Cell-cultured foods and related cells, compositions, methods, and systems

Abstract

Provided herein are culture media, methods, and systems for culturing aquatic animal cells to increase lipid uptake, cell viability, and / or cell differentiation in the presence of one or more lipids at a concentration of at least 10 μg / ml, as well as cells obtainable and obtained thereby, associated cell biomass, and food products. Also provided herein are culture media, methods, and systems for culturing terrestrial animal cells to increase lipid uptake, cell viability, and / or cell differentiation in the presence of one or more lipids at a concentration of at least 10 μg / ml, as well as cells obtainable and obtained thereby, associated cell biomass, and food products.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 174,444, filed April 13, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Cell-cultured foods are part of the alternative food market and have been the focus of development by many companies around the world as a means to address public health, environmental, and animal welfare issues associated with livestock and agriculture. Public health concerns include the relatively high saturated fatty acid content of terrestrial animal meat, such as beef, and essentially zero omega-3 polyunsaturated fatty acid content. Diets high in saturated fat and low in omega-3 have been linked to elevated LDL cholesterol levels and increased risk of heart disease.

[0003] Despite growing interest and efforts in producing animal-free alternative foods, challenges remain in developing optimized formulations and methods for producing cell- and tissue-culture derived foods, especially cell-cultured foods enriched with fatty acids and nutrients. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure relates to methods and systems for producing aquatic animal cell cultured foods, as well as related cells, compositions, cells and cell cultured foods, which may have a desired fatty acid profile. In embodiments, the methods disclosed herein provide improved cell growth in culture and enhanced incorporation of desired fatty acids in the cell biomass.

[0005] According to a first aspect, culture media, methods and systems for increasing the content of monounsaturated fatty acids in aquatic animal cells, as well as aquatic animal cells obtainable and / or obtained thereby, are described.

[0006] The culture medium comprises a basal cell medium for aquatic animal cells supplemented with monounsaturated fatty acids typically at a concentration of about 0.1 μg / ml to about 1000 μg / ml, preferably about 0.1 μg / ml to about 500 μg / ml, about 1 μg / ml to about 100 μg / ml, or about 5 μg / ml to about 50 μg / ml.

[0007] The method includes culturing aquatic animal cells in a culture medium for aquatic animal cells supplemented with monounsaturated fatty acids, typically at a concentration of about 0.1 μg / ml to about 1000 μg / ml, for a period of time and under conditions that allow for uptake of the monounsaturated fatty acids by the aquatic animal cells in accordance with the present disclosure.

[0008] The system includes a culture medium for aquatic animal cells in combination with monounsaturated fatty acids and / or aquatic animal cells for simultaneous, combined or sequential use in the methods for increasing the content of monounsaturated fatty acids in aquatic animal cells described herein.

[0009] According to a second aspect, culture media, methods and systems for increasing the content of polyunsaturated fatty acids, saturated fatty acids and / or sterols in aquatic animal cells, as well as aquatic animal cells obtainable and / or obtained thereby, are described.

[0010] The culture medium comprises a basal cell medium for aquatic animal cells supplemented with polyunsaturated fatty acids, saturated fatty acids, and / or sterols in combination with an effective amount of nervonic acid, in some embodiments, each of the supplemented lipids, e.g., polyunsaturated fatty acids, saturated fatty acids, and / or sterols, is present at a concentration of about 10 μg / ml or greater.

[0011] The method includes culturing aquatic animal cells in a culture medium for aquatic animal cells described herein supplemented with at least 10 μg / ml of polyunsaturated fatty acids, saturated fatty acids, and / or sterols, wherein the culture medium is further supplemented with an effective amount of nervonic acid, and the culturing is performed for a period of time and under conditions that allow for uptake of polyunsaturated fatty acids, saturated fatty acids, and / or sterols by the aquatic animal cells in accordance with the present disclosure.

[0012] The system includes a basal cell culture medium for aquatic animal cells in combination with polyunsaturated fatty acids, saturated fatty acids, and / or sterols, nervonic acid, and / or aquatic animal cells for simultaneous, combined, or sequential use in the methods for increasing the polyunsaturated fatty acid, saturated fatty acid, and / or sterol content in aquatic animal cells as described herein.

[0013] In a preferred embodiment, the polyunsaturated fatty acid is or comprises an unsaturated fatty acid, such as an omega-3 polyunsaturated fatty acid. According to a third aspect, culture media, methods and systems for increasing the lipid content in aquatic animal cells, as well as aquatic animal cells obtainable and / or obtained thereby, are described.

[0014] The culture medium can comprise a basal cell medium for aquatic animal cells supplemented with at least 10 μg / ml of lipid, and when the lipid is or comprises a polyunsaturated fatty acid, a saturated fatty acid, and / or a sterol, the culture medium further comprises nervonic acid in an amount effective to enable upload of the polyunsaturated fatty acid and / or sterol by the aquatic animal cells.

[0015] The method includes culturing aquatic animal cells in a culture medium for aquatic animal cells as described herein supplemented with at least 10 μg / ml of lipid, the culturing being carried out for a period of time and under conditions that allow for uptake of the lipid by the aquatic animal cells, in which, when the lipid is or comprises a polyunsaturated fatty acid and / or a sterol, the culture medium further comprises an effective amount of nervonic acid.

[0016] The system includes a culture medium for aquatic animal cells in combination with one or more lipids and / or aquatic animal cells for simultaneous, combined, or sequential use in the methods for increasing lipid content in aquatic animal cells described herein. In embodiments, when the one or more lipids are or include one or more polyunsaturated fatty acids and / or sterols, the system further includes nervonic acid in an amount effective to cause uptake of the polyunsaturated fatty acids and / or sterols into the aquatic animal cells.

[0017] In a preferred embodiment, the lipid is or comprises an unsaturated fatty acid, such as an omega-3 polyunsaturated fatty acid.

[0018] According to a fourth aspect, methods and systems for increasing lipid content in aquatic animal myoblasts and / or fibroblasts and associated culture media and aquatic animal myoblasts and / or fibroblasts obtainable and / or obtained thereby are described.

[0019] The method comprises culturing myoblasts and / or fibroblasts in a culture medium containing at least 10 μg / ml of lipid (and further containing an effective amount of nervonic acid if the lipid is or comprises a polyunsaturated fatty acid and / or a sterol), for a period and under conditions that allow for uptake of the lipid by the aquatic animal myoblasts and / or fibroblasts. In a preferred embodiment, the lipid is or comprises an unsaturated fatty acid, such as an omega-3 polyunsaturated fatty acid.

[0020] The system comprises a culture medium for aquatic animal cells, a lipid, and / or cells selected from aquatic animal myoblasts and / or fibroblasts for simultaneous, combined, or sequential use in the methods for increasing lipid content in aquatic animal myoblasts and / or fibroblasts described herein. When the lipid is or comprises a polyunsaturated fatty acid and / or a sterol, the system further comprises an effective amount of nervonic acid.

[0021] The culture medium comprises a basal medium supplemented with a lipid, where the lipid is or comprises nervonic acid in an amount effective to increase the lipid content of the myoblasts and / or fibroblasts.

[0022] According to a fifth aspect, a method and system for increasing the polyunsaturated fatty acid content in aquatic animal cells and associated culture media and aquatic animal cells obtainable thereby are described.

[0023] The method involves culturing aquatic animal cells in a culture medium comprising a polyunsaturated fatty acid and an effective amount of nervonic acid.

[0024] The system comprises nervonic acid and polyunsaturated fatty acids, optionally in combination with culture medium and / or aquatic animal cells, for simultaneous, combined or sequential use in the methods for increasing polyunsaturated fatty acid content in aquatic animal cells described herein.

[0025] The culture medium comprises a basal medium, a polyunsaturated fatty acid, and nervonic acid in an amount effective to increase uptake of the polyunsaturated fatty acid in the cells of the aquatic animal.

[0026] According to a sixth aspect, a method and system for increasing the omega-3 content in aquatic animal cells and related culture media and aquatic animal cells obtainable thereby are described.

[0027] The method involves culturing aquatic animal cells in a culture medium containing omega-3 and an effective amount of nervonic acid.

[0028] The system comprises nervonic acid and omega-3 fatty acids, optionally in combination with culture medium and / or aquatic animal cells, for simultaneous, combined or sequential use in the methods for increasing omega-3 fatty acid content in aquatic animal cells described herein.

[0029] The culture medium comprises a basal medium, an omega-3 fatty acid, and nervonic acid in an amount effective to increase the uptake of omega-3 fatty acid in the cells of an aquatic animal.

[0030] According to a seventh aspect, methods and systems for increasing the viability of aquatic animal cells in the presence of polyunsaturated fatty acids, saturated fatty acids, and / or sterols are described, as well as related culture media and aquatic animal cells obtainable thereby.

[0031] The method includes culturing aquatic animal cells in a culture medium comprising polyunsaturated fatty acids, saturated fatty acids, and / or sterols and an effective amount of nervonic acid.

[0032] The system includes nervonic acid in combination with polyunsaturated fatty acids, saturated fatty acids, and / or sterols, optionally in combination with culture medium and / or aquatic animal cells, for simultaneous, combined, or sequential use in the methods for increasing omega-3 fatty acid content in aquatic animal cells described herein.

[0033] The culture medium comprises a basal medium, a polyunsaturated fatty acid, a saturated fatty acid, and / or a sterol, and nervonic acid in an amount effective to increase cell viability of the cells of the aquatic animals described herein.

[0034] According to an eighth aspect, aquatic animal preadipocytes are described, the preadipocytes comprising desired lipids in an amount of 0.1% to 1% by weight of the cells. Typically, the total lipid content of the cells is greater than about 2.0% by weight. In a preferred embodiment, the lipid content of the preadipocytes described herein may contain about 50% SFA, 25% PUFA (preferably including omega-3), and 25% MUFA. It is preferred that the preadipocytes or adipocytes have low SFA, with the cells containing a relatively high percentage of unsaturated fatty acids as a percentage of total fat. For example, the SFA content may be about 30% or less, about 20% or less, about 10% or less, or about 5% or less.

[0035] According to a ninth aspect, aquatic animal myoblasts or myocytes and associated biomass comprising said cells are described, wherein the myoblasts contain the desired fatty acids in an amount of at least about 0.5% by weight. Typically, the total lipid content of the cells is greater than about 2.0% by weight. It is preferred that the myoblasts or myocytes have low SFA, and the cells contain a relatively high percentage of unsaturated fatty acids as a percentage of total fat. For example, the SFA content can be about 30% or less, about 20% or less, about 10% or less, or about 5% or less.

[0036] According to a tenth aspect, aquatic animal fibroblasts and associated biomass comprising said cells are described, wherein the fibroblasts contain desired fatty acids in an amount of at least about 0.5% by weight. Typically, the total lipid content of the cells is greater than about 2.0% by weight. It is preferred that the fibroblasts have low SFAs and contain a relatively high percentage of unsaturated fatty acids as a percentage of total fat. For example, the SFA content can be about 30% or less, about 20% or less, about 10% or less, or about 5% or less.

[0037] According to an eleventh aspect, an aquatic animal biomass is described comprising any one of the aquatic animal cells described herein alone or in any possible combination of different cell types.

[0038] According to a twelfth aspect, an aquatic animal cell culture food product is described, comprising any one of the aquatic animal cells described herein alone or in any possible combination of different cell types, and / or any one of the aquatic animal biomasses described herein. The aquatic animal cell culture food product may contain one, two, three, four or more cell types, such as myocytes, fibroblasts, adipocytes, endothelial cells, and any combination thereof. In other embodiments, the aquatic animal cell culture food product may contain one, two, three, four or more cell types, such as myocytes, myoblasts, fibroblasts, adipocytes, endothelial cells, epithelial cells, preadipocytes, keratinocytes, embryo-derived cells, induced pluripotent stem cells, mesenchymal stem cells, and any combination thereof. In a more preferred embodiment, the aquatic animal cell culture food product does not contain adipocytes. In a most preferred embodiment, the food product is a cell-cultured fish product.

[0039] According to a thirteenth aspect, an aquatic animal cell culture food product is described that includes any one of the aquatic animal cells described herein, the aquatic animal cells having a lipid content of at least 0.2% by weight, preferably more than about 2.0% by weight, for example, about 2.0% to about 90% by weight. In some embodiments, the cell culture food product described herein includes the fish cells, particularly white fish cells, described herein, that have a lipid (e.g., fatty acid) content of more than about 2.0% by weight. In some embodiments, the lipid (e.g., fatty acid) content is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, or about 70% to about 90%. In a preferred embodiment, the food product does not contain adipocytes. In a most preferred embodiment, the food product is a cell-cultured fish product.

[0040] According to a fourteenth aspect, an aquatic animal-based food product is described, comprising any one of the cells described herein having an omega-3 fatty acid content of at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% by weight. In a preferred embodiment, the food product does not comprise fat cells.

[0041] According to a fifteenth aspect, a composition for increasing lipid uptake and / or cell viability of aquatic animal cells and / or cell biomass is described. The composition comprises nervonic acid together with a suitable vehicle. In particular, nervonic acid may be present at a concentration of about 1 μg / ml or more, for example, about 1 μg / ml to about 10 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, and about 10 μg / ml.

[0042] Nervonic acid is about 10 μg / ml to about 50 μg / ml, for example, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, about 15 μg / ml, about 16 μg / ml, about 17 μg / ml, about 18 μg / ml, about 19 μg / ml, about 20 μg / ml, about 21 μg / ml, about 22 μg / ml, about 23 μg / ml, about 24 μg / ml, about 25 μg / ml, about 26 μg / ml, about 27 μg / ml, about 28 μg / ml, about 29 μg / ml, 35 μg / ml, about 36 μg / ml, about 37 μg / ml, about 38 μg / ml, about 39 μg / ml, about 40 μg / ml, about 41 μg / ml, about 42 μg / ml, about 43 μg / ml, about 44 μg / ml, about 45 μg / ml, about 46 μg / ml, about 47 μg / ml, about 48 μg / ml, about 49 μg / ml, or about 50 μg / ml.

[0043] According to a sixteenth aspect, methods and systems for uptake of lipids into aquatic animal cells are described, as well as related culture media, cells, cell biomass and cell culture food products obtainable therefrom.

[0044] The method involves culturing aquatic animal cells in the presence of vaccenic acid for a period of time and under conditions that result in the uptake of vaccenic acid by the aquatic animal cells.

[0045] The system comprises vaccenic acid in combination with aquatic animal cells and / or culture medium for use in combination in the methods for uptake of lipids into aquatic animal cells described herein.

[0046] The culture medium comprises a basal medium and an amount of vaccenic acid effective to effect lipid uptake by the aquatic animal cells.

[0047] The culture media, methods, and systems, and related compositions, cells, cell biomass, and cell culture foods described herein, in various embodiments, provide for increased lipid incorporation into aquatic animal cells, such as fish myoblasts, fibroblasts, and preadipocytes, which may have a controllable fat content of about 0.1% to about 90%, preferably about 2.5% to about 90%, e.g., about 2.5% to about 20%.

[0048] The culture media, methods, and systems, and associated compositions, cells, cell biomass, and cell cultured food products described herein, in various embodiments, allow for the lipid content in the cells of an aquatic animal and associated food products to be controlled by selecting one or more desired lipids, such as polyunsaturated fatty acids, more particularly omega-3 fatty acids, DHA, and EPA, as well as additional fatty acids identifiable by one of skill in the art.

[0049] The culture media, methods, and systems, and associated compositions, cells, cell biomass, and cell cultured foods described herein, in various embodiments, enable enhanced uptake of polyunsaturated fatty acids by aquatic animal cells, resulting in the production of cell cultured foods with high lipid content and increased levels of fatty acids, such as omega-3 fatty acids.

[0050] The compositions, methods, and systems, and associated cells, cell biomass, and cell-cultured foods described herein, in various embodiments, provide improved cell viability, enabling, for example, more rapid production of cell-cultured fish products with controlled fatty acid composition and levels.

[0051] The compositions, methods, and systems described herein, and associated cells, cell biomass, and cell-cultured foods, in various embodiments, provide for the production of cell-cultured foods comprising aquatic animal cells, particularly cell-cultured fish products, such as those that contain adequate levels of lipids without the use of fat cells (see, e.g., cell-cultured foods comprising white fish cells having a lipid content of about 2.0% to about 90% by weight, preferably greater than 2.0% to about 90% by weight).

[0052] The culture media, methods, and systems, and related compositions, cells, cell biomass, and cell culture foods described herein, in embodiments, enable increased viability, differentiation, and / or lipid uptake in cells of aquatic animals.

[0053] The culture media, methods, and systems, and related compositions, cells, cell biomass, and cell culture food products described herein may be practiced using culture media that do not require the presence of (and therefore may not include) dexamethasone, biotin, T3, pantothenate, IBMX, and / or insulin. Preferably, the culture media does not include at least one, and preferably all, of dexamethasone, biotin, T3, pantothenate, IBMX, and insulin.

[0054] The culture media, methods, and systems described herein, as well as the associated compositions, cells, cell biomass, and cell culture foods described herein, can be used in connection with a variety of applications in which cell viability, controlled growth, and lipid content in the cells and associated cell culture foods are desired. For example, the compositions, methods, and systems described herein, as well as the associated cells, cell biomass, and cell culture foods described herein, can be used to produce cell culture foods, such as foods with controlled lipid content. Thus, exemplary application areas include food manufacturing, food processing, and commercialization. Further exemplary applications include the use of the culture media, compositions, methods, and systems described herein, as well as the associated cells and cell biomass cell culture foods, in several fields, including basic biology research, applied biology, bioengineering, bioenergy, medical research, therapeutics, and additional fields that may be identified by one of skill in the art upon reading this disclosure.

[0055] For example, although aquatic animal cells are mentioned above, the media, methods and systems are generally suitable for culturing terrestrial animal cells and preparing cultured cell food products therefrom. The fatty acid profile of the cultured terrestrial animal cells may exhibit an increased omega-3 fatty acid content, a decreased saturated fatty acid content, or both.

[0056] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will become apparent from the description and drawings, and from the claims.

[0057] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the Detailed Description and Examples sections, serve to explain the principles and implementations of the present disclosure. Exemplary embodiments of the present disclosure will be more fully understood from the Detailed Description and the accompanying drawings. [Brief description of the drawings]

[0058] [Figure 1]1 shows micrographs showing the introduction of fatty acid mixture into silver carp preadipocytes in some embodiments. Carp preadipocytes were cultured in control medium or serum-reduced or serum-free medium containing 1% fatty acid mixture. LM shows Millipore Sigma Lipid Mixture 1000x, FBS shows fetal bovine serum. Bright field images show lipid accumulation on days 2 and 7 with characteristic cell rounding. [Figure 2A] Figure 1 illustrates the results of immunofluorescence staining of silver carp preadipocytes transfected with fatty acid mixture. Silver carp preadipocytes were cultured in serum-free lipid transfection medium for 7 days in the presence of control medium or lipid mixture. Cells were fixed and stained for nuclei (Hoechst), cytoskeletal protein F-actin (phalloidin), and lipid droplets (BODIPY). Fluorescence images show lipid transfection in treated conditions but not in control conditions. [Figure 2B] The fluorescent staining data shown in FIG. 2A was quantified and tabulated as the percentage of lipid uptake relative to total cell volume. [Figure 3A] 1 shows the introduction of a fatty acid mixture into bluefin tuna preadipocytes in some embodiments. Bluefin tuna preadipocytes were cultured in control medium or serum-reduced or serum-free medium containing 1% fatty acid mixture. Brightfield images show lipid accumulation on days 1 and 6 with characteristic cell rounding. The presence of lipid droplets is confirmed by intracellular BODIPY (green) staining, which is significantly enhanced under lipid treatment conditions when compared to the control. [Figure 3B] The fluorescent staining data shown in FIG. 3A was quantified and tabulated as the percentage of lipid uptake relative to total cell volume. [Figure 4] 1 shows the introduction of single fatty acids into silver carp preadipocytes in some embodiments. Silver carp preadipocytes were cultured in serum-reduced medium for 6 days in the presence of increasing concentrations of individual fatty acids (DHA, EPA, linoleic acid, or palmitoleic acid). Brightfield images show concentration-dependent lipid accumulation with characteristic cell rounding. Some concentration-related toxicity is observed due to cell number reduction in palmitoleic acid. [Figure 5A] Immunofluorescence staining of silver carp preadipocytes transfected with single fatty acids. Silver carp preadipocytes were cultured in serum-reduced medium for 6 days in the presence of increasing concentrations of individual fatty acids (DHA, EPA, linoleic acid, or palmitoleic acid). BODIPY staining of lipid droplets confirms that transfection increases with the concentration of DHA, EPA, and linoleic acid, while palmitoleic acid peaks at lower concentrations due to toxicity above 50 μg / ml. [Figure 5B] The fluorescent staining data shown in FIG. 5A was quantified and tabulated as the percentage of lipid incorporation relative to total cell volume. [Figure 6] FIG. 1 shows the introduction of a fatty acid mixture into Seriola fibroblasts in some embodiments. Seriola fibroblasts were cultured in control medium or serum-reduced medium containing 1% fatty acid mixture for up to 7 days. Brightfield microscopy images show lipid droplet accumulation on day 2 and that introduction was sustained through day 7. Control cultures do not show any lipid accumulation. [Figure 7A] Immunofluorescence staining of Seriola fibroblasts transfected with fatty acid mixture. Seriola fibroblasts were cultured for 7 days in control medium or serum-reduced medium containing 1% fatty acid mixture. BODIPY staining of lipid droplets confirms lipid accumulation in cells treated with lipid mixture-containing medium but not in control medium. [Figure 7B] The fluorescent staining data shown in FIG. 7A was quantified and tabulated as the fold increase in total cell volume compared to lipid-loaded controls. [Figure 8A] 1 shows the introduction of a fatty acid mixture into Seriola myoblasts in some embodiments. Seriola myoblasts were cultured in control medium or serum-reduced medium containing 1% fatty acid mixture. Brightfield images show that lipids are introduced into cells on day 2 and maintained through day 7 for cells cultured in the presence of lipid mixture, but not in control medium. Immunofluorescent staining of lipid droplets with BODIPY confirms lipid introduction and retention in treated cells on day 7. [Figure 8B]The fluorescent staining data shown in FIG. 8A was quantified and tabulated as the fold increase in total cell volume relative to lipid-loaded controls. [Figure 9A] Figure 1 shows the introduction of fatty acid mixture into mahi-mahi myoblasts in some embodiments.Mahi-mahi myoblasts are cultured in control medium or serum-reduced medium containing 1% fatty acid mixture.Bright field images show lipid introduction into cells by day 4 in the presence of fatty acid mixture.Fluorescent staining of cells with BODIPY confirms lipid accumulation in cells treated with lipid mixture but not in control cells. [Figure 9B] The fluorescent staining data shown in FIG. 9A was quantified and tabulated as the fold increase in total cell volume relative to lipid-loaded controls. [Figure 10A] 1 shows the introduction of a fatty acid mixture into bluefin tuna myoblasts in some embodiments. Bluefin tuna myoblasts were cultured in control medium or serum-reduced medium containing 1% fatty acid mixture. Brightfield images show that the fatty acid mixture is introduced into the cells by day 3 and maintained at day 6 in the presence of the fatty acid mixture. Fluorescent staining of the cells with BODIPY confirms lipid accumulation in cells treated with the lipid mixture but not in control cells. [Figure 10B] The fluorescent staining data shown in FIG. 10A was quantified and tabulated as the fold increase in total cell volume compared to lipid-loaded controls. [Figure 11A] 1 shows the introduction of individual saturated fatty acids into Seriola myoblasts in some embodiments. Seriola myoblasts were cultured in serum-reduced medium for 6 days in the presence of increasing concentrations of individual saturated fatty acids (lauric, myristic, palmitic, or stearic acid). Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. The highest lipid introduction is observed with lauric acid, which shows accumulation at all concentrations tested, with rounding and increased cell size at higher concentrations. Some lipid retention is observed with myristic, palmitic, and stearic acids, but only at concentrations above 50 μg / ml. [Figure 11B]The data shown in FIG. 11A was quantified and tabulated as a percentage of lipid uptake relative to total cell volume. [Figure 12A] 1 shows the introduction of individual monounsaturated fatty acids into Seriola myoblasts in some embodiments. Seriola myoblasts were cultured in serum-reduced medium for 6 days in the presence of increasing concentrations of individual saturated fatty acids (palmitoleic acid, oleic acid, vaccenic acid, or nervonic acid). Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. For palmitoleic acid, oleic acid, and vaccenic acid, introduction was observed at all concentrations, with increasing lipid concentration resulting in increased cell rounding and accumulation. No lipid introduction was observed with nervonic acid. [Figure 12B] The data shown in FIG. 12A was quantified and tabulated as a percentage of lipid uptake relative to total cell volume. [Figure 13A] Figure 1 shows the introduction of individual polyunsaturated fatty acids into Seriola myoblasts in some embodiments. Seriola myoblasts were cultured in serum-reduced medium for 6 days in the presence of increasing concentrations of individual saturated fatty acids (EPA, DHA, linolenic acid, or linoleic acid). Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. For linolenic acid, introduction was observed at all concentrations, with increasing lipid concentration resulting in increased cell rounding and accumulation. Lipid retention of EPA, DHA, and linoleic acid was only observed from 50 μg / ml, limiting the total introduction capacity. DHA showed some toxic effect at the highest concentration tested, as evidenced by lower cell numbers. [Figure 13B] The data shown in FIG. 13A was quantified and tabulated as a percentage of lipid uptake relative to total cell volume. [Figure 14A] 1 shows dose-dependent delivery of a complex fatty acid mixture into carp preadipocytes in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 14B]The data shown in Figure 14A was quantified and tabulated as percent lipid uptake relative to total cell volume, with the increase in lipid uptake ranging up to 40 percentage points higher than controls. [Figure 15A] 1 shows dose-dependent delivery of a complex fatty acid mixture into bluefin tuna preadipocytes in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 15B] The data shown in Figure 15A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 65 percentage points above controls. [Figure 16A] 1 shows dose-dependent delivery of a complex fatty acid mixture to lipidated bluefin tuna preadipocytes cultured in serum-free medium according to some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 16B] The data shown in Figure 16A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 60 percentage points above controls. [Figure 17A] 1 shows dose-dependent delivery of a complex fatty acid mixture into yellowfin tuna fibroblasts in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 17B] The data shown in Figure 17A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 45 percentage points above controls. [Figure 18A] 1 shows dose-dependent delivery of a complex fatty acid mixture into bluefin tuna fibroblasts in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 18B] The data shown in Figure 18A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 35 percentage points above controls. [Figure 19A] 1 shows dose-dependent delivery of a complex fatty acid mixture into bluegill fibroblasts in accordance with some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 19B] The data shown in Figure 19A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 35 percentage points above controls. [Figure 20A] 1 shows dose-dependent delivery of a complex fatty acid mixture into Mozambique tilapia brain-derived cells according to some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 20B] The data shown in Figure 20A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 25 percentage points above controls. [Figure 21A] 1 shows dose-dependent delivery of a complex fatty acid mixture into chicken embryo fibroblasts in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 21B] The data shown in Figure 21A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 35 percentage points above controls. [Figure 22A] 1 shows dose-dependent delivery of a complex fatty acid mixture into rabbit skin fibroblasts according to some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 22B] The data shown in Figure 22A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 35 percentage points above controls. [Figure 23A]1 shows dose-dependent delivery of a complex fatty acid mixture into red snapper myoblasts in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 23B] The data shown in Figure 23A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 20 percentage points above controls. [Figure 24A] 1 shows dose-dependent delivery of a complex fatty acid mixture into Mahi-Mahi myoblasts in accordance with some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 24B] The data shown in Figure 24A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 55 percentage points above controls. [Figure 25A] 1 shows dose-dependent delivery of a complex fatty acid mixture into bluefin tuna myoblasts in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 25B] The data shown in Figure 25A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 40 percentage points above controls. [Figure 26A] 1 shows dose-dependent delivery of a complex fatty acid mixture to lipid-loaded bluefin tuna myoblasts cultured in serum-free medium according to some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 26B] The data shown in Figure 26A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 30 percentage points above controls. [Figure 27A] 1 shows dose-dependent delivery of a complex fatty acid mixture into bovine satellite cells in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 27B] The data shown in Figure 27A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 15 percentage points higher than controls. [Figure 28A] 1 shows dose-dependent delivery of a complex fatty acid mixture into Atlantic salmon kidney epithelial cells according to some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 28B] The data shown in Figure 28A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 15 percentage points higher than controls. [Figure 29A] 1 shows dose-dependent delivery of a complex fatty acid mixture into rainbow trout gill epithelial cells in accordance with some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 29B] The data shown in Figure 29A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 45 percentage points above controls. [Figure 30A] 1 shows dose-dependent delivery of a complex fatty acid mixture into canine kidney epithelial cells in accordance with some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 30B] The data shown in Figure 30A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 35 percentage points above controls. [Figure 31A] 1 shows dose-dependent delivery of a complex fatty acid mixture into porcine kidney epithelial cells in accordance with some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 31B]The data shown in Figure 31A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 50 percentage points higher than controls. [Figure 32A] 1 shows dose-dependent delivery of a complex fatty acid mixture into fall armyworm epithelial cells in some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 32B] The data shown in Figure 32A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 20 percentage points higher than controls. [Figure 33A] 1 shows dose-dependent delivery of a complex fatty acid mixture into quail neural retina cells in accordance with some embodiments. Fluorescent images of cells stained with BODIPY confirm the accumulation of fatty acids as a function of concentration. [Figure 33B] The data shown in Figure 33A was quantified and tabulated as percent lipid uptake relative to total cell volume, with increases in lipid uptake ranging up to 30 percentage points higher than controls. [Diagram 34] Seriola myoblasts in control and serum-reduced medium. Seriola myoblasts cultured in control or serum-reduced medium for 6 days show no lipid uptake by bright field or fluorescent imaging. Staining with BODIPY showed no lipid accumulation in any condition. [Diagram 35] Seriola myoblasts in control medium without added fatty acids. Seriola myoblasts cultured in control medium for 6 days show no lipid uptake by brightfield or fluorescent imaging. Cells were stained with Hoechst for nuclei, Mitotracker for mitochondrial integrity, and phalloidin for F-actin cytoskeletal protein, but staining with BODIPY showed no lipid accumulation. [Diagram 36]Figure 1 shows transfection of Seriola myoblasts with chemically defined fatty acid combinations. The highest lipid content was observed in media 16, 22, and 25, as indicated by extensive BODIPY staining of lipid droplets. Comparable cell numbers and viability were observed compared to control conditions where no lipid transfection occurred. [Figure 37A] Plots depicting quantification of cell number following exposure to defined fatty acid combinations are shown. Seriola myoblasts grown in control media (media 28 and 29) or media containing defined fatty acid combinations (media 1-27) were quantified. Fluorescence intensity of each combination was quantified to assess effects on cell phenotype. Media 16, 22, and 25 showed comparable cell numbers and health to the control, and significantly higher lipid content. Increased lipid content was observed for most fatty acid combinations tested, with all three highest combinations containing the highest levels of nervonic acid tested. [Figure 37B] Plots depicting quantification of lipid incorporation following exposure to defined fatty acid combinations are shown. Seriola myoblasts grown in control media (media 28 and 29) or media containing defined fatty acid combinations (media 1-27) were stained with BODIPY for lipid droplets. Fluorescence intensity of each combination was quantified to assess effects on cell phenotype. Media 16, 22, and 25 showed comparable cell numbers and health to the control, and significantly higher lipid content. Increased lipid content was observed for most fatty acid combinations tested, with all three highest combinations containing the highest levels of nervonic acid tested. [Figure 37C]Plots depicting quantification of cell health following exposure to defined fatty acid combinations are shown. Seriola myoblasts grown in control media (media 28 and 29) or media containing defined fatty acid combinations (media 1-27) were stained with Mitotracker for mitochondrial health. Fluorescence intensity of each combination was quantified to assess effects on cell phenotype. Media 16, 22, and 25 showed comparable cell numbers and health to the control, and significantly higher lipid content. Increased lipid content was observed for most fatty acid combinations tested, with all three highest combinations containing the highest levels of nervonic acid tested. [Figure 38-1] Protective effect of nervonic acid (NA) on bluefin tuna myoblasts in the presence of toxic fatty acids (FA). A 70-fold increase in cell number is observed at a toxic concentration of 50 μg / mL DHA in the presence of nervonic acid. [Figure 38-2] Same as above. [Figure 39-1] Figure 1 shows the protective effect of nervonic acid on canine kidney cells in the presence of toxic fatty acids. A 5.5-fold increase in cell number is observed at a toxic concentration of 50 μg / mL DHA in the presence of nervonic acid. [Figure 39-2] Same as above. [Figure 40-1] Figure 2 shows the protective effect of nervonic acid on rabbit skin fibroblasts in the presence of toxic fatty acids. A 42-fold increase in cell number is observed at a toxic concentration of 50 μg / mL DHA in the presence of nervonic acid. [Figure 40-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Provided herein are compositions, methods, and systems, as well as associated cells, cell biomass, and cell culture foods, which in several embodiments enable improved growth and enhanced lipid incorporation in the cell biomass.

[0060] The cells and cell biomass may include cells derived from any animal defined herein as an organism of the kingdom Animalia. The cell-cultured food product may include cells derived from any animal except any member of the genus Homo. As used herein, the term "aquatic animal" refers to any animal, either vertebrate or invertebrate, that lives most or all of its life in water. Thus, an aquatic animal refers to an animal that exhales air or takes in dissolved oxygen in water through specialized organs called gills or directly through the skin. As will be understood by those skilled in the art, aquatic animals may live in fresh water (freshwater animals) or salt water (marine animals).

[0061] Exemplary aquatic animals include fish (gill-bearing aquatic cranial animals that lack digitated limbs) and Crustaceans (shell-bearing aquatic invertebrates that belong to the phyla Mollusca, Crustacea (Arthropoda), or Echinodermata).

[0062] In particular, aquatic animals in the sense of the present disclosure include fish such as cartilaginous fish, bony fish, ray-finned fish, lobe-finned fish, and seafood, such as various species of mollusks (e.g., bivalve mollusks such as clams, oysters, and mussels, and cephalopods such as octopus and squid), crustaceans (e.g., shrimp, crabs, and lobsters), and echinoderms (e.g., sea cucumbers and sea urchins). Such animals are composed of various cells with different morphologies and functions, such as myoblasts, myocytes, fibroblasts, adipocytes, preadipocytes, endothelial cells, stem cells, osteoblasts, osteocytes, keratinocytes, neurons, and other cells identifiable by one skilled in the art.

[0063] Exemplary aquatic animals include pangasius, flounder, hake, scup, smelt, rainbow trout, hard clams, blue crab, peekytoe crab, jumbo crab, cuttlefish, eastern oyster, Pacific oyster, anchovy, herring, golden mullet, moi, orange roughy, Atlantic perch, Lake Victoria perch, yellow perch, European oyster, Dover sole, sturgeon, tilefish, barracuda, yellowtail, sea urchin, Atlantic mackerel, sardine, black sea bass, European sea bass, hybrid striped bass, and the like. bass), bream, cod, drum, haddock, hoki, pollock, reef fish, pink salmon, snapper, tilapia, turbot, walleye, lake whitefish, wolffish, hard clams, cockles, jonah crab, snow crab, crayfish, mussels, Korean prawns, cod, Atlantic salmon, coho salmon, skate, Dungeness crab, king crab, purple mussel, green mussel, red shrimp, escolar, Chinook salmon, Chum salmon, American shad, alpine char, carp, catfish, John Dory, grouper, halibut, Japanese sablefish, koi These include horse mackerel, abalone, snails, stone crabs, American lobsters, spiny lobsters, octopus, black tiger shrimp, freshwater shrimp, Gulf shrimp, white shrimp, squid, barramundi, casque, dogfish, kingklip, mahi-mahi, sunfish, mako shark, swordfish, albacore tuna, yellowfin tuna, colts, squid lobsters, Magellan's mussels, mikazuki shrimp, barracuda, Chilean sea bass, cobia, croaker, eels, blue marlin, mullet, sockeye salmon, bluefin tuna, shrimp, crabs, lobsters, and echinoderms (e.g., sea cucumbers and sea urchins).Some preferred aquatic animals include yellowtail (e.g., Seriola lalandi), mahi-mahi (Coryphaena hippurus), red snapper (Lutjanus Campechanus), bluefin tuna (e.g., Thunnus orientalis and Thunnus thynnus), yellowfin tuna (Thunnus albacares), cod (e.g., Gadus morhua, Gadus Macrocephalus, Gadus ogac), flounder, halibut, herring, mackerel, trevally, salmon, sea bass, Patagonian toothfish (Dissostichus eleginoides), squid, clams, lobsters, crabs, scallops, shrimp, eels, bass (e.g., Micropterus salmoides), bluegill (Lepomis macrochirus), and carp (e.g., Hypophthalmichthys molitrix).

[0064] For example, the finned fish species may be a marine finned fish species or a freshwater finned fish species. Exemplary finned marine fish species include mahi mahi, bluefin tuna, Alaska pollock, albacore tuna, American shad, anchovy, alpine char, Atlantic mackerel, Atlantic perch, Atlantic salmon, barracuda, barramundi, bass, black sea bass, Atlantic marlin, bream, Chilean sea bass, Chinook salmon, chum salmon, cobia, cod, coho salmon, croaker, drum, casque, dogfish, John Dory, Dover sole, eel, escolar, European sea bass, flounder, grouper, Gulf shrimp, haddock, hake, halibut, herring, hoki, kingclip, golden cod, mackerel, mako shark, moi, scaup shark, mullet, sunfish, orange roughy, vannamei shrimp, Patagonian toothfish (Dissostichus eleginoides), pink salmon, trevally, rainbow trout, red snapper (Lutjanus campechanus), reef fish, sablefish, salmon, sardines, scup, sea bass, skate, smelt, snapper, sockeye salmon, sturgeon, swordfish, tilefish, turbot, wackefish, wolffish, yellowfin tuna, and Seriola quinqueradiata.

[0065] Exemplary freshwater finned fish include, but are not limited to, those selected from the group consisting of alpine char, barramundi, pangasius, bass, bluegill (Lepomis Macrochirus), bream, carp, catfish, croaker, drum, dogfish, bowfin, eel, freshwater shrimp, hybrid striped bass, Lake Victoria perch, lake whitefish, mullet, rainbow trout, salmon, smelt, sturgeon, tilapia, walleye, and yellow perch.

[0066] Exemplary crustacean species include shrimp, crabs, crayfish, and lobsters, such as, but not limited to, those selected from the group consisting of American lobster, black tiger shrimp, blue crab, Korean shrimp, crabs, crayfish, Dungeness crab, Jonah crab, red king crab, lobster, peachytoe crab, red shrimp, mikazuki shrimp, shrimp, snow crab, Japanese crab, spiny lobster, stone crab, and squid lobster.

[0067] Exemplary echinoderm species include, but are not limited to, those selected from the group consisting of sea cucumbers and sea urchins.

[0068] Exemplary cephalopod species include, but are not limited to, those selected from the group consisting of octopuses and squids.

[0069] Exemplary mollusc species include, but are not limited to, those selected from the group consisting of clams, oysters, mussels, abalone, mussels, blue mussels, cockles, clams, cuttlefish, eastern oysters, hard clams, Pacific oysters, European oysters, geoducks, green mussels, scallops, and cockles.

[0070] As used herein, the term "terrestrial animal" refers to an animal, either vertebrate or invertebrate, that lives most or all of its life outside of water. Terrestrial animals consume oxygen from the air, which is obtained by breathing through lungs, by entry of air into the trachea, or by other mechanisms known to those of skill in the art. Terrestrial animals include many mammals, birds, reptiles, amphibians, and insects, among others, known to those of skill in the art.

[0071] Exemplary terrestrial animals include, but are not limited to, armyworms, crickets, grasshoppers, frogs, toads, newts, salamanders, alligators, crocodiles, snakes, chickens, turkeys, ducks, geese, pheasants, game hen, quail, horses, rhinos, tapirs, cows, pigs, giraffes, camels, sheep, deer, goats, rabbits, dogs, and hippos.

[0072] Exemplary terrestrial insect species include, but are not limited to, those selected from the group consisting of armyworms, crickets, and grasshoppers.

[0073] Exemplary terrestrial amphibian species include, but are not limited to, those selected from the group consisting of frogs, toads, newts, and salamanders.

[0074] Exemplary terrestrial reptile species include, but are not limited to, those selected from the group consisting of alligators, crocodiles, and snakes.

[0075] Exemplary terrestrial bird species include, but are not limited to, those selected from the group consisting of chickens, turkeys, ducks, geese, pheasants, game hens, and quails.

[0076] Exemplary terrestrial mammalian species include, but are not limited to, those selected from the group consisting of horses, rhinos, tapirs, cows, pigs, giraffes, camels, sheep, deer, goats, rabbits, dogs, and hippos.

[0077] All animals, whether aquatic or terrestrial, are composed of a variety of cells with different morphologies and functions, such as myoblasts, myocytes, fibroblasts, adipocytes, preadipocytes, endothelial cells, epithelial cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, osteoblasts, osteocytes, keratinocytes, neurons, and other cells identifiable by one of skill in the art.

[0078] As used herein, an animal cell may be derived from more than one animal species, such as two, three, four or more aquatic and / or terrestrial animal species.

[0079] The term "myoblast" is a term of art that refers to the precursor of myocytes, also called muscle cells. As will be appreciated by those skilled in the art, myoblasts differentiate into muscle cells through myogenesis. Depending on the type of muscle cell they differentiate into, myoblasts can be classified as skeletal muscle myoblasts, smooth muscle myoblasts, and cardiac muscle myoblasts. Exemplary myoblasts from aquatic and terrestrial animals include skeletal muscle myoblasts and smooth muscle myoblasts.

[0080] The term "fibroblast" is a technical term referring to a type of cell found in animal connective tissues that synthesizes components of the extracellular matrix, such as collagen. Fibroblasts generate the structural framework of animal tissues and play a vital role in wound healing. Fibroblasts are the most abundant cell in animal connective tissues. Fibroblasts have a branched cytoplasm surrounding an oval, patchy nucleus with two or more nucleoli. Active fibroblasts can be recognized by their abundance of rough endoplasmic reticulum. Inactive fibroblasts, also called fibrocytes, are smaller, spindle-shaped, and have a reduced amount of rough endoplasmic reticulum. Fibroblasts are loosely scattered when they need to spread over a large space, but when they are crowded, they are often locally aligned in parallel clusters. Exemplary fibroblasts include those derived from muscle and other tissues, such as the brain, heart, or skin.

[0081] The term "adipocyte" is a technical term referring to adipose cells, also known as lipocytes. Adipocytes are cells that primarily compose adipose tissue and are specialized to store energy as fat. Adipocytes can be derived from mesenchymal stem cells that give rise to fat cells through adipogenesis. In cell culture, adipocytes can also form osteoblasts, myocytes, and other cell types. There are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT), also known as white fat and brown fat, respectively, and composed of two types of adipocytes. Adipocytes can arise either from preadipocytes present in adipose tissue or from bone marrow-derived precursor cells that migrate to adipose tissue. Cells as used herein typically include adipocytes derived from white adipose tissue.

[0082] The term "preadipocytes" is a term of art that refers to precursors of mature differentiated adipocytes that can be stimulated to form adipocytes. Preadipocytes can be isolated from subcutaneous or visceral adipose tissue of animals.

[0083] The preadipocytes can be grown in a preadipocyte growth medium that contains all the growth factors and supplements required for optimal growth of undifferentiated preadipocytes. For example, the preadipocytes can be grown in a preadipocyte growth medium that contains an endothelial cell growth supplement, epidermal growth factor, hydrocortisone, and / or heparin.

[0084] The formation of adipocytes from preadipocytes requires a tightly regulated cell differentiation process called adipogenesis, in which mesenchymal stem cells commit to preadipocytes and preadipocytes differentiate into adipocytes. The term "differentiate" or "differentiation" refers to the process of expression pattern change, in which pluripotent gene expression is converted to cell type specific gene expression. Transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT enhancer binding protein (C / EBP) are the main regulators of adipogenesis. As will be appreciated by those skilled in the art, differentiated adipocytes are characterized by growth arrest, morphological changes, high expression of adipogenic genes, and production of adipokines such as adiponectin, leptin, resistin (in mice, not in humans) and TNF-alpha.

[0085] In embodiments of the present disclosure, methods and systems for the composition and associated cells, cell biomass, and cell culture foods having controllable cellular lipid content and lipid uptake, and / or improved cell differentiation and / or cell viability associated with set lipid content and lipid uptake are described.

[0086] The term "lipid" is a term of art that refers to any organic compound that contains a linear or cyclic aliphatic chain of at least six carbon atoms and at least one oxygen or nitrogen atom bonded to one of the carbon atoms, and has a solubility of at least 20% by weight in ether or ethanol and 1% by weight or less in water at 20° C. As will be understood by those skilled in the art, lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, saccharolipids, and polyketides.

[0087] A "fatty acid" is a hydrophobic molecule consisting of a saturated or unsaturated aliphatic hydrocarbon chain terminating in a carboxylic acid moiety. A "saturated fatty acid" (SFA) does not contain a double bond. SFAs can be classified according to chain length, typically containing 4-22 carbon atoms. For example, exemplary SFAs can include lauric acid with 12 carbons, myristic acid with 14 carbons, palmitic acid with 16 carbons, stearic acid with 18 carbons, and capric acid with 10 carbons. Additionally, exemplary saturated fatty acids are well known in the art. An "unsaturated fatty acid" (UFA) contains one or more double bonds in the fatty acid chain. Unsaturated fatty acids can be classified based on the number of double bonds contained in the chain. UFAs include fatty acids with various numbers of double bonds at various positions along the carbon chain. For example, an unsaturated fatty acid is monounsaturated (MUFA) if it contains one double bond, or polyunsaturated (PUFA) if it contains two or more double bonds.

[0088] PUFA, as a class, contains many nutritionally important compounds such as essential fatty acids.Polyunsaturated fatty acids can be classified into groups based on the length of carbon skeleton, such as short-chain polyunsaturated fatty acids (SC-PUFA) with 16 or 20 carbon atoms and long-chain polyunsaturated fatty acids (LC-PUFA) with more than 18 carbon atoms.Polyunsaturated fatty acids can also be classified into methylene-interrupted polyenes, such as omega-3, omega-6 and omega-9, conjugated fatty acids, and other PUFAs based on chemical structure.

[0089] In particular, omega-3 fatty acids are polyunsaturated fatty acids (PUFAs) characterized by the presence of a double bond three atoms away from the terminal methyl group in their chemical structure. The three main omega-3 fatty acids are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

[0090] Unsaturated fatty acids can include fatty acids with various carbon chain lengths, from 12 to 22 carbons. Polyunsaturated fatty acids can include fatty acids with various carbon chain lengths, such as linoleic acid or α-linolenic acid with 18 carbons, EPA with 20 carbons, or DHA with 22 carbons. Monounsaturated fatty acids (MUFA) can include fatty acids with various carbon chain lengths, such as palmitoleic acid with 16 carbons, vaccenic acid with 18 carbons, oleic acid with 18 carbons, and nervonic acid with 24 carbons.

[0091] Exemplary unsaturated fatty acids include alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linoleylaidic acid, palmitoleic acid, vaccenic acid, oleic acid, nervonic acid, and others that can be identified by one of skill in the art. Additional exemplary SFAs, MUFAs, and PUFAs are known in the art and disclosed herein (see, e.g., Table 7 in Example 5).

[0092] Sterols are well known in the art and are steroids that have a hydroxyl group at the 3-position of the A ring. Thus, sterols include a fused tetracyclic core structure of steroids substituted at the A ring, as understood by those skilled in the art. Steroids can include 18 carbon (C18) steroids such as estrogens, C19 steroids such as androgens (e.g., testosterone and androsterone), C21 steroids such as progestogens, glucocorticoids, and mineralocorticoids, and secosteroids, including various forms of vitamin D, characterized by the truncation of the B ring of the core structure. Exemplary sterols include cholesterol and its derivatives, which are important components of membrane lipids, as well as glycerophospholipids and sphingomyelin, phytosterols (e.g., β-sitosterol, stigmasterol, and brassicasterol), ergosterol, and additional sterols that can be identified by those skilled in the art.

[0093] Phospholipids are well known in the art and include, for example, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, etc. Phospholipids are present in lecithin and can be extracted from lecithin.

[0094] Glycerolipids are well known in the art and are composed of mono-, di- and tri-substituted glycerol.Therefore, glycerolipids can include monoglycerides, diglycerides and triglycerides.In particular, triglycerides are fatty acid triesters of glycerol, in which the three hydroxyl groups of glycerol are typically esterified with different fatty acids.As will be understood by those skilled in the art, triglycerides are triesters of glycerol bound to three fatty acid molecules.As will be understood by those skilled in the art, triglycerides can be classified into saturated and unsaturated types.

[0095] In embodiments of the present disclosure, culture media, methods, and systems for culturing aquatic animal cells, and aquatic animal cells obtainable and / or obtained thereby, are described. In embodiments of the present disclosure, culture media, methods, and systems for culturing terrestrial animal cells, and aquatic animal cells obtainable and / or obtained thereby, are described. In embodiments of the present disclosure, culture media, methods, and systems for culturing aquatic or terrestrial animal cells, and aquatic or terrestrial animal cells obtainable and / or obtained thereby, are described.

[0096] As used herein, the term "culture medium" refers to a liquid, solid, or gelled composition containing organic, inorganic, and / or biological materials in which cells can live, remain viable, or grow. Culture media typically include a basal medium.

[0097] The term "basal medium" as used herein, as will be understood by those skilled in the art, refers to a culture medium that contains components essential for cell survival and growth, such as amino acids, glucose, and ions such as calcium, magnesium, potassium, sodium, and phosphate.

[0098] An example of a basal medium is Basal Media Formulation (www.sigmaaldrich.com / life-science / cell-culture / learning-center / media-formulations / basal.html). Further examples may be identifiable by one of skill in the art.

[0099] Exemplary biosources include serum. Media can be chemically defined. For example, Lipid Mixture 1 available from Sigma Aldrich (www.sigmaaldrich.com / catalog / product / sigma / l0288?lang=en®ion=US) contains non-animal derived fatty acids (2 μg / ml arachidonic acid, and 10 μg / ml each of linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, and stearic acid), 0.22 mg / ml New Zealand wool cholesterol, 2.2 mg / ml Tween®-80, 70 μg / ml tocopherol acetate, and 100 mg / ml Pluronic® F-68, dissolved in cell culture water.

[0100] A medium in the sense of the present disclosure may have a biological source including fetal bovine serum (FBS) or cod liver oil fatty acids. For example, Lipid Mixture (1000x) available from Sigma Aldrich (www.sigmaaldrich.com / catalog / product / sigma / l5146?lang=en®ion=US) contains 4.5 g / L cholesterol, 10 g / L cod liver oil fatty acids (methyl esters), 25 g / L polyoxyethylene sorbitan monooleate, and 2.0 g / L D-α-tocopherol acetate.

[0101] Embodiments of the present disclosure are based on the surprising discovery that lipids in culture media have different degrees of toxicity to aquatic animal cells, with MUFAs being non-toxic, whereas PUFAs, SFAs, and / or sterols are toxic as they reduced cell viability and / or proliferation compared to control media.

[0102] One way to detect toxicity in cell cultures is to detect the percentage of the area covered by cells under test conditions (e.g., cultures containing one or several lipids and / or other components of interest) compared to a control after a desired culture period. This can be achieved using any suitable method, for example by obtaining images, e.g., photomicrographs, of the culture vessel with attached cultured cells and calculating the area covered by the cultured cells using a suitable image processing and analysis program, such as ImageJ. In one example, when testing the introduction of lipids into cells, the lipid is considered to be slightly toxic if the cell-covered surface in the culture containing the test lipid is about 61-79% of the cell-covered surface in the control culture, and the lipid is considered to be toxic if the cell-covered surface is 60% or less of the cell-covered surface in the control culture.

[0103] Embodiments of the present disclosure are based on the surprising discovery that the addition of an effective amount of nervonic acid to culture medium for aquatic animal cells containing desired lipids (e.g., PUFAs, SFAs, and / or sterols) enables uptake of the desired lipids by the cells while reducing or even minimizing the associated levels of lipotoxicity to the cells.

[0104] "Nervonic acid" is known in the art and is a monounsaturated analog of lignoceric acid with the formula C24H46O2 and the IUPAC name (Z)-tetracos-15-enoic acid. As will be appreciated by those skilled in the art, nervonic acid is also known as selacholeate, nervonsaeure, cis-15-tetracosenoic acid, 24:1 cis, delta 15, or 24:1 omega 9. Nervonic acid is a cerebroside, a glycosphingolipid lipid. Nervonic acid has one double bond in the fatty acid chain, and all remaining carbon atoms are single bonds.

[0105] In embodiments, the present disclosure relates to a culture medium for culturing aquatic animal cells, such as myoblasts, myocytes, preadipocytes, adipocytes, and / or fibroblasts. The medium is a basal medium (preferably serum-free) supplemented with one or more lipids, such as PUFAs, SFAs, sterols, or any combination of PUFAs, SFAs, and / or sterols. Such a medium can be used, for example, to expand the cell population in culture through proliferation and / or to induce cell differentiation. Advantageously, the lipid content of the cultured cells can be altered using a medium supplemented with one or more lipids. For example, as described and exemplified herein, the relative amounts of desired fatty acids, such as palmitic acid, vaccenic acid, oleic acid, alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and any combination of the foregoing, may be increased in cells cultured in medium supplemented with these fatty acids compared to the same cells in which they occur naturally (i.e., as components of isolated aquatic animals; as components of isolated terrestrial animals) or the same cells cultured in medium without lipid supplements. Each desired lipid is typically present in the medium at a concentration of at least about 0.01 μg / ml. In examples, each desired lipid is present in the medium at a concentration of about 0.01 μg / ml to about 1000 μg / ml, about 0.01 μg / ml to about 100 μg / ml, about 0.1 μg / ml to about 100 μg / ml, about 1.0 μg / ml to about 100 μg / ml, about 10 μg / ml to about 200 μg / ml, about 10 μg / ml to about 150 μg / ml, and preferably about 10 μg / ml to about 100 μg / ml, or about 10 μg / ml to about 50 μg / ml. As described herein, some lipids (e.g., fatty acids) are toxic to cultured cells derived from aquatic animals at high concentrations. One skilled in the art would be able to identify the toxicity level of a lipid of interest and adjust the concentration of that lipid to achieve the desired degree of cellular lipid uptake, proliferation, and / or survival. One of skill in the art would be able to identify the toxicity level of a lipid of interest, a cell type of interest, and / or a cellular species of interest, and adjust the concentration of the lipid to achieve the desired degree of cellular lipid uptake, proliferation, and / or survival.

[0106] The media, methods, and systems disclosed herein can be used to create cells with a desired lipid content, for example, to enhance desired attributes such as flavor, aroma, shelf life, and / or nutrient content. For example, the media, methods, and systems disclosed herein can be used to create aquatic animal foods, such as the cells and seafood products disclosed herein, that contain the same or similar total fat content as that of wild-caught animals of the same species (i.e., the same type of cells, fillets, etc. as the wild-caught animals), but that contain less saturated fat as a percentage of total fat and / or by weight of the food. In one aspect, the media, methods, and systems disclosed herein can be used to create terrestrial animal foods, such as the cells and terrestrial animal foods disclosed herein, that contain the same or similar total fat content as that of captive and / or farmed animals of the same species (i.e., the same type of cells, fillets, etc. as the captive or farmed animals), but that contain less saturated fat as a percentage of total fat and / or by weight of the food. In an example, the disclosure relates to food products that can provide a particular health benefit, including foods that have a higher unsaturated fatty acid content and a lower saturated fatty acid content, for example, by increasing the amount of oleic acid and / or omega-3 fatty acids (by weight and / or as a percentage of total fat) compared to wild-caught seafood of the same species. In an example, the disclosure relates to food products that can provide a particular health benefit, including foods that have a higher unsaturated fatty acid content and a lower saturated fatty acid content, for example, by increasing the amount of oleic acid and / or omega-3 fatty acids (by weight and / or as a percentage of total fat) compared to wild-caught animals and / or farmed animals of the same species. Such foods are associated with particular health benefits, including reduced risk of cardiovascular disease. In another example, the disclosure relates to food products that have a phosphatidylserine content (by weight and / or as a percentage of total fat) compared to wild-caught seafood of the same species.In another example, the disclosure relates to food products having a phosphatidylserine content (by weight and / or as a percentage of total fat) relative to wild-caught and / or farmed animals of the same species, such foods being associated with a reduced risk of cognitive impairment or dementia.

[0107] In other examples, the media, methods, and systems disclosed herein can be used to produce cells and aquatic animal foods, such as seafood products, disclosed herein, with desired sensory attributes. In other examples, the media, methods, and systems disclosed herein can be used to produce cells and terrestrial animal foods, such as terrestrial animal foods, disclosed herein, with desired sensory attributes. For example, pleasant aroma is related to the fatty acid content, particularly the free fatty acid and PUFA content. Thus, the free fatty acid and PUFA content can be adjusted using the media and methods disclosed herein to achieve a desired aroma profile. Similarly, the lipid content can be varied to achieve a desired flavor profile, for example, by changing the omega-3 fatty acid content to make a seafood product more or less fishy. Other sensory attributes, such as mouthfeel and texture, cookability / moistness, and appearance, can also be adjusted by adjusting the lipid content of the cells. See, for example, Rosa et al., Nutrients 2020, 12, 3453; doi:10.3390 / nu12113454, on the sensory attributes and lipid composition of seafood. Similar considerations apply to terrestrial animal foods.

[0108] As described and exemplified herein, it has surprisingly been discovered that the inclusion of nervonic acid in media supplemented with lipids (e.g., fatty acids) promotes lipid uptake by cultured aquatic animal cells, including at concentrations that would otherwise be toxic to the cultured cells. This was a surprising discovery, in part, because aquatic animal cells did not substantially take up nervonic acid when cultured in media supplemented with nervonic acid but not other fatty acids. As described and exemplified herein, nervonic acid was the only MUFA tested that was not substantially taken up by aquatic animal cells cultured in media supplemented with the test MUFA. Without wishing to be bound by any particular theory, it is believed that nervonic acid promotes the uptake of other fatty acids and reduces or blocks fatty acid toxicity in cultured aquatic animal cells, such as fish cells.

[0109] Thus, in a preferred embodiment, the medium further comprises nervonic acid in an amount effective to increase the uptake of lipids (e.g., PUFAs, MUFAs, SFAs, and / or sterols) by the cultured aquatic animal cells and / or in an amount effective to reduce the toxic effects of PUFAs, MUFAs, SFAs, and / or sterols on the cultured aquatic animal cells, e.g., in an amount sufficient to improve the viability and / or growth of the cells in the culture. Typically, nervonic acid is included in the medium at a concentration of at least about 1 μg / ml, preferably from about 1 μg / ml to about 1000 μg / ml, from about 1 μg / ml to about 200 μg / ml, from about 1 μg / ml to about 150 μg / ml, and preferably from about 1 μg / ml to about 100 μg / ml, from about 1 μg / ml to about 50 μg / ml, from about 10 μg / ml to about 50 μg / ml, from about 50 μg / ml to about 100 μg / ml, or from about 50 μg / ml to about 75 μg / ml. In further embodiments, the medium described herein (e.g., but not necessarily limited to, medium supplemented with an effective amount of nervonic acid and / or one or more other lipids (e.g., PUFAs, MUFAs, SFAs, sterols, and combinations thereof)) is further supplemented with an antioxidant. When aquatic animal cells, such as fish myoblasts, myocytes, preadipocytes, adipocytes, fibroblasts, etc., are cultured in such media, the cells can take up lipids and antioxidants. This can reduce lipid oxidation in the cultured cells, which can contribute to loss of food quality of cells, particularly cells rich in unsaturated fatty acids such as PUFAs and MUFAs produced using the methods disclosed herein. Lipid oxidation in cells cultured according to the methods described herein can contribute to changes in flavor profile, fishiness, shortened shelf life, and reduced nutritional value. Thus, in these embodiments, the medium contains an effective amount of antioxidant to reduce or prevent lipid oxidation in the cultured cells, and a product containing the cultured cells. The effective amount of antioxidant in the culture is typically an amount of about 10 ng / ml to about 1000 mg / ml, depending on the particular antioxidant selected, and can be determined using a suitable method for any desired lipid.For example, the lipid oxidation product malondialdehyde (MDA) can be assessed in cells using suitable methods and expressed as thiobarbituric acid reactive substances (TBARS; μg MDA per mg cells). See, e.g., Secci, G. and Parisi, G. Italian Journal of Animal Science 15(1):124-136 (2016).

[0110] Antioxidants suitable for use in the media described herein include, for example, ascorbic acid, mitoquinol, creatine, pinostrobin, catalase, N-acetylcysteine, thiazolidinedione, lipoic acid, butylated hydroxyanisole, baicalein, epicatechin gallate, rutin, myricetin, apigenin, sautinone, propionyl-L-carnitine, tocopherols (including alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), te rt-Butylhydroquinone (TBHQ), phenols, carotenoids, anoxomers, dilauryl thiodipropionate, resveratrol, ethoxyquin, propyl gallate, 2,4,5-trihydroxybutyrophenone (THBP), quercetin, carnosol, thymol, catechin, morin, proanthocyanidin dimer B2, algae extracts, plant extracts (e.g., rosemary extract, grape seed extract, green tea extract, ginsing extract, blueberry extract, wolfberry extract), and the like, as well as combinations thereof. Tocopherols, such as alpha-tocopherol, are generally the preferred antioxidants.

[0111] Thus, in the embodiments described herein, the culture medium for aquatic animal cells comprises a basal medium supplemented with one or more lipids each in an amount of at least about 0.01 μg / ml, and when the one or more lipids are or include polyunsaturated fatty acids, saturated fatty acids, and / or sterols, the medium further comprises nervonic acid at a concentration of at least about 1 μg / ml. Preferably, each lipid supplement is present in the medium at a concentration of about 0.1 μg / ml to about 1000 μg / ml, preferably about 0.1 μg / ml to about 500 μg / ml, about 1 μg / ml to about 100 μg / ml, or about 5 μg / ml to about 50 μg / ml. Preferably, the total concentration of lipids in the culture medium is about 1 mg / ml or less, for example, the total concentration of lipids in the culture medium can be about 0.5 mg / ml or less or about 0.1 mg / ml or less.

[0112] In some embodiments, the culture medium may contain individual fatty acids or mixtures of fatty acids at a total concentration of 1 mg / ml of total fatty acids in the lipid-loaded culture. Table 1 below provides an exemplary list of types and ranges of fatty acids that can be included in lipid-loaded culture medium. [Table 1-1] [Table 1-2]

[0113] In some embodiments, culture medium methods and systems are described that allow for the incorporation of MUFAs at concentrations of at least 10 μg / ml or greater, particularly at concentrations between 10 μg / ml and 1000 μg / ml.

[0114] In some embodiments, the lipids included in the lipid induction medium are monounsaturated fatty acids (MUFAs) provided at concentrations between 100 ng / ml and 1 mg / ml that are non-toxic to naturally occurring fish cells.

[0115] Exemplary MUFAs that can be used in the culture medium methods and systems described herein include myristoleic acid (C14:1 ω-5), palmitoleic acid (C16:1 ω-7), sapienic acid (C16:1 ω-10), vaccenic acid (C18:1 ω-7), C18:1 ω9c, found in most phospholipids, oleic acid (C18:1 ω-9), petroselinic acid (C18:1 ω-12), paulic acid (C20:1 ω-7), gondoic acid (C20:1 ω-11), erucic acid (C22:1 ω-9c), brassicic acid (C22:1 ω-9t), nervonic acid (C24:1 ω-9), and any desired combinations thereof.

[0116] In some embodiments, MUFAs that can be used in the culture medium methods and systems described herein include MUFAs such as palmitoleic acid provided at a concentration of 50-100 μg / ml, MUFAs such as vaccenic acid provided at a concentration of 50-75 μg / ml, MUFAs such as oleic acid provided at a concentration of 75-100 μg / ml, and MUFAs such as nervonic acid provided at a concentration of 50-75 μg / ml.

[0117] In some embodiments, the lipids included in the lipid induction medium are polyunsaturated fatty acids (PUFAs) provided at concentrations that are toxic to naturally occurring fish cells when combined with nervonic acid, but are non-toxic. In these embodiments, the PUFAs are included at concentrations of at least 10 μg / ml or more, particularly between 10 μg / ml and 1000 μg / ml.

[0118] Exemplary PUFAs that can be used in connection with the culture medium methods and systems described herein include hexadecatrienoic acid (HTA) (C16:3 ω-3), linoleic acid (C18:2 ω-6), alpha linolenic acid (C18:3 ω-3), gamma linolenic acid (C18:3 ω-6), stearidonic acid (C18;4 ω-4), eicosadienoic acid (C20:2 ω-6), eicosatrienoic acid (ETE) (C20:3 ω-3), dimorpho-gamma-linolenic acid (C20:3 ω-6), mead acid (C20:3 ω-9), arachidonic acid (C20:4 ω-6), eicosapentaenoic acid (EPA) (C20:5 ω-3, C20:5 ω-6), and the like. omega-6), heneicosapentaenoic acid (HPA) (C21:5 omega-3), docosatetraenoic acid (C22:4 docosatetraenoic acid-6), DPA (C22:5 docosatetraenoic acid-3), docasahexaenoic acid (DHA) (C22:6 docasahexaenoic acid (DHA)-3), tetracosapentaenoic acid (C24:5 omega-3), and tetracosahexaenoic acid (herring acid) (C24:6 tetracosahexaenoic acid (herring acid) (C24:6 omega-3)), and any combination thereof.

[0119] In some embodiments, the PUFAs are provided in conjunction with the culture medium methods and systems described herein at a concentration between 100 ng / ml and 1 mg / ml. In some embodiments, the PUFAs can be provided in conjunction with the culture medium methods and systems described herein, for example, linoleic acid is provided at a concentration between 50 and 75 μg / ml, PUFAs such as alpha-linolenic acid are provided at a concentration between 50 and 100 μg / ml, PUFAs such as eicosapentaenoic acid (EPA) are provided at a concentration between 10 and 75 μg / ml, and PUFAs such as docasahexaenoic acid (DHA) are provided at a concentration between 10 and 25 μg / ml.

[0120] In some embodiments, the lipid contained in the lipid introduction medium is an SFA provided at a concentration that is toxic to naturally occurring fish cells when alone, particularly at a concentration of at least 10 μg / ml or more, more particularly at a concentration of 10 μg / ml to 1000 μg / ml, but which is non-toxic when combined with nervonic acid.

[0121] Exemplary SFAs that can be used in connection with the culture medium methods and systems described herein include capric acid (C10:0), undecylic acid (C11:0), lauric acid (C12:0), tridecylic acid (C13:0), myristic acid (C14:0), pentadecylic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), nonadecylic acid (C19:0), arachidic acid (C20:0), heneicosylic acid (C21:0), behenic acid (C22:0), tricosylic acid (C23:0), lignoceric acid (C24:0), and any desired combinations thereof.

[0122] In some embodiments of the culture medium methods and systems described herein, SFAs may be provided at concentrations between 100 ng / ml and 1 mg / ml, in some embodiments, SFAs such as lauric acid, myristic acid, and stearic acid are preferably provided at concentrations between 25 and 75 μg / ml, and SFAs such as palmitic acid are provided at concentrations between 25 and 50 μg / ml.

[0123] In some embodiments, the culture medium methods and systems described herein can be performed in the presence of one or more lipids (e.g., any one of the lipids described herein, alone or in any combination) in an amount of at least 10 μg / ml and can be used to achieve lipid induction, increased survival, and / or cell differentiation of one or more cell types of an aquatic animal.

[0124] In some embodiments, the culture medium methods and systems described herein can be performed in the presence of one or more lipids (e.g., any one of the lipids described herein, alone or in any combination) in an amount of at least 10 μg / ml and can be used to achieve lipid induction, increased viability, and / or cell differentiation of one or more cell types of terrestrial animals.

[0125] In particular, in some embodiments, the culture medium methods and systems described herein may include differentiation and lipid-induction media that function simultaneously to control cell differentiation and the lipid content of cells.

[0126] In some embodiments, the culture medium methods and systems described herein may include a cell culture medium comprising one or more fatty acids at a concentration between 25 μg / ml and 1000 μg / ml, preferably between 25 μg / ml and 100 μg / ml.

[0127] In some embodiments, the culture medium methods and systems described herein may include cell culture medium comprising nervonic acid at a concentration between 10 μg / ml and 1000 μg / ml, preferably between 10 μg / ml and 100 μg / ml.

[0128] In some embodiments, the culture medium methods and systems described herein may include cell culture medium comprising monounsaturated fatty acids at a concentration between 10 μg / ml and 1000 μg / ml, preferably between 10 μg / ml and 100 μg / ml.

[0129] In some embodiments, the culture medium methods and systems described herein may include a cell culture medium comprising linoleic acid, alpha linolenic acid, vaccenic acid, and palmitoleic acid at concentrations between 10 μg / ml and 50 μg / ml each.

[0130] In some embodiments, the culture medium methods and systems described herein may include a cell culture medium comprising linoleic acid, alpha linolenic acid, vaccenic acid, and palmitoleic acid at a concentration of less than 10 μg / ml each.

[0131] In some embodiments, the culture medium methods and systems described herein may include cell culture medium comprising omega-s polyunsaturated fatty acids at a concentration between 10 μg / ml and 50 μg / ml.

[0132] In some embodiments, the culture medium methods and systems described herein may include a cell culture medium comprising a basal medium and serum at a concentration of 4-10%.

[0133] In some embodiments, the methods and systems described herein may be implemented to increase the viability of cells in an aquatic animal cell biomass by culturing the cell biomass in the presence of a toxic fatty acid in the presence of an effective amount of nervonic acid. In these embodiments, nervonic acid is typically provided at a concentration of 10-1000 μg / ml, preferably 10-100 μg / ml, preferably 50-75 μg / ml.

[0134] In some embodiments, the culture media described herein are configured to provide a controllable set of lipids that are taken up by the cells of an aquatic animal within the meaning of the present disclosure.

[0135] In some embodiments, the culture media described herein are configured to provide a controllable set of lipids that are taken up by the cells of a terrestrial animal.

[0136] In some embodiments, the cells are cultured in the medium in the presence of serum. The term "serum" as used herein refers to the liquid fraction of whole blood collected after the blood is allowed to clot. The clot can be removed, for example, by centrifugation, and the resulting supernatant is referred to as serum. Serum can be provided at concentrations of 0% to 4%, or higher as needed. Serum suitable for culturing cells derived from aquatic animals is well known and includes bovine serum, such as fetal bovine serum (FBS).

[0137] It is well known that similar sera are suitable for culturing cells derived from terrestrial animals.

[0138] In some embodiments, the cells are cultured in a medium that is serum-free. In some embodiments, the cells described herein are cultured in a serum-free differentiation medium and / or lipid-loading medium.

[0139] In some embodiments, the culture medium methods and systems described herein can include cell culture media used for the survival and growth of cells derived from aquatic animals according to embodiments of the present disclosure, typically comprising a basal medium and optionally 4-10% serum depending on the species of origin of the cells.

[0140] In some embodiments, the culture medium methods and systems described herein can include cell culture media used for the survival and growth of cells derived from terrestrial animals according to embodiments of the present disclosure, typically comprising a basal medium and optionally 4-10% serum depending on the species of origin of the cells.

[0141] In some embodiments, the lipid-introducing medium used for the incorporation of lipids into cells described herein typically comprises a basal medium containing 0-4% serum in combination with one or more lipids to be introduced into the cells. In some embodiments, the medium contains the desired lipids at a concentration of at least 10 μg / ml, and the cells are cultured in the medium for about 6 or 7 days. If desired, the medium can contain lower concentrations of lipids (e.g., 1-2 μg / ml of each desired lipid), and the cells are cultured in the medium for a period of time to achieve the desired degree of lipid incorporation, which in embodiments is longer than 6 or 7 days. As will be appreciated by those of skill in the art, typically the lipids in the medium comprise fatty acids, such as a single fatty acid or a mixture of fatty acids including saturated, monounsaturated, and / or polyunsaturated fatty acids.

[0142] The present disclosure also relates to a differentiation medium used for cell differentiation according to embodiments of the present disclosure, which typically includes a basal medium containing 0-4% serum. In certain embodiments, dexamethasone, biotin, T3, pantothenate, IBMX, and insulin are not supplemented in the differentiation medium, as they are typically used in the medium to introduce desired lipids into undifferentiated cells such as myoblasts and preadipocytes. Compared to previous protocols that showed limited lipid introduction over the 14-day differentiation protocol period, differentiation and lipid introduction medium without dexamethasone, biotin, T3, pantothenate, IBMX, and insulin can generate fish cells with morphological changes from elongated to round to store large lipid droplets and increased cell size.

[0143] For example, as shown in Example 1, differentiation of preadipocytes into adipocytes in serum-reduced or serum-free medium in the presence of a complex fatty acid mixture and in the absence of dexamethasone, biotin, T3, pantothenate, IBMX, and insulin resulted in the uptake and storage of fatty acids in cultured carp preadipocytes (Example 1 and Figure 1). Notably, carp preadipocytes in the presence of fatty acids achieved morphological changes in 2 days that were maintained throughout the 7-day culture period.

[0144] In embodiments, the cells used in the disclosed methods and systems can be primary cells isolated from a desired aquatic species or a cell line derived from a desired aquatic species. In embodiments, the cells used in the disclosed methods and systems can be primary cells isolated from a desired terrestrial animal species or a cell line derived from a desired terrestrial animal species. Preferably, the cells are not genetically modified. In some embodiments, the aquatic animal cells can be harvested from any desired aquatic animal, particularly any fish, mollusc, and crustacean, using any suitable method. Similarly, in some embodiments, the terrestrial animal cells can be harvested from any desired terrestrial animal, such as any mammal, bird, reptile, amphibian, or insect, using any suitable method. As will be appreciated by those skilled in the art, many methods are well known in the art, such as enzymatic and mechanical dissociation of tissues. For example, detailed information on how to isolate preadipocytes from fish can be found in Vegusdal et al 2003, "An in vitro method for studying the proliferation and differentiation of Atlantic salmon preadipocytes", as will be appreciated by those skilled in the art. Information regarding the isolation of other cell types from fish, or any other animal, would be readily available to one of skill in the art.

[0145] As used herein, harvested cells can be grown in adherent 2D tissue culture or 3D cell suspension culture, as will be appreciated by those of skill in the art.

[0146] In the embodiments described herein, the aquatic animal derived harvested cells used herein are then cultured in a medium for proliferation, differentiation, and / or lipid incorporation, as will be understood by those skilled in the art.

[0147] In the embodiments described herein, the terrestrial animal derived harvested cells used herein are then cultured in a medium for proliferation, differentiation, and / or lipid incorporation, as will be understood by those of skill in the art.

[0148] Embodiments of the present disclosure also include cells obtained using any one of the methods and systems described herein.

[0149] In particular, the cells obtained after culturing in the lipid-introducing medium described herein contain increased amounts of desired lipids compared to cells taken directly from the corresponding live species. The amount of desired lipids in cells cultured in lipid-introducing medium is a function of the time of culturing, the concentration of lipids added to the medium, and the viability and growth of the cells. As described herein, the inclusion of nervonic acid in the medium enhances the uptake of lipids in aquatic animals. Typically, the cells will contain at least about twice the amount of desired lipids measured as g per g of total fat compared to cells of the corresponding aquatic animal species. Typically, the cells will contain at least about twice the amount of desired lipids measured as g per g of total fat compared to cells of the corresponding animal species. For example, myoblasts, myocytes, preadipocytes, adipocytes, or fibroblasts from a desired fish species cultured in a lipid-induction medium containing omega-3 fatty acids (e.g., EPA, DPA, and / or DHA) according to the present disclosure may contain about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 100-fold the amount of omega-3 fatty acids compared to the corresponding cells or meat of a wild-caught fish of the same species.

[0150] In embodiments, the cells are at least about 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, In some embodiments, the lipid composition may contain a desired percentage of lipid per gram of total fat that is 4, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 percentage points higher. (For clarity, a number of percentage points higher means that the percentage of total fat accounted for by the desired lipid is that number of percentage points higher.) By way of example, if the percentage of total fat accounted for by the desired lipid in a wild caught fish is 25% and the percentage of total fat accounted for by the desired lipid in cultured fish cells is 30%, then the cultured cells contain 5 percentage points more of the desired lipid. As a further example, if the percentage of total fat accounted for by the desired lipid in a farmed steer, bull, cow, or heifer is 0% and the percentage of total fat accounted for by the desired lipid in cultured bovine cells is 5%, then the cultured cells similarly contain 5 percentage points more of the desired lipid.

[0151] Additionally, for foods intended to resemble fillets, cuts, organs, or other foods that are not comprised of whole wild-caught or farmed animals, the comparison is made between the cultured cells and the animal fillets, cuts, organs, etc., rather than to whole animal carcasses.

[0152] Suitable methods for determining the fatty acid content of cells, meat, and other foods are well known in the art. For example, fatty acids can be extracted by hydrolysis methods. The fat is then extracted into ether and methylated to form fatty acid methyl esters (FAMEs). The FAMEs can then be quantitatively analyzed by gas chromatography (GC), with peaks representing each quantified fatty acid. Conveniently, lipid extraction kits are commercially available for extracting lipids from cells, body fluids, tissues, and the like. Lipids extracted from fish or cells using such kits or other suitable methods can then be analyzed and quantified using any suitable method, such as GC, HPL, mass spectrometry, and lipidomics. Lipids extracted from terrestrial animals or cells using such kits or other suitable methods can then be analyzed and quantified using any suitable method, such as GC, HPL, mass spectrometry, and lipidomics.

[0153] Tables 2A and 2B below list representative fatty acid profiles for naturally occurring aquatic animals, including fish, crustaceans, and mollusks. Tables 2C and 2D below list representative fatty acid profiles for naturally occurring terrestrial animals, including poultry, game, and farmed livestock. Data is presented as g fatty acid content per 100 g fillet (g / g fillet). The highest representative fatty acids include 16:0 (palmitic acid), 18:1 (vaccenic acid or oleic acid), and omega-3 fatty acids (EPA, DHA, and DPA) in aquatic animals, while terrestrial animals have almost no omega-3. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0154] In some embodiments, the methods and systems described herein increased the fatty acid content in cells of an aquatic animal by culturing the cells in a culture medium containing individual fatty acids and / or mixtures of fatty acids.

[0155] In some embodiments, the methods and systems described herein increased the fatty acid content in terrestrial animal cells by culturing the cells in a culture medium containing individual fatty acids and / or mixtures of fatty acids.

[0156] In some embodiments, the cells of an aquatic animal obtained using the methods and systems described herein have a fatty acid content of one or more fatty acids that is higher than the fatty acid content of a naturally occurring aquatic animal listed in Tables 2A and 2B.

[0157] In some embodiments, the cells of a terrestrial animal obtained using the methods and systems described herein have a fatty acid content of one or more fatty acids that is higher than the fatty acid content of a naturally occurring terrestrial animal listed in Tables 2C and 2D.

[0158] For example, flounder cells obtained using the methods and systems described herein may have a palmitic acid (C16:0) content of greater than 0.217 g per 100 g fillet, halibut cells obtained herein may have a palmitic acid (C16:0) content of greater than 0.174 g per 100 g fillet, herring cells obtained herein may have a palmitic acid (C16:0) content of greater than 0.172 g per 100 g fillet, and mackerel cells obtained herein may have a palmitic acid (C16:0) content of greater than 0.183 g per 100 g fillet (see Tables 2A and 2B).

[0159] Similarly, flounder cells obtained herein may have a vaccenic acid or oleic acid (C18:1) content of greater than 0.275 g per 100 g fillet, halibut cells obtained herein may have a vaccenic acid or oleic acid (C18:1) content of greater than 0.229 g per 100 g fillet, herring cells obtained herein may have a vaccenic acid or oleic acid (C18:1) content of greater than 0.193 g per 100 g fillet, and mackerel cells obtained herein may have a vaccenic acid or oleic acid (C18:1) content of greater than 0.191 g per 100 g fillet (see Tables 2A and 2B).

[0160] Similarly, flounder cells obtained herein may have an eicosapentaenoic acid (EPA) (C20:5 n-3) content of greater than 0.105 g per 100 g fillet, halibut cells obtained herein may have an EPA content of greater than 0.066 g per 100 g fillet, herring cells obtained herein may have an EPA content of greater than 0.090 g per 100 g fillet, and mackerel cells obtained herein may have an EPA content of greater than 0.075 g per 100 g fillet (see Tables 2A and 2B).

[0161] Similarly, flounder cells obtained herein may have a DPA (C22:5 n-3) content of greater than 0.022 g per 100 g fillet, halibut cells obtained herein may have a DPA content of greater than 0.016 g per 100 g fillet, herring cells obtained herein may have a DPA content of greater than 0.007 g per 100 g fillet, and mackerel cells obtained herein may have a DPA content of greater than 0.018 g per 100 g fillet (see Tables 2A and 2B).

[0162] Similarly, flounder cells obtained herein may have a DHA (C22:6 n-3) content of greater than 0.083 g per 100 g fillet, halibut cells obtained herein may have a DHA content of greater than 0.128 g per 100 g fillet, herring cells obtained herein may have a DHA content of greater than 0.110 g per 100 g fillet, and mackerel cells obtained herein may have a DHA content of greater than 0.121 g per 100 g fillet (see Tables 2A and 2B).

[0163] In some embodiments, the aquatic animal cells provided herein may have a higher total omega-3 polyunsaturated fatty acid (total n-3) content than that of naturally occurring aquatic animals. For example, flounder or halibut cells provided herein may have a total omega-3 polyunsaturated fatty acid content of greater than 0.210 g per 100 g fillet, herring cells provided herein may have a total omega-3 polyunsaturated fatty acid content of greater than 0.207 g per 100 g fillet, and mackerel cells provided herein may have a total omega-3 polyunsaturated fatty acid content of greater than 0.214 g per 100 g fillet (see Tables 2A and 2B).

[0164] In some embodiments, the cells of the terrestrial animals provided herein may have a higher total omega-3 polyunsaturated fatty acid (total n-3) content than that of naturally occurring terrestrial animals.

[0165] It will be understood that the described embodiments of flounder, halibut, herring, and mackerel cells having particular lipid contents are exemplary and not limiting in nature, and the present disclosure encompasses corresponding or similar embodiments of cells derived from other species.

[0166] In all naturally occurring species listed in Tables 2A and 2B, a significant proportion of the fatty acids were saturated fats, with a content of at least 15%, whereas the lipid-transfected cells provided herein can be transfected with only unsaturated or monounsaturated fats. The maximum concentration of each fatty acid is limited to about 30% in almost all naturally occurring aquatic animals in Tables 2A and 2B, whereas the cells provided herein can contain more than 30% of a single fatty acid as fat content.

[0167] In some of these embodiments, culture media, methods and systems for culturing preadipocytes are described herein.

[0168] In particular, in embodiments described herein relating to the culturing of adipocytes, the culture medium may comprise a basal medium supplemented with 25-1000 μg / ml lipid and 0-4% serum in amounts effective to control lipid content and increase preadipocyte viability, differentiation, and / or lipid uptake.

[0169] In some embodiments, the lipids included in the culture medium for preadipocytes of the present disclosure include one or more MUFAs, one or more PUFAs, one or more SFAs, and / or one or more sterols, alone or in any combination. In preferred embodiments, the adipocytes of the present disclosure include controlled amounts of PUFAs, more preferably omega-3 fatty acids and / or fat-soluble vitamins.

[0170] In exemplary embodiments described herein in relation to preadipocytes, a method of culturing aquatic preadipocytes includes culturing the preadipocytes in a preadipocyte culture medium according to the present disclosure comprising one or more fatty acids at effective concentrations, resulting in lipid uptake by the preadipocytes. Typically, in these embodiments, lipids may be provided at a concentration of 25 μg / ml up to 1000 μg / ml, preferably 25 μg / ml up to 100 μg / ml. Notably, in some of these embodiments, the selected fatty acid concentration is capable of inducing the round morphology with lipid droplets observed in fish cells without a reduction in cell confluency indicative of potential toxicity (see Example 2).

[0171] Similarly, terrestrial animal preadipocytes can be cultured by similar methods and using similar media.

[0172] In some embodiments, aquatic animal preadipocytes obtainable using the culture media, methods, and systems disclosed herein contain lipids in amounts between 0.1% and 90%. In a preferred embodiment, the fatty acids of the preadipocytes described herein contain about 50% SFA, 25% PUFA (preferably including omega-3), and 25% MUFA.

[0173] In some embodiments, the culture media, methods, and systems of the present disclosure may be used to increase the lipid content and / or cell viability of aquatic animal cells by culturing the cells in a culture medium comprising lipids and an effective amount of nervonic acid.

[0174] In particular, in preferred embodiments, the methods and systems and associated compositions, particularly culture media, may be used to control the lipid content and / or increase the survival rate of myoblasts and / or fibroblasts of aquatic animals.

[0175] In particular, in preferred embodiments, the methods and systems and associated compositions, particularly culture media, may be used to control the lipid content and / or increase the viability of myoblasts and / or fibroblasts in terrestrial animals.

[0176] In some of these embodiments, nervonic acid can be combined with saturated or polyunsaturated fatty acids, such as DHA and EPA, which show low lipid accumulation and some toxicity when used individually, but show high cell counts and high lipid accumulation when used in combination with nervonic acid (see Examples 6-7).

[0177] Thus, in some embodiments, nervonic acid, when combined with other fatty acids, can cause increased lipid uptake and lipid accumulation. Thus, the use of nervonic acid can not only enhance or enable lipid uptake of fatty acids, including fatty acids that have some degree of toxicity to cultured aquatic animal cells, but can also impart additional nutritional qualities to aquatic animal cell culture products, such as fish products, seafood products, and additional products identifiable by one of skill in the art.

[0178] Thus, in some embodiments, nervonic acid, when combined with other fatty acids, can cause increased lipid uptake and lipid accumulation. Thus, the use of nervonic acid can not only enhance or enable lipid incorporation of fatty acids, but can also impart additional nutritional quality to terrestrial animal cell culture products.

[0179] In some embodiments, nervonic acid is provided at a concentration of 10-1000 μg / ml, preferably 10-100 μg / ml, preferably 50 μg / ml.

[0180] In some embodiments, culture media, methods, systems, and compositions, including nervonic acid-containing culture media, may provide aquatic animal myoblasts and / or fibroblasts that contain a desired fatty acid (e.g., omega-3 fatty acids) in an amount of at least about 1% based on total fat. In some embodiments, culture media, methods, systems, and compositions, including nervonic acid-containing culture media, may provide terrestrial animal myoblasts and / or fibroblasts that contain a desired fatty acid (e.g., omega-3 fatty acids) in an amount of at least about 1% based on total fat. In some embodiments, culture media, methods, systems, and compositions, including nervonic acid-containing culture media, may provide aquatic animal myoblasts and / or fibroblasts that contain a desired fatty acid (e.g., omega-3 fatty acids) in an amount of at least about 1% based on total fat. For example, myoblasts, myocytes, preadipocytes, adipocytes, or fibroblasts from a desired fish species cultured in a lipid-induction medium containing a desired fatty acid according to the present disclosure and an effective amount of nervonic acid may contain about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 100-fold the amount of the desired fatty acid compared to the corresponding cells or meat of a wild-caught fish of the same species.

[0181] In some embodiments, culture media, methods, systems, and compositions, including nervonic acid-containing culture media, may provide terrestrial animal myoblasts and / or fibroblasts that contain desired fatty acids (e.g., omega-3 fatty acids) in an amount of at least about 1% relative to total fat.

[0182] In some embodiments, the disclosed methods and systems provide aquatic animal fibroblasts and associated biomass comprising said cells, wherein the fibroblasts comprise at least 1%-90% lipid. In some embodiments, the disclosed methods and systems provide terrestrial animal fibroblasts and associated biomass comprising said cells, wherein the fibroblasts comprise at least 1%-90% lipid.

[0183] In some embodiments, the disclosed methods and systems provide aquatic animal myoblasts and associated biomass comprising said cells, wherein the myoblasts comprise lipids in an amount of at least 1%. In some embodiments, the disclosed methods and systems provide terrestrial animal myoblasts and associated biomass comprising said cells, wherein the myoblasts comprise lipids in an amount of at least 1%.

[0184] In some embodiments, as will be understood by those of skill in the art upon reading this disclosure, nervonic acid may further be used to increase the viability of aquatic animal myoblasts, fibroblasts, and additional cells.

[0185] Thus, in some embodiments, the methods and systems described herein can increase the viability of cells in a cellular biomass of an aquatic animal by culturing the cellular biomass in the presence of an effective amount of nervonic acid. In some embodiments, the culture media, methods, and systems described herein increase the polyunsaturated fatty acid content in aquatic animal cells by culturing the cells in a culture medium comprising polyunsaturated fatty acids and an effective amount of nervonic acid. In some embodiments, the culture media, methods, and systems described herein increase the polyunsaturated fatty acid content in terrestrial animal cells by culturing the cells in a culture medium comprising polyunsaturated fatty acids and an effective amount of nervonic acid.

[0186] In some embodiments, the concentration of monounsaturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught animal (e.g., fish) of the same species. For example, the concentration of monounsaturated fatty acids such as palmitoleic acid, oleic acid, and vaccenic acid can be increased to include an amount greater than any one of the naturally occurring amounts such as those shown in Tables 2A and 2B. In some embodiments, the concentration of monounsaturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught or farmed terrestrial animal of the same species (e.g., those shown in Tables 2C and 2D).

[0187] In some embodiments, the concentration of polyunsaturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught animal (e.g., fish) of the same species. For example, the concentration of polyunsaturated linoleic acid and omega-3 polyunsaturated fatty acids can be increased to include amounts greater than any one of the naturally occurring amounts, such as those shown in Tables 2A and 2B. In some embodiments, the concentration of polyunsaturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught or farmed terrestrial animal of the same species (e.g., those shown in Tables 2C and 2D).

[0188] In some embodiments, the concentration of saturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught animal (e.g., fish) of the same species. For example, the concentration of lauric acid, myristic acid, palmitic acid, and / or stearic acid can be increased above any one of their naturally occurring amounts, such as those shown in Tables 2A and 2B. In some embodiments, the concentration of saturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught or farmed terrestrial animal of the same species (e.g., those shown in Tables 2C and 2D).

[0189] In some embodiments, culture media, methods and systems for uptake of vaccenic acid in aquatic animal cells are described, comprising culturing aquatic animal cells in the presence of vaccenic acid for a period and under conditions that result in the uptake of vaccenic acid by the aquatic animal cells.

[0190] In some embodiments, culture media, methods and systems for uptake of vaccenic acid in terrestrial animal cells are described, the methods comprising culturing terrestrial animal cells in the presence of vaccenic acid for a period and under conditions that result in the uptake of vaccenic acid by the aquatic animal cells.

[0191] The term "vaccenic acid" is also a term of art and refers to a compound known as C18:1, which exists as a trans stereoisomer ((11E)-11-octadecenoid acid) and a cis stereoisomer ((11Z)-11-octadecenoic acid). Vaccenic acid exists as a solid and is considered to be insoluble in water and relatively neutral. Vaccenic acid can be produced through biohydrogenation of linoleic and alpha-linolenic acids by microorganisms in the rumen and is found naturally in foods such as dairy and ruminant meat products.

[0192] In some embodiments, the cells provided herein comprise fish preadipocytes having increased concentrations of omega-3 polyunsaturated fatty acids, such as DHA and EPA, relative to naturally occurring amounts, such as those shown in Tables 2A and 2B.

[0193] In some embodiments, the cells provided herein comprise fish myoblasts and / or fibroblasts having increased concentrations of omega-3 polyunsaturated fatty acids, such as DHA and EPA, relative to naturally occurring amounts, such as those shown in Tables 2A and 2B.

[0194] In some embodiments, the concentration of omega-3 polyunsaturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught animal (e.g., fish) of the same species (e.g., the amounts shown in Tables 2A and 2B).

[0195] In some embodiments, the cells provided herein comprise terrestrial animal preadipocytes having increased concentrations of omega-3 polyunsaturated fatty acids, such as DHA and EPA, relative to naturally occurring amounts, such as those shown in Tables 2C and 2D.

[0196] In some embodiments, the cells provided herein comprise myoblasts and / or fibroblasts of terrestrial animals that have increased concentrations of omega-3 polyunsaturated fatty acids, such as DHA and EPA, relative to naturally occurring amounts, such as those shown in Tables 2C and 2D.

[0197] In some embodiments, the concentration of omega-3 polyunsaturated fatty acids is increased by at least about 1% to about 300% or more compared to the amount present in a wild-caught or farmed terrestrial animal of the same species (e.g., the amounts shown in Tables 2C and 2D).

[0198] In some embodiments, the terrestrial animal cells provided herein include myoblasts, fibroblasts, and / or preadipocytes that have an increased concentration of omega-3 polyunsaturated fatty acids compared to the amount present in the corresponding cell type of a wild caught or farmed terrestrial animal of the same species, and the increased concentration of omega-3 polyunsaturated fatty acids is at least about 1 percentage point or more, e.g., 2 percentage points, 3 percentage points, 4 percentage points, 5 percentage points, 6 percentage points, 7 percentage points, 8 percentage points, 9 percentage points, 10 percentage points, 11 percentage points, 12 percentage points, 13 percentage points, 14 percentage points, or more. , 15 percentage points, 16 percentage points, 17 percentage points, 18 percentage points, 19 percentage points, 20 percentage points, 21 percentage points, 22 percentage points, 23 percentage points, 24 percentage points, 25 percentage points, 26 percentage points, 27 percentage points, 28 percentage points, 29 percentage points, 30 percentage points points, 31 percentage points, 32 percentage points, 33 percentage points, 34 percentage points, 35 percentage points, 36 percentage points, 37 percentage points, 38 percentage points, 39 percentage points, 40 percentage points, 41 percentage points, 42 percentage points, 43 percentage points, 44 percentage points, 45 percentage points, 46 percentage points, 47 percentage points, 48 ​​percentage points, 49 percentage points, 50 percentage points, 51 percentage points, 52 percentage points, 53 percentage points, 54 percentage points, 55 percentage points, 56 percentage points, 57 percentage points, 58 percentage points, 59 percentage points, 60 percentage points, 61 percentage points,The percentage may be 62 percentage points, 63 percentage points, 64 percentage points, 65 percentage points, 66 percentage points, 67 percentage points, 68 percentage points, 69 percentage points, 70 percentage points, 71 percentage points, 72 percentage points, 73 percentage points, 74 percentage points, 75 percentage points, 76 percentage points, 77 percentage points, 78 percentage points, 79 percentage points, 80 percentage points, 81 percentage points, 82 percentage points, 83 percentage points, 84 percentage points, 85 percentage points, 86 percentage points, 87 percentage points, 88 percentage points, 89 percentage points, 90 percentage points, or more. The increase may be based on a comparison to the amounts set forth in Tables 2C and 2D.

[0199] Generally, terrestrial animal cells not obtained by the media, methods, and systems disclosed herein are essentially free of omega-3 polyunsaturated fatty acids, and therefore an increase of 1 or more percentage points in omega-3 polyunsaturated fatty acids can be achieved by adding omega-3 polyunsaturated fatty acids without changing the concentration of fatty acids already present.

[0200] In some embodiments, the terrestrial animal cells provided herein include myoblasts, fibroblasts, and / or preadipocytes that have increased concentrations of monounsaturated fatty acids compared to the amounts present in the corresponding cell types of a wild caught or farmed terrestrial animal of the same species. The increase in monounsaturated fatty acid concentration can be at least about 1 percentage point or more, e.g., 2 percentage points, 3 percentage points, 4 percentage points, 5 percentage points, 6 percentage points, 7 percentage points, 8 percentage points, 9 percentage points, 10 percentage points, 11 percentage points, 12 percentage points, 13 percentage points, 14 percentage points, 15 percentage points, 16 percentage points, 17 percentage points, 18 percentage points, 19 percentage points, 20 percentage points, 21 percentage points, 22 percentage points, 23 percentage points, 24 percentage points, 25 percentage points, 26 percentage points, 27 percentage points, 28 percentage points, 29 percentage points, 30 percentage points, 31 percentage points, 32 percentage points, 33 percentage points, 34 percentage points, 35 percentage points, 36 percentage points, 37 percentage points, 38 percentage points, 39 percentage points, 40 percentage points, 41 percentage points, 42 percentage points, 43 percentage points, 44 percentage points, 45 percentage points, 46 percentage points, 47 percentage points, 48 ​​percentage points, 49 percentage points, 50 percentage points, 51 percentage points, 52 percentage points, 53 percentage points, 54 percentage points, 55 percentage points, 56 percentage points, 57 percentage points, 58 percentage points, 59 percentage points, 60 percentage points, 61 percentage points, 62 percentage points, 63 percentage points, 64 percentage points, 65 percentage points, 66 percentage points, 67 percentage points, 68 percentage points, 69 percentage points, 70 percentage points, 71 percentage points, 7 percentage points, 16 percentage points, 17 percentage points, 18 percentage points, 19 percentage points, 20 percentage points, 21 percentage points, 22 percentage points, 23 percentage points, 24 percentage points, 25 percentage points, 26 percentage points, 27 percentage points, 28 percentage points, 29 percentage points, 30 percentage points, 31 percentage points percentage points, 32 percentage points, 33 percentage points, 34 percentage points, 35 percentage points, 36 percentage points, 37 percentage points, 38 percentage points, 39 percentage points, 40 percentage points, 41 percentage points, 42 percentage points, 43 percentage points, 44 percentage points, 45 percentage points, 46 percentage points, 47 percentage points, 48 ​​percentage points, 49 percentage points, 50 percentage points, 51 percentage points, 52 percentage points, 53 percentage points, 54 percentage points, 55 percentage points, 56 percentage points, 57 percentage points, 58 percentage points, 59 percentage points, 60 percentage points, 61 percentage points, 62 percentage points,The increase may be 63 percentage points, 64 percentage points, 65 percentage points, 66 percentage points, 67 percentage points, 68 percentage points, 69 percentage points, 70 percentage points, 71 percentage points, 72 percentage points, 73 percentage points, 74 percentage points, 75 percentage points, 76 percentage points, 77 percentage points, 78 percentage points, 79 percentage points, 80 percentage points, 81 percentage points, 82 percentage points, 83 percentage points, 84 percentage points, 85 percentage points, 86 percentage points, 87 percentage points, 88 percentage points, 89 percentage points, 90 percentage points, or more. The increase may be based on a comparison to the amounts set forth in Tables 2C and 2D. ,

[0201] In some embodiments, the terrestrial animal cells provided herein include myoblasts, fibroblasts, and / or preadipocytes that have an increased concentration of unsaturated fatty acids compared to the amount present in the corresponding cell type of a wild caught terrestrial animal or farmed terrestrial animal of the same species, and the increase in the concentration of unsaturated fatty acids is at least about 1 percentage point or more, e.g., 2 percentage points, 3 percentage points, 4 percentage points, 5 percentage points, 6 percentage points, 7 percentage points, 8 percentage points, 9 percentage points, 10 percentage points, 11 percentage points, 12 percentage points, 13 percentage points, 14 percentage points, 15 percentage points, or more. 16 percentage points, 17 percentage points, 18 percentage points, 19 percentage points, 20 percentage points, 21 percentage points, 22 percentage points, 23 percentage points, 24 percentage points, 25 percentage points, 26 percentage points, 27 percentage points, 28 percentage points, 29 percentage points, 30 percentage points, 31 percentage points percentage points, 32 percentage points, 33 percentage points, 34 percentage points, 35 percentage points, 36 percentage points, 37 percentage points, 38 percentage points, 39 percentage points, 40 percentage points, 41 percentage points, 42 percentage points, 43 percentage points, 44 percentage points, 45 percentage points, 46 percentage points, 47 percentage points, 48 ​​percentage points, 49 percentage points, 50 percentage points, 51 percentage points, 52 percentage points, 53 percentage points, 54 percentage points, 55 percentage points, 56 percentage points, 57 percentage points, 58 percentage points, 59 percentage points, 60 percentage points, 61 percentage points, 62 percentage points,The increase may be 63 percentage points, 64 percentage points, 65 percentage points, 66 percentage points, 67 percentage points, 68 percentage points, 69 percentage points, 70 percentage points, 71 percentage points, 72 percentage points, 73 percentage points, 74 percentage points, 75 percentage points, 76 percentage points, 77 percentage points, 78 percentage points, 79 percentage points, 80 percentage points, 81 percentage points, 82 percentage points, 83 percentage points, 84 percentage points, 85 percentage points, 86 percentage points, 87 percentage points, 88 percentage points, 89 percentage points, 90 percentage points, or more. The increase may be based on a comparison to the amounts set forth in Tables 2C and 2D. ,

[0202] In some embodiments, the terrestrial animal cells provided herein include myoblasts, fibroblasts, and / or preadipocytes that have a reduced concentration of saturated fatty acids compared to the amount present in the corresponding cell type of a wild-caught or farmed terrestrial animal of the same species. The reduction in the concentration of saturated fatty acids may be at least about 1 percentage point or more, e.g., 2 percentage points, 3 percentage points, 4 percentage points, 5 percentage points, 6 percentage points, 7 percentage points, 8 percentage points, 9 percentage points, 10 percentage points, 11 percentage points, 12 percentage points, 13 percentage points, 14 percentage points, 15 percentage points, 16 percentage points, 17 percentage points, 18 percentage points, 19 percentage points, 20 percentage points, 21 percentage points, 22 percentage points, 23 percentage points, 24 percentage points, 25 percentage points, 26 percentage points, 27 percentage points, 28 percentage points, 29 percentage points, 30 percentage points, 31 percentage points, 32 percentage points, 33 percentage points, 34 percentage points, 35 percentage points, 36 percentage points, 37 percentage points, 38 percentage points, 39 percentage points, 40 percentage points, 41 percentage points, 42 percentage points, 43 percentage points, 44 percentage points, 45 percentage points, 46 percentage points, 47 percentage points, 48 ​​percentage points, 49 percentage points, 50 percentage points, 51 percentage points, 52 percentage points, 53 percentage points, 54 percentage points, 55 percentage points, 56 percentage points, 57 percentage points, 58 percentage points, 59 percentage points, 60 percentage points, 61 percentage points, 62 percentage points, 63 percentage points, 64 percentage points, 65 percentage points, 66 percentage points, 67 percentage points, 68 percentage points, 69 percentage points, 70 percentage points, 71 percentage points, 72 percentage points, 73 percentage points, 74 percentage points, 75 percentage points, 76 percentage points, 77 percentage points, 78 percentage points, 79 percentage points, 80 percentage points, 81 percentage points, 82 percentage points, 83 percentage points, The reduction may be 29 percentage points, 30 percentage points, 31 percentage points, 32 percentage points, 33 percentage points, 34 percentage points, 35 percentage points, 36 percentage points, 37 percentage points, 38 percentage points, 39 percentage points, 40 percentage points, 41 percentage points, 42 percentage points, 43 percentage points, 44 percentage points, 45 percentage points, 46 percentage points, 47 percentage points, 48 ​​percentage points, 49 percentage points, 50 percentage points, 51 percentage points, 52 percentage points, 53 percentage points, 54 percentage points, 55 percentage points, or more. The reduction may be based on a comparison to the amounts set forth in Tables 2C and 2D.

[0203] In some embodiments, the terrestrial animal cells provided herein include myoblasts, fibroblasts, and / or preadipocytes that have an increased concentration of omega-3 polyunsaturated fatty acids by up to 90 percentage points or more and a decreased concentration of saturated fatty acids by up to 55 percentage points or more compared to the amount present in the corresponding cell type of a wild-caught or farmed terrestrial animal of the same species.

[0204] A food product containing the terrestrial animal cells described above may have an omega-3 PUFA content that is at least one percentage point higher and / or a UFA content that is at least one percentage point lower than a food product of identical composition (except where the terrestrial animal cells are derived from a wild-caught or farmed terrestrial animal of the same species).

[0205] In the methods and systems described herein, lipid-transduced cells (e.g., aquatic animal cells, terrestrial animal cells) can be harvested by cell separation techniques such as sedimentation or tangential flow filtration. The harvested cells can then be assembled into cell-cultured food products to form cubes, strips, or fillets using a variety of methods, including extrusion and bioprinting, as will be appreciated by those skilled in the art. The cubes, strips, or fillets are comprised of myoblasts, adipocytes, fibroblasts, or combinations of these cell types, and optionally a suitable matrix.

[0206] The cells obtained using the media, methods, and systems described herein can be used to produce a variety of cell-cultured food products, including, for example, products that have a substantially similar appearance, texture, and flavor to whole animals and / or wild-caught or farmed fish, seafood, beef, pork, or poultry fillets of the same species. Suitable methods for preparing such foods are known in the art and include combining the desired type of cells (e.g., myocytes, adipocytes, fibroblasts) and, optionally, plant cells, fungal cells, other non-animal cells, plant proteins, fungal proteins, other non-animal proteins, and / or a suitable matrix to produce a product that resembles, for example, a fish fillet, steak, or other beef fillet, pork fillet, poultry fillet, etc. Such products may be homogenous (e.g., having different types and species of cells, proteins from different sources, and / or matrix material distributed evenly throughout the product) or may be heterogeneous (e.g., having different types of cells preferentially located in one or more portions of the product, e.g., a layered structure).

[0207] Generally, a cell-cultured food product is an aquatic or terrestrial animal food product that contains cells cultured according to the present disclosure.

[0208] In an embodiment, the cell-cultured food product is a fish food product containing cells cultured according to the present disclosure. For example, the cell-cultured food product may contain aquatic animal cells, preferably fish cells. For example, the cell-cultured food product may consist essentially of or consist of fish cells and optionally a suitable matrix, the fish cells being selected from the group consisting of myoblasts, myocytes, fibroblasts, preadipocytes, adipocytes, keratinocytes, and combinations thereof, into which one or more desired lipids have been introduced, for example, by culturing the cells according to the present disclosure. Thus, the food product may have a lower amount of saturated fat (g of saturated fat per g of total fat) compared to the same species of wild-caught fish. The food product may have a higher amount of unsaturated fat (e.g., g of PUFAs and / or MUFAs per g of total fat) compared to the same species of wild-caught fish. In embodiments, the food product may have a higher amount of omega-3 fatty acids (g of omega-3 per g of total fat), such as DHA, EPA, ALA, and combinations thereof, compared to wild-caught fish of the same species, and may further include a higher amount of nervonic acid compared to wild-caught fish of the same species. Optionally, the food product of such embodiments also has a higher amount of palmitoleic acid, vaccenic acid, oleic acid, linoleic acid, lauric acid, myristic acid, palmitic acid, steric acid, and any combination thereof, compared to wild-caught fish of the same species. In embodiments, the food product consists essentially of or consists of aquatic animal cells derived from or obtained from a single animal species, and optionally a suitable matrix. Preferred food products consist essentially of or consist of cells derived from aquatic species disclosed herein, including carp, yellowtail, mahi-mahi, bluefin tuna, yellowfin tuna, red snapper, cod, Patagonian toothfish, and the like.

[0209] In particular, cell-cultured fish foods contain higher levels of total lipids than conventionally sourced fish, and the resulting fish products contain higher levels of polyunsaturated fatty acids, including omega-3 fatty acids, than conventionally sourced fish.

[0210] In a more specific example, the cell-cultured fish food contains less C16:00 and / or C18:00 fatty acids (as a percentage of total fat or by weight of product) compared to wild-caught fish of the same species. Optionally, or in addition, the cell-cultured fish food contains more C18:01 fatty acids and / or omega-3 fatty acids, such as ALA, EPA, and / or DHA (as a percentage of total fat or by weight of product) compared to wild-caught fish of the same species. The embodiments described herein also include a system for carrying out the methods described herein. In particular, in some embodiments, the system may include one or more MUFAs in combination with a basal culture medium and / or one or more cell types of an aquatic animal in the sense of the present disclosure that may be contained within an aquatic cell biomass. In some embodiments, the system may include a culture medium of the present disclosure that includes one or more MUFAs in combination with one or more cell types of an aquatic animal in the sense of the present disclosure that may be contained within an aquatic cell biomass.

[0211] In some embodiments, the system may include one or more PUFAs, SFAs, and / or sterols in combination with a basal culture medium and / or one or more cell types of an aquatic animal within the meaning of the present disclosure that may be contained within an aquatic cell biomass. In some embodiments, the system may include a culture medium of the present disclosure that includes one or more PUFAs, SFAs, and / or sterols in combination with one or more cell types of an aquatic animal within the meaning of the present disclosure that may be contained within an aquatic cell biomass.

[0212] In some embodiments, the system may include one or more PUFAs, SFAs, and / or sterols in combination with nervonic acid, and further in combination with a basal culture medium, and / or one or more cell types of an aquatic animal.

[0213] In some embodiments, the system may include one or more PUFAs in combination with nervonic acid and further in combination with a basal culture medium and / or one or more cell types of an aquatic animal within the meaning of the disclosure that may be contained within an aquatic cell biomass. In some embodiments, the system may include a culture medium of the present disclosure that includes one or more PUFAs in combination with nervonic acid and / or one or more cell types of an aquatic animal within the meaning of the disclosure that may be contained within an aquatic cell biomass. In some embodiments, the system may include a culture medium of the present disclosure that includes one or more PUFAs and nervonic acid in combination with one or more cell types of an aquatic animal within the meaning of the disclosure that may be contained within an aquatic cell biomass.

[0214] In some embodiments, the system may include omega-3 in combination with nervonic acid and further in combination with a basal culture medium and / or one or more cell types of an aquatic animal within the meaning of the disclosure that may be contained within an aquatic cell biomass. In some embodiments, the system may include a culture medium of the present disclosure that includes omega-3 in combination with nervonic acid and / or one or more cell types of an aquatic animal within the meaning of the present disclosure that may be contained within an aquatic cell biomass. In some embodiments, the system may include a culture medium of the present disclosure that includes omega-3 and nervonic acid in combination with one or more cell types of an aquatic animal within the meaning of the present disclosure that may be contained within an aquatic cell biomass.

[0215] In some embodiments, the system may include one or more lipids in combination with the basal culture medium and / or one or more cell types of an aquatic animal within the meaning of the present disclosure that may be contained within the aquatic cell biomass. In embodiments where the lipids include or consist of one or more PUFAs, SFAs, and / or sterols, the system further includes nervonic acid. In some embodiments, the system may include a culture medium of the present disclosure that includes one or more lipids as described herein in combination with one or more cell types of an aquatic animal within the meaning of the present disclosure that may be contained within the aquatic cell biomass. In embodiments where the lipids include or consist of one or more PUFAs, SFAs, and / or sterols, the culture medium and / or system further includes nervonic acid.

[0216] Although the above discussion has focused on media, methods, and systems for culturing cells for cell culture food products, the media, methods, and systems can be used to culture cells for any purpose. For example, the media, methods, and systems described herein are suitable for a wide variety of life science applications, including but not limited to pharmaceutical applications requiring in vitro fertilization and cell culture of eukaryotic cells (e.g., production of antibodies and therapeutic peptides and proteins, production of CAR-T cells, and stem cell culture, IPSC, and primary stem cells). The cells can be derived from species such as human, Chinese hamster, African green monkey, dog, Trichoplusia ni, or Spodoptera frugiperda, among others.

[0217] The suitability of media, methods, and systems for specific life science applications is evaluated by comparing the performance of the relevant cell type in conventional media, methods, and systems with their performance in the media, methods, and systems described and claimed herein. Exemplary cell types and applications are shown in Table 3 below. [Table 6]

[0218] In some embodiments, the system described herein can be provided in the form of a kit of parts, in which the culture medium, nervonic acid, and / or vaccenic acid can be provided in various combinations with each other and with lipids and / or cells. In the kit of parts, the components can be included in the kit independently, or can be included in a composition, optionally with a suitable vehicle carrier or auxiliary.

[0219] Additional components may also be included, including reference standards and further components identifiable by one of skill in the art upon reading this disclosure.

[0220] In some embodiments, the kit may include fish myoblasts and / or fibroblasts and nervonic acid. The kit-of-parts further includes one or more other fatty acids described herein, such as omega-3 polyunsaturated fatty acids.

[0221] In some embodiments, the kit comprises fish preadipocytes and one or more fatty acids as described herein. In some embodiments, the kit comprises preadipocytes from one or more terrestrial animals and one or more fatty acids as described herein. As will be appreciated by one of skill in the art, the kit of parts further comprises a basal medium, a culture medium, a differentiation medium, and / or a lipid introduction medium required to introduce one or more fatty acids into the preadipocytes as described herein.

[0222] In the embodiments described herein, the components of the kit may be provided with instructions suitable for carrying out the methods disclosed herein and other necessary reagents. The kit typically includes compositions in separate containers. Instructions, such as written or audio instructions, paper or electronic support (e.g., tape, CD-ROM, flash drive), or by indication of a uniform resource locator (URL) containing a PDF, HTML, or other electronic copy of instructions for carrying out the assay, are typically included in the kit. The kit may also contain other packaged reagents and materials (e.g., washing buffers, etc.), depending on the particular method used.

[0223] Further details regarding the compositions, methods and systems described herein will now become more apparent from detailed disclosure of the following examples, by way of illustration only, with reference to the Experimental Section. EXAMPLES

[0224] The cell cultured foods, and related cells, compositions, methods, and systems described herein are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

[0225] Materials and Methods Cell lines and culture conditions Cell lines were obtained from commercial vendors or developed by applicant as shown in Table 4. Culture conditions are as shown in Table 5. [Table 7-1] [Table 7-2] [Table 8-1] [Table 8-2]

[0226] reagent Fatty Acid Stock Solutions A 50 mg / mL fatty acid stock solution was prepared using either powdered fatty acids or concentrated fatty acid solutions. Powdered fatty acids were dissolved in 200 proof ethanol (not 100% denatured) at a concentration of 50 mg / mL. Concentrated fatty acid solutions were diluted to a final concentration of 50 mg / mL using 200 proof ethanol.

[0227] BSA-conjugated fatty acid stock solution 1 mL aliquots of a 10 mg / ml stock solution of BSA-conjugated fatty acid were prepared as follows. - Add 800μl of 7.5% BSA to a 1.5ml tube and place in a 37°C water bath until it reaches temperature. - Place 50mg / ml FA stock in a 37°C water bath and warm. - Add 25 μl of FA stock at a time to the BSA tube until the FA is dissolved, then repeat until a maximum of 200 μl has been added. - Add all the FA to the BSA tube and incubate the solution in a 37°C water bath for 1 hour after completely dissolving. - Filter through a 0.2μm filter and syringe into a 1.5mL tube - Store at -20℃ (recommended if not used within 48 hours)

[0228] Cell culture media Media for cell lines (EACC, ECACC) obtained from ATCC and Sigma were prepared as per the instructions on the accompanying product sheets. CO2-independent medium bases were prepared as shown in Table 6. In-house media formulations were prepared according to in-house methods. [Table 9]

[0229] BCA protein assay The Pierce BCA Protein Assay Kit was obtained from ThermoScientific (San Diego, California, Catalog Nos. 23225 and 23227, Document Part No. 2161296, Publication No. MAN0011430v.B.0) and the assay was performed according to the protocol provided with the kit.

[0230] Lipidomic analysis Harvesting cells and fatty acids for lipidomic analysis Media was manually removed from cells grown in 6-well plates. 1 ml of PBS was added to each well to gently wash and then manually removed. Then, while holding the plate at an angle, 0.5 ml of 10% methanol was added to each well. Samples were scraped starting at the top left and scraped to the bottom while moving the well from left to right or right to left. Remaining cells were scraped to the bottom of the well and then collected.

[0231] The samples were then pipetted into labeled amber glass 2 ml vials. An additional 0.5 ml of 10% methanol was then added to each well and any remaining sample was collected and collected in the same vial.

[0232] Samples were pipetted to ensure complete cell lysis and fractions were removed for BCA analysis according to the Pierce BCA Protein Assay referenced above, then flash frozen and stored at -80°C until lipidomic analysis.

[0233] Lipidomic Analysis Protocols The fatty acid content of lipid-transfected and control cells was analyzed by Quehenberger et al. and Lofgren et al. (Quehenberger O, Armando AM, Dennis EA. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim Biophys Acta. 2011 Nov;1811(11):648-56. doi:10.1016 / j.bbalip.2011.07.006. Epub 2011 Jul 20. PMID:21787881; PMCID:PMC3205314) (Lofgren, L., Forsberg, GB. & Stahlman, M. The BUME method: a new rapid and simple chloroform-free method for total lipid extraction of animal tissue. Sci Rep 6,27688(2016).https: / / doi.org / 10.1038 / srep27688).

[0234] Briefly, cells were homogenized in 1 mL of 10% methanol and 200 μL of the homogenate was extracted with modified BUME. The extract was dried and saponified using a 1:1 MeOH:KOH solution at 37 °C for 1 h. Fatty acids were extracted with a biphasic solution of acidified methanol and isooctane, derivatized using PFBB, and analyzed by GC-MS on an Agilent 6890N gas chromatograph equipped with an Agilent 7683 autosampler. Fatty acids were separated using a 15 m ZB-1 column (Phenomenex) and monitored using SIM identification. Analysis was performed using MassHunter software.

[0235] Example 1: Introduction of complex fatty acid mixtures into fish adipose-derived cells In this example, the introduction of a complex fatty acid mixture (including omega-3) composed of saturated and unsaturated fatty acids into adipose tissue-derived cells (preadipocytes) was demonstrated for silver carp (Hypophthalmichthys molitrix) and bluefin tuna (Thunnus orientalis).

[0236] Carp cell derived Silver carp (4-6 pounds total) were procured for isolation of preadipocytes. Cells were harvested from the visceral adipose tissue of silver carp by enzymatic and mechanical dissociation. Typically, 24 g of tissue was processed and cells were seeded from 1 g of tissue per well. Cells were cultured in growth medium and lipid uptake was tested up to passage 16.

[0237] Bluefin tuna cell-derived Pacific bluefin tuna (12-100 lbs) were wild caught in a regional area and identified visually and by genomic sequencing. Preadipocytes were harvested from subcutaneous fat. Typically, 6-24 g of tissue were processed via enzymatic and mechanical dissociation and seeded as 0.5-1 g of tissue per well.

[0238] Preadipocyte differentiation / lipid transduction A fish adipocyte differentiation protocol found in the literature [see Todorcevic et al. 2010] was initially used to test the differentiation of silver carp preadipocytes into adipocytes with lipid storage capacity. Cells were plated 1 cm 2 Cells were seeded at 5-7000 cells per well for 2 days, then treated with "adipogenic medium" (differentiation medium) for 2 days, followed by lipid introduction for 14 days with test medium containing 0.2% Sigma Lipid Mix (replenished every 2 days). Differentiation media found in the literature consisted of the following: basal medium supplemented with dexamethasone, biotin, T3, pantothenate, IBMX, insulin, and lipid mixture.

[0239] The cells were then stained with Oil Red O and Hoechst using the following protocol: The cells were washed with 1x PBS and fixed in 4% PFA for 10 minutes at room temperature. The cells were washed twice with PBS and incubated with 60% isopropanol for 20 seconds. The cells were then incubated with Oil Red O for 10 minutes. Once Oil Red O was removed, the cells were washed with 60% isopropanol to remove excess Oil Red O and then incubated with fresh 60% isopropanol for 30 seconds. The cells were then washed with distilled water for 20 seconds and stained with Hoechst for 15 minutes. After a final wash with distilled water, the cells were imaged.

[0240] Carp preadipocytes were plated on tissue culture polystyrene at 1 cm 2 Cells were seeded at a density of 5000 cells per well. Cells were cultured in the presence of control medium without fatty acid mixture or test medium with fatty acid mixture (Sigma Lipid Mix, 1%) at different serum levels (4, 2, 0%). Cell morphology was observed by bright field microscopy for characteristic rounding and lipid incorporation over a period of 6 days.

[0241] The fatty acid mixture used in this example is a commercial product purchased from Millipore Sigma, catalog number L5146, and consists of 4.5 g / L cholesterol, 10 g / L cod liver oil fatty acids (methyl esters), 25 g / L polyoxyethylene sorbitan monooleate, and 2 g / L D-α-tocopherol acetate in ethanol. The serum used in this example is produced from fetal bovine blood and processed for use in cell culture. As will be appreciated by those skilled in the art, serum is an undefined mixture that can vary from lot to lot.

[0242] On tissue culture polystyrene, 1 cm of bluefin tuna preadipocytes were 2Cells were seeded at a density of 10,000 cells per well. Cells were cultured in the presence of control medium without fatty acid mixture or test medium with fatty acid mixture (Sigma Lipid Mix, 1%) at different serum levels (4, 2, or 0%). Cell morphology was observed by bright field microscopy for characteristic rounding and lipid incorporation over a period of 6 days.

[0243] Immunofluorescence staining of cells was performed with Hoechst for cell nuclei, with phalloidin for cytoskeleton, and with BODIPY for lipid droplets.

[0244] The medium was aspirated and the cells were washed with 1×PBS. The PBS was aspirated and the cells were then fixed with 4% PFA for 10 minutes at room temperature. The PFA was removed and then gently washed with 1×PBS. The cells were then permeabilized with 0.1% Triton®-X-100 for 5 minutes and then blocked for 1 hour in 0.1% TBS / T with 5% chicken serum. The cells were then incubated with phalloidin, BODIPY, and Hoechst in basal medium without FBS for 1 hour. The cells were imaged in PBS. If phalloidin was not used, after fixation the cells were incubated with BODIPY and Hoechst for 1 hour before staining.

[0245] result Preadipocytes from freshwater carp or marine bluefin tuna were isolated and expanded in vitro. The proliferating cells showed elongated cell morphology and cell numbers increased over time. Initial testing with carp demonstrated that previously used methods for lipid transfer of preadipocytes incorporating insulin or cAMP stimulation were not effective. Existing methods showed limited lipid transfer over the 14 days of this differentiation protocol. Although a small number of cells stored some lipid (identified by Oil Red O staining), not all cells were morphologically round and stored large lipid droplets as shown by the process according to this example.

[0246] Here, serum-reduced or serum-free medium in the presence of a complex fatty acid mixture allowed carp preadipocytes to uptake and store fatty acids (Figure 1). The continuous presence of cell monolayers demonstrated that viability was maintained. As characteristic of mature adipocytes, cell morphology changed from elongated to round with increased cell size and the presence of lipid droplets. Compared to the control culture, which showed no morphological changes over 7 days, carp preadipocytes in the presence of fatty acids achieved morphological changes in 2 days that were maintained throughout the entire 7-day culture period. This suggests that fatty acid accumulation is more directly related to the addition of fatty acids rather than cell density and culture time. To confirm that the morphological changes in carp preadipocytes were caused by lipid droplet accumulation, immunofluorescence was performed (Figures 2A-2B). Carp preadipocytes cultured in the presence of fatty acid mixtures were positive for both lipid droplets and the cytoskeletal protein F-actin, whereas cells cultured in control conditions expressed the cytoskeletal protein F-actin but were negative for lipid accumulation.

[0247] Bluefin tuna-derived preadipocytes accumulated lipids in the presence of fatty acid mixtures, similar to carp-derived preadipocytes (Figures 3A-3B). Associated morphological changes included cell rounding and accumulation of lipid droplets. Both carp and tuna are apparently derived from freshwater or saltwater environments, but are used commercially as food in the seafood industry. Importantly, cells from both species accumulated lipids from complex fatty acid mixtures containing a mixture of saturated and unsaturated fatty acids, including omega-3s. Although carp and tuna are not closely related taxonomically, both are in the superclass of bony fishes, Osteichthyes, which may provide commonalities in preadipocyte behavior.

[0248] Example 2: Introduction of single fatty acids into fish adipose-derived cells Here, we demonstrate the introduction of individual fatty acids with different chemical structures in fish adipose tissue-derived cells, where the accumulation of specific fatty acids, such as omega-3, relative to saturated fatty acids allows for the tailoring of the nutritional composition in the cells and the final 3D seafood product.

[0249] Freshwater carp-derived preadipocytes as described in Example 1 were assessed for their ability to incorporate individual fatty acids. The cells were plated on tissue culture polystyrene at 1 cm 2 Cells were seeded at a density of 5-7000 cells per well. Cells were cultured in the presence of fatty acid-free control medium or test medium containing individual fatty acids at 25, 50, 75, or 100 μg / ml in the absence of serum. Individual fatty acids tested included saturated (palmitoleic acid) and unsaturated (linoleic acid). Cell morphology was observed over 6 days by bright field microscopy for characteristic rounding and lipid incorporation. Immunofluorescent staining of cells was performed as in Example 1 with DAPI for cell nuclei, phalloidin for cytoskeleton, and BODIPY for lipid droplets.

[0250] result Carp preadipocytes showed varying degrees of morphological changes and lipid accumulation depending on the concentration and type of fatty acid used. Similar to Example 1, where a complex mixture of fatty acids was used, single fatty acids at sufficient concentrations induced a round morphology that was maintained over 6 days (Figure 4). No toxicity was observed at the lowest concentration used, 25 μg / ml, but at higher concentrations up to 100 μg / ml palmitoleic acid showed a reduction in cell confluency, indicating that it was potentially toxic.

[0251] Confirmation of morphological changes correlating with lipid accumulation was observed by BODIPY staining of lipid droplets (Figures 5A-5B). Lipid droplets were observed at 25 μg / ml, the lowest concentration tested, and tended to increase in frequency at higher concentrations, if no toxicity was observed. Of note, both nutritionally important omega-3 polyunsaturated fatty acids, DHA and EPA, could be readily introduced into carp preadipocytes. In comparison with what has been described in the literature for the culture of primary fish preadipocytes, Sigma lipid mixtures are typically used for the differentiation process, rather than individual fatty acids or combinations of fatty acids. Palmitoleic acid, a monounsaturated fatty acid, showed effective introduction at 50 μg / ml, despite reduced cell numbers. Considering the naturally occurring ratio of fatty acids in carp, which contains significant amounts of monounsaturated acids including palmitoleic acid, the efficiency of incorporation of the various fatty acids is remarkable (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4325063 / and https: / / www.agriculturejournals.cz / publicFiles / 84796.pdf). These data suggest a process- and formulation-dependent effect of lipid accumulation in in vitro grown cells compared to in vivo grown cells.

[0252] Example 3: Introduction of complex fatty acid mixtures into fish connective tissue-derived cells In this example, the introduction of a complex fatty acid mixture (including omega-3) composed of saturated and unsaturated fatty acids into connective tissue cells (fibroblasts) of yellowtail fish (Seriola lalandi) was demonstrated.

[0253] Seriola fibroblasts were isolated from muscle tissue by enzymatic and mechanical dissociation. The cells did not fuse or differentiate into muscle, as was done with myoblasts. The cells were expanded in 2D tissue culture in the presence of 10% FBS for several passages to produce cell cultures with stable proliferation rates. The stabilized cell cultures were used for lipid incorporation experiments.

[0254] Fibroblasts were seeded on tissue culture polystyrene at a density of 5000 cells per cm2. Cells were cultured in the presence of control medium without fatty acid mixture or test medium containing fatty acid mixture (Sigma Lipid Mix, 1%) in the presence of 4% serum. Cell morphology was observed by bright field microscopy for characteristic rounding and lipid incorporation over a period of 7 days.

[0255] Immunofluorescence staining of cells was performed as described in Example 1 with Hoechst for cell nuclei, with phalloidin for cytoskeleton, and with BODIPY for lipid droplets.

[0256] result Control fibroblast cultures showed typical morphology with elongated cells throughout the 7-day culture period (Figure 6). Confluent monolayers at day 7 show a decrease in cell size as cells become more densely packed. In contrast, fibroblasts treated with fatty acid mixtures show a round morphology with the appearance of lipid droplets as early as day 2 and throughout the culture period. Immunofluorescence staining for lipids confirmed lipid accumulation in fibroblasts after treatment with fatty acid mixtures (Figures 7A-7B). In contrast to this example, previous lipid introduction studies did not evaluate fibroblasts as a storage mechanism for fatty acids. On the other hand, the approach according to embodiments of the present disclosure exemplified in this example shows a unique method to introduce lipids into cells and incorporate fatty acids into cell-cultured seafood products without adipose tissue-derived cells.

[0257] Example 4: Introduction of complex fatty acid mixtures into fish muscle-derived cells In this example, the introduction of a complex fatty acid mixture (including omega-3) composed of saturated and unsaturated fatty acids into muscle precursor cells (myoblasts) was demonstrated for yellowtail (Seriola lalandi), mahi-mahi (Coryphaena hippurus), and Pacific bluefin tuna (Thunnus orientalis).

[0258] Seriola quinqueradiata, Mahi-mahi, or Bluefin tuna myoblasts were isolated from muscle tissue by enzymatic and mechanical / or manual dissociation. Cells were expanded in 2D tissue culture in the presence of 10% FBS for several passages to produce cell cultures with stable proliferation rates. The myogenic potential of the cells was confirmed by differentiation assays and the formation of elongated multinucleated cells upon switching to low serum conditions. Stable cell cultures with confirmed myogenic function were used for lipid loading experiments.

[0259] Myoblasts were seeded at a density of 5000-10,000 cells per cm2 on tissue culture polystyrene. Cells were cultured in the presence of control medium without fatty acid mixture or test medium containing fatty acid mixture (Sigma Lipid Mix, 1%) in the presence of 4% serum. Cell morphology was observed by bright field microscopy for characteristic rounding and lipid incorporation for up to 7 days. Medium was replaced with fresh lipid every other day.

[0260] Immunofluorescence staining of cells was performed on days 6 or 7 as described in Example 1 with Hoechst for cell nuclei and BODIPY for lipid droplets.

[0261] result The introduction of the complex fatty acid mixture was performed on myoblasts from three different fish species: Seriola quinqueradiata, Mahi-Mahi, and Bluefin Tuna. Prior to evaluating lipid introduction, differentiation experiments confirmed myogenic potential. Similar to the other cell types tested, morphological changes were observable 1 day after lipid addition to the medium and could be maintained for several days (Figures 8A, 8B, 9A, 9B, 10A, and 10B). Over the culture period, cells continued to round and expand and filled with lipid droplets. Large lipid droplets were observable by day 6 using brightfield microscopy and could be confirmed as lipid droplets by staining with BODIPY. Interestingly, some spontaneous differentiation into myotubes was observed in both Mahi-Mahi and Bluefin Tuna myoblasts, which were then introduced into cells with multinucleated or elongated myocyte-like morphology.

[0262] These studies confirm the capacity of muscle-derived cells from a wide range of species for lipid transduction, with or without differentiation to a muscle cell phenotype. The ability to transduce multinucleated cells suggests that fatty acid transduction may occur at various stages of muscle differentiation and may be incorporated into mature muscle fibers in cell-cultured seafood products. All three species tested are in different classes, most closely related to the suborder Actinopterygii, which includes the ray-finned fish, indicating the broad applicability of muscle-derived fish cells for lipid transduction.

[0263] Example 5: Introduction of single fatty acids into fish muscle-derived cells Here, we demonstrate the introduction of individual fatty acids with different chemical structures in fish muscle-derived cells, where the accumulation of specific fatty acids, such as omega-3, relative to saturated fatty acids allows for the tailoring of the nutritional composition in the cells and the final 3D seafood product.

[0264] Myoblasts from Seriola spp., as described in Example 4, were assessed for their ability to take up individual fatty acids. The cells were plated on tissue culture polystyrene at 1 cm 2 Cells were seeded at a density of 6000 cells per 1000 ml. Cells were cultured in the presence of control medium without fatty acids or test medium containing 25, 50, 75, or 100 μg / ml of individual fatty acids conjugated to BSA in the presence of 4% serum. Individual fatty acids tested included saturated, monounsaturated, and unsaturated fatty acids shown in Table 7, which differ in carbon chain length and double bond position along the carbon chain. Cell morphology was observed over 6 days by bright field microscopy for characteristic rounding and lipid incorporation. Immunofluorescence staining of cells was performed with DAPI for cell nuclei, phalloidin for cytoskeleton, and BODIPY for lipid droplets, as in Example 1. [Table 10]

[0265] result The amount of fatty acid accumulation and subsequent nutritional composition of the cells were controlled by the chemical nature of the fatty acid, the concentration, and the time. A literature search on the nutritional composition of Seriola was conducted, and the top four fatty acids for each of saturated, unsaturated, and monounsaturated fatty acids were compiled and selected for this example. Each of the major types of fatty acids could be introduced into Seriola myoblasts and maintained for up to 6 days, as confirmed by BODIPY immunofluorescence staining (Figures 11A, 11B, 12A, 12B, 13A, and 13B). Negative control cultures cultured in the presence of serum and without additional lipids showed no lipid accumulation (Figure 34).

[0266] The saturated fatty acid lauric acid showed significant accumulation at all concentrations tested, while the remaining SFAs, myristic acid, palmitic acid, and stearic acid, showed limited accumulation and only a small range of incorporation even at the highest concentration of 100 μg / ml. Most of the MUFAs tested showed concentration-dependent lipid accumulation, with significant lipid accumulation even at the lowest concentration tested. The exception to MUFA incorporation was nervonic acid, which did not show lipid accumulation at any of the concentrations tested. PUFAs also showed concentration-driven lipid accumulation, especially with respect to linoleic acid. Of note, DHA, a related nutritional fatty acid, showed limited incorporation at all concentrations and toxicity as evidenced by changes in cell morphology and reduced cell numbers at >25 μg / ml.

[0267] These data suggest that carbon length is not a critical mediator of fatty acid incorporation, as each of the 18 carbon fatty acids tested, stearic acid, vaccenic acid, oleic acid, linoleic acid, and linolenic acid, had different concentration-dependent incorporation outcomes. Fatty acids with carbon lengths between 12 and 22 were successfully incorporation across a range of chemicals.

[0268] Cis-vaccenic acid was taken up very easily by fish cells, even though it is not a major component of most fish. This fatty acid is a common fatty acid in bacterial lipids and is normally present in most plant and animal tissues. The ability of fish cells to take up significant amounts of fatty acids beyond those normally present supports the use of targeted fatty acid incorporation to alter the nutritional profile of cells and / or cell-cultured seafood products via lipid incorporation.

[0269] Example 6: Introduction of chemically defined fatty acid mixtures into fish muscle-derived cells In this example, the introduction of chemically defined fatty acid mixtures was demonstrated in fish muscle-derived cells using mixtures of fatty acids of various chemical structures and concentrations.

[0270] Accumulation of specific fatty acids, such as omega-3, relative to saturated fatty acids allows for tailoring of the nutritional composition in the cells and the final 3D seafood product. Fatty acids not identified as contributors to lipid accumulation have shown synergistic abilities to enhance lipid accumulation or improve cell health, allowing fatty acid accumulation beyond that of the individual fatty acids when used individually.

[0271] Myoblasts from Seriola spp., as described in Example 4, were evaluated for their ability to take up fatty acids from a defined chemical composition. The cells were plated on tissue culture polystyrene at 1 cm 2 The cells were seeded at a density of 6000 cells per 1000 cells. The cells were cultured for 2 or 6 days in the presence of control medium without fatty acids or test medium containing fatty acids conjugated to BSA in the presence of 4% serum. For each individual fatty acid, a concentration of 0, 10, 25, or 50 μg / ml was used. The fatty acid combinations were determined using an experimental design using Taguchi L-27 design (Table 8). The fatty acids tested included monounsaturated and polyunsaturated fatty acids shown in Table 7, Example 5, with different carbon chain lengths and double bond positions along the carbon chain. In contrast to existing fish cells that contain SFA, especially palmitic and stearic acids, the experimental design did not include SFA.

[0272] Cells were fixed, stained, and imaged on day 2 or day 6. Cells were treated with NucBlue, BODIPY, and Mitotracker for 1.5 hours, and live imaging was performed. Cells were then fixed in 4% PFA for 15 minutes, then blocked overnight at 4°C in blocking buffer containing 5% goat serum, 1% BSA, and 0.2% fish gelatin in PBS. Cells were incubated with phalloidin in blocking buffer (phalloidin 1:800) for 1 hour at room temperature. Plates were washed twice with PBS before imaging. The effect of each fatty acid on cell number, fatty acid accumulation, and cell health was quantified using JMP statistical software. [Table 11]

[0273] result As in Examples 4 and 5 above, no fatty acid accumulation was detected in the control condition without added fatty acids (Figure 35). As shown by BODIPY staining, all fatty acids tested resulted in some degree of lipid accumulation, with the highest levels being reached in conditions 16, 22, and 25 (Figure 36, Table 9). The common factor in each of the combinations with the highest fatty acid accumulation was the presence of nervonic acid at 50 μg / ml. As shown in Example 5, the high accumulation of fatty acids in the presence of nervonic acid was surprising based on previous results showing that nervonic acid itself is not introduced when used individually. Importantly, at least one unsaturated omega-3 fatty acid was present in each of the mixtures with high lipid accumulation. As shown in Example 4, lipid accumulation of each individual fatty acid was minimal at 25 μg / ml, but when the same fatty acids were used in combination at low concentrations, the introduction was observed to be significant for total lipid droplet accumulation. Of note, combinations 22 and 25 use very low levels of fatty acids with very low introduction observed at any individual concentration tested, while this combination resulted in relatively high lipid accumulation. Additionally, the combination with the highest total lipid loading also showed higher cell counts, indicating that cell viability was maintained compared to other conditions (FIG. 37). Statistical analysis of lipid accumulation identified nervonic acid as a key factor in increasing lipid accumulation and cell count. As shown in Table 9, all combinations with high fatty acid accumulation contained 50 μg / ml nervonic acid. Compared to the individual fatty acids tested in Example 5, which showed low lipid accumulation and some toxicity of DHA when used individually, when used in combination with nervonic acid, it shows high cell counts and high lipid accumulation.

[0274] The saturated fatty acid content in fish can be approximately 40% of the total lipid content. As the Western diet also has a very high SFA intake, limiting the amount of SFA content in the product of the present invention will provide higher quality nutritional value to the consumer.

[0275] One of the relevant components of the combination that promoted high lipid uptake was the presence of nervonic acid. Nervonic acid is important in cerebrosides, fatty acids that are components of muscle and the central and peripheral nervous system. Nervonic acid is also one of the major fatty acids found in brain sphingolipids. Nervonic acid is found in breast milk and is recommended for pregnant and lactating women. Nervonic acid may also have neuroprotective effects and is commonly found in energy supplements. The use of nervonic acid in enhancing lipid uptake into cells is, to the inventor's knowledge, not yet known. The use of nervonic acid may allow lipid uptake of normally toxic fatty acids as well as impart additional nutritional quality to seafood products. [Table 12]

[0276] Example 7: Introduction of fatty acid mixtures and single fatty acids into mammalian cells In this example, the introduction of complex fatty acid mixtures (including omega-3s) composed of saturated and unsaturated fatty acids, as well as single fatty acids, including polyunsaturated fatty acids such as omega-3s, into muscle precursor cells (myoblasts) and fibroblasts was demonstrated in mammalian cells.

[0277] Bovine, porcine, caprine, lamb, or deer myoblasts or fibroblasts are isolated from muscle tissue by enzymatic and mechanical / or manual dissociation. Cells are expanded in 2D tissue culture or 3D cell suspension culture.

[0278] Cells are cultured in the presence of control medium without fatty acids or non-reduced or reduced serum test medium containing individual fatty acids at 1, 5, 10, 15, 25, 50, 75, or 100 μg / ml. The fatty acids are conjugated or unconjugated to BSA. The individual fatty acids include saturated, monounsaturated, and polyunsaturated fatty acids that vary in carbon chain length and position of the double bond along the carbon chain.

[0279] In control conditions where no fatty acids are added, no lipid droplet accumulation is detected. All fatty acids tested result in some degree of lipid accumulation, but the lipid accumulation of each individual fatty acid is negligible. However, when low concentrations of the same fatty acids are used in various combinations, significant lipid induction is observed in terms of total lipid accumulation.

[0280] Nervonic acid increases cell proliferation when added to growth medium as the only fatty acid. When combined with other fatty acids, Nervonic acid causes an increase in lipid uptake and lipid accumulation. Omega-3 polyunsaturated fatty acids, DHA and EPA, are not well uptaken when tested alone. When Nervonic acid is added to this mixture, cells are able to uptake and store higher levels of these polyunsaturated fatty acids, accelerating proliferation.

[0281] Example 8: Introduction of fatty acid mixtures and single fatty acids into cells from avian species In this example, the introduction of complex fatty acid mixtures (including omega-3s) composed of saturated and unsaturated fatty acids, as well as single fatty acids, including polyunsaturated fatty acids such as omega-3s, into muscle precursor cells (myoblasts) and fibroblasts was demonstrated in mammalian cells.

[0282] Chicken, duck, goose, or turkey myoblasts or fibroblasts are isolated from muscle tissue by enzymatic and mechanical / or manual dissociation. Cells are expanded in 2D tissue culture or cell suspension culture.

[0283] Cells are cultured in the presence of control medium without fatty acids or non-reduced or reduced serum test medium containing individual fatty acids at 1, 5, 10, 15, 25, 50, 75, or 100 μg / ml. The fatty acids are conjugated or unconjugated to BSA. The individual fatty acids include saturated, monounsaturated, and polyunsaturated fatty acids that vary in carbon chain length and position of the double bond along the carbon chain.

[0284] In the control condition where no fatty acid is added, no lipid accumulation is detected. All fatty acids tested result in some lipid accumulation, but the lipid accumulation of each individual fatty acid is negligible. However, when low concentrations of the same fatty acids are used in various combinations, the lipid induction is observed to be significant in terms of total lipid accumulation.

[0285] Nervonic acid increases cell proliferation when added to growth medium as the only fatty acid. When combined with other fatty acids, Nervonic acid causes an increase in lipid uptake and lipid accumulation. Omega-3 polyunsaturated fatty acids, DHA and EPA, are not well uptaken when tested alone. When Nervonic acid is added to this mixture, cells are able to uptake and store higher levels of these polyunsaturated fatty acids, accelerating proliferation.

[0286] Example 9: Fortification of fish products with lipids Fish myoblasts, pre-adipocytes, or fibroblasts are grown in 2D tissue culture or cell suspension culture. Growth medium is supplemented with a complex fatty acid mixture (including omega-3s) consisting of saturated, monounsaturated, and polyunsaturated fatty acids, or single fatty acids including polyunsaturated fatty acids such as omega-3s. Lipid-transduced cells are harvested by cell separation techniques such as sedimentation or tangential flow filtration. Harvested cells are assembled using various methods including extrusion and bioprinting to form cubes, strips, or fillets. The cubes, strips, or fillets are composed of myoblasts or adipocytes or fibroblasts, or combinations of these cell types. The resulting fish food product contains higher levels of total lipids than conventionally sourced fish. The resulting fish product contains higher levels of polyunsaturated fatty acids such as omega-3 fatty acids than conventionally sourced fish.

[0287] In another embodiment, fish myoblasts, preadipocytes, or fibroblasts are grown in 2D tissue culture or cell suspension culture and then harvested by centrifugation. The cells are then mixed with differentiation medium supplemented with a complex fatty acid mixture (including omega-3) consisting of saturated, monounsaturated, and polyunsaturated fatty acids, or a single fatty acid including polyunsaturated fatty acids such as omega-3.

[0288] Example 10: Dose-dependent transfer of complex FA mixtures into adipose, muscle, and connective tissue-derived cells of multiple species In this example, we demonstrate the introduction of a complex fatty acid mixture (including omega-3) composed of saturated and unsaturated fatty acids into muscle, adipose, and connective tissue-derived cells from several terrestrial and aquatic species (Table 10 below).

[0289] Silver carp and bluefin tuna preadipocytes were prepared as described in Example 1 above. Bluefin tuna fibroblasts were prepared similarly to bluefin tuna myoblasts. Seriola quinqueradiata fibroblasts were prepared as described in Example 3 above. Mahi-mahi myoblasts were prepared as described in Example 4 above. The remaining cells were obtained from ATCC or commercial sources and cultured according to information provided by the supplier (Tables 4-5 above).

[0290] Cell culture and lipid transfection Cells were seeded onto polystyrene 6-well plates. Myoblasts were plated at 1 cm 2 Preadipocytes were seeded at a density of 5,000–10,000 cells per cm. 2 The cells were seeded at a density of 5-7000 cells per well.

[0291] Cells were then cultured in the presence of control medium without the fatty acid mixture, or in the presence of test medium containing the fatty acid mixture (Sigma Lipid Mix, 1%) in the presence of control (10% FBS) or serum-reduced (4% FBS) medium. For all cell types and species, the medium was replaced with fresh lipids every other day.

[0292] Cell morphology and immunofluorescence staining Cell morphology was observed for characteristic rounding and lipid incorporation by bright field microscopy over a period of up to 7 days. Immunofluorescence staining of cells was performed on days 6 or 7 with Hoechst for cell nuclei and BODIPY for lipid droplets as described in Example 1.

[0293] result Immunofluorescence images and corresponding lipid quantification data for each sample are shown in Figures 14A-33B (Table 10 below). Quantification of lipid incorporation shows increased incorporation compared to control and naturally occurring levels for all species. In fibroblasts and myoblasts, where fat content is typically less than 1%, an increase of over 70 percentage points is observed over 3 days. As shown in previous examples, longer durations have been shown to potentially increase even further to 90% of cell volume. Surprisingly, using the methods described herein, fat could be infused as effectively as preadipocytes into many cell types that do not normally naturally accumulate fat, such as cell types derived from muscle, brain, skin, and embryonic tissues. [Table 13]

[0294] Example 11: Lipidomic analysis of lipid-transduced cells In this example, the fatty acid content of control and lipid-transduced cells was determined using the collection and lipid analysis protocol described above to demonstrate the range of lipid profiles achievable for various cell types derived from both terrestrial and aquatic species. Twelve unique cell lines from 11 species, both aquatic and terrestrial, across seven different cell types, including bluefin tuna myoblasts, bluefin tuna preadipocytes, carp preadipocytes, red snapper muscle-derived cells, yellowfin tuna cardiac fibroblasts, bluegill fibroblasts, tilapia brain-derived cells, salmon kidney cells, mahi-mahi myoblasts, chicken embryo fibroblasts, porcine kidney cells, and dog kidney cells, were evaluated for their ability to transduce lipids and create fatty acid custom profiles. Cells were cultured and transduced with lipids using the protocol described above. Lipid transduction was performed using the various condition concentrations shown in Table 11 below. Conditions 3-6 were selected to demonstrate tailoring of profiles to existing food profiles for cells derived from other species. Conditions 2 and 7-13 were chosen to demonstrate the range of total saturated fatty acids (SFA) or omega-3 that could be achieved using lipid incorporation. [Table 14]

[0295] result The fatty acid composition of the samples tested is shown below in Tables 12-23. Data are expressed as the relative percent concentration of total fatty acids for each condition tested, and as the natural concentration for the species of interest. In addition to the fatty acids shown, 17:1 heptadecenoic acid was measured but not detected in any of the samples, and 22:3 was detected as a minor fraction in a few samples, so data are not shown for either fatty acid. Data for each species is shown in the following tables: bluefin tuna myoblasts (Table 12A, B, C), bluefin tuna preadipocytes (Table 13A, B, C), carp preadipocytes (Table 14A, B, C), red snapper muscle-derived cells (Table 15A, B, C), yellowfin tuna cardiac fibroblasts (Table 16A, B, C), bluegill fibroblasts (Table 17A, B, C), tilapia brain-derived cells (Table 18A, B, C), salmon kidney cells (Table 19A, B, C), mahi-mahi myoblasts (Table 20A, B, C), chicken embryo fibroblasts (Table 21A, B, C), porcine kidney cells (Table 22A, B, C), and dog kidney cells (Table 23A, B, C). Changes in fatty acid composition compared to the control and naturally occurring were observed in all species. The percentage point changes from untransfected control cells of all species are shown in Tables 24A,B and 25A,B for saturated fatty acids (SFAs) and omega-3 fatty acids, respectively. Notably, saturated fatty acids were significantly decreased by over 55 percentage points, while omega-3 fatty acids were significantly increased by over 70 percentage points. For terrestrial animals, where omega-3 is typically less than 0-10%, the presence and significant increase of healthy fats represents an incredible ability to create unique compositional changes not found in nature. [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2] [Table 17-1]

Table 17-2

Table 18-1

Table 18-2

Table 19-1

Table 19-2

Table 20-1

Table 20-2

Table 21-1

Table 21-2

Table 22-1

Table 22-2

Table 23-1

Table 23-2

Table 24-1

Table 24-2

Table 24-3

Table 24-4

Table 25-1

Table 25-2

Table 26-1

Table 26-2

Table 27-1

Table 27-2

Table 28-1

Table 28-2

Table 29-1

Table 29-2

Table 30-1

Table 30-2

Table 31-1

Table 31-2

Table 32-1

Table 32-2

Table 33-1

Table 33-2

Table 34-1

Table 34-2

Table 35-1

Table 35-2

Table 36-1

Table 36-2

Table 37-1

Table 37-2

Table 38-1

Table 38-2

Table 39-1

Table 39-2

Table 40-1

Table 40-2

Table 41-1

Table 41-2

Table 42-1

Table 42-2

Table 43-1

Table 43-2

Table 44-1

Table 44-2

Table 45-1

Table 45-2

Table 46-1

Table 46-2

Table 47-1

Table 47-2

Table 48-1

Table 48-2

Table 49-1

Table 49-2

Table 50-1

[0296] Example 12: Effect of Nervonic Acid on FA Toxicity The concentration effects of fatty acids previously shown to be toxic to aquatic animal adipose-derived and muscle-derived cells (Examples 2, 5, and 6, respectively) were evaluated alone and in combination with Nervonic acid. EPA, DHA, stearic acid, palmitic acid, and Nervonic acid were tested at concentrations of 0, 10, 25, and 50 ug / mL. Concentrations of EPA, DHA, stearic acid, and palmitic acid were also tested in combination with Nervonic acid at 10, 25, and 50 ug / ml.

[0297] Cell lines were seeded into 4 x 96-well plates following standard passaging protocols. Cells were grown to approximately 80% confluence and then transferred to lipid loading medium containing 0 / 4% FBS and the appropriate lipid composition, 6 wells / condition. Cells were monitored for 24-48 hours to assess any protective effects of the addition of nervonic acid.

[0298] Cells from terrestrial cell lines were imaged on an IncuCyte for a one-time scan, images were collected, and confluence was quantified using the IncuCyte software. After imaging, cell viability was determined by a commercial ATP assay (CellTiter-Glo 2.0, Promega), where the luminescent signal is proportional to cell number.

[0299] Cells from aquatic species were placed into an IncuCyte and scanned every 6 h for up to 48 h. Once imaging was completed, cell viability was determined by a commercially available ATP assay (CellTiter-Glo 2.0, Promega), in which the luminescent signal is proportional to cell number.

[0300] result Upon addition of nervonic acid to the introduction of another fatty acid, two distinct phenomena were observed. First, in the presence of toxic concentrations of DHA, nervonic acid had a beneficial effect on viability, resulting in an increase in cellular ATP, an indicator of cell number. Figures 38-40 show that for various species, in combination with DHA, nervonic acid is able to rescue cells from toxicity compared to other fatty acids. The extent of improvement was a 70-fold, 5.5-fold, and 42-fold increase in bluefin tuna myoblast, canine kidney, and rabbit skin fibroblast cells, respectively. Notably, this was also observed in Example 6, where all combinations that showed a significant increase in cell number had both DHA and nervonic acid. The above examples are provided to give those of skill in the art a complete disclosure and description of the methods of making and using embodiments of the introduction of various types of fatty acids, alone or in combination with others, to obtain fatty acid-rich meat cells with high cell numbers and increased lipid uptake and lipid accumulation, as well as the related compositions, methods, and systems of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Those skilled in the art will recognize how to adapt the features of the exemplary cells, compositions, methods, and systems disclosed herein to other cells, compositions, methods, and systems in accordance with the various embodiments and claims.

[0301] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains.

[0302] The entire disclosure of each document cited in the Background Art, Summary of the Invention, Detailed Description of the Invention, and Examples (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) is incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety. However, in the event of any inconsistency between the cited documents and this disclosure, this disclosure shall prevail.

[0303] The terms and expressions used in this specification are used as terms of description and not of limitation, and the use of such terms and expressions does not have the intention of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the present disclosure as claimed. Thus, although the present disclosure has been specifically disclosed by embodiments, exemplary embodiments, and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present disclosure as defined by the appended claims.

[0304] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 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. The term "plurality" includes two or more referents unless the content clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0305] When a Markush group or other grouping is used herein, it is intended that all individual members of the group, as well as all combinations and possible subcombinations of the group, are individually included in the disclosure. Unless otherwise stated, the disclosure can be practiced using any combination of components or materials described or exemplified herein. Those skilled in the art will understand that methods, system elements, and materials other than those specifically exemplified can be used in the practice of the disclosure without undue experimentation. All art-known functional equivalents of any such methods, device elements, and materials are intended to be included in the disclosure. Whenever a range is given herein (e.g., a temperature range, frequency range, time range, or composition range), all intermediate ranges and all subranges, as well as all individual values ​​included in the given range, are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from the claims of the disclosure. The disclosure illustratively described herein can suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein.

[0306] Many embodiments of the present disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the present disclosure, and it will be apparent to those skilled in the art that the present disclosure can be implemented using many variations of the genetic circuits, genetic molecular components, and method steps described in this description. As will be apparent to those skilled in the art, the methods and systems useful in the methods and systems of the present invention may include many optional configurations and processing elements and steps.

[0307] In particular, it is to be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

[0308] References Anne Vegusdal,Hilde Sundvold,Tor Gjoen,and Bente Ruyter.An in vitro Method for Studying the Proliferation and Differentiation of Atlantic Salmon Preadipocytes.2003.Lipids,Vol.38,no 3,289-296.

Claims

1. A cultured cell of an animal, the cultured cell comprising: a) a lower saturated fat (SFA) content (g saturated fat per g total fat), and / or b) has a higher unsaturated fat (UFA) content (g of polyunsaturated fatty acids (PUFA) and / or monounsaturated fats (MUFA) per g of total fat); A cultured cell of an animal, wherein each of a) and b) is in comparison to a wild-caught or farmed animal of the same species as the cultured cell.

2. the cells comprising one or more of myoblasts, myocytes, preadipocytes, adipocytes, fibroblasts, keratinocytes, epithelial cells, endothelial cells, embryo-derived cells, induced pluripotent stem cells, mesenchymal stem cells, and combinations thereof from the animal; and optionally i) the adipocytes contain one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols in an amount between 0.01% and 1% by weight of the cells; and / or ii) the fibroblasts contain one or more polyunsaturated fatty acids, one or more saturated fatty acids, one or more monounsaturated fatty acids, and / or one or more sterols in an amount of at least 1% by weight of the cells; and / or iii) the myoblasts comprise one or more polyunsaturated fatty acids, one or more saturated fatty acids, one or more monounsaturated fatty acids, and / or one or more sterols in an amount of at least 1% by weight of the cells; The cultured cell according to claim 1.

3. The animal is i) is an aquatic animal, or ii) contains fish and / or crustaceans; or iii) includes one or more of the following: cartilaginous fish, bony fish, ray-finned fish, lophophoran fish, mollusks, mussels, cephalopods, crustaceans, and echinoderms; or iv) Basa, flounder, hake, scup, smelt, rainbow trout, hard clam, blue crab, peeky toe crab, Asahi crab, cuttlefish, eastern oyster, Pacific oyster, anchovy, herring, golden cod, moi, orange roughy, Atlantic perch, Lake Victoria perch, yellow perch, European oyster, Dover sole, sturgeon, tilefish, barracuda, yellowtail, sea urchin, Atlantic mackerel, sardine, black sea bass, European sea bass, hybrid striped bass bass), bream, cod, drum, haddock, hoki, pollock, reef fish, pink salmon, snapper, tilapia, turbot, walleye, lake whitefish, wolffish, hard clams, cockles, jonah crab, snow crab, crayfish, mussels, Korean prawns, sablefish, Atlantic salmon, coho salmon, skate, Dungeness crab, king crab, purple mussels, green mussels, red shrimp, escolar, Chinook salmon, Chum salmon, American shad, alpine char, carp, catfish, John Dory, grouper, halibut, squirt Shark, trevally, abalone, snails, stone crabs, American lobsters, spiny lobsters, octopus, black tiger shrimp, freshwater shrimp, Gulf shrimp, white shrimp, squid, barramundi, casque, dogfish, kingklip, mahi-mahi, sunfish, mako shark, swordfish, albacore tuna, yellowfin tuna, geoduck shells, squid lobsters, Magellan's mussels, mikazuki shrimp, barracuda, Chilean sea bass, cobia, croaker, eel, blue marlin, mullet, sockeye salmon, bluefin tuna, shrimp, crabs, lobsters, and echinoderms; or v) one or more of Thunnus orientalis, Lutjanus campechanus, Coryphaena hippurus, Seriola lalandi, Micropterus salmoides, Thunnus albacares, Lepomis macrochirus, Hypophthalmichthys molitrix, Salmo salar, Oncorhynchus mykiss, and Oreochromis massambicus; or vi) containing one or more of fish, clams, oysters, octopus, squid, shrimp, crabs, lobsters, sea cucumbers, and sea urchins; or vii) a terrestrial animal, optionally the terrestrial animal is a mammal, a bird, a reptile, an amphibian, or an insect; or the terrestrial animal is an armyworm, a cricket, a grasshopper, a frog, a toad, a newt, a salamander, an alligator, a crocodile, a snake, a chicken, a turkey, a duck, a goose, a pheasant, a game hen, a quail, a horse, a rhino, a tapir, a cow, a pig, a giraffe, a camel, a sheep, a deer, a goat, a rabbit, a dog, or a hippopotamus; or the terrestrial animal is selected from the group consisting of armyworms, crickets, and grasshoppers; or the terrestrial animal is selected from the group consisting of frogs, toads, newts, and salamanders; or the terrestrial animal is selected from the group consisting of alligators, crocodiles, and snakes; or the terrestrial animal is selected from the group consisting of chickens, turkeys, ducks, geese, pheasants, game hens, and quails; or The terrestrial animal is selected from the group consisting of horses, rhinos, tapirs, cows, pigs, giraffes, camels, sheep, deer, goats, rabbits, dogs, and hippos; The cultured cell according to claim 2.

4. The animal is a terrestrial animal, and the cell is i) an omega-3 PUFA content that is at least one percentage point higher than the same type of cell derived from a wild-caught or farmed terrestrial animal of the same species; and / or ii) an SFA content that is at least one percentage point lower and / or an MUFA content that is at least one percentage point higher than the same type of cell derived from a wild-caught or farmed terrestrial animal of the same species; The cultured cell according to any one of claims 1 to 3, comprising:

5. The cultured cells, a) a lower saturated fat (SFA) content (g saturated fat per g total fat), and / or b) has a higher unsaturated fat (UFA) content (g of polyunsaturated fatty acids (PUFA) and / or monounsaturated fats (MUFA) per g of total fat); 4. The cultured cells of any one of claims 1 to 3, comprising adipocytes, fibroblasts or myoblasts, each of a) and b) being in comparison to adipocytes, fibroblasts or myoblasts of a wild-caught or farmed animal of the same species as the cultured cells.

6. 13. A biomass comprising one or more cultured cells of claim 1.

7. 10. A method for producing a culture medium comprising the steps of: one or more cultured cells according to claim 1 and / or a biomass according to claim 6, and optionally a suitable matrix, Optionally, the food product further comprises increased nervonic acid content as compared to a wild-caught or farmed animal of the same species as said cultured cells.

8. 8. The food product of claim 7, wherein the food product is a homogenous mixture of two or more cell types.

9. The food product is i) has an omega-3 PUFA content that is at least one percentage point higher than a food of identical composition that contains terrestrial animal cells, except that said terrestrial animal cells are derived from the same species of wild-caught terrestrial animal or farmed terrestrial animal; or ii) has an SFA content that is at least one percentage point lower than a food of identical composition that contains terrestrial animal cells (except that said terrestrial animal cells are derived from the same species of wild-caught terrestrial animal or farmed terrestrial animal); or iii) comprises aquatic animal cells and has an omega-3 PUFA content that is at least one percentage point higher than a food of identical composition (except that said aquatic animal cells are derived from a wild-caught aquatic animal of the same species); or iv) comprising aquatic animal cells and having an SFA content that is at least one percentage point lower than a food of identical composition (except that said aquatic animal cells are derived from a wild-caught aquatic animal of the same species); The food product of claim 7.

10. A culture medium comprising a basal cell culture medium for animal cells supplemented with at least 0.1 μg / ml of one or more monounsaturated fatty acids.

11. The culture medium of claim 10 , wherein the animal is an aquatic animal or a terrestrial animal.

12. i) the monounsaturated fatty acid comprises one or more monounsaturated fatty acids having a carbon chain length of 12 to 22 carbons, and optionally the monounsaturated fatty acids include myristoleic acid (C14:1 ω-5), palmitoleic acid C16:1 ω-7, sapienic acid C16:1 ω-10, vaccenic acid (C18:1 ω-7), C18:1 ω9c found in most phospholipids, oleic acid (C18:1 ω-9), petroselinic acid (C18:1 ω-12), paulic acid (C20:1 ω-7), gondoic acid (C20:1 ω-11), erucic acid (C22:1 ω-9c), brassidic acid (C22:1 ω-9t), and / or nervonic acid (C24:1 ω-9); or ii) the monounsaturated fatty acids include palmitoleic acid, vaccenic acid, oleic acid, and / or nervonic acid; The culture medium of claim 10.

13. the monounsaturated fatty acid is at a concentration of 0.1 μg / ml to 1000 μg / ml; Optionally, the monounsaturated fatty acid is at a concentration of 1-100 μg / ml or 50-100 μg / ml; Optionally, the monounsaturated fatty acid is at a concentration of 50-75 μg / ml; Optionally, the monounsaturated fatty acid is at a concentration of 75 to 100 μg / ml.

14. 11. The culture medium of claim 10, wherein the monounsaturated fatty acids comprise palmitoleic acid at a concentration of 1-100 μg / ml or 50-100 μg / ml, vaccenic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml, oleic acid at a concentration of 1-100 μg / ml or 75-100 μg / ml, and / or MUFA (e.g. nervonic acid) at a concentration of 1-100 μg / ml or 50-75 μg / ml.

15. The culture medium of claim 10 , wherein the culture medium further comprises up to 4% serum.

16. 11. The culture medium of claim 10, wherein the culture medium does not contain any additional components such as dexamethasone, biotin, T3, pantothenate, IBMX, and / or insulin.

17. A method for culturing cells, comprising culturing the cells of an animal in a culture medium containing at least 0.1 μg / ml of one or more monounsaturated fatty acids for a period of time and under conditions that allow uptake of the monounsaturated fatty acids by the cells of the animal.

18. 18. The method of claim 17, wherein the animal is an aquatic animal or a terrestrial animal.

19. i) the monounsaturated fatty acid comprises one or more monounsaturated fatty acids having a carbon chain length containing 12 to 22 carbons; Optionally, the monounsaturated fatty acids include myristoleic acid (C14:1 ω-5), palmitoleic acid C16:1 ω-7, sapienic acid C16:1 ω-10, vaccenic acid (C18:1 ω-7), C18:1 ω9c found in most phospholipids, oleic acid (C18:1 ω-9), petroselinic acid (C18:1 ω-12), paulic acid (C20:1 ω-7), gondoic acid (C20:1 ω-11), erucic acid (C22:1 ω-9c), brassidic acid (C22:1 ω-9t), and / or nervonic acid (C24:1 ω-9); ii) the monounsaturated fatty acids include palmitoleic acid, vaccenic acid, oleic acid, and / or nervonic acid; 20. The method of claim 17.

20. the monounsaturated fatty acid is at a concentration of 0.1 μg / ml to 1000 μg / ml or 10 μg / ml to 1000 μg / ml; Optionally, the monounsaturated fatty acid is at a concentration of 1-100 μg / ml or 50-100 μg / ml; Optionally, the monounsaturated fatty acid is at a concentration of 50-75 μg / ml; Optionally, the monounsaturated fatty acid is at a concentration of 75-100 μg / ml.

21. 18. The method of claim 17, wherein the monounsaturated fatty acids comprise palmitoleic acid at a concentration of 1-100 μg / ml or 50-100 μg / ml, vaccenic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml, oleic acid at a concentration of 1-100 μg / ml or 75-100 μg / ml, and / or MUFA (e.g. nervonic acid) at a concentration of 1-100 μg / ml or 50-75 μg / ml.

22. The method according to claim 17, wherein the culture medium is a culture medium according to any one of claims 10 to 16.

23. 20. A cell of an animal, said cell being obtainable and / or obtained by the method of claim 17.

24. 24. A biomass comprising the cells of claim 23.

25. 25. A food product comprising one or more of the cells of claim 23 and / or the biomass of claim 24.

26. the cells have a monounsaturated fatty acid content of at least 0.2% by weight; Optionally, the cells have a monounsaturated fatty acid content of 0.2% to 90% by weight.

27. The cells, i) fish cells, in particular white fish cells, said cells comprising a monounsaturated fatty acid content of 0.2% to 90% by weight, or ii) fish cells, in particular white fish cells, said cells comprising a monounsaturated fatty acid content of 1% to 90% by weight; 26. The food product of claim 25.

28. 26. The food product of claim 25, wherein the food product has a monounsaturated fatty acid content of between 10% and 20%.

29. 26. The food product of claim 25, wherein the food product is free of adipocytes.

30. 26. The food product of claim 25, wherein the food product is a cell-cultured fish product.

31. 1. A system for culturing animal cells, comprising: One or more monounsaturated fatty acids, 20. The method according to claim 17 for increasing the content of one or more monounsaturated fatty acids in cells, comprising the system in combination with a culture medium for animal cells and / or animal cells for simultaneous, combined or sequential use.

32. 1. A culture medium for animal cells, comprising: the culture medium comprises a basal cell medium for animal cells supplemented with at least 0.1 μg / ml or at least 10 μg / ml of one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols; A culture medium for cells of said animal, said culture medium further comprising nervonic acid at a concentration of at least 1 μg / ml or at least 10 μg / ml.

33. the polyunsaturated fatty acid comprises one or more polyunsaturated fatty acids having a carbon chain length of 12 to 24 carbons; Optionally, the polyunsaturated fatty acid is selected from the group consisting of hexadecatrienoic acid (HTA) (C16:3 ω-3), linoleic acid (C18:2 ω-6), alpha linolenic acid (C18:3 ω-3), gamma linolenic acid (C18:3 ω-6), stearidonic acid (C18;4 ω-4), eicosadienoic acid (C20:2 ω-6), eicosatrienoic acid (ETE) (C20:3 ω-3), dimorpho-gamma-linolenic acid (C20:3 ω-6), mead acid (C20:3 ω-9), arachidonic acid (C20:4 ω-6), eicosapentaenoic acid (EPA) (C20:5 ω-3, C20:5 ω-6), heneicosapentaenoic acid (HPA) (C21:5 ω-3, C21:5 ω-6), ω-3), docosatetraenoic acid (C22:4 docosatetraenoic acid-6), DPA (C22:5 docosatetraenoic acid-3), docasahexaenoic acid (DHA) (C22:6 docasahexaenoic acid (DHA)-3), tetracosapentaenoic acid (C24:5 ω-3), and tetracosahexaenoic acid (herring acid) (C24:6 tetracosahexaenoic acid (herring acid) (C24:6 ω-3)); or Optionally, the polyunsaturated fatty acids include alpha linolenic acid (ALA), eicosapentaenoic acid (EPA), and / or docosahexaenoic acid (DHA).

33. The culture medium of claim 32.

34. the polyunsaturated fatty acid is at a concentration of 0.1 μg / ml to 1000 μg / ml; Optionally, the polyunsaturated fatty acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 50-75 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 75-100 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 10-25 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 10-75 μg / ml; or Optionally, the polyunsaturated fatty acid comprises linoleic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml; or Optionally, the polyunsaturated fatty acids include alpha-linolenic acid at a concentration of 1-100 μg / ml or 50-100 μg / ml; or Optionally, the polyunsaturated fatty acid comprises eicosapentaenoic acid (EPA) at a concentration of 1-100 μg / ml or 10-75 μg / ml; or Optionally, the polyunsaturated fatty acid comprises docasahexaenoic acid (DHA) at a concentration of 1-100 μg / ml or 10-25 μg / ml.

33. The culture medium of claim 32.

35. the saturated fatty acid comprises one or more saturated fatty acids having a carbon chain length of 10 to 24 carbons; 33. The culture medium of claim 32, optionally wherein the saturated fatty acids comprise one or more of capric acid (C10:0), undecylic acid (C11:0), lauric acid (C12:0), tridecylic acid (C13:0), myristic acid (C14:0), pentadecylic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), nonadecylic acid (C19:0), arachidic acid (C20:0), heneicosylic acid (C21:0), behenic acid (C22:0), tricosylic acid (C23:0), and lignoceric acid (C24:0).

36. the saturated fatty acid is at a concentration of 0.1 μg / ml to 1000 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 50-75 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 75-100 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 10-25 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 10-75 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 25-75 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 25-50 μg / ml.

37. 33. The culture medium of claim 32, wherein the sterol comprises at least one of glycerophospholipids, sphingomyelin; phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; ergosterol and secosteroids, including various forms of Vitamin D.

38. the nervonic acid is at a concentration of 0.1 to 1000 μg / ml or 10 to 1000 μg / ml; Optionally, the nervonic acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; or Optionally, the nervonic acid is at a concentration of 50-75 μg / ml.

39. 33. The culture medium of claim 32, wherein the culture medium further comprises up to 4% serum.

40. 33. The culture medium of claim 32, wherein the culture medium does not contain any additional components such as dexamethasone, biotin, T3, pantothenate, IBMX, and / or insulin.

41. 1. A method for culturing animal cells, comprising the steps of: Culturing said cells of an animal in a culture medium comprising at least 0.1 μg / ml or 10 μg / ml of one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols, and further comprising nervonic acid at a concentration of at least 1 μg / ml or 10 μg / ml; The method, wherein said culturing is carried out for a period and under conditions that allow for uptake of monounsaturated fatty acids by said cells of said animal.

42. the polyunsaturated fatty acid comprises one or more polyunsaturated fatty acids having a carbon chain length of 12 to 24 carbons; Optionally, the polyunsaturated fatty acid is selected from the group consisting of hexadecatrienoic acid (HTA) (C16:3 ω-3), linoleic acid (C18:2 ω-6), alpha linolenic acid (C18:3 ω-3), gamma linolenic acid (C18:3 ω-6), stearidonic acid (C18;4 ω-4), eicosadienoic acid (C20:2 ω-6), eicosatrienoic acid (ETE) (C20:3 ω-3), dimorpho-gamma-linolenic acid (C20:3 ω-6), mead acid (C20:3 ω-9), arachidonic acid (C20:4 ω-6), eicosapentaenoic acid (EPA) (C20:5 ω-3, C20:5 ω-6), heneicosapentaenoic acid (HPA) (C21:5 ω-3, C21:5 ω-6), ω-3), docosatetraenoic acid (C22:4 docosatetraenoic acid-6), DPA (C22:5 docosatetraenoic acid-3), docasahexaenoic acid (DHA) (C22:6 docasahexaenoic acid (DHA)-3), tetracosapentaenoic acid (C24:5 ω-3), and tetracosahexaenoic acid (herring acid) (C24:6 tetracosahexaenoic acid (herring acid) (C24:6 ω-3)).

43. the polyunsaturated fatty acid is at a concentration of 0.1 μg / ml to 1000 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 50-75 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 75-100 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 10-25 μg / ml; or Optionally, the polyunsaturated fatty acid is at a concentration of 10-75 μg / ml.

44. The polyunsaturated fatty acid is i) containing linoleic acid in a concentration of 1-100 μg / ml or 50-75 μg / ml; and / or ii) containing alpha-linolenic acid in a concentration of 1-100 μg / ml or 50-100 μg / ml; and / or iii) eicosapentaenoic acid (EPA) in a concentration of 1-100 μg / ml or 10-75 μg / ml; and / or iv) docasahexaenoic acid (DHA) in a concentration of 1-100 μg / ml or 10-25 μg / ml; Optionally, the polyunsaturated fatty acids include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

42. The method of claim 41.

45. the saturated fatty acid comprises one or more saturated fatty acids having a carbon chain length of 10 to 24 carbons; Optionally, the saturated fatty acids comprise one or more of capric acid (C10:0), undecylic acid (C11:0), lauric acid (C12:0), tridecylic acid (C13:0), myristic acid (C14:0), pentadecylic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), nonadecylic acid (C19:0), arachidic acid (C20:0), heneicosylic acid (C21:0), behenic acid (C22:0), tricosylic acid (C23:0), and lignoceric acid (C24:0).

46. the saturated fatty acid is at a concentration of 0.1 μg / ml to 1000 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 50-75 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 75-100 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 10-25 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 10-75 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 25-75 μg / ml; or Optionally, the saturated fatty acid is at a concentration of 25-50 μg / ml.

47. 42. The method of claim 41, wherein the sterols include at least one of glycerophospholipids and sphingomyelins; phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; ergosterol and secosteroids, including various forms of vitamin D.

48. the nervonic acid is at a concentration of 1-100 μg / ml or 10-1000 μg / ml; or Optionally, the nervonic acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; or Optionally, the nervonic acid is at a concentration of 50-75 μg / ml.

49. 42. The method of claim 41 , wherein the culture medium is the culture medium of claim 30 .

50. 42. A cell of an animal, said cell being obtainable and / or obtained by the method of claim 41, Optionally, the cells are cells of an animal, comprising one or more of myoblasts, myocytes, preadipocytes, adipocytes, fibroblasts, keratinocytes, epithelial cells, endothelial cells, embryo-derived cells, induced pluripotent stem cells, and mesenchymal stem cells of the animal.

51. The cells, i) comprising adipocytes comprising one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols in an amount of 0.1% to 1% by weight of said cells; and / or ii) comprising fibroblasts comprising one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols, in an amount of at least 1% by weight of said cells; and / or iii) myoblasts comprising one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols in an amount of at least 1% by weight of said cells; 51. A cell of the animal according to claim 50.

52. the animal is an aquatic animal, Optionally, the aquatic animals include fish and / or crustaceans; or Optionally, the aquatic animals include one or more of Chondrichthyes, Osteichthyes, Actinopterygii, Lophophora, Mollusca, Mussels, Cephalopods, Crustaceans, and Echinoderms; or Optionally, the aquatic animal is selected from the group consisting of pangasius, flounder, hake, scup, smelt, rainbow trout, hard clam, blue crab, peachytoe crab, jumbo crab, cuttlefish, eastern oyster, Pacific oyster, anchovy, herring, golden cod, moi, orange roughy, Atlantic perch, Lake Victoria perch, yellow perch, European oyster, Dover sole, sturgeon, tilefish, barracuda, or yellowtail. Fish, sea urchins, Atlantic mackerel, sardines, black sea bass, European sea bass, hybrid striped bass, bream, cod, drum, haddock, hoki, pollock, reef fish, pink salmon, snapper, tilapia, turbot, walleye, lake whitefish, wolffish, hard clams, cockles, Jonah crab, snow crab, crayfish, mussels, Korean prawns, sablefish , Atlantic salmon, Coho salmon, Skate ray, Dungeness crab, King crab, Purple mussel, Green mussel, Red shrimp, Escolar, Chinook salmon, Chum salmon, American shad, Alpine char, Carp, Catfish, John dory, Grouper, Halibut, Scutellaria, Koban trevally, Abalone, Conch, Stone crab, American lobster, Spiny lobster, Octopus, Black tiger shrimp, Freshwater shrimp, Gulf shrimp, or one or more of the following: whitenamen shrimp, squid, barramundi, casque, dogfish, kingklip, mahi-mahi, sunfish, mako shark, swordfish, albacore tuna, yellowfin tuna, geoduck, squid lobster, Magellanic mussel, mikazuki shrimp, barracuda, Chilean sea bass, cobia, croaker, eel, blue marlin, mullet, sockeye salmon, bluefin tuna, shrimp, crabs, lobsters, and echinoderms; or Optionally, the aquatic animal comprises one or more of Thunnus orientalis, Lutjanus campechanus, Coryphaena hippurus, Seriola lalandi, Micropterus salmoides, Thunnus albacares, Lepomis macrochirus, Hypophthalmichthys molitrix, Salmo salar, Oncorhynchus mykiss, and Oreochromis massambicus; or Optionally, the aquatic animal comprises one or more of fish, clams, oysters, octopus, squid, shrimp, crabs, lobsters, sea cucumbers, and sea urchins.

53. the animal is a terrestrial animal, Optionally, the terrestrial animal is a mammal, a bird, a reptile, an amphibian, or an insect; or Optionally, said terrestrial animal is an armyworm, a cricket, a grasshopper, a frog, a toad, a newt, a salamander, an alligator, a crocodile, a snake, a chicken, a turkey, a duck, a goose, a pheasant, a game hen, a quail, a horse, a rhino, a tapir, a cow, a pig, a giraffe, a camel, a sheep, a deer, a goat, a rabbit, a dog, or a hippopotamus; or Optionally, the terrestrial animal is selected from the group consisting of armyworms, crickets, and grasshoppers; or Optionally, the terrestrial animal is selected from the group consisting of frogs, toads, newts, and salamanders; or Optionally, the terrestrial animal is selected from the group consisting of alligators, crocodiles, and snakes; or Optionally, the terrestrial animal is selected from the group consisting of chickens, turkeys, ducks, geese, pheasants, game hens, and quails; or Optionally, the terrestrial animal is selected from the group consisting of horse, rhinoceros, tapir, cow, pig, giraffe, camel, sheep, deer, goat, rabbit, dog, and hippopotamus.

54. The cells, i) has an omega-3 PUFA content that is at least one percentage point higher than the same type of cell derived from the same species of wild-caught or farmed terrestrial animal; and / or ii) have a UFA content that is at least one percentage point lower than the same type of cell derived from a wild-caught or farmed terrestrial animal of the same species; A cell of a terrestrial animal according to claim 53.

55. 51. A biomass comprising one or more cells of claim 50.

56. 57. The method according to claim 55, comprising: Optionally, the cells of the animal have a polyunsaturated fatty acid, saturated fatty acid, and / or sterol content of at least 0.2% by weight; or Optionally, the food product, wherein said cells of an animal have a polyunsaturated fatty acid, saturated fatty acid, and / or sterol content of between 0.2% and 50% by weight.

57. the cells of an animal include fish cells, in particular white fish cells, and the cells have a polyunsaturated fatty acid, saturated fatty acid and / or sterol content of 0.2% to 2% by weight; or 57. The food product of claim 56, wherein the cells of an animal comprise fish cells, in particular white fish cells, and the cells comprise a polyunsaturated fatty acid, saturated fatty acid and / or sterol content of 1% to 2% by weight.

58. the food product has a polyunsaturated fatty acid, saturated fatty acid, and / or sterol content of 10% to 20%; Optionally, the food product is free of adipocytes, and / or Optionally, the food product of claim 56 is a cell-cultured fish product.

59. 1. A system for culturing animal cells, comprising:

42. The system comprising one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols in combination with nervonic acid, a culture medium for the cells of an animal, and / or cells of an animal for simultaneous, combined or sequential use in the method for increasing the content of monounsaturated fatty acids in cells of an animal as described in claim 41.

60. 60. The system of claim 59, wherein the sterols include at least one of glycerophospholipids and sphingomyelins; phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; ergosterol and secosteroids, including various forms of Vitamin D.

61. the nervonic acid is at a concentration of 1-100 μg / ml or 10-1000 μg / ml; or Optionally, the nervonic acid is in an amount of 10-100 μg / ml; or Optionally, the nervonic acid is in an amount of 50-75 μg / ml.

62. 60. The system of claim 59, wherein the cells of the animal comprise one or more of myoblasts, myocytes, preadipocytes, adipocytes, fibroblasts, keratinocytes, epithelial cells, endothelial cells, embryo-derived cells, induced pluripotent stem cells, and mesenchymal stem cells of the animal.

63. the animal is an aquatic animal, Optionally, the aquatic animals include fish and / or crustaceans; Optionally, the aquatic animals include one or more of: Chondrichthyes, Osteichthyes, Actinopterygii, Lophophora, Mollusca, Mussels, Cephalopods, Crustaceans, and Echinoderms; Optionally, the aquatic animal is selected from the group consisting of pangasius, flounder, hake, scup, smelt, rainbow trout, hard clams, blue crab, peachytoe crab, jumbo crab, cuttlefish, eastern oyster, Pacific oyster, anchovy, herring, golden cod, moi, orange roughy, Atlantic perch, Lake Victoria perch, yellow perch, European oyster, Dover sole, sturgeon, tilefish, wahoo, yellowtail, Japanese amberjack ... Fish of the genus, sea urchin, Atlantic mackerel, sardine, black sea bass, European sea bass, hybrid striped bass, bream, cod, drum, haddock, hoki, pollock, reef fish, pink salmon, snapper, tilapia, turbot, walleye, lake whitefish, wolffish, hard clams, cockles, Jonah crab, snow crab, crayfish, mussels, Korean shrimp, silver clams Pollock, Atlantic salmon, Coho salmon, Skate ray, Dungeness crab, King crab, Purple mussel, Green mussel, Red shrimp, Escolar, Chinook salmon, Chum salmon, American shad, Alpine char, Carp, Catfish, John dory, Grouper, Halibut, Scutellaria, Koban trevally, Abalone, Conch, Stone crab, American lobster, Spiny lobster, Octopus, Black tiger shrimp, Freshwater shrimp, Gulf prawns , whitefish, squid, barramundi, casque, dogfish, kingklip, mahi-mahi, sunfish, mako shark, swordfish, albacore tuna, yellowfin tuna, cob mussels, squid lobsters, Magellanic mussels, mikazuki shrimp, barracuda, Chilean sea bass, cobia, croaker, eel, blue marlin, mullet, sockeye salmon, bluefin tuna, shrimp, crabs, lobsters, and echinoderms; Optionally, the aquatic animal comprises one or more of Thunnus orientalis, Lutjanus campechanus, Coryphaena hippurus, Seriola lalandi, Micropterus salmoides, Thunnus albacares, Lepomis macrochirus, Hypophthalmichthys molitrix, Salmo salar, Oncorhynchus mykiss, and Oreochromis massambicus; Optionally, the aquatic animals include one or more of fish, clams, oysters, octopus, squid, shrimp, crabs, lobsters, sea cucumbers, and sea urchins; 60. The system of claim 59.

64. the animal is a terrestrial animal, Optionally, the terrestrial animal is a mammal, a bird, a reptile, an amphibian, or an insect; Optionally, the terrestrial animal is an armyworm, a cricket, a grasshopper, a frog, a toad, a newt, a salamander, an alligator, a crocodile, a snake, a chicken, a turkey, a duck, a goose, a pheasant, a game hen, a quail, a horse, a rhino, a tapir, a cow, a pig, a giraffe, a camel, a sheep, a deer, a goat, a rabbit, a dog, or a hippopotamus; Optionally, the terrestrial animal is selected from the group consisting of armyworms, crickets, and grasshoppers. Optionally, the terrestrial animal is selected from the group consisting of frogs, toads, newts, and salamanders. Optionally, the terrestrial animal is selected from the group consisting of an alligator, a crocodile, and a snake. Optionally, the terrestrial animal is selected from the group consisting of chickens, turkeys, ducks, geese, pheasants, game hens, and quails; or Optionally, the terrestrial animal is selected from the group consisting of a horse, a rhino, a tapir, a cow, a pig, a giraffe, a camel, a sheep, a deer, a goat, a rabbit, a dog, and a hippopotamus.

60. The system of claim 59.

65. said cells of said terrestrial animal have an omega-3 PUFA content that is at least one percentage point higher than the same type of cell derived from a wild-caught or farmed terrestrial animal of the same species; or said cells of said terrestrial animal having a UFA content that is at least one percentage point lower than the same type of cells derived from a wild-caught or farmed terrestrial animal of the same species; 65. The system of claim 64.

66. 60. The system of claim 59, wherein the culture medium is a basal medium, optionally further comprising up to 4% serum.

67. 60. The system of claim 59, wherein the culture medium does not contain any additional components such as dexamethasone, biotin, T3, pantothenate, IBMX, and / or insulin.

68. 1. A culture medium for animal cells, comprising: a basal cell culture medium for animal cells supplemented with at least 0.1 μg / ml or 10 μg / ml of lipid; When the lipid comprises one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols, the culture medium further comprises nervonic acid at a concentration of at least 0.1 μg / ml or 10 μg / ml; Optionally, the lipid is at a concentration of 1-1000 μg / ml or 10 μg / ml-1000 μg / ml; Optionally, the lipid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; Optionally, the lipid is at a concentration of 50-75 μg / ml; Optionally, the lipid is at a concentration of 75-100 μg / ml; Optionally, the lipid is at a concentration of 10-25 μg / ml; Optionally, the lipid is at a concentration of 10-75 μg / ml; Optionally, the lipid is at a concentration between 100 ng / ml and 1 mg / ml.

69. The lipid is i) one or more monounsaturated fatty acids having a carbon chain length of 12 to 22 carbons; Optionally, the monounsaturated fatty acids include myristoleic acid (C14:1 ω-5), palmitoleic acid C16:1 ω-7, sapienic acid C16:1 ω-10, vaccenic acid (C18:1 ω-7), C18:1 ω9c found in most phospholipids, oleic acid (C18:1 ω-9), petroselinic acid (C18:1 ω-12), paulic acid (C20:1 ω-7), gondoic acid (C20:1 ω-11), erucic acid (C22:1 ω-9c), brassidic acid (C22:1 ω-9t), and / or nervonic acid (C24:1 ω-9); and / or ii) containing palmitoleic acid, vaccenic acid, oleic acid, and / or nervonic acid; and / or iii) palmitoleic acid in a concentration of 50-100 μg / ml, vaccenic acid in a concentration of 50-75 μg / ml, oleic acid in a concentration of 75-100 μg / ml, and / or nervonic acid in a concentration of 50-75 μg / ml; and / or iv) one or more polyunsaturated fatty acids having a carbon chain length of 12 to 24 carbons; Optionally, the polyunsaturated fatty acid is selected from the group consisting of hexadecatrienoic acid (HTA) (C16:3 ω-3), linoleic acid (C18:2 ω-6), alpha linolenic acid (C18:3 ω-3), gamma linolenic acid (C18:3 ω-6), stearidonic acid (C18;4 ω-4), eicosadienoic acid (C20:2 ω-6), eicosatrienoic acid (ETE) (C20:3 ω-3), dimorpho-gamma-linolenic acid (C20:3 ω-6), mead acid (C20:3 ω-9), arachidonic acid (C20:4 ω-6), eicosapentaenoic acid (EPA) (C20:5 ω-3, C20:5 ω-6), heneicosapentaenoic acid (HPA) (C21:5 ω-3, C21:5 ω-6), ω-3), docosatetraenoic acid (C22:4 docosatetraenoic acid-6), DPA (C22:5 docosatetraenoic acid-3), docasahexaenoic acid (DHA) (C22:6 docasahexaenoic acid (DHA)-3), tetracosapentaenoic acid (C24:5 ω-3), and tetracosahexaenoic acid (herring acid) (C24:6 tetracosahexaenoic acid (herring acid) (C24:6 ω-3)); Optionally, the polyunsaturated fatty acid comprises eicosapentaenoic acid (EPA) at a concentration of 1-100 μg / ml or 10-75 μg / ml. Optionally, the polyunsaturated fatty acid comprises docasahexaenoic acid (DHA) at a concentration of 1-100 μg / ml or 10-25 μg / ml. Optionally, the polyunsaturated fatty acid comprises linoleic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml. Optionally, the polyunsaturated fatty acid comprises alpha-linolenic acid at a concentration of 1-100 μg / ml or 50-100 μg / ml. Optionally, the polyunsaturated fatty acids include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); and / or v) one or more saturated fatty acids having a carbon chain length of 10 to 24 carbons; Optionally, the saturated fatty acids comprise one or more of capric acid (C10:0), undecylic acid (C11:0), lauric acid (C12:0), tridecylic acid (C13:0), myristic acid (C14:0), pentadecylic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), nonadecylic acid (C19:0), arachidic acid (C20:0), heneicosylic acid (C21:0), behenic acid (C22:0), tricosylic acid (C23:0), and lignoceric acid (C24:0); 69. The culture medium of claim 68.

70. 69. The culture medium of claim 68, wherein the sterols comprise at least one of glycerophospholipids and sphingomyelins; phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; ergosterol and secosteroids, including various forms of Vitamin D.

71. The nervonic acid has a concentration of 0.1 to 1000 μg / ml; Optionally, the nervonic acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; Optionally, the nervonic acid is at a concentration of 50-75 μg / ml. The culture medium according to any one of claims 68 to 70.

72. the culture medium further comprises up to 4% serum; and / or The culture medium according to any one of claims 68 to 70, wherein the culture medium does not contain any additional components such as dexamethasone, biotin, T3, pantothenate, IBMX, and / or insulin.

73. 1. A method for culturing animal cells, comprising the steps of: Culturing said cells of the animal in a culture medium containing at least 0.1 μg / ml or 10 μg / ml lipid; said culturing being carried out for a period and under conditions that allow for uptake of fatty acids of said lipids by said cells of said animal; When the lipid comprises one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols, the culture medium further comprises nervonic acid at a concentration of at least 0.1 μg / ml or 10 μg / ml; Optionally, the lipid is at a concentration of 0.1 μg / ml to 1000 μg / ml; Optionally, the lipid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; Optionally, the lipid is at a concentration of 50-75 μg / ml; Optionally, the lipid is at a concentration of 75-100 μg / ml; Optionally, the lipid is at a concentration of 10-25 μg / ml; Optionally, the lipid is at a concentration of 10-75 μg / ml.

74. The lipid is i) one or more monounsaturated fatty acids having a carbon chain length of 12 to 22 carbons; Optionally, the monounsaturated fatty acids include myristoleic acid (C14:1 ω-5), palmitoleic acid C16:1 ω-7, sapienic acid C16:1 ω-10, vaccenic acid (C18:1 ω-7), C18:1 ω9c found in most phospholipids, oleic acid (C18:1 ω-9), petroselinic acid (C18:1 ω-12), paulic acid (C20:1 ω-7), gondoic acid (C20:1 ω-11), erucic acid (C22:1 ω-9c), brassidic acid (C22:1 ω-9t), and / or nervonic acid (C24:1 ω-9); and / or ii) containing palmitoleic acid, vaccenic acid, oleic acid, and / or nervonic acid; and / or iii) palmitoleic acid at a concentration of 1-100 μg / ml or 50-100 μg / ml, vaccenic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml, oleic acid at a concentration of 1-100 μg / ml or 75-100 μg / ml, and / or nervonic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml; and / or iv) one or more polyunsaturated fatty acids having a carbon chain length of 12 to 24 carbons; Optionally, the polyunsaturated fatty acid is selected from the group consisting of hexadecatrienoic acid (HTA) (C16:3 ω-3), linoleic acid (C18:2 ω-6), alpha linolenic acid (C18:3 ω-3), gamma linolenic acid (C18:3 ω-6), stearidonic acid (C18;4 ω-4), eicosadienoic acid (C20:2 ω-6), eicosatrienoic acid (ETE) (C20:3 ω-3), dimorpho-gamma-linolenic acid (C20:3 ω-6), mead acid (C20:3 ω-9), arachidonic acid (C20:4 ω-6), eicosapentaenoic acid (EPA) (C20:5 ω-3, C20:5 ω-6), heneicosapentaenoic acid (HPA) (C21:5 ω-3, C21:5 ω-6), ω-3), docosatetraenoic acid (C22:4 docosatetraenoic acid-6), DPA (C22:5 docosatetraenoic acid-3), docasahexaenoic acid (DHA) (C22:6 docasahexaenoic acid (DHA)-3), tetracosapentaenoic acid (C24:5 ω-3), and tetracosahexaenoic acid (herring acid) (C24:6 tetracosahexaenoic acid (herring acid) (C24:6 ω-3)); Optionally, the polyunsaturated fatty acid comprises eicosapentaenoic acid (EPA) at a concentration of 1-100 μg / ml or 10-75 μg / ml; Optionally, the polyunsaturated fatty acid comprises docasahexaenoic acid (DHA) at a concentration of 1-100 μg / ml or 10-25 μg / ml; Optionally, the polyunsaturated fatty acid comprises linoleic acid at a concentration of 1-100 μg / ml or 50-75 μg / ml; Optionally, the polyunsaturated fatty acid comprises alpha-linolenic acid at a concentration of 1-100 μg / ml or 50-100 μg / ml; Optionally, the polyunsaturated fatty acids include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); and / or v) one or more saturated fatty acids having a carbon chain length of 10 to 24 carbons; Optionally, the saturated fatty acids comprise one or more of capric acid (C10:0), undecylic acid (C11:0), lauric acid (C12:0), tridecylic acid (C13:0), myristic acid (C14:0), pentadecylic acid (C15:0), palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0), nonadecylic acid (C19:0), arachidic acid (C20:0), heneicosylic acid (C21:0), behenic acid (C22:0), tricosylic acid (C23:0), and lignoceric acid (C24:0); 74. The method of claim 73.

75. 74. The method of claim 73, wherein the sterols include at least one of glycerophospholipids and sphingomyelins; phytosterols, such as β-sitosterol, stigmasterol, and brassicasterol; ergosterol and secosteroids, including various forms of vitamin D.

76. The nervonic acid has a concentration of 0.1 to 1000 μg / ml; Optionally, the nervonic acid is at a concentration of 1-100 μg / ml or 10-100 μg / ml; Optionally, the nervonic acid is at a concentration of 50-75 μg / ml.

77. A cell of an animal, said cell being obtainable and / or obtained by a method according to any one of claims 73 to 75.

78. 78. A biomass comprising one or more cells of claim 77.

79. 79. A food product comprising one or more of the cells of an animal according to claim 76 and / or the biomass according to claim 78, Optionally, said cells of an animal have a lipid content of at least 0.2% by weight; Optionally, said cells of an animal have a lipid content of between 0.2% and 90% by weight; Optionally, said cells of an animal comprise fish cells comprising a lipid content of 0.2% to 2% by weight; Optionally, said cells of an animal comprise fish cells comprising a lipid content of 1% to 2% by weight; Optionally, the food product has a lipid content of between 10% and 20%; Optionally, the food product is free of fat cells; Optionally, the food product is a cell-cultured fish product. food.

80. 1. A system for culturing animal cells, comprising: A lipid in combination with a culture medium for said cells of an animal and / or with the cells of an animal for simultaneous, combined or sequential use in a method for increasing the content of monounsaturated fatty acids in cells of an animal according to any one of claims 73 to 76, When the lipid comprises one or more polyunsaturated fatty acids, one or more saturated fatty acids, and / or one or more sterols, the system further comprises nervonic acid.

81. 8. The cultured cells of claim 1, the biomass of claim 6, or the food product of claim 7, wherein the cells comprise adipocytes, fibroblasts, and / or myoblasts.

82. the cells of the animal have a lipid content of at least 0.2% by weight; or the cells of the animal have a lipid content of between 0.2% and 50% by weight; or 8. The cultured cells of claim 1, the biomass of claim 6, or the food of claim 7, wherein the cells of an animal have a lipid content of 50% to 90% by weight.

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