step of inducing steatosis or lipid accumulation in the hepatocytes. In some cases, steatosis is induced at least in part by the use of an adipose medium formulation. For example, the adipose medium formulation sometimes includes at least a specific lipid concentration, as described elsewhere herein.

[0097] In some cases, the media formulation includes at least one nutrient or nutritional agent to enhance the nutrient content of the finished food product. The nutrients may be macronutrients or micronutrients. Macronutrients are nutrients required in large amounts and include proteins, fats, and carbohydrates. Micronutrients are required in small amounts and include vitamins, minerals, some amino acids, and specific compounds such as flavonoids. In some instances, at least one nutrient is added to the media formulation for uptake by a population of cultured cells. For example, fatty liver cells used to produce foie gras typically have high lipid accumulation in the cytoplasm. Cultivating hepatocytes in a media formulation with a specific lipid composition (e.g., omega-3 fatty acids) induces the resulting fatty liver cells to have an altered lipid profile that partially reflects the lipid composition of the media. Some methods provide for the production of cultured tissues with increased nutrient content for human consumption. For example, some such methods include: a) culturing a population of cells in a culture medium having at least one nutrient; b) manipulating lipid metabolic pathways to induce steatosis in the population of differentiated cells such that the cells accumulate a high lipid content; and c) processing the population of differentiated cells into a homogenously textured tissue for human consumption. Another method includes: a) culturing a population of cells in a culture medium having at least one nutrient; b) manipulating lipid metabolic pathways to induce steatosis in the population of differentiated cells such that the cells accumulate a high lipid content; and c) processing the population of differentiated cells into a homogenously textured tissue for human consumption.

[0098] In some cases, media formulations are generated that contain specific growth factors, proteins, lipids, hormones, or any combination thereof required for culturing cells. Often, mammalian cell overexpression systems are used to generate any of the aforementioned media components. In some instances, transgene expression in yeast, specific fungal, bacterial, algal, or insect cell (baculovirus) systems is utilized. In some instances, the expressed media components are then isolated and / or purified. Sometimes, media formulations are generated using media conditioning techniques. Alternatively, cells may be cultured using a coculture model. However, in various cases, cells are cultured without coculture or without coculture with xenobiotic cells, such as yeast (e.g., organisms or cells that do not belong to the same kingdom, phylum, and / or species as the cells being cultured to produce food). For example, some avian cells are cocultured with non-yeast organisms, such as mouse feeder cells.

[0099] Successful reduction or elimination of fetal bovine serum from cell culture media has been demonstrated. Figure 17 shows a graph plotting cell number from an immortalized cell line derived from adult duck hepatocytes. These immortalized cells were cultured in gradually decreasing concentrations of fetal bovine serum (FBS) in the presence of soy hydrolysate (10 g / L). Supplementing the medium with soy hydrolysate allowed for a 92% reduction in the serum requirements of cultured hepatocytes.

[0100] Figure 18 shows duck fibroblasts successfully grown in 10% shiitake mushroom extract after serial reduction of fetal bovine serum from the cell culture medium. In some cases, the medium is supplemented with at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% mushroom extract. In some examples, the medium is supplemented with no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% mushroom extract. Medium supplemented with mushroom extract may utilize reduced serum concentrations or no serum. In some cases, the supplemented medium has 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or less serum (e.g., animal serum such as FBS). Such serum-reduced or serum-free medium formulations can be utilized to grow or culture any of the cells described herein, including cells derived from various species, such as avian cells, fish cells, porcine cells, bovine cells, and cells of other edible species. In some cases, the cells are derived from domesticated species (e.g., cows, pigs, chickens, ducks, etc.). In other cases, the cells are derived from non-domesticated species (e.g., trout, salmon, lobster, crab, etc.). Various cell types, including pluripotent cells, multipotent cells, embryonic stem cells, induced pluripotent stem cells, myocytes, adipocytes, satellite cells, preadipocytes, mesenchymal stem cells, fibroblasts, hepatocytes, and other cell types, can be cultured in the serum-reduced or serum-free medium formulations described herein.

[0101] In some cases, cell populations or cell lines are suitable for growth in serum-reduced or serum-free media formulations without the need for supplementation. Figure 19A shows duck fibroblasts grown in serum-free media with no additional supplements; Figure 19B shows a control culture grown in DMEM supplemented with 10% fetal bovine serum.

[0102] Scalable generation of cultured cells Various methods are optionally used to scale up the production of cultured cells for human consumption. The systems and methods disclosed herein enable large-scale production of cultured foods (Figures 4A-4B). One method is to use two-dimensional surfaces such as tissue culture dishes or their functional equivalents (e.g., cell culture chambers). A typical example is a cell culture chamber with a polystyrene surface treated to increase hydrophilicity to enhance the attachment of adherent cells. Sometimes, cell culture chambers are coated with a protein composition that serves as a substrate for the cultured cells. Cell culture chambers often use media formulations such as those described herein. In many cases, the 2D surface approach is scaled up by combining multiple cell culture chambers. Sometimes, multiple cell culture chambers are stacked and arranged side by side. In some embodiments, cell culture chambers are stacked to a height of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 chambers. Stacks of cell culture chambers are typically positioned side-by-side. For example, in some instances, stacks of cell culture chambers are positioned side-by-side and / or behind-and-behind to maximize space utilization. In many cases, the chambers are physically coupled (e.g., fabricated as a single unit) and have common inlet and vent ports connected by channels that allow for the flow of liquids and gases.

[0103] In certain embodiments, bioreactor systems for culturing cells are provided herein. Certain bioreactor systems facilitate the production of cultured tissue suitable for human consumption. For example, some bioreactor systems include: a) a reactor chamber including a plurality of microscaffolds that provide an adhesive surface for cell attachment; b) a population of self-renewing cells cultured in the bioreactor; c) a first source providing at least one maintenance medium containing components for maintaining the population of self-renewing cells without spontaneous differentiation; and d) a second source providing at least one differentiation medium containing components for differentiating the population of self-renewing cells into a specific lineage; wherein the reactor chamber receives the maintenance medium from the first source for culturing the population of cells and the differentiation medium from the second source for differentiating the population of cells, and the population of cells produced in a single batch comprises a cultured tissue suitable for human consumption and having a dry weight of at least 1 kg.

[0104] Bioreactor systems are typically scalable for large-scale cell culture. Figure 20 illustrates a schematic diagram of one embodiment of a bioreactor system, including a reactor chamber (2001) for culturing cells. Often, bioreactor systems include a stirring element (2003) for agitating the contents of the reactor chamber (2001). Continuous or periodic agitation helps maintain cells, cell clumps, and / or microscaffolds in suspension. Fresh medium is added to the reactor chamber through at least one input port (2002). The fresh medium is sometimes a maintenance medium, differentiation medium, adipose medium, proliferation medium, or other medium formulation disclosed herein. Depleted medium or effluent is removed from the reactor chamber through at least one output port (2007). Optionally, oxygen, carbon dioxide, and / or other gases are introduced through at least one input gas port (2006). The input gas port (2006) is optionally connected to an aerator located within the reactor chamber. Often, bioreactor systems include at least one sensor (2004) for monitoring the reactor chamber. The at least one sensor (2004) is typically in communication with a controller (2008) (e.g., a computer). In many cases, the reactor chamber is seeded with a plurality of microscaffolds (2005). The microscaffolds (2005) allow the attachment of certain adherent cells, such as hepatocytes. For example, some methods for producing cultured fish meat for human consumption include: a) obtaining a population of self-renewing cells from fish; b) culturing the population of self-renewing cells in a medium containing the microscaffolds; c) inducing differentiation in the population of cells to form at least one of myocytes and adipocytes; and d) processing the population of cells into fish meat for human consumption.

[0105] Figure 21 illustrates an exemplary process in which a bioreactor system is used for meat production. In this example, specialized cells, such as embryonic, pluripotent, or multipotent cells, are isolated from eggs and adapted for growth in a bioreactor (e.g., using the hanging drop method to form spheroid bodies, as shown in Figure 22). The cells are grown using a medium containing water and plant-derived nutrients (e.g., using a plant-based alternative to animal-derived serum, such as soy hydrolysate or mushroom extract). The cells are grown in the sterile environment of the bioreactor for 4-6 weeks. Optionally, the cells differentiate and are then harvested and / or processed into meat products.

[0106] The transfer of cells from 2D cell culture dishes to 3D bioreactors can be carried out using various methods, such as the hanging drop method shown in FIG. 22A. The hanging drop method involves placing cells in a hanging drop culture and incubating them under physiological conditions until they form 3D spheroids in direct cell-to-cell contact and in contact with extracellular matrix components. The illustrative example shown in FIG. 22A involves suspending duck liver cells in a hanging drop to initiate the formation of spheroids (left panel). The spheroids are then transferred into 3D culture in a bioreactor (FIG. 22A right panel). 3D culture allows for more rapid proliferation and / or growth of cultured cells. Another illustrative bioreactor is shown in FIG. 22B (left panel). Cells from the spheroids can be expanded in 3D culture (FIG. 22B right panel). In an illustrative embodiment of the hanging drop method, adherent cell cultures are washed with PBS and incubated with a 0.05% trypsin / 1 mM EDTA solution to detach the cells. The trypsin is then neutralized by the addition of medium, and the cells are digested with DNAse at room temperature for 5 minutes. The cells are centrifuged at 250 Gs for 5 minutes. The supernatant is then discarded, and the cells are resuspended in medium. Hanging drops are formed by pipetting a large volume of medium (e.g., 10 μl) containing the cells onto the bottom of the tissue culture dish lid. Multiple hanging drops can be formed on a single lid. A few milliliters of PBS can be added to the bottom of the tissue culture dish to prevent dehydration of the hanging drops. The lid is then placed on the tissue culture dish, followed by incubation under standard cell culture conditions (e.g., 5% CO2, 37°C, 95% humidity) until spheroids are observed.

[0107] Sometimes, a bioreactor system includes at least one bioreactor, bioreactor tank, or reactor chamber (2001). For example, in certain instances, a bioreactor system includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, or 100 reactor chambers. In some cases, a bioreactor system includes from about 1 reactor chamber to about 1,000 reactor chambers. Sometimes, a bioreactor system includes about 1 reactor chamber. A bioreactor system typically includes up to about 1,000 reactor chambers.

[0108] In some embodiments, the bioreactor system has from about 1 reactor chamber to about 5 reactor chambers, from about 1 reactor chamber to about 10 reactor chambers, from about 1 reactor chamber to about 20 reactor chambers, from about 1 reactor chamber to about 50 reactor chambers, from about 1 reactor chamber to about 100 reactor chambers, from about 1 reactor chamber to about 200 reactor chambers, from about 1 reactor chamber to about 300 reactor chambers, from about 1 reactor chamber to about 400 reactor chambers, reactor chamber, about 1 reactor chamber to about 500 reactor chambers, about 1 reactor chamber to about 1,000 reactor chambers, about 5 reactor chambers to about 10 reactor chambers, about 5 reactor chambers to about 20 reactor chambers, about 5 reactor chambers to about 50 reactor chambers, about 5 reactor chambers to about 100 reactor chambers, about 5 reactor chambers to about 200 reactor chambers, about 5 reactor chambers to about 300 reactor chambers, about 5 reactor chambers from about 5 reactor chambers to about 400 reactor chambers, from about 5 reactor chambers to about 500 reactor chambers, from about 5 reactor chambers to about 1,000 reactor chambers, from about 10 reactor chambers to about 20 reactor chambers, from about 10 reactor chambers to about 50 reactor chambers, from about 10 reactor chambers to about 100 reactor chambers, from about 10 reactor chambers to about 200 reactor chambers, from about 10 reactor chambers to about 300 reactor chambers, from about 10 reactor chambers to about 400 reactor chambers , about 10 reactor chambers to about 500 reactor chambers, about 10 reactor chambers to about 1,000 reactor chambers, about 20 reactor chambers to about 50 reactor chambers, about 20 reactor chambers to about 100 reactor chambers, about 20 reactor chambers to about 200 reactor chambers, about 20 reactor chambers to about 300 reactor chambers, about 20 reactor chambers to about 400 reactor chambers, about 20 reactor chambers to about 500 reactor chambers, about 20 reactor chambers to about 1,000 reactor chambers, about 50 reactor chambers to about 100 reactor chambers, about 50 reactor chambers to about 200 reactor chambers, about 50 reactor chambers to about 300 reactor chambers, about 50 reactor chambers to about 400 reactor chambers, about 50 reactor chambers to about 500 reactor chambers, about 50 reactor chambers to about 1,000 reactor chambers, about 100 reactor chambers to about 200 reactor chambers, about 100 reactor chambers to about 300 reactor chambers, about 100 reactor chambers to about 400 reactor chambers, about 100 reactor chambers to about 500 reactor chambers, about 100 reactor chambers to about 1,0 00 reactor chambers, about 200 reactor chambers to about 300 reactor chambers, about 200 reactor chambers to about 400 reactor chambers, about 200 reactor chambers to about 500 reactor chambers, about 200 reactor chambers to about 1,000 reactor chambers, about 300 reactor chambers to about 400 reactor chambers, about 300 reactor chambers to about 500 reactor chambers, about 300 reactor chambers to about 1,000 reactor chambers, about 400 reactor chambers to about 500 reactor chambers, about 400 reactor chambers to about 1,000 reactor chambers, or about 500 reactor chambers to about 1,000 reactor chambers.

[0109] In some cases, at least one reactor chamber has an internal volume suitable for large-scale cell culture. In some cases, the reactor chamber has an internal volume of about 1 L to about 100,000 L. In most instances, the reactor chamber has an internal volume of at least about 1 L. Sometimes, the reactor chamber has an internal volume of up to about 100,000 L.

[0110] Often, the reactor chamber is between about 1 L and about 10 L, between about 1 L and about 50 L, between about 1 L and about 100 L, between about 1 L and about 500 L, between about 1 L and about 1,000 L, between about 1 L and about 5,000 L, between about 1 L and about 10,000 L, between about 1 L and about 50,000 L, between about 1 L and about 100,000 L, between about 10 L and about 50 L, between about 10 L and about 100 L, between about 10 L and about 500 L, between about 10 L and about 1,000 L, 10L~Approx. 5,000L, Approx. 10L~Approx. 10,000L, Approx. 10L~Approx. 50,000L, Approx. 10L~Approx. 100,000L, Approx. 50L~Approx. 100L, Approx. 50L~Approx. 500L, Approx. Approx. 1,000L, Approx. 50L~Approx. 5,000L, Approx. 50L~Approx. 10,000L, Approx. 50L~Approx. 50,000L, Approx. 50L~Approx. 100,000L, Approx. 1,000L, approximately 100L to approximately 5,000L, approximately 100L to approximately 10,000L, approximately 100L to approximately 50,000L, approximately 100L to approximately 100,000L, approximately 500L to approximately 1,000L, approximately 500L to approximately 5,000L, approximately 500L to approximately 10,000L, approximately 500L to approximately 50,000L, approximately 500L to approximately 100,000L, approximately 1,000L to approximately 5,000L, approximately 1,000L It has an internal volume of about 10,000 L, about 1,000 L to about 50,000 L, about 1,000 L to about 100,000 L, about 5,000 L to about 10,000 L, about 5,000 L to about 50,000 L, about 5,000 L to about 100,000 L, about 10,000 L to about 50,000 L, about 10,000 L to about 100,000 L, or about 50,000 L to about 100,000 L.

[0111] In some cases, bioreactor systems suitable for large-scale production of cultured cells for food production are disclosed herein. Often, cells are cultured in a batch mode. Alternatively, or in combination, cells are cultured continuously. In both batch and continuous culture methods, fresh nutrients are typically supplied to ensure appropriate nutrient concentrations for producing the desired food product. As an example, in a fed-batch culture, nutrients (e.g., fresh medium) are supplied to the bioreactor, and the cultured cells remain in the bioreactor until ready to be processed into a finished food product. In a fed-batch culture, a basal medium is supplied to the bioreactor to support the primary cell culture, after which additional feed medium is supplied to replenish depleted nutrients. Sometimes, bioreactor systems produce at least a certain amount of cells per batch. In some cases, bioreactor systems produce batches of about 1 billion cells to about 10 quadrillion cells. Often, bioreactor systems produce batches of at least about 1 billion cells. Bioreactor systems typically produce up to about 10 quadrillion cells.

[0112] Sometimes bioreactor systems are used to culture cells at a density of approximately 1 billion cells to approximately 10 billion cells, approximately 1 billion cells to approximately 50 billion cells, approximately 1 billion cells to approximately 100 billion cells, approximately 1 billion cells to approximately 500 billion cells, approximately 1 billion cells to approximately 1 trillion cells, approximately 1 billion cells to approximately 5 trillion cells, approximately 1 billion cells to approximately 10 trillion cells, approximately 1 billion cells to approximately 100 trillion cells, approximately 1 billion cells to approximately 1 quadrillion cells, approximately 1 billion cells to approximately 10 quadrillion cells, approximately 10 billion cells to approximately 50 billion cells, approximately 10 billion cells to approximately 100 billion cells, approximately 10 0 billion cells to approximately 500 billion cells, approximately 10 billion cells to approximately 1 trillion cells, approximately 10 billion cells to approximately 5 trillion cells, approximately 10 billion cells to approximately 10 trillion cells, approximately 10 billion cells to approximately 100 trillion cells, approximately 10 billion cells to approximately 1 quadrillion cells, approximately 10 billion cells to approximately 1 quadrillion cells, approximately 50 billion cells to approximately 100 billion cells, approximately 50 billion cells to approximately 500 billion cells, approximately 50 billion cells to approximately 1 trillion cells, approximately 50 billion cells to approximately 5 trillion cells, approximately 50 billion cells to approximately 10 trillion cells, approximately 50 billion cells to approximately 100 trillion cells, approximately 50 billion cells to approximately 1,000 trillion cells, approximately 50 billion cells to approximately 1 quadrillion cells, approximately 50 billion cells to approximately 10 quadrillion cells, approximately 100 billion cells to approximately 500 billion cells, approximately 100 billion cells to approximately 1 trillion cells, approximately 100 billion cells to approximately 5 trillion cells, approximately 100 billion cells to approximately 10 trillion cells, approximately 100 billion cells to approximately 100 trillion cells, approximately 100 billion cells to approximately 1,000 trillion cells, approximately 100 billion cells to approximately 1 quadrillion cells, approximately 100 billion cells to approximately 10 quadrillion cells, approximately 500 billion cells ~approximately 1 trillion cells, approximately 500 billion cells ~ approximately 5 trillion cells, approximately 500 billion cells ~ approximately 10 trillion cells, approximately 500 billion cells ~ approximately 100 trillion cells, approximately 500 billion cells ~ approximately 1,000 trillion cells, approximately 500 billion cells ~ approximately 1 quadrillion cells, approximately 500 billion cells ~ approximately 10 quadrillion cells, approximately 1 trillion cells ~ approximately 5 trillion cells, approximately 1 trillion cells ~ approximately 10 trillion cells, approximately 1 trillion cells ~ approximately 100 trillion cells, approximately 1 trillion cells ~ approximately 1,000 trillion cells, approximately 1 trillion cells ~ approximately 1 quadrillion cells, approximately 1 trillion cells ~ approximately 1 quadrillion cells, approximately 5 trillion cells ~ approximately 10 trillion cells,Generate a batch of about 5 trillion cells to about 100 trillion cells, about 5 trillion cells to about 1 quadrillion cells, about 5 trillion cells to about 1 quadrillion cells, about 5 trillion cells to about 1 quadrillion cells, about 10 trillion cells to about 100 trillion cells, about 10 trillion cells to about 1 quadrillion cells, about 10 trillion cells to about 1 quadrillion cells, about 10 trillion cells to about 1 quadrillion cells, about 100 trillion cells to about 1 quadrillion cells, about 100 trillion cells to about 1 quadrillion cells, about 100 trillion cells to about 1 quadrillion cells, about 1 quadrillion cells to about 1 quadrillion cells, about 1 quadrillion cells to about 1 quadrillion cells, about 1 quadrillion cells to about 1 quadrillion cells, 1 quadrillion cells to about 1 quadrillion cells, or about 1 quadrillion cells to about 1 quadrillion cells.

[0113] In some cases, the bioreactor system generates batches of cultured cells over a period of time. For example, in some cases, the bioreactor system generates at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 batches of cultured cells.

[0114] In some cases, the bioreactor system produces batches of cultured cells having at least a certain mass. Sometimes, the mass is measured as a dry weight after excess medium or supernatant has been removed. Typically, the bioreactor system produces batches of cultured cells ranging from about 1 kg to about 10,000 kg. In certain instances, the bioreactor system produces batches of at least about 1 kg. Often, the bioreactor system produces batches of up to about 10,000 kg.

[0115] In particular examples, the bioreactor system may be configured to produce a bioreactor having a capacity of about 1 kg to about 5 kg, about 1 kg to about 10 kg, about 1 kg to about 20 kg, about 1 kg to about 30 kg, about 1 kg to about 40 kg, about 1 kg to about 50 kg, about 1 kg to about 100 kg, about 1 kg to about 500 kg, about 1 kg to about 1,000 kg, about 1 kg to about 5,000 kg, about 1 kg to about 10,000 kg, about 5 kg to about 10 kg, about 5 kg to about 20 kg, about 5 kg to about 30 kg, about 5 kg to about 40 kg, about 5 kg to about 50 kg, about 5 kg to about 100 kg, about 5 kg to about 500 kg, about 5 kg to about 5 kg ~ approx. 1,000 kg, approx. 5 kg ~ approx. 5,000 kg, approx. 5 kg ~ approx. 10,000 kg, approx. 10 kg ~ approx. 20 kg, approx. 10 kg ~ approx. 30 kg, approx. 0kg to 1,000kg, 10kg to 5,000kg, 10kg to 10,000kg, 20kg to 30kg, 20kg to 40kg, 20kg to 50kg, 20kg to 100kg, 20kg to 500kg, 20kg to 1,000kg 0kg, about 20kg to about 5,000kg, about 20kg to about 10,000kg, about 30kg to about 40kg, about 30kg to about 50kg, about 30kg to about 100kg, about 30kg to about 500kg, about 30kg to about 1,000kg, about 30kg to about 5,000kg, about 30kg to about 10,000kg, about 40kg to about 50kg, about 40kg to about 100kg, about 40kg to about 500kg, about 40kg to about 1,000kg, about 40kg to about 5,000kg, about 40kg to about 10,000kg, about 50kg to about 100kg, about 50kg to Produce batches of about 500 kg, about 50 kg to about 1,000 kg, about 50 kg to about 5,000 kg, about 50 kg to about 10,000 kg, about 100 kg to about 500 kg, about 100 kg to about 1,000 kg, about 100 kg to about 5,000 kg, about 100 kg to about 10,000 kg, about 500 kg to about 1,000 kg, about 500 kg to about 5,000 kg, about 500 kg to about 10,000 kg, about 1,000 kg to about 5,000 kg, about 1,000 kg to about 10,000 kg, or about 5,000 kg to about 10,000 kg.

[0116] Cells grown in bioreactor systems are typically grown in suspension. Bioreactor systems often have components that allow for automated cell culture expansion and maintenance. A bioreactor includes at least one reactor chamber or tank in which the cultured cells grow. In some cases, a bioreactor system has at least one pump for circulating the culture medium, introducing new medium, and / or removing unwanted medium. Often, a bioreactor system includes multiple medium tanks for introducing various types of medium, such as growth medium, maintenance medium (e.g., for maintaining self-renewal capacity), differentiation medium, and adipose medium. Occasionally, a bioreactor system includes at least one of an oxygenator, a carbon dioxide regulator, and a central controller that regulates the components of the bioreactor system. In many instances, a bioreactor has stirring elements to maintain the cultured cells in suspension and / or to keep the medium mixed. Bioreactors typically include at least one sensor for monitoring the environment inside the reactor chamber. The sensors are typically biosensors, chemical sensors, or optical sensors for monitoring parameters important to cell culture. In some cases, the sensors are configured to monitor at least one of pH, temperature, oxygen, carbon dioxide, glucose, lactate, ammonia, hypoxanthine, amino acids, dopamine, and lipids. Often, the at least one sensor is in communication with a control unit (e.g., a computer system) that monitors the sensor parameters. In certain examples, the control unit provides the sensor parameters to a user, such as on a display screen. Often, the control unit includes at least one input source for receiving commands from a user.

[0117] In some cases, cells are cultured in suspension in cell culture flasks. The cell culture flasks are optionally stacked and / or arranged side-by-side, as described for 2D surface cell culture. Cells cultured in suspension are typically non-adherent. However, in some cases, adherent cells are cultured on scaffolds in suspension. The scaffold provides structural support and a physical environment for cells to attach, grow, and migrate. In addition, the scaffold typically imparts mechanical properties such as elasticity and tensile strength. 3D scaffolds are often used to culture adherent cells to allow for 3D cell growth. Scaffolds sometimes have specific shapes or sizes to guide the growth of cultured cells. In some cases, scaffolds are composed of one or more different materials. Some scaffolds are solid, while others are porous. Porous scaffolds allow cell migration or infiltration into the pores. The scaffold is composed of a biocompatible material to guide appropriate cell recognition. Additionally, the scaffold is made of a material with appropriate mechanical properties and degradation kinetics for the desired tissue type to be generated from the cells. In certain instances, the scaffold includes a degradable material to allow for remodeling and / or elimination of the scaffold in the cultured food. For example, in some cases, a 3D scaffold that molds cultured hepatocytes into the shape of a liver biodegrades after the stem cells expand to fill the interior space of the scaffold. In other instances, the scaffold includes a material that remains in the cultured food. For example, sometimes at least a portion of the collagen scaffold that provides support to cultured muscle cells continues to provide texture and sustained structural support in the cultured food. In some cases, the scaffold includes a biomaterial such as a hydrogel, an extracellular matrix molecule (ECM) or chitosan, or a biocompatible synthetic material (e.g., polyethylene terephthalate). ECM molecules are typically proteoglycans, non-proteoglycan polysaccharides, or proteins.Possible ECM molecules for use in scaffolding include collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin, and fibronectin. Plant-based scaffolds are sometimes used for 3D culture. Non-limiting examples of plant-based scaffolds include scaffolds derived from plants such as apple, seaweed, or jackfruit. Plant-based scaffolds often contain at least one plant-based material, such as cellulose, hemicellulose, pectin, lignin, alginate, or any combination thereof. Sometimes, plant-based scaffolds are decellularized. In some cases, scaffolds are not required for 3D culture. In various examples, the scaffold used in the methods and compositions described herein comprises at least one of hydrogel, chitosan, polyethylene terephthalate, collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin, fibronectin, cellulose, hemicellulose, pectin, lignin, alginate, glucomannan, polycaprolactone (PCL), textured vegetable protein (TVP), and acrylate. One example of a textured vegetable protein is textured soy protein (TSP), which typically contains a high percentage of soy protein, soy flour, or soy concentrate. TVP and TSP can be used to impart a meat-like texture and firmness to the meat products described herein. In some cases, meat products containing TVP or TSP are seasoned (e.g., using various salts, herbs, and / or spices) to create a meat-like flavor.

[0118] In some cases, cells are cultured on microscaffolds that allow cell adhesion. Microscaffolds are typically microscaffolds that allow adherent cells to grow in suspension bioreactor systems. For example, microscaffolds provide a surface for adherent cells, such as hepatocytes, to adhere to while the microscaffold itself is in suspension. In certain instances, scaffolds and / or microscaffolds are produced by 3D printing of a suitable material (e.g., collagen). Microscaffolds often have a porous structure. Occasionally, microscaffolds have a solid, non-porous structure. Microscaffolds are smaller than traditional scaffolds. Scaffolds are typically used to provide a macroscopic structure and / or shape for cell populations, while microscaffolds provide a seed or core structure for adherent cells to attach to while remaining small enough to remain in suspension with agitation. The use of microscaffolds allows for the culture of adherent cells in suspension culture. Cultivation of cells using microscaffolds in suspension culture in bioreactor systems allows for the large-scale production of adherent cells. For example, adherent cells such as hepatocytes, myocytes, and adipocytes can be grown on a large scale in bioreactor suspension culture using microscaffolds. This allows for the production of premium food items such as foie gras or sushi-grade salmon meat. Alternatively, cultured fish cells are sometimes processed into surimi, such as salmon, tuna, or trout surimi.

[0119] Figure 23A shows trout satellite cells grown on glucomannan microscaffolds that successfully differentiated into myotubes in three-dimensional culture. Figure 23B shows the negative regulation of undifferentiated satellite cells. Figure 24A shows duck fibroblasts (arrowheads) successfully grown on glucomannan microscaffolds (arrows). Figure 24B shows a representative glucomannan microscaffold. Alternative materials can be used to generate microscaffolds. The microscaffolds can include at least one of glucomannan, alginate, chitosan, polycaprolactone (PCL), matrix proteins (e.g., collagen, fibronectin, laminin), textured vegetable protein (TVP), textured soy protein (TSP), and acrylates. In some cases, polycaprolactone (PCL) is used to generate PCL-woven scaffolds or PCL-fibrin composites. In some cases, the microscaffold is modified. In certain instances, the microscaffold is conjugated to one or more factors related to cell adhesion, proliferation, differentiation, or a combination thereof. For example, a glucomannan microscaffold is conjugated to FGF2 to promote the proliferation and differentiation of muscle satellite cells into myotubes. In some cases, the microscaffold is conjugated to one or more growth or differentiation factors via covalent bonding, such as chemical crosslinking or other reactions, to immobilize the factors in the microscaffold structure.

[0120] Alternatively, in some cases, a scaffold or microscaffold is not required for culturing cells in suspension. Sometimes, the cells are non-adherent cells and do not require a substrate or surface for adhesion. In certain instances, the cells are modified or engineered so that they no longer require an adhesive substrate. For example, hepatocytes are typically adherent cells, but in some instances, hepatocytes are modified so that they no longer require an extracellular matrix for adhesion for survival and proliferation. Other adherent cells include muscle cells and adipocytes, which are sometimes cultured to produce cultivated meat products. For example, some methods for producing cultivated meat for human consumption include: a) obtaining a population of self-renewing cells capable of growth in suspension culture; b) culturing the population of self-renewing cells in suspension; c) inducing differentiation of the population of cells to form at least one of muscle cells and adipocytes; and d) processing the population of cells into meat for human consumption.

[0121] The cell culture systems described herein enable the cultivation of cells for food production in a pathogen-free environment. Generally, cells are grown in a culture environment free of dangerous impurities that affect human health. Cell culture plates, flasks, and bioreactors typically provide cell culture conditions free of dangerous pathogens (e.g., H1N1), parasites, heavy metals, and toxins (e.g., endotoxins, pesticides, etc.). In some cases, the methods described herein do not utilize antibiotics. Occasionally, the methods use an inducer such as tetracycline for a one-time induction of cell differentiation and / or cell phenotype, after which the inducer is removed before the cells are processed into food.

[0122] Tissue processing Provided herein are systems and methods for processing cultured cells to produce the appropriate taste, texture, consistency, or other desired qualities in a food product. The cells are typically differentiated cell populations, such as, for example, muscle cells, adipocytes, or hepatocytes.

[0123] In various cases, the cells are animal cells. Sometimes, the cells are fish, animal, or avian cells. Examples of avian cells include cells derived from geese, ducks, chickens, Cornish pheasants, turkeys, guinea fowl, quails, pigeons, nuphar, emus, ostriches, hen, ptarmigans, swans, doves, woodcocks, partridges, and snipes. As an example, Figure 25 shows an image of duck muscle tissue produced according to the methods disclosed herein. Successful production of the duck muscle tissue in Figure 25 was confirmed by the ability of the muscle tissue to contract spontaneously.

[0124] Some methods disclosed herein enable the production of detextured cultured muscle tissue for human consumption. Detextured muscle tissue includes certain fish muscle tissues. Certain methods for producing detextured muscle tissue include: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation of the population of cells to form detextured muscle tissue; and d) processing the detextured cultured muscle tissue for human consumption. Additionally, some methods produce cultured fish tissue with enhanced nutrient content for human consumption. For example, certain methods include: a) culturing a population of fish muscle cells in a culture medium with at least one nutrient; b) expanding the population of muscle cells; and c) processing the population of muscle cells into fish tissue for human consumption. The systems and methods described herein often enable the production of edible compositions containing fish tissue generated from cultured muscle cells and adipocytes.

[0125] In some cases, the fish fat cells and / or muscle cells are processed into fish meat, such as salmon meat. In various examples, the fish fat cells and / or muscle cells are processed into finished fish meat products. Other examples of processed fish meat products include minced fish meat, fish fillets, fish cutlets, and fish steaks. These various shapes and sizes of fish meat products are obtained by processing the muscle cells and / or fat cells with various additional ingredients, such as binders, fillers, or extenders, to provide structural adhesion and / or texture. In some examples, the meat product is cooked or cured. Processing the cultured cells into meat products may include at least one of smoking, fermenting, salting, marinating, poaching, baking, barbecuing, casseroling, pan-frying, oven-frying, grilling, and microwaving. In some cases, the cells are processed into sushi-grade fish meat suitable for raw consumption without refrigeration. In certain cases, the fish meat is not cooked during processing. Examples of sushi-grade fish meat produced according to the systems and methods disclosed herein include salmon and tuna. As used herein, sushi-grade meat is produced free of parasites and bacteria. The cells typically do not contain pathogens, parasites, toxins, heavy metals (e.g., mercury), antibiotics, or any combination thereof. Certain systems and methods described herein provide for the production of cultured foods without exposure to impurities. Some methods enable the production of cultured cells for human consumption without the use of antibiotics. For example, certain methods include: a) culturing a population of cells without the use of antibiotics; b) inducing differentiation within the population of cells; c) inducing high lipid accumulation within the population of cells; and d) processing the population of cells for human consumption. Also disclosed herein are methods for producing cultured cells for human consumption without exposure to pathogens. Some such methods include: a) culturing a population of cells in a pathogen-free culture environment; b) inducing differentiation within the population of cells; c) inducing high lipid accumulation within the population of cells; and d) processing the population of cells for human consumption.Certain methods allow for the production of cultured foods without exposure to toxins. For example, some such methods include: a) culturing a population of cells in a toxin-free culture environment; b) inducing differentiation within the population of cells; c) inducing high lipid accumulation within the population of cells; and d) processing the population of cells for human consumption.

[0126] In certain cases, cultured meat contains a mixed population of muscle cells and adipocytes. Often, preadipocytes and satellite cells are isolated from sources such as small fish. Preadipocytes and satellite cells are useful because they have a degree of self-renewal capability. Preadipocytes and satellite cells are typically cultured and expanded, and then differentiated. In some cases, preadipocytes and satellite cells are cultured together. Typically, preadipocytes and satellite cells are cultured separately until, after differentiation, they are co-cultured together at a specific ratio to achieve the desired ratio in the final fish meat product. Alternatively, populations of cells are sometimes induced to differentiate into different cell types in the same culture. For example, in this scenario, some cells form into adipocytes and some cells form into muscle cells. Typically, muscle cells and adipocytes are cultured separately and then mixed. Sometimes, muscle cells and adipocytes are homogeneously mixed in equal proportions. In other cases, muscle cells and adipocytes are heterogeneously mixed in unequal proportions. For co-culture or treatment, muscle cells and adipocytes are typically combined at a specific ratio or proportion. For example, in some cases, muscle cells and adipocytes are combined at a ratio of at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, or at least 100:1, respectively. In many cases, the muscle cells and / or fat cells are fish cells. In some instances, the muscle cells and / or fat cells are derived from salmon. Sometimes, the muscle cells and / or fat cells are derived from sea bass, tuna, mackerel, Atlantic blue marlin, swordfish, yellowtail, salmon, trout, eel, abalone, squid, clam, blood clam, sweetfish, scallop, sea bream, halfbeak, shrimp, flounder, cockle, octopus, or crab. Examples of tuna include yellowfin tuna, southern bluefin tuna, longfin tuna, albacore tuna, Atlantic bluefin tuna, and bigeye tuna.In certain cases, instead of combining muscle cells and adipocytes, muscle cells are induced to undergo steatosis to provide the desired lipid or fat content found in conventional salmon meat without the need for adipocytes.

[0127] Systems and methods are provided herein for producing meat with a specific ratio of fast-twitch muscle cells and / or fibers to slow-twitch muscle cells and / or fibers. Meat produced according to the systems and methods disclosed herein typically contains muscle cells or skeletal muscle cells with a specific ratio of fast (type II) and slow (type I) muscle fibers. Slow-twitch muscle fibers exhibit low-intensity contractions activated by oxidative pathways and demonstrate relatively high endurance, while fast-twitch muscle fibers are associated with high-intensity contractions activated by glycolysis. Fast-twitch muscle fibers are characterized by highly glycolytic and anaerobic muscle fibers. The ratio of fast-twitch muscle fibers to slow-twitch muscle fibers in muscle tissue plays a role in the taste, color, texture, and other cooking characteristics of meat. For example, fish meat is characterized by a higher proportion of fast-twitch muscle fibers compared to animal meat, which plays a role in the cooking differences between the two categories of meat. Occasionally, muscle cells, such as salmon muscle cells, are cultured to contain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more fast-twitch fibers (e.g., highly glycolytic and anaerobic muscle fibers). The percentage of fast-twitch fibers can be characterized by evaluating tissue samples using various examination techniques, such as microscopy-based imaging (e.g., contacting tissue sections with fast-twitch fiber markers). In some cases, muscle cells produced according to the methods described herein have a diameter of at least about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 3 The length may be 50 μm, about 400 μm, about 450 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, about 1000 μm, about 2000 μm, about 3000 μm, about 4000 μm, about 5000 μm, about 6000 μm, about 7000 μm, about 8000 μm, about 9000 μm, about 10000 μm, or longer.

[0128] Certain muscle tissues lack the texture of animal skeletal muscle, such as beef, pork, or chicken. For example, fish muscle tissue tends to have a textured or textureless consistency. Fish muscle tissue, such as salmon and tuna, is often described as having a delicate, soft-tasting texture and / or a uniform consistency. Other examples of textureless meats include squid and octopus muscle tissue, which have a consistency without a distinct texture compared to animal skeletal muscle. Liver foods, such as foie gras, are also characterized as lacking texture. Certain methods described herein enable the production of textureless cultured tissue. Some such methods include: a) culturing a population of cells; b) inducing differentiation in the population of cells; c) manipulating lipid metabolic pathways to induce steatosis in the population of cells so that the cells accumulate a high lipid content; and d) processing the population of cells into a textureless tissue.

[0129] Certain methods disclosed herein produce textureless cultured muscle tissue for human consumption. Some such methods include: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation of the population of cells to form textureless muscle tissue; and d) processing the cultured textureless muscle tissue for human consumption. In some cases, the cells are hepatocytes. The hepatocytes are harvested after cultivation for processing into food products. Occasionally, the hepatocytes are processed into foie gras.

[0130] The systems and methods disclosed herein enable the production of culinary foie gras compositions comprising tissue-cultured hepatocytes that have a high lipid content and are processed for human consumption. Some food compositions include cultured organ cells that are processed into textureless, non-muscle food products for human consumption. The food products sometimes include cultured avian fatty hepatocytes and seasonings. Foie gras compositions often include cultured hepatocytes with a high lipid content and hepatocytes with a low lipid content. Edible compositions are occasionally produced that include avian hepatocytes grown in cell culture and processed for human consumption. In some cases, the food products are packaged with an optional label. For example, some packaged foie gras compositions include cultured hepatocytes and a package with a label indicating that the foie gras composition was produced without force-feeding. Other packaged foie gras compositions include cultured hepatocytes and a package with a label indicating that the foie gras was produced in a pathogen-free environment. In certain embodiments, the packaged edible composition includes cultured cells that have been processed into a food product and a label indicating that the composition was produced without exposure to toxins. Figure 26 shows exemplary food products produced from duck hepatocytes. The left panel shows duck liver pâté produced using duck fatty hepatocytes. The right panel shows foie gras butter produced using duck fatty hepatocytes. Figure 27 shows exemplary food products for human consumption produced according to the methods disclosed herein. The left panel shows salmon pâté produced using salmon muscle cells. The right panel shows duck meat pâté produced using duck muscle cells. In addition, chicken meat pâté has also been developed using chicken muscle cells.

[0131] In some cases, the foie gras has a texture and / or consistency substantially identical to traditional foie gras. Often, the foie gras is ranked as Grade A, Grade B, or Grade C foie gras. In some cases, the foie gras is free of blemishes. Foie gras typically has only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 blemishes. Occasionally, liver cells are processed as a single foie gras composition. For example, a single foie gras composition is typically liver or liver-shaped. Processing methods for harvested cells for human consumption include centrifugation and compression. In some cases, the harvested cells receive some structural integrity from the scaffold and / or microscaffold to which the cells are attached during culture. Occasionally, the harvested cells are combined with at least one other ingredient. Harvested cells are often combined with at least one other ingredient to obtain a food product with a desired texture, moisture retention, product adhesion, or any combination thereof. The ingredients are typically binders, fillers, or bulking agents. Fillers or binders are often non-meat substances containing carbohydrates, such as starch. Examples of fillers and binders include potato starch, wheat flour, eggs, gelatin, carrageenan, and tapioca flour. Alternatively, bulking agents tend to have a high protein content. Examples of bulking agents include soy protein, dairy protein, and meat-derived protein. Specific ingredients that contribute flavor, texture, or other cooking properties are added in some instances. For example, extracellular matrix proteins are sometimes used to adjust structural firmness and texture. Certain proteins, such as heme and collagen, are sometimes incorporated into the extracellular matrix to contribute to the taste and texture of the final food product.

[0132] Cells are often grown in suspension culture on microscaffolds containing at least one natural protein with texture-modifying properties. Microscaffolds of various compositions can be used to impart a desired texture and / or firmness to the final food product. Occasionally, textured plant proteins, such as soy protein, are used. Microscaffolds optionally contain at least one filler or binder material to provide texture to the food product. Sometimes, microscaffolds are fabricated from biodegradable materials so that no further microscaffold structure remains in the finished food product. For example, a population of cells is seeded onto the microscaffold in a bioreactor. As the cells attach and grow on the microscaffold, the microscaffold gradually biodegrades until only a group of cells, now attached to each other, and the extracellular matrix material they secrete remain. Thus, microscaffolds (and also larger 3D scaffolds) can be used to guide the structure of the resulting cultured food product, but not the food product intended for human consumption. Alternatively, microscaffolds and 3D scaffolds include materials that do not biodegrade and / or remain in culture for consumption. For example, certain materials described herein can be used to generate scaffolds to impart particular structure, texture, taste, or other desired properties.

[0133] In certain instances, the foie gras composition weighs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 28, 32, 36, 42, 48, 52, 56, 60, or 64 ounces. Often, the foie gras composition weighs from about 1 ounce to about 64 ounces. Often, the foie gras composition weighs at least about 1 ounce. Typically, the foie gras composition weighs up to about 64 ounces.

[0134] In some embodiments, the foie gras composition is about 1 ounce to about 2 ounces, about 1 ounce to about 4 ounces, about 1 ounce to about 8 ounces, about 1 ounce to about 12 ounces, about 1 ounce to about 16 ounces, about 1 ounce to about 20 ounces, about 1 ounce to about 24 ounces, about 1 ounce to about 30 ounces, about 1 ounce to about 36 ounces, about 1 ounce to about 48 ounces, about 1 ounce to about 64 ounces, about 2 ounces to about 4 ounces, about 2 ounces to about 8 ounces, about 2 ounces to about 12 ounces, about 2 ounces to about 16 ounces, or about 2 ounces to about 20 ounces. , approximately 2 ounces to approximately 24 ounces, approximately 2 ounces to approximately 30 ounces, approximately 2 ounces to approximately 36 ounces, approximately 2 ounces to approximately 48 ounces, approximately 2 ounces to approximately 64 ounces, approximately 4 ounces to approximately 8 ounces, approximately 4 ounces to approximately 12 ounces, approximately 4 ounces to approximately 16 ounces, approximately 4 ounces to approximately 20 ounces, approximately 4 ounces to approximately 24 ounces, approximately 4 ounces to approximately 30 ounces, approximately 4 ounces to approximately 36 ounces, approximately 4 ounces to approximately 48 ounces, approximately 4 ounces to approximately 64 ounces, approximately 8 ounces to approximately 12 ounces, approximately 8 ounces to approximately 16 ounces, approximately 8 ounces to approximately 20 ounces, approximately 8 ounces to approximately 2 4 oz, approximately 8 oz to approximately 30 oz, approximately 8 oz to approximately 36 oz, approximately 8 oz to approximately 48 oz, approximately 8 oz to approximately 64 oz, approximately 12 oz to approximately 16 oz, approximately 12 oz to approximately 20 oz, approximately 12 oz to approximately 24 oz, approximately 12 oz to approximately 30 oz, approximately 12 oz to approximately 36 oz, approximately 12 oz to approximately 48 oz, approximately 12 oz to approximately 64 oz, approximately 16 oz to approximately 20 oz, approximately 16 oz to approximately 24 oz, approximately 16 oz to approximately 30 oz, approximately 16 oz to approximately 36 oz, approximately 16 oz to approximately 48 oz, approximately 16 oz The weight is from about 1 ounce to about 64 ounces, from about 20 ounces to about 24 ounces, from about 20 ounces to about 30 ounces, from about 20 ounces to about 36 ounces, from about 20 ounces to about 48 ounces, from about 20 ounces to about 64 ounces, from about 24 ounces to about 30 ounces, from about 24 ounces to about 36 ounces, from about 24 ounces to about 48 ounces, from about 24 ounces to about 64 ounces, from about 30 ounces to about 36 ounces, from about 30 ounces to about 48 ounces, from about 30 ounces to about 64 ounces, from about 36 ounces to about 48 ounces, from about 36 ounces to about 64 ounces, or from about 48 ounces to about 64 ounces.

[0135] In some instances, the cultured cells are processed into additional foods in addition to foie gras and salmon or fish meat. For example, the cells are sometimes processed into liver slices or whole livers for cooking purposes. In some cases, other tissues are produced for human consumption, such as yakitori or other chicken organ products. Often, other organs are produced for birds and other species (e.g., thymus or pancreas for sweetbread). In some cases, fatty or fatty liver cells are produced and mixed with healthy liver cells to make foie gras pâté or other terrines. The stem cell isolation techniques described herein are optionally used to grow chicken, duck, or other animal meats. In certain instances, the techniques described herein are also applicable to the production of other animal-based meats.

[0136] Some of the systems and methods disclosed herein enable the production of cultured hepatocytes for human consumption. Certain methods enable the production of a textureless cultured tissue with a high lipid content, the method comprising: a) culturing a population of cells; b) inducing differentiation of the population of cells; c) manipulating lipid metabolic pathways to induce steatosis in the population of cells so that the cells accumulate a high lipid content; and d) processing the population of cells into a textureless tissue with a high lipid content.

[0137] Certain cultured cells and / or tissues produced using the methods described herein are processed into food products. In some cases, the cultured food products are packaged and / or labeled. The cultured cells and / or tissues can be processed into multiple slices (e.g., foie gras or salmon slices) to form the cultured food products. The multiple slices can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 slices. Sometimes, the plurality of sections is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or less sections. In certain examples, the cultured food is processed into shaped portions. The shaped portions can be individually or integrally packaged. The shaped portions can be any number of shapes, such as rectangular, square, circular, triangular, donut-shaped, tubular, pyramidal, or other shapes. The square portions can have a flattened shape (e.g., thin sections). For example, the thickness of the cultured food portion can be about 5 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, or about 100 mm or less. Sometimes, the thickness of the cultured food portion is at least about 5 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm, about 80 mm, about 90 mm, or about 100 mm.

[0138] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0139] Detailed Description of Drawings Figure 1 illustrates one embodiment of a process for producing cells for human consumption using self-renewing cells. In this process, a population of self-renewing cells is obtained (101) and then cultured (e.g., on a microscaffold) (102). Differentiation is induced in the cell population (103), followed by induction of lipid accumulation (104). Finally, the population of cells is processed for human consumption (105).

[0140] Figure 2 illustrates one embodiment of a process for culturing muscle tissue for human consumption. In this example, first and second populations of self-renewing cells are obtained (201)(204). The two populations of cells are cultured to a desired population size (202)(205). Differentiation of the two populations is then induced to generate myocytes (203) and adipocytes (206). Finally, the two populations of cells are processed for human consumption (207).

[0141] Figure 3 outlines a typical process for preparing cultivated meat for consumption. First, stem cells are identified, isolated, and characterized. These cells are then grown in two-dimensional culture, such as on a feeder cell layer. These cells are then transferred to suspension culture in a bioreactor. After transfer to suspension culture, the cells are differentiated into muscle cells. The meat is then harvested and finally prepared and cooked.

[0142] Figure 4A shows an exemplary approach for growing meat products for human consumption, with avian hepatocytes as a typical example for the production of foie gras. First, a progenitor cell line is obtained. In one approach, embryonic stem cells are isolated from avian embryos (401). Induced pluripotent stem cells can also be generated and differentiated from avian skin fibroblasts (402). Embryonic germ cells can also be isolated from avian embryos (403). Direct reprogramming of avian skin fibroblasts into hepatocytes is an option (404). Another method involves immortalizing adult avian differentiated hepatocytes (405), such as by viral transduction. Optionally, the adult avian differentiated hepatocytes are serially passaged and grown, and selected for spontaneously transformed cells (e.g., cells spontaneously immortalized by random mutation) (406). Primitive liver stem cells in adult avian liver tissue can also be isolated (407). Additionally, cultured hepatocytes can be cultured and exposed to toxins or injury to enhance their proliferative potential (408). Isolated stem cells (411) are then differentiated into differentiated hepatocytes (412) and subjected to induction of steatosis (413). Steatosis can be induced using a variety of methods, including genetic intervention and exogenous treatment (409). Finally, these processes are evaluated to augment strategies to enable large-scale production (410).

[0143] Figure 4B shows an exemplary approach for growing fish meat products for human consumption. First, a primitive cell line is obtained. In one approach, embryonic stem cells are isolated from fish embryos (421). Induced pluripotent stem cells can also be generated and differentiated from fish somatic cells (422). Primordial germ cells can also be generated from fish somatic cells (423). Self-renewing fibroblasts can be selected (424), such as through repeated passaging and selection of growing cell colonies. For example, differentiated fibroblasts from adult fish are serially passaged and grown, and spontaneously transformed cells (e.g., cells spontaneously immortalized by random mutation) are selected for. Another method involves direct reprogramming of fibroblasts into muscle cells (425). Another method involves immortalizing fish cells, such as muscle satellite cells or adipocyte precursor cells, using various methods, such as by viral transduction (e.g., TERT, SV40, large T antigen) (426). Pluripotent cells (e.g., embryonic stem cells (421), pluripotent stem cells (422), and primordial germ cells (423)) can be grown to desired quantities (433) during the food production process. The pluripotent cells can then be differentiated into precursor cells, such as preadipocytes (430) and / or muscle satellite cells (429). In some cases, cells can be induced to differentiate into desired cell types (e.g., muscle and / or adipocytes) using genetic techniques and / or exogenous treatments (427). For example, preadipocytes (adipocyte precursors) (430) and muscle satellite cells (429) can be induced to differentiate into adipocytes (432) and muscle cells (431), respectively, using genetic manipulations such as gene editing and / or construct expression. Exogenous treatments can include small molecule treatments to induce differentiation. This may also include exposing cells to extracellular structures and / or signals, such as microscaffolds optionally conjugated with differentiation / growth factors (e.g., FGF2). Finally, these processes may be evaluated to augment strategies to enable large-scale production. (428) Various combinations of the techniques described in Figures 4A-4B can be used for the production of cultured foods.In a typical embodiment, adult differentiated cells, such as fish (e.g., bass or salmon) fibroblasts, are serially passaged to identify cells that have undergone spontaneous immortalization (424). These immortalized cells can be cultured to a desired volume and then directly transdifferentiated from the differentiated lineage into a desired lineage, such as adipocytes and / or myocytes (or hepatocytes) (425). Transdifferentiation can be achieved using a variety of genetic modification techniques (427), such as using expression constructs described herein.

[0144] Another approach, not explicitly shown in Figures 4A-4B, utilizes mesenchymal stem cells (MSCs) for cultivated food production. Mesenchymal stem cells are multipotent stromal cells that can differentiate into various cell types, including osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchymal stem cells can be derived from various sources, such as bone marrow and adipose tissue. For example, MSCs from bone marrow can be isolated using flow cytometry or by direct seeding on cell culture plates to form colony-forming units (fibroblasts). MSCs can be grown to a desired mass in culture before being induced to differentiate into target differentiated cell types, such as myocytes, adipocytes, hepatocytes, or any combination thereof. In some cases, MSCs are split into separate cultures and differentiated separately before being combined to form meat products (e.g., myocytes, adipocytes, or a mixture of hepatocytes and adipocytes). MSCs can be cultured using various cell culture methods and grown using serum-supplemented basal media. In some cases, MSCs are cultured using serum-free media. Occasionally, MSCs are cultured using serum-free or serum-reduced media supplemented with plant-based supplements such as mushroom-derived extracts or soybean hydrolysate.

[0145] Figures 5A-5D show the isolation and characterization of satellite cells isolated from trout. Where present, insets magnify images in detail, and the scale bar equals 10 μm in all micrographs. A substantially pure population of fish satellite cells is shown in Figure 5, with satellite cells accounting for approximately 80% of the isolated cells. Figure 5B shows RT-PCR results confirming the presence of hallmark genes (Mstn1a, Myf5) expressed in these isolated satellite cells. Figure 5C shows mature myocytes generated by satellite cell differentiation. Figure 5D shows a sheet of trout myotubes differentiated from satellite cells.

[0146] Figure 6A shows the co-culture of salmon muscle satellite cells (arrowheads) and salmon preadipocytes (arrows) (scale bar is 500 µm). Figure 6B shows successful myocyte differentiation into mature myocytes within the co-culture (scale bar is 10 µm).

[0147] Figure 7A shows salmon fibroblasts induced to form spheroids for propagation in a bioreactor (scale bar is 500 µm). Figure 7B shows spheroids returned to 2D culture conditions to assess viability, as evidenced by cells from the spheroid migrating to the periphery to form colonies (scale bar is 500 µm).

[0148] FIG. 8 shows a culture of bath muscle satellite cells that was successfully cultured according to the cell culture techniques disclosed herein.

[0149] Figure 9 shows embryonic stem cell colonies derived from duck eggs. ESCs formed colonies growing on a monolayer of mouse embryonic fibroblast (MEF) feeder cells as shown in Figure 9.

[0150] Figure 10A shows a population of duck hepatocytes in culture. These duck hepatocytes were analyzed for markers of hepatocyte differentiation using reverse transcriptase polymerase chain reaction (RT-PCR). Figure 10B shows the results of an RT-PCR assay comparing undifferentiated cell controls (left lane) with duck hepatocyte samples (right lane) for expression of the hepatocyte differentiation markers L-FABP, alpha-fetoprotein, and HNF3b. β-actin was used as a control.

[0151] Figure 11A shows self-renewing cells generated by culturing primary fibroblasts from duck and harvesting colonies of dividing cells after 6-8 weeks. Figure 11B shows self-renewing cells generated by culturing primary fibroblasts from trout and harvesting colonies of dividing cells after 6-8 weeks. Both duck and trout self-renewing cell colonies were characterized for morphology, growth rate, and proliferation potential (changes in morphology, rate of growth, and number of passages achieved without genetic instability).

[0152] Figure 12 shows an exemplary embodiment of a construct that can be introduced into cells to provide inducible differentiation into hepatocytes. The construct contains a tetracycline response element (TRE) and ORFs for hepatocyte reprogramming factors HNF1A, FOXA1, and HNF4A. The construct can be stably transformed into target cells, such as pluripotent or multipotent cells. In some cases, the construct can be stably transformed into terminally differentiated cells, such as fibroblasts. The TRE represses expression of the ORF but allows the ORF to be transcribed in the presence of tetracycline or doxycycline. Thus, cell lines that stably integrate this construct can be induced to differentiate into hepatocytes via treatment with tetracycline / doxycycline.

[0153] Figure 13 shows an exemplary embodiment of a construct that can be introduced into cells to enable inducible expression of one or more genes that cause the cells to become steatotic. The construct contains a tetracycline response element (TRE) and ORF for one or more genes involved in lipid metabolism, such as ZFP423 (zinc finger protein transcription factor) and / or ATF4 (activating transcription factor 4). The construct can be stably transformed into target cells, such as pluripotent or multipotent cells. In some cases, the construct can be stably transformed into terminally differentiated cells, such as fibroblasts. The TRE represses expression of the ORF but allows the ORF to be transcribed in the presence of tetracycline or doxycycline. Thus, cell lines that stably integrate this construct can be induced to undergo or become steatotic via treatment with tetracycline / doxycycline. In some cases, the construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 genes selected from the group consisting of ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, and KLF6.

[0154] Figure 14 shows an exemplary embodiment of a DNA construct system that can be introduced into cells to enable a proliferation / differentiation switch from a pluripotent phenotype to a differentiated phenotype. This system includes a first construct containing a pluripotency cassette that provides constitutive expression of ORFs for pluripotency factors (e.g., Oct4, Sox2, Klf4, I-Myc). The pluripotency factors in the first construct are flanked by pLox sites. The system has a second construct containing a differentiation cassette that provides tetracycline-inducible expression of MyoD and Cre recombinase. Addition of an inducer, such as tetracycline or doxycycline, can induce expression of MyoD and Cre recombinase. MyoD expression can help drive cells to undergo differentiation into muscle cells. The Cre recombinase enzyme can catalyze the excision of the pluripotency factors flanked by the pLox sites. The inducer can then be removed to stop the induction of MyoD and Cre recombinase expression. An advantage of this system is the low footprint left by the system after excision of the pluripotency factors and removal of the inducer.

[0155] Figure 15 shows an exemplary construct that can be introduced into cells to provide an inducible "off switch." This construct contains one or more genes of interest flanked by pLox sites and an expression cassette containing a TRE and Cre recombinase. Addition of an inducer can cause cell lines that stably integrate this construct to express Cre recombinase to catalyze excision of the intervening sequences flanking the pLox sites. Thus, one or more genes (e.g., genes that promote differentiation) and the TRE and Cre recombinase expression cassette are removed, resulting in footprint-free excision of the gene of interest.

[0156] Figure 16A shows cultured duck hepatocytes. The top panel shows a negative control culture of duck hepatocytes not treated with linoleic acid. A magnification of the top panel shows the cell morphology of the hepatocytes. The bottom panel shows duck hepatocytes treated with 2 μM linoleic acid. A magnification of the bottom panel shows that the linoleic acid-treated hepatocytes were successfully induced to undergo steatosis (accumulation of lipid-containing vesicles—indicated by arrowheads). Figure 16B shows a dose-response graph of linoleic acid treatment on the percentage of hepatocytes with steatosis. As can be seen in the graph, increasing concentrations of linoleic acid (0, 0.1, 0.25, 0.5, 1, 2 μM) correlate with a corresponding increase in the percentage of hepatocytes with steatosis. At 2 μM linoleic acid, the percentage of fatty hepatocytes was greater than 85%. Similar results were achieved with oleic acid and using alternative protocols with the following components: IBMX (methylxanthine), rosiglitazone (thiazolidinedione), increased glucose concentrations, other fatty acid species, and / or corticosteroids such as dexamethasone.

[0157] Figure 17 shows a graph plotting the number of cells from an immortalized cell line derived from adult duck hepatocytes by selecting for rapidly proliferating hepatocytes after serial passage. These immortalized cells were cultured in decreasing concentrations of fetal bovine serum (FBS) in the presence of soybean hydrolysate (10 g / L). The number of hepatocytes and the percentage of FBS were graphed over time, with hepatocytes starting at less than 4 million cells and 10% serum, and gradually increasing in number over a 20-day period until the number of cells reached just over 10 million at less than 2% serum (0.8%). Supplementing the medium with soybean hydrolysate allowed the serum requirements of the cultured cells to be reduced by 92%.

[0158] FIG. 18 shows duck fibroblasts successfully grown in 10% shiitake extract after successive reductions of fetal bovine serum from the cell culture medium.

[0159] FIG. 19A shows duck fibroblasts grown in serum-free medium with no additional supplements; FIG. 19B shows a control culture grown in DMEM supplemented with 10% fetal bovine serum.

[0160] FIG. 20 shows one embodiment of a bioreactor system used for cell culture. The bioreactor system includes a reactor chamber (2001) for culturing cells and a stirring element (2003) for stirring the contents of the reactor chamber (2001). Culture medium is added to the reactor chamber through at least one input port (2002). The culture medium is sometimes a maintenance medium, a differentiation medium, an adipose medium, a proliferation medium, or any other medium formulation disclosed herein. Culture medium is removed from the reactor chamber through at least one output port (2007). Optionally, oxygen, carbon dioxide, and / or other gases are introduced through at least one input gas port (2006). The input gas port (2006) is optionally connected to an aeration device positioned inside the reactor chamber. Often, the bioreactor system includes at least one sensor (2004) for monitoring the reactor chamber. At least one sensor (2004) is typically in communication with a control unit (2008) (e.g., a computer). Often, the reactor chamber is seeded with a plurality of microscaffolds (2005). The microscaffolds (2005) allow for the attachment of specific adherent cells, such as hepatocytes.

[0161] Figure 21 shows an exemplary process in which a bioreactor system is used for meat production. Specific cells are isolated from eggs and grown in a bioreactor. The cells are grown using a medium containing water and plant-derived nutrients (e.g., instead of serum). The cells are grown in the sterile environment of the bioreactor for 4-6 weeks. Finally, the cells are harvested and / or processed into meat products.

[0162] Figures 22A and 22B show spheroids formed from duck liver cells grown in hanging drops and spinner flasks, where the spheroids can be transferred for three-dimensional suspension culture. As shown in Figure 22A, cells are grown in "hanging drops" of medium and developed into spheroids, which are then transferred to spinner flasks and grown in three-dimensional suspension culture, allowing for scale-up of cell production.

[0163] Figure 23A shows fish satellite cells grown on glucomannan microscaffolds (10% w / v) differentiated to form three-dimensional myotubes, and Figure 23B shows the downregulation of undifferentiated satellite cells from the same preparation grown under identical cell culture conditions.

[0164] Figure 24A shows duck fibroblasts (arrowheads) successfully grown on glucomannan microscaffolds (arrows), and Figure 24B shows a representative glucomannan microscaffold.

[0165] Figure 25 shows duck muscle tissue produced by differentiation of muscle satellite cells. This figure represents a still from a movie demonstrating spontaneous muscle cell contraction.

[0166] Figure 26 shows additional exemplary food products for human consumption produced according to the methods disclosed herein. The left panel shows duck liver pâté produced using duck fatty hepatocytes. The right panel shows foie gras butter produced using duck fatty hepatocytes.

[0167] Figure 27 shows exemplary food products for human consumption produced according to the methods disclosed herein. The left panel shows a salmon pate. The right panel shows a duck pate. In addition, a chicken pate has also been developed.

[0168] Figure 28A shows an exemplary embodiment of a method for Cre delivery for the purpose of activating / silencing specific genes, and Figure 28B shows various ways in which Cre can be used to induce a "switch" between activated gene sets related to meat production (e.g., proliferation and differentiation).

[0169] Specific Definitions Unless otherwise specified, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Any reference to "or" is intended to include "and / or" unless otherwise specified.

[0170] As used herein, "hepatocytes" refers to liver cells, hepatocytes, and hepatocyte-like cells. Hepatocytes can be derived from animals and from a variety of bird species, including, but not limited to, ducks (e.g., Mallard ducks, Barbary ducks), geese (e.g., Greyland geese), chickens, turkeys, emus, Cornish quails, Plymouth Rocks, and ostriches.

[0171] As used herein, "high lipid accumulation" refers to the formation of lipid-containing vacuoles or vesicles within at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a population of cells. In some cases, "high lipid accumulation" refers to the formation of lipid-containing vacuoles or vesicles within a majority (e.g., more than half) of the cells in a population of cells.

[0172] As used herein, "self-renewal" or "self-renewing" refers to cell division or proliferation while maintaining a particular cell type (e.g., an undifferentiated state). Examples of self-renewing cells include embryonic stem cells, induced pluripotent stem cells, and differentiated pluripotent stem cells (e.g., muscle satellite cells, hepatoblasts, and other progenitor cells). In some cases, self-renewing cells include differentiated cells that have been immortalized (e.g., via spontaneous immortalization).

[0173] As used herein, "about" refers to a range of about 10% of a particular amount, unless otherwise specified. For example, about 10 liters (L) refers to 9-11 L.

[0174] Whenever the term "about" is used in connection with a number or range, it is intended to mean approximately, or optionally replace "about" with "exactly."

[0175] Numbered Embodiments The following embodiments detail non-limiting permutations of feature combinations disclosed herein. Other permutations of feature combinations are also contemplated. In particular, each of these numbered embodiments is contemplated as dependent on or related to any preceding or following numbered embodiment, regardless of the order in which they are listed. 1. A method for producing cultured cells having high fat accumulation for human consumption, the method comprising culturing a population of cells; inducing differentiation in the population of cells; inducing high fat accumulation in the population of cells; and processing the population of cells for human consumption. 2. The method of embodiment 1, wherein the differentiated population of cells comprises hepatocytes. 3. The method of embodiment 1, wherein the processing comprises preparing the population of cells as foie gras. 4. The method of embodiment 1, wherein the population of cells is derived from duck or goose. 5. The method of embodiment 1, wherein the population of cells is derived from at least one of poultry and livestock. 6. The method of embodiment 1, wherein the inducing high fat accumulation comprises inducing steatosis. 7. The method of embodiment 1, wherein high fat accumulation is characterized by excessive accumulation of cytoplasmic lipid droplets. 8. The method of embodiment 1, wherein the inducing high fat accumulation comprises exposing the population of cells to an exogenous compound that modulates at least one lipid metabolic pathway. 9. The method of embodiment 1, wherein inducing high fat accumulation comprises exposing the population of cells to at least one of a toxin and a high lipid concentration. 10. The method of embodiment 1, wherein inducing high fat accumulation comprises modulating at least one lipid metabolic pathway to enhance lipid retention in the population of cells. 11. The method of embodiment 1, wherein the population of cells is modified to express at least one gene to induce differentiation into hepatocytes after treatment with an induction agent. 12. The method of embodiment 11, wherein the at least one gene to induce differentiation into hepatocytes comprises at least one of hepatocyte nuclear factor 1 alpha (HNF1A), Forkhead Box A2 (FOXA2), and hepatocyte nuclear factor 4 alpha (HNF4A). 13. The method of embodiment 1, wherein inducing high fat accumulation comprises modifying the population of cells to generate a modified cell line configured to express at least one gene for enhanced steatosis.14. The method of embodiment 13, wherein the modified cell line is configured to express at least one gene for enhanced steatosis after treatment with the induction agent. 15. The method of embodiment 13, wherein the modified cell line is stably transformed with a construct comprising an open reading frame (ORF) encoding ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, or KLF6. 16. The method of embodiment 13, wherein the modified cell line is stably transformed with a construct that promotes expression of at least one gene for enhanced steatosis when the modified cell line is exposed to tetracycline or a derivative thereof. 17. The method of embodiment 1, wherein inducing hyperlipidemia comprises modifying at least one gene in at least one cell in the population of cells to regulate lipid metabolism. 18. The method of embodiment 1, wherein the population of cells comprises differentiated liver, heart, kidney, stomach, intestine, lung, diaphragm, esophagus, thymus, pancreas, or tongue cells. 19. The method of embodiment 1, wherein processing the population of cells for human consumption comprises mixing the population of cells with cells having low lipid accumulation. 20. The method of embodiment 1, wherein the population of cells is isolated as embryonic stem cells. 21. The method of embodiment 1, wherein the population of cells has been modified to induce pluripotency. 22. The method of embodiment 1, wherein the population of cells is isolated as pluripotent adult stem cells. 23. The method of embodiment 1, wherein culturing comprises growing and expanding the population of cells in cell culture. 24. The method of embodiment 1, wherein inducing differentiation comprises exposing the population of cells to culture conditions that stimulate differentiation. 25. The method of embodiment 1, wherein inducing differentiation comprises exposing the population of cells to at least one growth factor that stimulates differentiation. 26. The method of embodiment 1, wherein the culturing step comprises growing a population of cells on a two-dimensional surface.27. The method of embodiment 1, wherein the culturing step comprises growing the population of cells on a three-dimensional scaffold. 28. The method of embodiment 1, wherein the culturing step comprises growing the population of cells on a microscaffold in a bioreactor, the microscaffold allowing cell attachment. 29. The method of embodiment 1, wherein the microscaffold comprises glucomannan or alginate. 30. The method of embodiment 1, wherein the population of cells does not require an adhesive substrate for survival and proliferation. 31. The method of embodiment 1, wherein the population of cells is suitable for suspension culture. 32. The method of embodiment 1, wherein the population of cells forms a tissue without texture after differentiation. 33. The method of embodiment 1, wherein the population of cells forms a non-muscle tissue after differentiation. 34. The method of embodiment 1, wherein the culturing step comprises growing the population of cells in a media formulation comprising at least one nutrient agent. 35. The method of embodiment 34, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 36. The method of embodiment 34, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 37. The method of embodiment 34, wherein at least one nutrient comprises a monounsaturated fatty acid. 38. The method of embodiment 34, wherein at least one nutrient comprises linoleic acid, oleic acid, or a combination thereof. 39. The method of embodiment 1, wherein the population of cells is cultured using a serum-free media formulation. 40. The method of embodiment 1, wherein the population of cells is cultured using a mushroom-based media formulation. 41. The method of embodiment 1, wherein the population of cells is cultured using a media formulation comprising soy hydrolysate. 42. A method for producing a detexturized cultured tissue having a high lipid content, the method comprising obtaining a population of self-renewing differentiated cells; culturing the population of differentiated cells; manipulating at least one lipid metabolic pathway to induce steatosis in the population of differentiated cells such that the cells accumulate a high lipid content; and processing the population of differentiated cells into a detexturized tissue.43. The method of embodiment 42, wherein obtaining a population of differentiated cells capable of self-renewal comprises transforming the differentiated cells into immortalized cells. 44. The method of embodiment 42, wherein obtaining a population of differentiated cells capable of self-renewal comprises culturing the differentiated cells until spontaneous mutations occur in the immortalized cells. 45. The method of embodiment 42, wherein the population of differentiated cells comprises hepatocytes. 46. The method of embodiment 42, wherein the processing comprises using the population of differentiated cells as an ingredient in foie gras. 47. The method of embodiment 42, wherein the population of differentiated cells is derived from duck or goose. 48. The method of embodiment 42, wherein the population of differentiated cells is derived from at least one of poultry and livestock. 49. The method of embodiment 42, wherein steatosis is characterized by excessive accumulation of cytoplasmic lipid droplets. 50. The method of embodiment 42, wherein manipulating at least one lipid metabolic pathway comprises exposing the population of cells to an exogenous compound. 51. The method of embodiment 42, wherein manipulating at least one lipid metabolic pathway comprises exposing the population of differentiated cells to at least one of a toxin and a high lipid concentration. 52. The method of embodiment 42, wherein manipulating at least one lipid metabolic pathway comprises modifying at least one gene in the population of differentiated cells to regulate lipid metabolism. 53. The method of embodiment 42, wherein manipulating at least one lipid metabolic pathway comprises modifying the population of differentiated cells with a genetic construct to generate a modified cell line configured to express at least one gene for enhanced steatosis. 54. The method of embodiment 53, wherein the modified cell line is configured to express at least one gene for enhanced steatosis after treatment with an induction agent. 55. The method of embodiment 53, wherein the modified cell line is stably transformed with a construct comprising an open reading frame (ORF) encoding ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, or KLF6.56. The method of embodiment 53, wherein the modified cell line is stably transformed with a construct that promotes expression of at least one gene for enhanced steatosis when the modified cell line is exposed to tetracycline or a derivative thereof. 57. The method of embodiment 42, wherein the population of differentiated cells comprises liver, heart, kidney, stomach, intestine, lung, diaphragm, esophagus, thymus, pancreas, or tongue cells. 58. The method of embodiment 42, wherein treating the population of differentiated cells comprises mixing the population of cells with cells having low lipid accumulation. 59. The method of embodiment 42, wherein culturing comprises growing and expanding the population of cells in cell culture. 60. The method of embodiment 42, wherein culturing comprises growing the population of cells on a two-dimensional surface. 61. The method of embodiment 42, wherein culturing comprises growing the population of cells on a three-dimensional scaffold. 62. The method of embodiment 42, wherein the culturing step comprises growing the population of cells on a microscaffold in a bioreactor, the microscaffold allowing cell adhesion. 63. The method of embodiment 62, wherein the microscaffold comprises glucomannan or alginate. 64. The method of embodiment 42, wherein the population of cells does not require an adhesive substrate for survival and proliferation. 65. The method of embodiment 42, wherein the population of cells is suitable for suspension culture. 66. The method of embodiment 42, wherein the population of differentiated cells forms tissue without texture. 67. The method of embodiment 42, wherein the population of cells forms non-muscle tissue. 68. The method of embodiment 42, wherein the culturing step comprises growing the population of cells in a media formulation comprising at least one nutrient agent. 69. The method of embodiment 68, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 70. The method of embodiment 68, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 71. The method of embodiment 68, wherein the at least one nutrient agent comprises a monounsaturated fatty acid.72. The method of embodiment 68, wherein the at least one nutrient comprises linoleic acid, oleic acid, or a combination thereof. 73. The method of embodiment 42, wherein the population of cells is cultured using a serum-free media formulation. 74. The method of embodiment 42, wherein the population of cells is cultured using a mushroom-based media formulation. 75. The method of embodiment 42, wherein the population of cells is cultured using a media formulation comprising soy hydrolysate. 76. A method for producing cultured non-muscle tissue for human consumption, the method comprising the steps of obtaining a population of self-renewing cells; culturing the population of self-renewing cells; forming non-muscle tissue. 76. A method for producing cultured non-muscle tissue for human consumption, comprising: inducing differentiation of a population of cells to form a non-muscle tissue; and processing the cultured non-muscle tissue for human consumption. 77. A method for producing cultured tissue for human consumption, comprising: obtaining a population of self-renewing cells; adapting the population of self-renewing cells to a suspension culture; culturing the population of self-renewing cells; inducing differentiation of the population of cells to form a cultured tissue; and processing the cultured tissue for human consumption. 78. A method for producing cultured muscle tissue without texture for human consumption, comprising: obtaining a population of self-renewing cells; culturing the population of self-renewing cells; inducing differentiation of the population of cells to form a non-textured muscle tissue; and processing the cultured non-muscle tissue for human consumption. 79. The method of embodiment 78, wherein the non-textured muscle tissue is octopus, squid, or cuttlefish muscle. 80. The method of embodiment 78, wherein inducing differentiation of a population of cells comprises generating myotubes. 81. The method of embodiment 80, wherein the population of differentiated cells comprises myotubes that are at least 50 μm in length. 82. The method of embodiment 78, wherein the de-textured muscle tissue is fish muscle tissue. 83. The method of embodiment 82, wherein the fish muscle tissue comprises highly glycolytic and anaerobic muscle fibers. 84. The method of embodiment 83, wherein highly glycolytic and anaerobic muscle fibers comprise at least 80% of the fish muscle tissue. 85. The method of embodiment 82, wherein the population of cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. 86. The method of embodiment 82, wherein the de-textured muscle tissue is combined with adipose tissue. 87. The method of embodiment 87, wherein the fish muscle tissue and adipose tissue are sushi-quality grade. 88. The method of embodiment 78, wherein the population of cells is isolated as embryonic stem cells. 89. The method of embodiment 78, wherein the population of cells has been modified to induce pluripotency.90. The method of embodiment 78, wherein the population of cells has been modified to incorporate a genetic construct comprising an open reading frame (ORF) of at least one gene configured to induce differentiation of the population of cells into muscle cells. 91. The method of embodiment 90, wherein the at least one gene configured to induce differentiation comprises Myogenin (MyoG), Myogenic Differentiation 1 (MyoD), Myogenic Factor 6 (MRF4), Myogenic Factor 5 (MYF5), or any combination thereof. 92. The method of embodiment 89, wherein the population of cells has been modified to incorporate a first genetic construct comprising an open reading frame (ORF) of at least one pluripotency gene configured to promote cell division; and a second genetic construct comprising an open reading frame (ORF) of a regulatory factor configured to inactivate the at least one pluripotency gene. 93. The method of embodiment 92, wherein the at least one pluripotency gene is configured to promote at least 50 cell divisions. 94. The method of embodiment 92, wherein the regulatory factor is a recombinase, and the open reading frame (ORF) of the at least one pluripotency gene is flanked by recombination sequences recognized by the recombinase, such that expression of the recombinase catalyzes excision of the open reading frame (ORF) of the at least one pluripotency gene. 95. The method of embodiment 92, wherein the second genetic construct comprises an open reading frame (ORF) of at least one cell lineage gene for differentiating the cell line, and an inducible promoter controlling expression of the at least one cell lineage gene open reading frame (ORF) and the regulatory factor open reading frame (ORF). 96. The method of embodiment 95, wherein inducing differentiation comprises exposing the population of cells to a transducing agent to induce expression of the at least one cell lineage gene open reading frame (ORF) and the regulatory factor open reading frame (ORF).97. The method of embodiment 96, further comprising removing the induction agent after exposing the population of cells to the induction agent and before processing the detextured cultured muscle tissue for human consumption. 98. The method of embodiment 78, wherein the population of cells is isolated as pluripotent adult stem cells. 99. The method of embodiment 78, wherein the culturing step comprises growing and expanding the population of cells in cell culture. 100. The method of embodiment 78, wherein the inducing differentiation step comprises exposing the population of cells to culture conditions that stimulate differentiation. 101. The method of embodiment 78, wherein the inducing differentiation step comprises exposing the population of cells to at least one growth factor that stimulates differentiation. 102. The method of embodiment 78, wherein the culturing step comprises growing the population of cells on a two-dimensional surface. 103. The method of embodiment 78, wherein the culturing step comprises growing the population of cells on a three-dimensional scaffold. 104. The method of embodiment 78, wherein the culturing step comprises growing the population of cells on a microscaffold in a bioreactor, the microscaffold allowing cell adhesion. 105. The method of embodiment 104, wherein the microscaffold comprises glucomannan or alginate. 106. The method of embodiment 78, wherein the population of cells does not require an adhesive substrate for survival and proliferation. 107. The method of embodiment 78, wherein the population of cells is suitable for suspension culture. 108. The method of embodiment 78, wherein the population of cells forms a tissue without texture after differentiation. 109. The method of embodiment 78, wherein the population of cells forms a non-muscle tissue after differentiation. 110. The method of embodiment 78, wherein the culturing step comprises growing the population of cells in a media formulation comprising at least one nutrient agent. 111. The method of embodiment 110, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 112. The method of embodiment 110, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid.113. The method of embodiment 110, wherein the at least one nutritional agent comprises a monounsaturated fatty acid. 114. The method of embodiment 110, wherein the at least one nutritional agent comprises linoleic acid, oleic acid, or a combination thereof. 115. The method of embodiment 78, wherein the population of cells is cultured using a serum-free media formulation. 116. The method of embodiment 78, wherein the population of cells is cultured using a mushroom-based media formulation. 117. The method of embodiment 78, wherein the population of cells is cultured using a media formulation comprising soy hydrolysate. 118. A method for preparing foie gras comprising cultured avian liver tissue, the method comprising obtaining a population of self-renewing avian-derived cells; differentiating the population of avian-derived cells into hepatocytes; and inducing steatosis in the hepatocytes to generate cultured avian liver tissue having a high lipid content; and preparing the cultured avian liver tissue as foie gras. 119. The method of embodiment 118, wherein the avian-derived cells are duck cells. 120. The method of embodiment 118, wherein the avian-derived cells are goose cells. 121. A foie gras composition for cooking, comprising tissue cultured hepatocytes having a high lipid content and processed for human consumption. 122. The composition of embodiment 121, wherein the composition is processed into a plurality of pieces. 123. The composition of embodiment 122, wherein each piece weighs about 5 ounces or less. 124. The composition of embodiment 122, wherein each piece is individually packaged. 125. The composition of embodiment 121, wherein the foie gras composition weighs at least about 1.5 pounds and is round, firm, and free of blemishes. 126. The composition of embodiment 121, wherein the foie gras composition has a packaging label indicating a Grade Classification A for the foie gras composition. 127. The composition of embodiment 121, wherein the foie gras composition weighs about 0.75 to about 1.5 pounds. 128. The composition of embodiment 121, wherein the foie gras composition has a packaging label indicating a grade classification B for the foie gras composition.129. The composition of embodiment 121, wherein the foie gras composition weighs less than about 1 pound and has no more than three blemishes. 130. The composition of embodiment 121, wherein the foie gras composition has a packaging label indicating a grade classification of C for the foie gras composition. 131. The composition of embodiment 121, wherein the tissue culture hepatocytes are steatotic. 132. The composition of embodiment 121, wherein the tissue culture hepatocytes are characterized by excessive accumulation of cytoplasmic lipid droplets. 133. The composition of embodiment 121, wherein the high lipid content is achieved by exposure to an exogenous compound that modulates at least one lipid metabolic pathway. 134. The composition of embodiment 121, wherein the high lipid content is achieved by exposure to at least one of a toxin and a high lipid concentration. 135. The composition of embodiment 121, wherein the high lipid content is achieved by modulation of at least one lipid metabolic pathway to enhance lipid retention within the population of cells. 136. The composition of embodiment 121, wherein the high lipid content is obtained by modification of at least one gene in the tissue culture hepatocytes. 137. The composition of embodiment 121, wherein the foie gras composition further comprises cells with low lipid accumulation. 138. The composition of embodiment 121, wherein the tissue culture hepatocytes are differentiated from isolated embryonic stem cells. 139. The composition of embodiment 121, wherein the tissue culture hepatocytes are differentiated from induced pluripotent stem cells. 140. The composition of embodiment 121, wherein the tissue culture hepatocytes are differentiated from isolated pluripotent adult stem cells. 141. The composition of embodiment 121, wherein the tissue culture hepatocytes are generated by differentiation in a population of self-renewing cells. 142. The composition of embodiment 141, wherein differentiation comprises exposing the population of cells to culture conditions that stimulate differentiation. 143. The composition of embodiment 141, wherein differentiation comprises exposing the population of cells to at least one growth factor that stimulates differentiation. 144. The composition of embodiment 121, wherein the tissue culture hepatocytes are grown on a two-dimensional surface.145. The composition of embodiment 121, wherein the tissue culture hepatocytes are grown on a three-dimensional scaffold. 146. The composition of embodiment 121, wherein the tissue culture hepatocytes are grown on a microscaffold in a bioreactor, the microscaffold allowing cell adhesion. 147. The composition of embodiment 121, wherein the tissue culture hepatocytes do not require an adhesive substrate for survival and proliferation. 148. The composition of embodiment 121, wherein the tissue culture hepatocytes are suitable for suspension culture. 149. The composition of embodiment 121, wherein the tissue culture hepatocytes form a non-textured tissue. 150. The tissue culture hepatocytes are non-muscular. 151. The composition of embodiment 121, wherein the tissue culture hepatocytes are cultured in a media formulation comprising at least one nutrient agent. 152. The composition of embodiment 151, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 153. The composition of embodiment 151, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 154. The composition of embodiment 151, wherein the at least one nutrient agent comprises a monounsaturated fatty acid. 155. A composition comprising cultured organ cells that is processed into a textureless non-muscle food product for human consumption. 156. The composition of embodiment 155, wherein the cultured organ cells comprise hepatocytes. 157. The composition of embodiment 155, wherein the cultured organ cells comprise avian cells. 158. The food product of embodiment 155, wherein the food product is processed into a plurality of pieces. 159. The composition of embodiment 158, wherein each piece weighs about 5 ounces or less. 160. The composition of embodiment 158, wherein each piece is individually wrapped. 161. The composition of embodiment 155, wherein the food product is foie gras. 162. The composition of embodiment 161, wherein the foie gras weighs at least about 1.5 pounds and is round, firm, and free of blemishes. 163. The composition of embodiment 161, wherein the foie gras has a packaging label indicating grade classification A. 164. The composition of embodiment 161, wherein the foie gras weighs from about 0.75 to about 1.5 pounds. 165. The composition of embodiment 161, wherein the foie gras has a packaging label indicating grade classification B. 166. The composition of embodiment 161, wherein the foie gras weighs less than about 1 pound and has no more than three blemishes. 167. The composition of embodiment 161, wherein the foie gras has a packaging label indicating grade classification C. 168. The composition of embodiment 161, wherein the tissue culture hepatocytes are fatty. 169. The composition according to embodiment 161, wherein the foie gras is characterized by a high fat content.170. The composition of embodiment 169, wherein the high lipid content is achieved by exposure to an exogenous compound that modulates at least one lipid metabolic pathway. 171. The composition of embodiment 169, wherein the high lipid content is achieved by exposure to at least one of a toxin and a high lipid concentration. 172. The composition of embodiment 169, wherein the high lipid content is achieved by modulation of at least one lipid metabolic pathway to enhance lipid retention within the population of cells. 173. The composition of embodiment 169, wherein the high lipid content is achieved by modification of at least one gene in tissue culture hepatocytes. 174. The composition of embodiment 169, wherein the foie gras composition further comprises cells with low lipid accumulation. 175. The composition of embodiment 155, wherein the cultured organ cells are grown on a two-dimensional surface. 176. The composition of embodiment 155, wherein the cultured organ cells are grown on a three-dimensional scaffold. 177. The composition of embodiment 155, wherein the cultured organ cells are grown on a microscaffold in a bioreactor, the microscaffold allowing cell adhesion. 178. The composition of embodiment 155, wherein the cultured organ cells do not require an adhesive substrate for survival and proliferation. 179. The composition of embodiment 155, wherein the cultured organ cells are suitable for suspension culture. 180. The composition of embodiment 155, wherein the cultured organ cells form tissue without texture. 181. The composition of embodiment 155, wherein the cultured organ cells form non-muscle tissue. 182. The composition of embodiment 155, wherein the cultured organ cells are cultured in a media formulation comprising at least one nutrient agent. 183. The composition of embodiment 182, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 184. The composition of embodiment 182, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 185. The composition of embodiment 182, wherein the at least one nutrient agent comprises a monounsaturated fatty acid. 186. The composition of embodiment 182, wherein the cultured organ cells are cultured using a serum-free media formulation.187. The composition of embodiment 182, wherein the cultured organ cells are cultured using a mushroom-based medium formulation. 188. An edible foie gras composition, comprising cultured avian fatty hepatocytes and a seasoning. 189. The composition of embodiment 188, wherein the seasoning comprises at least one of salt, pepper, and sugar. 190. A foie gras composition, comprising cultured hepatocytes having a high lipid content and hepatocytes having a low lipid content. 191. The composition of embodiment 190, wherein the cultured hepatocytes having a high lipid content and the hepatocytes having a low lipid content are mixed together. 192. The composition of embodiment 190, wherein the foie gras composition is suitable as an ingredient for preparing one of a mousse, a parfait, and a pâté. 193. The composition of embodiment 190, wherein the hepatocytes having a low lipid content are cultured cells. 194. The composition of embodiment 190, wherein the hepatocytes having a low lipid content are non-cultured cells. 195. An edible composition comprising avian hepatocytes grown in cell culture and processed for human consumption. 196. A packaged foie gras composition comprising cultured hepatocytes and a package bearing a label indicating that the foie gras composition was not produced by force-feeding. 197. A packaged foie gras composition comprising cultured hepatocytes and a package bearing a label indicating that the foie gras was produced in a pathogen-free environment. 198. The composition of embodiment 197, wherein the label indicates that the composition was produced without infection with avian influenza viruses. 199. A packaged edible composition comprising cultured cells processed into a food product and a label indicating that the composition was produced without exposure to a toxin. 200. The composition of embodiment 199, wherein the toxin is one of an insecticide, a herbicide, and a fungicide. 201. A method for producing cultured cells for human consumption without the use of antibiotics, the method comprising: culturing a population of cells without the use of antibiotics; inducing differentiation in the population of cells; inducing high lipid accumulation in the population of cells; and processing the population of cells for human consumption.202. A method for producing cultured cells for human consumption without exposure to pathogens, the method comprising: culturing a population of cells in a pathogen-free culture environment; inducing differentiation in the population of cells; inducing high lipid accumulation in the population of cells; and processing the population of cells for human consumption. 203. A method for producing cultured cells for human consumption without exposure to toxins, the method comprising: culturing a population of cells in a toxin-free culture environment; inducing differentiation in the population of cells; inducing high lipid accumulation in the population of cells; and processing the population of cells for human consumption. 204. A method for producing a cultured tissue with a high lipid content and no vascularization, the method comprising: culturing a population of cells; inducing differentiation in the population of cells; manipulating lipid metabolic pathways to induce steatosis in the population of differentiated cells such that the cells accumulate a high lipid content; and processing the population of differentiated cells into a tissue with ... texture. 205. A method for producing cultured tissue with increased nutrient content for human consumption, the method comprising: culturing a population of cells in a culture medium having at least one nutrient agent; manipulating lipid metabolic pathways to induce steatosis in the population of differentiated cells such that the cells accumulate a high lipid content; and processing the population of differentiated cells into avascular, textureless tissue for human consumption. 206. The method of embodiment 205, wherein at least one nutrient agent comprises an omega-3 fatty acid. 207. The method of embodiment 205, wherein at least one nutrient agent comprises a polyunsaturated fatty acid. 208. The method of embodiment 205, wherein at least one nutrient agent comprises a monounsaturated fatty acid. 209. A method for producing cultured organ tissue for human consumption, the method comprising culturing a population of self-renewing cells; inducing differentiation of the population of cells to produce organ tissue; and processing the organ tissue for human consumption. 210. The method of embodiment 209, wherein the organ tissue is liver, heart, kidney, stomach, intestine, lung, diaphragm, esophagus, thymus, pancreas, or tongue tissue.211. The method of embodiment 210, wherein the organ tissue is liver tissue. 212. The method of embodiment 211, wherein the processing step comprises mixing the organ tissue with additional cellular tissue. 213. The method of embodiment 212, wherein the additional cellular tissue comprises non-steatotic liver cells. 214. A method for producing cultured fish tissue with enhanced nutrient content for human consumption, the method comprising culturing a population of fish muscle cells in a culture medium having at least one nutrient; expanding the population of muscle cells; and processing the population of muscle cells into fish tissue for human consumption. 215. The method of embodiment 214, wherein the fish tissue comprises fast muscle fibers. 216. The method of embodiment 214, further comprising combining the population of fat cells and the population of muscle cells. 217. The method of embodiment 214, wherein the fish muscle cells are salmon muscle cells. 218. The method of embodiment 214, wherein the fish muscle cells are tuna muscle cells. 219. The method of embodiment 214, wherein the fish muscle cells are trout muscle cells. 220. An edible composition comprising fish tissue generated from cultured muscle cells and adipocytes. 221. A method for producing cultured fish meat for human consumption, the method comprising obtaining a population of self-renewing cells; culturing the population of self-renewing cells in a medium comprising a microscaffold; inducing differentiation of the population of cells to form at least one of muscle cells and adipocytes; and processing the population of cells into fish meat for human consumption. 222. The method of embodiment 221, wherein the microscaffold comprises glucomannan or alginate. 223. The method of embodiment 221, wherein at least a subset of the population of cells has been modified to incorporate a genetic construct comprising an open reading frame (ORF) of at least one gene configured to induce differentiation of the population of cells into muscle cells.224. The method of embodiment 223, wherein the at least one gene configured to induce differentiation comprises Myogenin (MyoG), Myogenic Differentiation 1 (MyoD), Myogenic Factor 6 (MRF4), Myogenic Factor 5 (MYF5), or any combination thereof. 225. The method of embodiment 221, wherein at least a subset of the population of cells has been modified to incorporate a genetic construct comprising an open reading frame (ORF) of at least one gene configured to induce differentiation of the population of cells into adipocytes. 226. At least a subset of the population of cells has been modified to incorporate an open reading frame (ORF) of at least one pluripotency gene configured to promote cell division. 226. The method of embodiment 221, wherein the cell line is modified to incorporate a first genetic construct comprising an open reading frame (ORF) of at least one pluripotency gene; and a second genetic construct comprising an open reading frame (ORF) of a regulatory factor configured to inactivate at least one pluripotency gene. 227. The method of embodiment 226, wherein the regulatory factor is a recombinase, and the open reading frame (ORF) of the at least one pluripotency gene is flanked by recombination sequences recognized by the recombinase, such that expression of the recombinase catalyzes excision of the at least one pluripotency gene open reading frame (ORF). 228. The method of embodiment 226, wherein the second genetic construct comprises an open reading frame (ORF) of at least one cell lineage gene for differentiating the cell line, and an inducible promoter controlling expression of the at least one cell lineage gene open reading frame (ORF) and the regulatory factor open reading frame (ORF). 229. The method of embodiment 228, wherein inducing differentiation comprises exposing the population of cells to an induction agent to induce expression of at least one open reading frame (ORF) of a cell lineage gene and an open reading frame (ORF) of a regulatory factor. 230. The method of embodiment 229, further comprising removing the induction agent after exposing the population of cells to the induction agent and before processing the population of cells into fish meat for human consumption. 231. The method of embodiment 221, wherein the fish meat is sushi. 232. The method of embodiment 221, wherein the fish meat is surimi. 233. The method of embodiment 221, wherein the fish meat is suitable for raw consumption. 234. The method of embodiment 221, wherein the fish meat is cooked. 235. The method of embodiment 221, wherein the fish meat is salmon meat. 236. The method of embodiment 221, wherein the fish meat is sushi-grade salmon meat. 237. The method of embodiment 221, wherein the fish meat is tuna meat.238. The method of embodiment 221, wherein the fish meat is sushi-grade tuna meat. 239. The method of embodiment 221, wherein the fish meat is trout meat. 240. The method of embodiment 221, wherein the inducing differentiation step (c) causes the population of cells to form myocytes and adipocytes. 241. The method of embodiment 240, wherein the fish meat is composed of at least 50% highly glycolytic and anaerobic muscle fibers. 242. The method of embodiment 221, wherein the population of cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. 243. The method of embodiment 221, wherein the treating step (d) comprises combining the population of cells with a second population of cells composed of myocytes or adipocytes. 244. The method of embodiment 221, wherein the population of cells is isolated as embryonic stem cells. 245. The method of embodiment 221, wherein the population of cells has been modified to induce pluripotency. 246. The method of embodiment 221, wherein the population of cells is isolated as pluripotent adult stem cells. 247. The method of embodiment 221, wherein the culturing step comprises growing and expanding the population of cells in cell culture. 248. The method of embodiment 221, wherein the inducing differentiation step comprises exposing the population of cells to culture conditions that stimulate differentiation. 249. The method of embodiment 221, wherein the inducing differentiation step comprises exposing the population of cells to at least one growth factor that stimulates differentiation. 250. The method of embodiment 221, wherein the culturing step comprises growing the population of cells on a two-dimensional surface. 251. The method of embodiment 221, wherein the culturing step comprises growing the population of cells on a three-dimensional scaffold. 252. The method of embodiment 221, wherein the culturing step comprises growing the population of cells on a microscaffold in a bioreactor, the microscaffold allowing cell attachment. 253. The method of embodiment 221, wherein the population of cells forms a tissue without texture after differentiation.254. The method of embodiment 221, wherein the culturing step comprises growing the population of cells in a media formulation comprising at least one nutrient agent. 255. The method of embodiment 254, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 256. The method of embodiment 254, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 257. The method of embodiment 254, wherein the at least one nutrient agent comprises a monounsaturated fatty acid. 258. The method of embodiment 221, wherein the population of cells is cultured using a serum-free media formulation. 259. The method of embodiment 221, wherein the population of cells is cultured using a mushroom-based media formulation. 260. A method of producing cultured meat for human consumption, the method comprising obtaining a population of self-renewing cells capable of growth in suspension culture; culturing the population of self-renewing cells in suspension; inducing differentiation of the population of cells to form at least one of myocytes and adipocytes; and processing the population of cells into meat for human consumption. 261. The method of embodiment 260, wherein the meat is fish meat. 262. The method of embodiment 261, wherein the fish meat is sushi. 263. The method of embodiment 261, wherein the fish meat is surimi. 264. The method of embodiment 261, wherein the fish meat is suitable for raw consumption. 265. The method of embodiment 221, wherein the fish meat is cooked. 266. The method of embodiment 261, wherein the fish meat is salmon meat. 267. The method of embodiment 221, wherein the fish meat is sushi-grade salmon meat. 268. The method of embodiment 261, wherein the fish meat is tuna meat. 269. The method of embodiment 268, wherein the population of self-renewing cells is derived from a tuna selected from yellowfin tuna, southern bluefin tuna, longfin tuna, albacore tuna, Atlantic bluefin tuna, and bigeye tuna. 270. The method of embodiment 268, wherein the population of self-renewing cells is derived from bluefin tuna. 271. The method of embodiment 221, wherein the fish meat is sushi-grade tuna meat.272. The method of embodiment 261, wherein inducing differentiation of the population of cells causes the population of cells to form muscle cells and adipocytes. 273. The method of embodiment 261, wherein the fish meat is composed of at least 50% highly glycolytic and anaerobic muscle fibers. 274. The method of embodiment 261, wherein the population of cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. 275. The method of embodiment 261, wherein processing the population of cells into meat for human consumption comprises combining the population of cells with a second population of cells composed of muscle cells or adipocytes. 276. The method of embodiment 261, wherein the population of cells is isolated as embryonic stem cells. 277. The method of embodiment 261, wherein the population of cells has been modified to induce pluripotency. 278. The method of embodiment 261, wherein the population of cells is isolated as pluripotent adult stem cells. 279. The method of embodiment 261, wherein the culturing step comprises growing and expanding the population of cells in cell culture. 280. The method of embodiment 261, wherein the inducing differentiation step comprises exposing the population of cells to culture conditions that stimulate differentiation. 281. The method of embodiment 261, wherein the inducing differentiation step comprises exposing the population of cells to at least one growth factor that stimulates differentiation. 282. The method of embodiment 261, wherein the culturing step comprises growing the population of cells on a two-dimensional surface. 283. The method of embodiment 261, wherein the population of cells forms a tissue without texture after differentiation. 284. The method of embodiment 261, wherein the culturing step comprises growing the population of cells in a media formulation comprising at least one nutrient agent. 285. The method of embodiment 284, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 286. The method of embodiment 284, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 287. The method of embodiment 284, wherein the at least one nutritional agent comprises a monounsaturated fatty acid.288. The method of embodiment 261, wherein the population of cells is cultured using a serum-free media formulation. 289. The method of embodiment 261, wherein the population of cells is cultured using a mushroom-based media formulation. 290. A system for producing a cultured tissue suitable for human consumption, the system comprising: a reactor chamber comprising a plurality of microscaffolds providing an adhesive surface for cell attachment; a population of self-renewing cells cultivated in the bioreactor; a first source providing at least one maintenance medium comprising ingredients for maintaining the population of self-renewing cells without spontaneous differentiation; and a second source providing at least one differentiation medium comprising ingredients for differentiating the population of self-renewing cells towards a specific lineage; wherein the reactor chamber receives the maintenance medium from the first source for cultivating the population of cells and the differentiation medium from the second source for differentiating the population of cells, wherein the population of cells produced in a single batch comprises a cultured tissue suitable for human consumption and having a dry weight of at least 1 kg. 291. The system of embodiment 290, further comprising at least one sensor for monitoring the reactor chamber. 292. The system of embodiment 290, wherein the at least one sensor is a biosensor, a chemical sensor, or an optical sensor. 293. The system of embodiment 290, wherein the at least one sensor is configured to monitor at least one of pH, temperature, oxygen, carbon dioxide, glucose, lactate, ammonia, hypoxanthine, amino acids, dopamine, and lipids. 294. The system of embodiment 290, further comprising at least one additional reactor chamber. 295. The system of embodiment 290, wherein a single batch has a dry weight of at least 5 kg. 296. The system of embodiment 290, further comprising a plurality of microscaffolds. 297. The system of embodiment 296, wherein the plurality of microscaffolds comprises glucomannan or alginate. 298. The system of embodiment 290, further comprising at least one 3D scaffold.299. The system of embodiment 290, further comprising a third source providing at least one lipid-based medium comprising a component for inducing steatosis or lipid accumulation in the population of cells. 300. The system of embodiment 290, wherein the component for inducing steatosis or lipid accumulation comprises linoleic acid, oleic acid, or a combination thereof. 301. The system of embodiment 290, wherein the population of cells is cultured in a medium comprising at least one nutrient. 302. The system of embodiment 301, wherein the at least one nutritional agent comprises mushroom extract, soy hydrolysate, or a combination thereof. 303. A method for producing cultured fish tissue, the method comprising culturing a population of fish preadipocytes and a population of fish satellite cells; inducing differentiation of the population of fish preadipocytes to form adipocytes; inducing differentiation of the population of fish satellite cells to generate myocytes; co-culturing the adipocytes and myocytes; and processing the adipocytes and myocytes into fish tissue for human consumption. 304. The method of embodiment 303, wherein the fish tissue comprises fast-twitch muscle fibers. 305. The method of embodiment 303, wherein the fish tissue is salmon tissue. 306. The method of embodiment 303, wherein the fish tissue is tuna tissue. 307. The method of embodiment 303, wherein the fish tissue is trout tissue. 308. The method of embodiment 303, wherein the fish tissue is surimi. 309. The method of embodiment 303, wherein the fish tissue is sushi. 310. The method of embodiment 303, wherein the fish tissue is prepared for raw human consumption. 311. The method of embodiment 303, wherein the fish tissue is cooked for human consumption. 312. The method of embodiment 303, wherein adipocytes and muscle cells are co-cultured in a media formulation comprising at least one nutrient agent. 313. The method of embodiment 312, wherein the at least one nutrient agent comprises an omega-3 fatty acid. 314. The method of embodiment 312, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid. 315. The method of embodiment 312, wherein the at least one nutrient agent comprises a monounsaturated fatty acid. 316. The method of embodiment 261, wherein a serum-free media formulation is used for cell culture. 317. The method of embodiment 261, wherein a mushroom-based media formulation is used for cell culture.318. A method for producing cultured fish tissue, the method comprising culturing a population of fish preadipocytes and a population of fish satellite cells suitable for suspension culture; inducing differentiation of the population of fish preadipocytes to form adipocytes; inducing differentiation of the population of fish satellite cells to form myocytes; co-culturing the adipocytes and myocytes; and processing the adipocytes and myocytes into fish tissue for human consumption. 319. An edible composition comprising fish tissue produced from co-cultured myocytes and adipocytes. 320. An edible composition comprising fish tissue produced from preadipocytes and satellite cells. 321. A method for producing cultured fish meat for human consumption, the method comprising obtaining a population of preadipocytes and a population of satellite cells; adapting the population of preadipocytes and the population of satellite cells to suspension culture; inducing differentiation into the population of preadipocytes and the population of satellite cells; co-culturing the populations in suspension culture; and processing the populations into fish meat for human consumption. 322. The method of embodiment 321, wherein the fish meat is sushi. 323. The method of embodiment 321, wherein the fish meat is surimi. 324. The method of embodiment 321, wherein the fish meat is suitable for raw consumption. 325. The method of embodiment 321, wherein the fish meat is cooked. 326. The method of embodiment 321, wherein the fish meat is salmon meat. 327. The method of embodiment 321, wherein the fish meat is sushi-grade salmon meat. 328. The method of embodiment 321, wherein the fish meat is tuna meat. 329. The method of embodiment 321, wherein the fish meat is sushi-quality tuna meat. 330. The method of embodiment 321, wherein the fish meat is trout meat. 331. The method of embodiment 321, wherein the fish meat is composed of at least 50% highly glycolytic and anaerobic muscle fibers. 332. The method of embodiment 321, wherein the population of preadipocytes is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout.333. The method of embodiment 321, wherein the population of satellite cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. 334. The method of embodiment 321, wherein the co-culturing step comprises growing and expanding the population in cell culture. 335. The method of embodiment 321, wherein the inducing step comprises exposing the population of preadipocytes to at least one growth factor that stimulates differentiation into adipocytes. 336. The method of embodiment 321, wherein the inducing step comprises exposing the population of satellite cells to at least one growth factor that stimulates differentiation into myocytes. 337. The method of embodiment 321, wherein the culturing step comprises growing the population of cells in a bioreactor. 338. The method of embodiment 321, wherein the myocytes and adipocytes form tissue without texture after differentiation. 339. The method of embodiment 321, wherein the myocytes and adipocytes are cultured in a media formulation comprising at least one nutrient. 340. The method of embodiment 339, wherein at least one nutritional agent comprises an omega-3 fatty acid. 341. The method of embodiment 339, wherein at least one nutritional agent comprises a polyunsaturated fatty acid. 342. The method of embodiment 339, wherein at least one nutritional agent comprises a monounsaturated fatty acid. 343. The method of embodiment 321, wherein a serum-free media formulation is used for cell culture. 344. The method of embodiment 321, wherein a mushroom-based media formulation is used for cell culture. 345. A processed fish product suitable for human consumption, comprising fish meat produced from cultured muscle cells and adipocytes. 346. A processed fish product suitable for human consumption, comprising fish meat derived from cultured satellite cells and pre-adipocytes. 347. A processed fish product suitable for human consumption, comprising fish meat produced from muscle cells and adipocytes grown in suspension culture.348. A method for producing cultured fish meat for human consumption, the method comprising the steps of obtaining a population of fish preadipocytes growable in suspension culture; obtaining a population of fish satellite cells growable in suspension culture; inducing differentiation of the population of fish preadipocytes and the population of fish satellite cells to form adipocytes and myocytes; co-culturing the adipocytes and myocytes in suspension culture with at least one nutrient; and processing the population of cells into fish meat for human consumption. 349. The method of embodiment 348, wherein the fish meat is sushi. 350. The method of embodiment 348, wherein the fish meat is surimi. 351. The method of embodiment 348, wherein the fish meat is suitable for raw consumption. 352. The method of embodiment 348, wherein the fish meat is cooked. 353. The method of embodiment 348, wherein the fish meat is salmon meat. 354. The method of embodiment 348, wherein the fish meat is sushi-grade salmon meat. 355. The method of embodiment 348, wherein the fish meat is tuna meat. 356. The method of embodiment 348, wherein the fish meat is sushi-grade tuna meat. 357. The method of embodiment 348, wherein the fish meat is composed of at least 50% highly glycolytic and anaerobic muscle fibers. 358. The method of embodiment 348, wherein the population of cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. 359. The method of embodiment 348, wherein inducing differentiation in (c) comprises exposing the population of preadipocytes and the population of satellite cells to culture conditions that stimulate differentiation. 360. The method of embodiment 348, wherein inducing differentiation in (c) comprises exposing the population of preadipocytes to at least one growth factor that stimulates differentiation. 361. The method of embodiment 348, wherein inducing differentiation in (c) comprises exposing the population of satellite cells to at least one growth factor that stimulates differentiation. 362. The method of embodiment 348, wherein adipocytes and muscle cells form tissue without texture.363. The method of embodiment 348, wherein at least one nutritional agent comprises an omega-3 fatty acid. 364. The method of embodiment 348, wherein at least one nutritional agent comprises a polyunsaturated fatty acid. 365. The method of embodiment 348, wherein at least one nutritional agent comprises a monounsaturated fatty acid. 366. The method of embodiment 348, wherein a serum-free media formulation is used for cell culture. 367. The method of embodiment 348, wherein a mushroom-based media formulation is used for cell culture. 368. A method for producing cultured liver tissue for human consumption, the method comprising obtaining a population of cells; modifying the population of cells to generate a modified cell line configured to express at least one hepatocyte differentiation factor after treatment with an induction agent; culturing the modified cell line; and treating the modified cell line with an induction agent to generate cultured liver tissue; and processing the cultured liver tissue for human consumption. 369. A method for producing fatty liver tissue for human consumption, the method comprising: obtaining a hepatocyte cell line that is modified to express at least one fatty factor after treatment with an induction agent; culturing the hepatocyte cell line; and treating the hepatocyte cell line with the induction agent to produce fatty liver tissue; and processing the fatty liver tissue into a food product for human consumption. 370. A genetically modified cell line suitable for meat production, the cell line comprising: a first genetic construct comprising at least one pluripotency gene to promote cell cycle progression; and a second genetic construct comprising at least one lineage gene to promote differentiation, a regulatory factor configured to inactivate the at least one pluripotency gene, and an inducible promoter controlling expression of the at least one lineage gene and the regulatory factor. 371. A method for producing cultured tissue for human consumption, the method comprising the steps of obtaining a population of self-renewing cells; culturing the population of self-renewing cells; inducing differentiation of the population of cells to form the cultured tissue; and processing the cultured tissue for human consumption.372. The method of embodiment 371, wherein obtaining the population of self-renewing cells comprises transitioning the population of cells from a two-dimensional adherent culture to a three-dimensional culture in a bioreactor. 373. The method of embodiment 371, wherein culturing comprises seeding the population of self-renewing cells on a three-dimensional microscaffold. 374. The method of embodiment 3, wherein the three-dimensional microscaffold promotes cell proliferation, adhesion, differentiation, or a combination thereof. 375. The method of embodiment 3, wherein the three-dimensional microscaffold is conjugated to at least one factor that promotes cell proliferation, adhesion, differentiation, or a combination thereof. 376. The method of embodiment 375, wherein the microscaffold comprises glucomannan, alginate, collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin, fibronectin, or a combination thereof. 377. The population of self-renewing cells undergoes induced differentiation. 378. The method of any one of embodiments 371 to 376, wherein the population of self-renewing cells comprises at least one cell modified for expression in a pluripotent cell line. 378. The method of embodiment 377, wherein the at least one cell is modified to incorporate a first genetic construct comprising an open reading frame (ORF) of at least one pluripotency gene; and a second genetic construct comprising an open reading frame (ORF) of a regulatory factor configured to inactivate the at least one pluripotency gene. 379. The method of embodiment 378, wherein the population of self-renewing cells comprises at least one cell that has undergone at least 50 cell divisions in culture. 380. The method of embodiment 378, wherein the regulatory factor is a recombinase, and the open reading frame (ORF) of the at least one pluripotency gene is flanked by recombination sequences recognized by the recombinase, such that expression of the recombinase catalyzes excision of the open reading frame (ORF) of the at least one pluripotency gene. 381. The method of embodiment 378, wherein the at least one pluripotency gene comprises at least one of Hepatocyte Nuclear Factor 1 alpha (HNF1A), Forkhead Box A2 (FOXA2), and Hepatocyte Nuclear Factor 4 alpha (HNF4A). 382. The method of embodiment 378, wherein the at least one pluripotency gene comprises Myogenin (MyoG), Myogenic Differentiation 1 (MyoD), Myogenic Factor 6 (MRF4), Myogenic Factor 5 (MYF5), or any combination thereof. 383. The method of embodiment 378, wherein the second gene construct further comprises an open reading frame (ORF) of at least one differentiation gene; and an inducible promoter controlling expression of the open reading frame (ORF) of the at least one differentiation gene and the open reading frame (ORF) of the regulatory factor. 384. The method of embodiment 383, wherein the step of inducing differentiation comprises exposing at least one cell to an inducer to induce expression of at least one cell lineage gene ORF and a regulatory factor ORF.385. The method of embodiment 384, further comprising removing the inducer after the population of self-renewing cells has been treated with the inducer and before processing the cultured tissue for human consumption. 386. The method of embodiment 371, wherein inducing differentiation comprises generating myotubes within the population of self-renewing cells. 387. The method of embodiment 386, wherein inducing differentiation further comprises generating adipocytes within the population of self-renewing cells. 388. The method of any one of embodiments 371-387, wherein the population of self-renewing cells comprises a first subset of cells that differentiate into myocytes and a second subset of cells that differentiate into adipocytes during inducing differentiation of the population of cells to form the cultured tissue. 389. The method of embodiment 371, wherein inducing differentiation comprises generating hepatocytes within the population of self-renewing cells. 390. The method of embodiment 389, wherein the population of self-renewing cells is derived from an avian species selected from duck, goose, chicken, and turkey. 391. The method of embodiment 389, further comprising inducing steatosis in at least one of the hepatocytes. 392. The method of embodiment 391, wherein the population of self-renewing cells comprises at least one cell modified to express at least one gene to enhance steatosis after treatment with the induction agent. 393. The method of embodiment 392, wherein at least one cell is stably transformed using a construct comprising at least one open reading frame (ORF) encoding ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, KLF6, or any combination thereof. 394. The method of any one of embodiments 389 to 393, wherein inducing steatosis comprises incubating the hepatocytes in a culture medium comprising at least a nutrient agent. 395. The method of embodiment 394, wherein the at least one nutrient agent comprises a polyunsaturated fatty acid, a monounsaturated fatty acid, or a combination thereof.396. The method of embodiment 394, wherein the at least one nutrient agent comprises palmitic acid, oleic acid, docosahexaenoic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, or a combination thereof. 397. The method of any one of embodiments 371-376, wherein the cultured tissue comprises muscle cells from octopus, squid, or cuttlefish. 398. The method of any one of embodiments 371-376, wherein the cultured tissue comprises fish muscle tissue. 399. The method of embodiment 398, wherein the population of self-renewing cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout. 400. The method of embodiment 398, wherein the fish muscle tissue is combined with separately cultured fish adipose tissue during processing of the cultured tissue for human consumption. 401. The method of embodiment 371, wherein the population of cells is cultured using a serum-free media formulation. 402. The method of embodiment 401, wherein the serum-free media formulation comprises mushroom extract or soy hydrolysate. 403. A cultured food product for human consumption, comprising a cultured tissue produced according to the method of any one of embodiments 371-402. 404. The cultured food product of embodiment 403, wherein the cultured food product comprises a package having a label indicating that the cultured tissue was produced in a pathogen-free environment, a toxin-free environment, a non-force-fed animal environment, or any combination thereof. 405. The cultured food product of embodiment 403, wherein the cultured tissue is processed into a plurality of slices to form the cultured food product and packaged. 406. A method for producing a cultured tissue for human consumption, the method comprising: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation of the population of self-renewing cells to form the cultured tissue; and d) processing the cultured tissue for human consumption. 407. The method of embodiment 406, wherein the step of obtaining the population of self-renewing cells comprises transferring the population of cells from a two-dimensional adherent culture to a three-dimensional culture in a bioreactor.408. The method of embodiment 406, wherein the population of self-renewing cells comprises immortalized differentiated cells. 409. The method of any one of embodiments 406-408, wherein inducing differentiation of the population of self-renewing cells comprises inducing transdifferentiation of cells in the population into myocytes, adipocytes, or a combination thereof. 410. The method of embodiment 406, wherein culturing comprises seeding the population of self-renewing cells on the three-dimensional microscaffold. 411. The method of embodiment 410, wherein the three-dimensional microscaffold promotes cell proliferation, adhesion, differentiation, or a combination thereof. 412. The method of embodiment 410, wherein the three-dimensional microscaffold is conjugated to at least one factor that promotes cell proliferation, adhesion, differentiation, or a combination thereof. 413. The method of any one of embodiments 410-412, wherein the microscaffold comprises at least one of hydrogel, chitosan, polyethylene terephthalate, collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin, fibronectin, cellulose, hemicellulose, pectin, lignin, alginate, glucomannan, polycaprolactone (PCL), textured vegetable protein (TVP), textured soy protein (TSP), and acrylates. 414. The method of any one of embodiments 406-413, wherein the population of self-renewing cells comprises at least one cell modified to undergo directed differentiation. 415. The method of embodiment 414, wherein at least one cell has been modified to incorporate a first genetic construct comprising an open reading frame (ORF) of at least one pluripotency gene; and a second genetic construct comprising an open reading frame (ORF) of a regulatory factor configured to inactivate the at least one pluripotency gene. 416. The method of embodiment 415, wherein the population of self-renewing cells comprises at least one cell that has undergone at least 50 cell divisions in culture.417. The method of embodiment 415, wherein the regulatory factor is a recombinase, and the open reading frame (ORF) of the at least one pluripotency gene is flanked by recombination sequences recognized by the recombinase, such that expression of the recombinase catalyzes excision of the open reading frame (ORF) of the at least one pluripotency gene. 418. The method of embodiment 415, wherein the second genetic construct comprises an ORF of at least one hepatocyte differentiation factor selected from Hepatocyte Nuclear Factor 1 alpha (HNF1A), Forkhead Box A2 (FOXA2), and Hepatocyte Nuclear Factor 4 alpha (HNF4A). 419. The method of embodiment 415, wherein the second genetic construct comprises at least one myogenic factor selected from Myogenin (MyoG), Myogenic Differentiation 1 (MyoD), Myogenic Factor 6 (MRF4), and Myogenic Factor 5 (MYF5). 420. The method of embodiment 415, wherein the second genetic construct comprises at least one adipogenic factor selected from fatty acid binding protein 4 (FABP4), insulin-responsive glucose transporter type 4 (GLUT4), adiponectin, C1Q, and collagen domain-containing (ADIPOQ), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), perilipin 1 (PLIN1), leptin (LEP), and lipoprotein lipase (LPL). 421. The method of embodiment 415, wherein the second genetic construct further comprises: a) an open reading frame (ORF) of at least one differentiation gene; and b) an inducible promoter controlling expression of i) the open reading frame (ORF) of the at least one differentiation gene and ii) the open reading frame (ORF) of the regulatory factor. 422. The method of embodiment 421, wherein the step of inducing differentiation comprises exposing at least one cell to an inducer to induce expression of at least one cell lineage gene ORF and a regulatory factor ORF.423. The method of embodiment 421, further comprising removing the inducer after the population of self-renewing cells has been treated with the inducer and before processing for human consumption in step d). 424. The method of embodiment 406, wherein the step of inducing differentiation comprises generating myotubes within the population of self-renewing cells. 425. The method of embodiment 424, wherein the step of inducing differentiation further comprises generating adipocytes within the population of self-renewing cells. 426. The method of any one of embodiments 406 to 425, wherein the population of self-renewing cells comprises pluripotent cells induced to differentiate into myocytes and adipocytes during step c). 427. The pluripotent cells are a first portion of muscle satellite cells. The method of embodiment 426, wherein the population of self-renewing cells comprises a first subpopulation of hepatocytes and a second subpopulation of preadipocytes. 428. The method of embodiment 406, wherein inducing differentiation comprises generating hepatocytes within the population of self-renewing cells. 429. The method of embodiment 428, wherein the population of self-renewing cells is derived from an avian species selected from duck, goose, chicken, and turkey. 430. The method of embodiment 429, further comprising inducing steatosis in at least one of the hepatocytes. 431. The method of embodiment 430, wherein the population of self-renewing cells comprises at least one cell modified to express at least one gene to enhance steatosis after treatment with an induction agent. 432. The method of embodiment 431, wherein at least one cell is stably transformed with a construct comprising an open reading frame (ORF) encoding ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, or KLF6. 433. The method of any one of embodiments 430-432, wherein inducing steatosis comprises incubating the hepatocytes in a culture medium comprising at least a nutrient. 434. The method of embodiment 433, wherein the at least one nutrient comprises a polyunsaturated fatty acid, a monounsaturated fatty acid, or a combination thereof. 435. The method of embodiment 433 or 434, wherein the at least one nutrient comprises palmitic acid, oleic acid, docosahexaenoic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, or a combination thereof. 436. The method of any one of embodiments 406-427, wherein the cultured tissue comprises muscle cells of an octopus, squid, or cuttlefish. 437. The method of any one of embodiments 406-427, wherein the cultured tissue comprises muscle tissue of a fish. 438. The method of any one of embodiments 406 to 427, wherein the population of self-renewing cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout.439. The method of embodiment 437, wherein fish muscle tissue is combined with separately cultured fish adipose tissue during step d). 440. The method of any one of embodiments 406 to 439, wherein the population of cells is cultured using a serum-free media formulation. 441. The method of any one of embodiments 406 to 440, wherein the serum-free media formulation comprises mushroom extract or soy hydrolysate. 442. A cultured food product for human consumption, comprising a cultured tissue produced according to the method of any one of embodiments 406 to 441. 443. The cultured food product of embodiment 442, wherein the cultured food product comprises a package having a label indicating that the cultured tissue was produced in a pathogen-free environment, a toxin-free environment, a non-force-fed environment, or any combination thereof. 444. The cultured food product of embodiment 442 or 443, wherein the cultured tissue is processed into a plurality of sections and packaged to form the cultured food product. [Example]

[0176] The following illustrative examples represent embodiments of the systems, methods, and compositions described herein and are not intended to be limiting in any way.

[0177] Example 1 - Cultured fish meat produced using embryonic stem cells

[0178] Embryonic stem cells are isolated from salmon embryos. Initially, the embryonic stem cells are cultured using an optimized media substrate and media formulation to achieve sustained cell proliferation and maintenance of a dedifferentiated state. The media formulation utilizes a synthetic, serum-free medium. The cells are cultured in a pathogen-free cell culture system. The embryonic stem cells are then induced to differentiate into muscle satellite cells and preadipocytes. The muscle satellite cells and preadipocytes are cultured and expanded to a desired cell mass. The muscle satellite cells and preadipocytes are then differentiated into muscle cells and adipocytes, which are then harvested and processed by centrifugation and compression to produce the texture and firmness of fish meat.

[0179] Example 2 - Cultured fish meat produced using induced pluripotent stem cells

[0180] Fish fibroblasts were isolated from salmon. An episomal reprogramming strategy was used to create induced pluripotent stem cells from the isolated fish fibroblasts without using classical viral reprogramming techniques. First, the induced pluripotent stem cells were cultured using an optimized media substrate and medium formulation to achieve sustained cell proliferation and maintenance of a dedifferentiated state. The medium formulation utilized a synthetic, serum-free medium. The cells were cultured in a pathogen-free cell culture system. Next, the iPS cells were expanded to the desired cell mass and then induced to differentiate into myocytes and adipocytes. Finally, the myocytes and adipocytes were harvested and processed by centrifugation and compression to produce the texture and firmness of fish meat.

[0181] Example 3 - Cultured fish meat produced using direct cell reprogramming

[0182] Fish fibroblasts are isolated from salmon. They are serially passaged until a cell line with the capacity for continuous self-renewal (e.g., immortalization) is selected. The immortalized fibroblasts are grown to a desired mass in culture and then transdifferentiated. A reprogramming strategy using overexpression of a selectable gene is utilized to directly reprogram fibroblasts into myocytes and adipocytes without creating an intermediate pluripotent cell type. Thus, transdifferentiation allows immortalized fibroblasts to be converted into a desired cell type without the need for stem cells.

[0183] Example 4 - Microscaffolding system for culturing synthetic foods

[0184] The cells are cultured using any of the techniques described in Examples 1-6, using a bioreactor containing a microscaffold that allows the attachment and growth of adherent hepatocytes to generate small cellular structures that can be grown in suspension. The microscaffold is composed of a biocompatible material that biodegrades over time so that the cultured hepatocyte structures eventually leave no more scaffolding material. The hepatocyte structures are subsequently processed to produce foie gras.

[0185] Example 5 - 3D scaffolding system for culturing synthetic foods

[0186] The cells are cultured using any of the techniques described in Examples 1-6, which use a 3D scaffold to induce adherent hepatocyte growth and generate a cellular structure approximating the size and shape of a conventional avian liver. The 3D scaffold is composed of a biocompatible material, such as alginate, that biodegrades over time so that the finished foie gras product contains no more scaffolding material. As a result, the hepatocytes grow to a size similar to that of a conventional avian liver without the need for centrifugation and processing, thereby producing foie gras with the desired texture and firmness.

[0187] Example 6 - Foie gras produced using conventional techniques

[0188] Geese chicks are raised and spend the first four weeks of their lives feeding and growing. They are then transferred to cages and fed a high-protein, high-starch diet for another four weeks. At approximately eight to ten weeks, the chicks are force-fed via gavage, where a feeding tube is used to force two to four pounds of grain and fat down the bird's throat on a daily basis. Due to excessive food consumption, the birds' livers undergo steatosis, an enlargement of the liver that can reach more than ten times its normal size. During this process, the birds are exposed to various pathogens in crowded and unhealthy conditions. Finally, the geese are slaughtered, and their livers are harvested and sold as foie gras.

[0189] Example 7 - Sushi-quality salmon produced using embryonic stem cells

[0190] Embryonic stem cells are isolated from salmon embryos. Initially, embryonic stem cells are cultured using an optimized media substrate and formulation to achieve sustained cell proliferation and maintenance of a dedifferentiated state. The media formulation utilizes a synthetic, serum-free medium. The cells are cultured in a pathogen-free cell culture system without exposure to toxins or heavy metals, such as mercury, often found in fish meat. Separate populations of embryonic stem cells are then induced to differentiate into myocytes and adipocytes, respectively. In this case, differentiation of myocytes results in the desired ratio of approximately 80% fast-twitch muscle fibers and approximately 20% slow-twitch muscle fibers. The myocytes and adipocytes are cultured and expanded to the desired cell mass. The myocytes and adipocytes are then harvested and processed by centrifugation and compression to produce salmon of a quality suitable for traditional sushi, or alternatively, the texture and consistency of surimi-like salmon.

[0191] Example 8 - Sushi-grade salmon

[0192] Preadipocytes and satellite cells are isolated from fingerling salmon and subsequently characterized and cultivated as separate cell lines in cell culture. Each cell line is cultured using an optimized media formulation to adapt the cell line to suspension culture. Adipocyte and myocyte differentiation is then induced in the preadipocyte and satellite cell lines, respectively. The cell lines are then co-cultured at an optimal ratio to achieve the desired final myocyte-to-adipocyte ratio. The media formulation utilizes a synthetic serum-free medium containing a mushroom extract to replace fetal bovine serum. The cells are cultured in a pathogen-free cell culture system without exposure to toxins or heavy metals, such as mercury, often found in fish meat. The culture medium is supplemented with high concentrations of free fatty acids, such as oleic acid, which induces adipocyte uptake and storage of excess extracellular fatty acids. The co-cultured myocytes and adipocytes are then harvested and processed by centrifugation and compression to produce the texture and firmness of salmon surimi.

[0193] Example 9 - Isolation and culture of salmon stem cells (adipocyte precursor cells and muscle satellite cells)

[0194] Fish myocytes and adipocytes were targeted for the development of fish-related food based on their intrinsic regenerative potential during early development. We characterized and optimized ex vivo cultures of relevant salmon tissues, including myocytes, adipocytes, hepatocytes, fibroblasts, and undifferentiated pluripotent cell lineages. First, trout preadipocytes and satellite cells (capable of differentiating into myocytes) were isolated, cultured, and characterized. Trout satellite cells were isolated and subsequently characterized as shown in Figures 5A-5D. Where present, insets magnify the images in detail, and the scale bar equals 10 μm in all micrographs. A substantially pure population of fish satellite cells was successfully isolated, as shown in Figure 5, where satellite cells accounted for approximately 80% of the isolated cells. These cells were then characterized with relevant transcriptional markers. Figure 5B shows RT-PCR results confirming the presence of hallmark genes (Mstn1a, Myf5) expressed in these isolated cells. Next, the culture conditions were optimized for these cell lines. Using the culture medium protocol, satellite cells were successfully differentiated into mature myocytes (Figure 5C). Myotube sheets differentiated from satellite cells are shown in Figure 5D. Preadipocytes were also differentiated into adipocytes.

[0195] Additionally, salmon muscle satellite cells (arrowheads) were co-cultured with salmon preadipocytes (arrows) to produce a food product containing both muscle and adipocytes or tissues, as shown in Figure 6A (scale bar is 100 μm). As shown in Figure 6B, the preadipocytes differentiated into adipocytes, and the muscle satellite cells differentiated into myocytes (arrowheads) (scale bar is 10 μm).

[0196] Example 10 - De novo generation of induced pluripotent stem cells using episomal reprogramming

[0197] Pluripotent stem cells are of interest for the development of cultured foods based on their lack of lineage commitment and high versatility for directed differentiation. For example, myocytes, adipocytes, fibroblasts, and other tissue components can be generated from a single pool of pluripotent stem cells.

[0198] Induced pluripotent stem cells (iPSCs) are generated using episomal (non-integrated) reprogramming. iPSCs form colonies that grow on a monolayer of mouse embryonic fibroblast (MEF) feeder cells. These cells are then characterized for pluripotency markers, proliferation capacity, and differentiation efficiency. Culture conditions are further optimized for the generated cell lines with respect to cost, large-scale synthesis, and maintenance of genomic / phenotypic stability.

[0199] Example 11 - Stem Cell Adaptation to Suspension Culture

[0200] Stem cell-based meat production represents an approach with several advantages over alternative cellular agriculture methods. First, the use of pluripotent stem cells is preferable to traditional muscle satellite cultures due to the uncertain replicative potential of the former; this characteristic obviates the need to repeatedly obtain muscle biopsies from animals during the production process. Second, pluripotent stem cells are preferable to characterized immortalized cell lines because they typically do not harbor perturbations in cell cycle gene networks or other associated mutations. Finally, although the genetic and phenotypic characteristics of cell lines change over time in culture, the use of pluripotent stem cells with established differentiation protocols ensures the reproducibility of the final product. However, one obstacle to stem cell-based meat production is that stem cells are typically grown in two-dimensional culture on feeder cell lines. Successful adaptation of stem cells to three-dimensional suspension culture for meat production would dramatically reduce resource costs for cell culture.

[0201] We will implement and optimize the transition of cells from two-dimensional suspension culture (e.g., cell culture dishes) to three-dimensional suspension culture to validate methods for scaling up cultivated food production. Three-dimensional suspension culture represents the first step in scaling for food production, as it presents the opportunity to grow significantly larger quantities of biomaterial along with more efficient growth medium utilization.

[0202] Growth of induced pluripotent stem cells (iPSCs) in suspension cult...

Claims

1. 1. A method for producing cultured tissue for human consumption, the method comprising: a) obtaining a population of self-renewing cells; b) culturing the population of self-renewing cells; c) inducing differentiation in the population of self-renewing cells to form a cultured tissue; and d) processing the cultured tissue for human consumption.

2. 10. The method of claim 1, wherein obtaining the population of self-renewing cells comprises transitioning the population of cells from a two-dimensional adherent culture to a three-dimensional culture in a bioreactor.

3. The method of claim 1 , wherein the population of self-renewing cells comprises immortalized differentiated cells.

4. 4. The method of any one of claims 1-3, wherein inducing differentiation in the population of self-renewing cells comprises inducing transdifferentiation of cells in the population into muscle cells, adipocytes, or a combination thereof.

5. 10. The method of claim 1, wherein the culturing step comprises seeding a population of self-renewing cells on the three-dimensional microscaffold.

6. 4. The method of claim 3, wherein the three-dimensional microscaffold promotes cell proliferation, adhesion, differentiation, or a combination thereof.

7. 4. The method of claim 3, wherein the three-dimensional microscaffold is conjugated to at least one factor that promotes cell growth, adhesion, differentiation, or a combination thereof.

8. 8. The method of claim 7, wherein the microscaffold comprises at least one of hydrogel, chitosan, polyethylene terephthalate, collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin, fibronectin, cellulose, hemicellulose, pectin, lignin, alginate, glucomannan, polycaprolactone (PCL), textured vegetable protein (TVP), textured soy protein (TSP), and acrylates.

9. 9. The method of claim 8, wherein the population of self-renewing cells comprises at least one cell that has been modified to undergo induced differentiation.

10. At least one cell comprises: a) a first genetic construct comprising the open reading frame (ORF) of at least one pluripotency gene; and b) a second genetic construct comprising an open reading frame (ORF) of a regulatory element configured to inactivate at least one pluripotency gene; 10. The method of claim 9, wherein the nucleic acid sequence is modified to incorporate

11. 11. The method of claim 10, wherein the population of self-renewing cells comprises at least one cell that undergoes at least 50 cell divisions in culture.

12. 11. The method of claim 10, wherein the regulatory factor is a recombinase and the open reading frame (ORF) of the at least one pluripotency gene is flanked by recombination sequences recognized by the recombinase such that expression of the recombinase catalyzes excision of the open reading frame (ORF) of the at least one pluripotency gene.

13. The method of claim 10, wherein the second gene construct comprises an ORF of at least one hepatocyte differentiation factor selected from hepatocyte nuclear factor 1 alpha (HNF1A), forkhead box A2 (FOXA2), and hepatocyte nuclear factor 4 alpha (HNF4A).

14. 11. The method of claim 10, wherein the second gene construct comprises at least one myogenic factor selected from myogenin (MyoG), myogenic differentiation 1 (MyoD), myogenic factor 6 (MRF4), and myogenic factor 5 (MYF5).

15. 11. The method of claim 10, wherein the second genetic construct comprises at least one adipogenic factor selected from fatty acid binding protein 4 (FABP4), insulin-responsive glucose transporter type 4 (GLUT4), adiponectin, C1Q and collagen domain-containing (ADIPOQ), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), perilipin 1 (PLIN1), leptin (LEP), and lipoprotein lipase (LPL).

16. The second gene construct is a) an open reading frame (ORF) of at least one differentiation gene; and b) i. an open reading frame (ORF) of at least one differentiation gene; and ii. Regulatory factor open reading frame (ORF) Inducible promoter controlling the expression of The method of claim 10 further comprising:

17. 17. The method of claim 16, wherein inducing differentiation comprises exposing at least one cell to an inducer to induce expression of at least one cell lineage gene ORF and a regulatory factor ORF.

18. 18. The method of claim 17, further comprising removing the inducer after the population of self-renewing cells is treated with the inducer in step d) and before being processed for human consumption.

19. 2. The method of claim 1, wherein inducing differentiation comprises generating myotubes within the population of self-renewing cells.

20. 20. The method of claim 19, wherein inducing differentiation further comprises generating adipocytes within the population of self-renewing cells.

21. The method of any of claims 1-20, wherein the population of self-renewing cells comprises pluripotent cells that are induced to differentiate into muscle cells and adipocytes during step c).

22. 21. The method of claim 20, wherein the pluripotent cells comprise a first subpopulation of muscle satellite cells and a second subpopulation of preadipocytes.

23. The method of claim 1 , wherein inducing differentiation comprises generating hepatocytes within the population of self-renewing cells.

24. 24. The method of claim 23, wherein the population of self-renewing cells is derived from an avian species selected from ducks, geese, chickens, and turkeys.

25. 24. The method of claim 23, further comprising inducing steatosis in at least one of the hepatocytes.

26. 26. The method of claim 25, wherein the population of self-renewing cells comprises at least one cell modified to express at least one gene to enhance steatosis upon treatment with an induction agent.

27. 27. The method of claim 26, wherein at least one cell is stably transformed with a construct comprising an open reading frame (ORF) encoding ATF4, ZFP423, LPIN1, PPAR, APOC3, APOE, ORL1, PEMT, MTTP, SREBP, STAT3, or KLF6.

28. 28. The method of claim 27, wherein inducing steatosis comprises incubating the hepatocytes in a medium comprising at least a nutrient.

29. 30. The method of claim 28, wherein the at least one nutritional agent comprises a polyunsaturated fatty acid, a monounsaturated fatty acid, or a combination thereof.

30. 30. The method of claim 28, wherein the at least one nutritional agent comprises palmitic acid, oleic acid, docosahexaenoic acid, stearic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, or a combination thereof.

31. 17. The method of claim 16, wherein the cultured tissue comprises muscle cells from an octopus, squid, or cuttlefish.

32. 17. The method of claim 16, wherein the cultured tissue comprises fish muscle tissue.

33. 33. The method of claim 32, wherein the population of self-renewing cells is derived from sea bass, tuna, mackerel, blue marlin, swordfish, yellowtail, salmon, or trout.

34. 33. The method of claim 32, wherein fish muscle tissue is combined with separately cultured fish fatty tissue during step d).

35. 10. The method of claim 1, wherein the population of cells is cultured using a serum-free media formulation.

36. 36. The method of claim 35, wherein the serum-free media formulation comprises mushroom extract or soy hydrolysate.

37. A cultured food product for human consumption comprising a cultured tissue produced according to the method of any one of claims 1-36.

38. 38. The cultured food product for human consumption of claim 37, wherein the cultured food product comprises a package having a label indicating that the culture was produced in a pathogen-free environment, a toxin-free environment, an environment where animals are not force-fed, or any combination thereof.

39. 38. The cultured food product for human consumption of claim 37, wherein the cultured tissue is processed into a plurality of sections and packaged to form the cultured food product.

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