Methods of increasing differentiation and / or fat accumulation in cells

Abstract

The present invention relates to a method for increasing the differentiation and / or lipid accumulation in cells.

Description

[0001] Methods of increasing differentiation and / or fat accumulation in cells.

[0002] Field of the invention

[0003] The present invention relates to methods of increasing differentiation and / or fat accumulation in cells.

[0004] Background of the invention

[0005] According to the most recent United Nations estimations, the current world population is 7.9 billion in July 2022 [https: / / www.worldometers.info / es / poblacion-mundial / #ref-1] and it is expected to reach 10 billion around the year 2056. This increase will be heterogeneously distributed around the globe, with nine countries covering half the projected growth of the global population in the next 30 years, including India, Nigeria, Pakistan, Egypt, and the United States of America. Population and economic growth are major drivers of increased meat consumption. According to the United Nations Food and Agricultural Organization (FAO, https: / / www.oecd- ilibrary.org / agriculture-and-food / oecd-fao-agricultural-outlook-2022-2031_f1 b0b29c-en), an estimated growth of 15% in global meat consumption is projected by 2031 . On the other hand, the correlation between income growth and higher meat consumption is clearly demonstrable at lower income rates but once consumers reach an adequate standard of living, they become more sensitive to environmental, ethical, and animal welfare and health concerns.

[0006] For this reason, there is a growing interest in finding alternative protein sources which ideally will be sustainable and will contain the nutrients normally provided by meat in the human diet. Cultured meat arises as another alternative to traditional animal agriculture that aims to produce the skeletal muscle and adipose tissues that normally comprise animal meats, except using in vitro tissue and biological engineering techniques. Despite efforts to develop robust protocols for scalable generation of animal cell types from easily accessible and renewable sources, the differentiation of animal (pluripotent) stem cells into specific cell types often remains cumbersome, lengthy, and difficult to reproduce and / or has not been established yet.

[0007] Additionally, to date, plant-based and cultured meat alternatives have focused mostly on mimicking the muscle component of meat. However, fat is also a crucial component of meat, contributing to sensory / flavor, textural attributes and palatability (Zhang, Shu, et al. "DNA polymorphisms in bovine fatty acid synthase are associated with beef fatty acid composition 1." Animal genetics 39.1 (2008): 62-70.).

[0008] Differentiation of adipocytes in culture has previously been described. However these protocols have several limitation including heterogeneous adipocyte degree of maturation, lack of scalable process and are not food safe.

[0009] Accordingly there remains a need in the art for the production and culturing of mature adipocytes that are suitable for human consumption and that can be produced in a scalable and cost effective manner. Summary of the invention

[0010] In a first aspect, the invention relates a method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises, an mTor agonist and a component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist ; at least one growth factor, preferably wherein the growth factor is an ERK- modulator; at least one carbon source; and at least one thiol-containing antioxidant.

[0011] In some embodiments, the mTor agonist is selected from the group consisting of4,6-di-4- morpholinyl-N-(4-nitrophenyl)-1 ,3,5-triazin-2-amine (MHY1485) or derivatives thereof, 3-benzyl-5- ((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO), TSC1 knockdown agents (miRNA, siRNA, shRNA for example), PDK1 activators, AKT1 activators, TSC2 knockdown agents, PTEN knockdown agents and 2 hydroxy 2 methyl butyrate or a salt thereof, such as the calcium salt of 2 hydroxy 2 methyl butyrate (HMB-Ca), preferably the mTor agonist is 2 hydroxy 2 methyl butyrate or the calcium salt of 2 hydroxy 2 methyl butyrate (HMB-Ca).

[0012] In some embodiments, wherein the thiol-containing antioxidant is selected from the group consisting of glutathione, N-acetyl-L-cysteine, alpha lipoic acid, L-cysteine, dihydrolipoic acid, thioredoxin, mercaptoethanol and derivatives thereof, preferably the thiol-containing antioxidant is N-acetyl-L-cysteine.

[0013] In some embodiments, the carbon source is a sugar and / or a component involved in metabolic pathway preferably the citric acid pathway.

[0014] In some embodiments, the carbon source is a sugar preferably a monosaccharide or a disaccharide, preferably selected from the group consisting of galactose, fructose, xylose, sucrose, lactose, 1 ,6-bisphosphate maltose, isomaltulose, trehalose glucose, glucose 6-phosphate, fructose 6-phosphate, preferably fructose.

[0015] In some embodiments, the carbon source is a component involved in metabolic pathway preferably the citric acid pathway selected from the group consisting of succinyl-CoA, acetyl-CoA, succinate, fumarate, L-malate, oxaloacetate preferably wherein the component is citrate.

[0016] In some embodiments, the growth factor is a ERK modulator selected from the group consisting of FGF2, GF-EGF, AMD3100, RLX-33 and ACA-28, preferably wherein the growth factor is FGF2.

[0017] In some embodiments, the at least one PPARy agonist and / or a CEBPa agonist is selected from the group consisting of: Indomethacin, magnolol, amorfrutins (comprising, for example, amorfrutin 1 , amorfrutin 2, amorfrutin A, amorfrutin B, amorfrutin C and amorfrutin D), honokiol, lecithine (such as L-a-lecithine from soy beans), formononetin, bixin, norbixin, catechin, A9- tetrahydrocannabinol, (9S, 13R)-12-oxo-phytodienoic acid, odoratin, hydroxy unsaturated fatty acids from Coix lacrymajobi, commipheric acid, kaempferol-3-O-B-glucopyranoside, citral, alkamides from Echinacae purpera, tocotrienols, deoxyelephantopin, acetylated flavonol glycosides, kampferol, , 5’-formulglabridin, (2R,3R)-3,4’,7-trihydroxy-3’-prenylflavane, echinatin, (3R)-2’,3’,7-trihydroxy-4’- methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, glabrone, licochalcone E, flavonoids from Glycyrrhiza uralensis, 3-arylcoumarins from Glycyrrhiza uralensis, meranzin, fatty acids from Lycium chinense, lunularin, fatty acids from Melarnpyrum pratense, cucurbitane-type triterpene glycosides, polyacetylenes from Notopterygium incisum, biochanin A, ginsenoside 20(S)- protopanaxatriol, ginsenoside Rbi, fatty acids from Pineilia ternata, oleaninic acid, pseudolaric acid B, daidzein, amorphastilbol, carnosic acid, carnosol, 12-O-methul carnosic acid, u-linolenic acid, inoleic acid, naringenin, saurufuran A, isosilybin A, gallotannins, carvacrol, isoflavones from Trifolium pratense, ellagic acid, epicatechin gallate, flavonoids from Vitis vinifera, dehydrotrametenolic acid and 6-shogaol, preferably the agonist is magnolol and / or catechin.

[0018] In some embodiments, wherein the culture medium is a basal medium that is supplemented with said medium components.

[0019] In some embodiments, the cell is capable of lipid accumulation such as adipocytes, hepatocytes, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells mesenchymal cells, fibro-adipogenic progenitor cells, lipoblasts, adipoblasts, pre-adipocytes, hepatoblasts, pre-heptatocytes

[0020] In some embodiments, the culture medium further comprises at least one of the following compounds:

[0021] - at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid;

[0022] - at least one vitamin B;

[0023] - at least one additional growth factor; and

[0024] - at least one chromium (III) salt.

[0025] In some embodiments, the at least PUFA is an omega 3 fatty acid and the at least one short chain fatty acid is butyric acid;

[0026] - the at least one vitamin B is selected from the group consisting of thiamine, riboflavin, niacin, niacinamide, nicotinamide riboside, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, Biotin, folate and cobalamins, preferably biotin ;

[0027] - the least one additional growth factor is EGF; and

[0028] - the least one chromium (III) salt is chromium(lll) chloride.

[0029] In some embodiments, the method further comprises the step of:

[0030] - incorporating the cultured cell into a food product for animal, preferably human, consumption.

[0031] In a second aspect, the invention provides for a cell obtainable by or obtained by the method according to any of the preceding claims.

[0032] In a third aspect, the invention provides for a medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises an mTor agonist as defined herein and at least one component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARg) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as defined herein; at least one growth factor, preferably wherein the growth factor is an ERK-modulator as defined herein; at least one carbon source as defined herein; and at least one thiol-containing antioxidant as defined herein; and optionally comprising:

[0033] - at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid as defined herein;

[0034] - at least one vitamin B as defined herein;

[0035] - at least one additional growth factor as defined herein; and

[0036] - at least one chromium (III) salt as defined in herein.

[0037] In a fourth aspect the invention provides for a cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method as described herein, optionally further comprising (cultured) mammalian myocytes.

[0038] In a fifth the invention provides for a cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method as defined herein.

[0039] Description of the invention

[0040] Definitions

[0041] Unless defined otherwise, 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. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

[0042] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0043] As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0044] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.

[0045] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.

[0046] The terms ‘differentiating’ and “differentiation”’ as used herein includes reference to the process of specialization of cells. During this process, progenitor cells (which may also be referred to as precursor cells or stem cells) change from one cell type to another. Differentiation may be activated and regulated by hormones, growth factors or other signaling molecules. Preferably, the differentiation as referred to herein is adipogenic differentiation or hepatocytic differentiation , i.e. the process wherein adipogenic progenitor cells or hepatocytic progenitor cells ultimately differentiate into adipocytes (fat cells) or hepatocytes (liver cells).

[0047] The term “lipid accumulation” as used herein is defined as an increase in the level of at least one form of fatty acid in a cell or tissue. Lipid accumulation is detected, for example, by Nile red staining, by C1-BODIPY-C12 staining, by lipid extraction and gas chromatography / mass spectroscopy, or by any other method known in the art. Lipid accumulation refers to, for example, an increase in the level of total lipids or an increase in at least one type of fatty acid (e.g., very long chain fatty acids).

[0048] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo- derived stem cells, epiblast-derived stem cells (EpiSC), induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using, methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

[0049] As used herein, a “medium” or “cell culture medium” refers to an aqueous based solution that provides for the growth, viability, or storage of cells. A medium as contemplated herein can be supplemented with one or more nutrients to promote the desired cellular activity, such as cell viability, growth, proliferation, differentiation of the cells cultured in the medium. A medium, as used herein, includes a serum replacement, a medium supplement, a complete medium or a cryopreservation medium. The pH of a culture medium should be suitable to the microorganisms that will be grown. Most bacteria grow in from around pH 6.5 to about pH 7.0 while most animal cells thrive in from about pH 7.2to around pH 7 .4.

[0050] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0051] It has been observed that traditional ways of culturing cells that are capable of lipid accumulation leads to cells that have a lower total lipid amount than cells that are isolated from their natural environment. Because of this lower lipid accumulation, it is required to add more of these cells per food product which increases the costs of the food product and limits scalability of cultured meat to a broader audience. Additionally, cells with lower lipid accumulation have a less efficient metabolism and therefore make less efficient use of the nutrient supplied in the culture media. The inventors have now surprisingly found a culture medium that is capable of boosting (i.e. increasing) differentiation and / or lipid accumulation in cells such that these cells more closely resemble the naturally grown cells. Additionally, it has been observed that use of this boosting medium not only increases the triglyceride content of adipocytes but that the composition is may be more comparable to the triglyceride content of animal meat, for example porcine meat.

[0052] Accordingly in a first aspect, the invention provides for a method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises an mTor agonist, and at least one further component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist; at least one growth factor, preferably wherein the growth factor is an ERK- modulator as; at least one carbon source; and at least one thiol-containing antioxidant.

[0053] As used herein, the term “mTORCI agonist” refers to a substance that activates mTOR in a reprogramming mechanism after introduction of a reprogramming factor. Through the mechanism of activating mTORCI , the mTORCI activator upregulates the expression of CXCR2 and cMYC and decreases autophagy activity. With the mechanism, the mTORCI activator exhibits an effect of enhancing reprogramming efficiency. Therefore, so long as it activates mTORCI in a reprograming mechanism subsequent to the introduction of a reprogramming factor, any mTORCI activator may be used without limitations. Examples of activators of mTORCI signaling are well known in the art and include, but are not limited to: 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1 ,3,5- triazin-2-amine (MHY1485) or derivatives thereof, 3-benzyl-5-((2-nitrophenoxy) methyl)- dihydrofuran-2(3H)-one (3BDO), TSC1 knockdown agents (miRNA, siRNA, shRNA for example), PDK1 activators, AKT1 activators, TSC2 knockdown agents, PTEN knockdown agents, 2 hydroxy 2 methyl butyrate or a salt thereof, such as a calcium salt of 2 hydroxy 2 methyl butyrate (HMB-Ca).

[0054] In certain embodiments, the mTor activator is present in a concentration of about 8,0, 8,5,

[0055] 9,0, 9,5, 10,0, 10,5, 11 ,0, 11 ,5, 12,0, 12,5, 13,0, 13,5, 14,0, 14,5, 15,0 or 15,5

[0056] |j.g / mL(micrograms / mL). In certain embodiments the concentration of the mTor activator is about 8,0 to 15,5 |j.g / mL, about 8,5 to 15,0 |j.g / mL, about 9,0 to 15,0 |j.g / mL such as about 14,5 |j.g / mL.

[0057] In certain preferred embodiments, the mTor agonist is 2 hydroxy 2 methyl butyrate. In alternative preferred embodiments, the mTor agonist is 2 hydroxy 2 methyl butyrate or a salt thereof, such as a calcium salt of 2 hydroxy 2 methyl butyrate (HMB-Ca).

[0058] In a first alternative embodiment of the first aspect of the invention, the invention provides for a method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises a thiol-containing antioxidant and at least one further component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as described herein; at least one growth factor, preferably wherein the growth factor is an ERK-modulator as described herein; at least one carbon source as described herein; and an mTor agonist as described herein.

[0059] In certain embodiments, thiol-containing antioxidant is present in a concentration of about 0,10, 0,15, 0,20, 0,25, 0,30, 0,35, or 0,40 mM. In certain embodiments the concentration of thiol- containing antioxidant is about 0,10 to 0,40mM, preferably about 0,10 to 0,35 mM, more preferably about 0,15 to 0,35 mM, even more preferably about 0,20 to 0,30 mM such as about 0,25mM.

[0060] In certain embodiments, the thiol-containing antioxidant is selected from the group consisting of L-glutathione, N-acetyl-L-cysteine, alpha lipoic acid, L-cysteine, dihydrolipoic acid, thioredoxin, dithiothreitol, mercaptoethanol and derivatives thereof. In preferred embodiments, the thiol-containing antioxidant is acetylcysteine.

[0061] The term "acetyl-cysteine" as used herein may be N-acetyl-L-cysteine (NAC) (e.g. unmodified NAC), or a derivative thereof, such as N-acetylcysteine-amide (NACA).

[0062] In certain embodiments, the term "acetyl-cysteine" as used herein means N-acetyl-L- cysteine (NAC).

[0063] In certain embodiments, the medium further comprises an mTor agonist as described herein, preferably the, the mTor agonist is 2 hydroxy 2 methyl butyrate or a salt thereof such as a calcium salt of 2 hydroxy 2 methyl butyrate (HMB-Ca).

[0064] In yet a further alternative embodiment of the first aspect of the invention, the invention provides for a method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises at least citrate and magnolol and at least one further component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as described herein; at least one growth factor, preferably wherein the growth factor is an ERK- modulator as described herein; at least one carbon source as described herein; an mTor agonist as described herein; and at least one thiol-containing antioxidant.

[0065] In certain embodiments, the medium further comprises an mTor agonist as described herein, preferably the mTor agonist is 2 hydroxy 2 methyl butyrate or a salt thereof such as a calcium salt of 2 hydroxy 2 methyl butyrate (HMB-Ca).

[0066] The term “increasing” as used herein means having a greater quantity, for example a quantity only slightly greater than the original quantity, or for example a quantity in large excess compared to the original quantity, and including all quantities in between. Alternatively, “increased” may refer to a quantity that is at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the quantity or activity for which the increased quantity or activity is being compared. In certain embodiment, the method as disclosed herein results in an increased lipid accumulation that is at least 15% increased than the baseline lipid accumulation capacity. In some embodiments, the lipid accumulation is at least 30%, 40%, 50% 100%, at least 150%, at least 200%, at least 250%, or at least 300% increased over the than the baseline lipid accumulation capacity. The terms “increased”, “greater than”, and “improved” are used interchangeably herein.

[0067] In certain embodiments, the carbon source as used in the various embodiments of the invention as described herein is a sugar and / or a component involved in metabolic pathway preferably the citric acid pathway.

[0068] In embodiments where the carbon source is a component involved in the citric acid pathway, the component involved in the citric acid pathway is present in a concentration of about 300, 310, 320, 330, 340, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 |ig / mL (micrograms / mL). In certain embodiments component involved in the citric acid pathway is present in a concentration of about 320 to 580 |ig / mL, such as about 330 |ig / mL.

[0069] In embodiments where the carbon source is a component involved in the citric acid pathway such as a component selected from the group consisting of: succinyl-CoA, acetyl-CoA, succinate, fumarate, L-malate, Oxaloacetate. Preferably the component is citrate.

[0070] In embodiments where the carbon source is a sugar, preferably a monosaccharide or a disaccharide.

[0071] In embodiments where the carbon source is a sugar, the sugar is present in a concentration of about 1 ,0, 1 ,1 , 1 ,2, 1 ,3, 1 ,4, 1 ,5, 1 ,6, 1 ,7, 1 ,8, 1 ,9, 2,0, 2,1 , 2,2, 2,3, 2,4, 2,5, 2,6, 2,7, 2,8, 2,9, 3,0 mg / mL (milligrams / mL). In certain embodiments, the sugar is present in a concentration of about 1 ,3 to 2,3 mg / mL such as 1 ,8 mg / mL.

[0072] In certain embodiments, the sugar is selected from the group consisting of: galactose, xylose, sucrose, lactose, 1 ,6-bisphosphate maltose, isomaltulose, trehalose glucose, glucose 6- phosphate, fructose 6-phosphate, and fructose. Preferably, the sugar is fructose.

[0073] In certain embodiments, the medium comprises a sugar and a component involved in the citric acid pathway, preferably the medium comprises citrate and fructose.

[0074] In certain embodiments, the growth factor is an ERK modulator. In certain embodiments, the ERK modulator is present in a concentration of about 2x10-4, 3x10-4, 4x10-4, 5x10-4, or 6x10-4|j.g / mL (micrograms / mL). In certain embodiments the concentration of the ERK modulator is about 2x1 O'4to 6x1 O'4|j.g / mL, about 3x1 O'4to 6x1 O'4|j.g / mL, or about 4x1 O'4, 6x1 O'4|j.g / mL such as 5x1 O'4|j.g / mL.

[0075] In some embodiments the ERK modulator is selected from the group consisting of: FGF2, GF-EGF, AMD3100, RLX-33 and ACA-28 and is preferably, FGF2.

[0076] The terms ‘fibroblast growth factor’ and ‘FGF’, as used herein, include reference to proteins that can stimulate growth and differentiation of animal cells, amongst other functions. FGFs bind to a fibroblast growth factor receptor (FGFR). FGFs are preferably recombinantly produced. Preferably, the FGF as disclosed herein is a recombinant FGF. Preferably, the FGF as disclosed herein is FGF2. It is to be understood that the terms ‘fibroblast growth factor’ and ‘FGF, as used herein, may also include reference to fragments of FGF that retain the biological function of FGF, such as FGF replacement peptides. In certain embodiments, the medium comprises a FGF activator selected from the group consisting of: an FGF21 analog for Type 2 diabetes (T2DM) such as PF-0523 1023, an indole derivative such as ID-8, an activator of the Wnt pathway, for example an inhibitor of glycogen synthase kinase 3p (GSK3p), such as 1-Azakenpaullone, a macrolide antibiotic with an immunosuppressive property such as Tacrolimus (FK-506), and a target of a negative regulator of a downstream pathway of FGF signaling, FGF2 modifications such as described in WO2017089016 and US8772460B2 or a combination thereof. In some embodiments, the FGF2 is of human origin. In an alternative embodiment, the FGF2 is of bovine origin.

[0077] In some embodiments, the target of a negative regulator of a downstream pathway of FGF signaling is an inhibitor that hyperactivates FGF pathway by activating ERK pathway. In some embodiments, the inhibitor is a Duspo inhibitor. In some embodiments, the Duspo inhibitor is (E / Z)- BCI hydrochloride.

[0078] In certain embodiments, the medium comprises at least one, at least two, at least three or at least four or more PPARy and / or a CEPBa agonists.

[0079] In certain embodiments, the at least one PPARy agonist and / or a CEPBa agonist is selected from the group consisting of: Indomethacin, magnolol, amorfrutins (comprising, for example, amorfrutin 1 , amorfrutin 2, amorfrutin A, amorfrutin B, amorfrutin C and amorfrutin D), honokiol, lecithine (such as L-a-lecithine from soy beans), formononetin, bixin, norbixin, (-)-catechin, A9- tetrahydrocannabinol, (9S, 13R)-12-oxo-phytodienoic acid, odoratin, hydroxy unsaturated fatty acids from Coix lacrymajobi, commipheric acid, kaempferol-3-O-B-glucopyranoside, citral, alkamides from Echinacae purpera, tocotrienols, deoxyelephantopin, acetylated flavonol glycosides, kampferol, , 5’-formulglabridin, (2R,3R)-3,4’,7-trihydroxy-3’-prenylflavane, echinatin, (3R)-2’,3’,7-trihydroxy-4’- methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, glabrone, licochalcone E, flavonoids from Glycyrrhiza uralensis, 3-arylcoumarins from Glycyrrhiza uralensis, meranzin, fatty acids from Lycium chinense, lunularin, fatty acids from Melarnpyrum pratense, cucurbitane-type triterpene glycosides, polyacetylenes from Notopterygium incisum, biochanin A, ginsenoside 20(S)- protopanaxatriol, ginsenoside Rbi, fatty acids from Pineilia ternata, oleaninic acid, pseudolaric acid B, daidzein, amorphastilbol, carnosic acid, carnosol, 12-O-methul carnosic acid, u-linolenic acid, inoleic acid, naringenin, saurufuran A. isosilybin A. gallotannins, carvacrol, isoflavones from Trifolium pratense, ellagic acid, epicatechin gallate, flavonoids from Vitis vinifera, dehydrotrametenolic acid and 6-shogaol.

[0080] Preferably the agonist is magnolol and / or (-)-catechin. In certain embodiments, the medium comprises both (-)-catechin and magnolol.

[0081] In certain embodiments, the concentration of (-)-catechin is about 2,0, 2,5, 3,0, 3,5, 4,0, 4,5, 5,0, or 5,5 |j.g / mL(micrograms / mL). In certain embodiments, the concentration of (-)-catechin is about 2,0 to 5,5 |j.g / mL, or about 2.0 to 4,0 |j.g / mL such as for example 3,0 |j.g / mL.

[0082] In certain embodiments, the concentration of magnolol is about 2,0, 2,5, 3,0, 3,5, 4,0, 4,5, 5,0, 5,5, 6,0, or 6,5 |j.g / mL(micrograms / mL). In certain embodiments, the concentration of magnolol is about 3,0 to 5,5 |j.g / mL, or about 3.5 to 5,0 |j.g / mL such as for example 3,7 |j.g / mL. In certain embodiments, the culture medium is a basal medium that is supplemented with said medium components.

[0083] The term ‘basal medium’, as used herein, includes reference to a liquid medium that supports cellular growth by providing essential components for growth. A basal medium may be provided in liquid or powdered format. A basal medium that is not supplemented with any compound may enable cellular growth, but supplementation may be required for growth depending on the cell type. A basal medium may be supplemented with one or more components selected from the nonlimiting group consisting of amino acids, lipids, sugars, carbohydrates, anions, cations, buffering agents, colorants, vitamins, antioxidants, hormones, enzymes, proteins and trace elements. In some embodiments, the basal medium as disclosed herein is a commercially available basal medium such as DMEM (Dulbecco’s Modified Eagle Medium) and Ham’s F- 12. In some embodiments, the basal medium is the medium described in any of International patent application nos. W02024 / 084082, WO2024 / 170696 and WO2024 / 170702, which are incorporated by reference herein. Any of the medial described in any one of those patent applications may be used as basal medium in the methods of the invention.

[0084] In certain embodiments, the medium as described in the various embodiments of the invention further comprises doxycycline.

[0085] Culturing the cells as described herein can be performed under so called 2D culturing conditions, which is considered the conventional approach to culturing cells. However, the method as described can also easily be adapted to allow culturing under 3D conditions as shown in the examples below.

[0086] 3D cell culture is an artificially-created environment which enables cells to grow or interact with their surroundings in three dimensions. In such culture, cells typically form 3D colonies, which may be referred to as "spheroids". The 3D culture approach may more accurately model the cells' in vivo growth and behaviour. The skilled person is readily able to carry out 3D cell culture, for example by taking advantage of any of a number of commercially-available culturing tools. For example, the 3D culture may be carried out using scaffold or scaffold-free techniques. Scaffoldbased techniques make use of supports such as solid scaffolds and hydrogels to enable the cells to form a 3D culture. Such scaffolds may aim to mimic the natural extracellular matrix (ECM), which is present in vivo. Scaffold-free techniques dispense with the use of the scaffold on which to grow the cells. Instead, 3D spheroids may be established through the use of, for example, low-adhesion plates, hanging-drop plates, micro-patterned surfaces, rotating bioreactors, magnetic levitation and magnetic 3D bioprinting.

[0087] In some embodiments, the cells as described herein can be cultured in a bioreactor. Preferably the cells are culture in a bioreactor of up to about 500 litres, up to about 1000 litres, up to about 5000 litres, up to about 10000 litres or more.

[0088] The cell as used in the various embodiments of the invention as described herein, is a cell that is capable of lipid accumulation. In certain embodiments, the cell is from a mammal, preferably a non-human mammal. Preferably, the cell is an animal cell. In certain embodiments cell according to the invention is from an edible animal species. In certain embodiments, the pluripotent stem cells are of a livestock or poultry species.

[0089] Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons. In certain embodiments, the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare.

[0090] Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits.

[0091] In certain embodiments, the cell is a cell that is naturally capable of lipid accumulation such as a fat cells (adipocytes) or precursor cells thereof (such as fibro-adipogenic progenitor cells, lipoblasts, adipoblasts and, pre-adipocytes) or liver cells (hepatocyte) or precursor cells thereof (such as pre-adipocytes, hepatoblasts and pre-heptatocytes).

[0092] In certain embodiments, the cells are derived from an immortalized cell line. Exemplary cell lines include, but are not limited to, 3T3-L1 (mouse pre-adipocytes), buffalo rat liver cells (BRL 3A), chicken liver cells (LMH),

[0093] In certain embodiments, the cell is an adipocyte originating from a domestic pig a domestic cattle (such as a porcine adipocyte or a bovine adipocyte or a precursor of either thereof) or a goose.

[0094] In certain embodiments, the cell is an hepatocyte originating from a duck or a goose.

[0095] In certain embodiments, the cell is a pluripotent cell. Preferably the pluripotent cell is selected from the group consisting of embryonic stem cells, epiblast-derived stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. Preferably, when a pluripotent cell is cultured, the pluripotent stem cell is a porcine or a bovine pluripotent stem cell. Most preferably, a porcine pluripotent stem cell. In certain embodiments, the stem cell according to the invention is a porcine epiblast stem cell (pEpiSCs) or bovine epiblast stem cell (pEpiSCs).

[0096] In certain embodiments, the cell is a modified pluripotent cell that comprise an expression construct for expression of a PPAR-y protein and an expression construct for expression of a CEBPa protein such as described in International patent application no. W02024 / 084082.

[0097] In certain embodiments, the cells as used in the methods as described herein are described in International patent application nos. W02024 / 084082, WO2024 / 170696 and W02024 / 170702. Any cell described in any one of those patent applications may be useful in the methods of the invention for culturing cells.

[0098] In certain embodiments, the medium further comprises at least one of the following compounds:

[0099] - at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid;

[0100] - at least one vitamin B;

[0101] - at least one additional growth factor; and

[0102] - at least one chromium (III) salt.

[0103] In certain embodiments, at least one polyunsaturated fatty acid (PUFA) is an omega 3 fatty acid selected from the group consisting of a-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Preferably, the medium further comprises DHA.

[0104] In certain embodiments the medium comprises both DHA and butyric acid. In certain embodiments, the DHA is present in a concentration of about 0,008, 0,009, 0,010, 0,011 , 0,012, 0,013, 0,014, 0,015, 0,016, 0,017, 0,018, 0,019, 0,020 mg / mL. In certain embodiments, the concentration of DHA is about 0,008 to 0,020 mg / mL, or about 0,014 to 0,017 mg / mL such as for example 0.0164 mg / mL.

[0105] In certain embodiments, the short chain fatty acid is butyric acid. In certain embodiments, the butyric acid is present in a concentration of about 0,10, 0,11 , 0,12, 0,13, 0,14, 0,15, 0,16, 0,17, 0,18, 0,2, 0,3, 0,4 mg / mL. In certain embodiments, the concentration of butyric acid is about 0,10 to 0,4 mg / mL, or about 0,12 to 0,3 mg / mL such as for example 0.1322 mg / mL.

[0106] In certain embodiments, the medium further comprises at least one vitamin B selected from the group consisting of thiamine, riboflavin, niacin, niacinamide, nicotinamide riboside, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, biotin, folate and cobalamins. Preferably, the medium comprises biotin.

[0107] In certain embodiments, the biotin is present in a concentration of about 0,001 , 0,002, 0,004, 0,005, 0,006, 0,008, 0,009, 00,01 , 0,0022, 0,0023, 0,0024 0.0025, 0,0026, 0,0027, 0,0028, 0,0029 mg / mL. In certain embodiments, the concentration of biotin is about 0,001 to 0,01 mg / mL, or about 0,002 to 0,008 mg / mL such as for example 0.004 mg / mL.

[0108] In certain embodiments, the additional growth factor is epidermal growth factor (EGF).

[0109] In certain embodiments, the EGF is present in a concentration of about 1x10-4, 1x10-51x10_61x10-7, 1x10-8mg / mL. In certain embodiments, the concentration of EGF is about 1x10-5mg / mL.

[0110] In certain embodiments, the at least one chromium (III) salt is chromium(lll) chloride. In certain embodiments, the chromium(lll) chloride is present in a concentration of about 0,0018, 0,002, 00,0021 , 0,0022, 0,0023, 0,0024 0.0025, 0,0026, 0,0027, 0,0028, 0,0029, 0,0030, 0,0035 mg / mL. In certain embodiments, the concentration of chromium(lll) chloride is about 0,0018 to 0,0035 mg / mL, or about 0,0022 to 0,0025 mg / mL such as for example 0.0027 mg / mL.

[0111] In certain embodiments, the methods as described in the various embodiments of the invention further comprises the step of:

[0112] - incorporating the cultured cell into a food product for animal, preferably human, consumption.

[0113] In certain embodiments, the invention provides for a method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises:

[0114] - (-)-catechin, preferably in a concentration of about 2.0 to 4,0 pg / mL such as for example 3,0 pg / mL;

[0115] - magnolol, preferably in a concentration of about 3.5 to 5,0 pg / mL such as for example 3,7 pg / mL;

[0116] - fructose, preferably in a concentration of about 1 ,3 to 2,3 mg / mL such as 1 ,8 mg / mL;

[0117] - citrate, preferably in a concentration of about 320 to 580 pg / mL, such as about 330 pg / mL;

[0118] - FGF2, preferably in a concentration of about 4x10-4, 6x10-4pg / mL such as 5x10-4pg / mL;

[0119] - 2 hydroxy 2 methyl butyrate preferably in a concentration of about 0,20 to 0,30 mM such as about 0,25mM; and - acetyl cysteine preferably in a concentration of about 30,0 to 50,0 |j.g / mL such as about 40,0 P-g / mL, optionally, the method further comprises

[0120] DHA, preferably in a concentration of about 0,008 to 0,020 mg / mL, such as 0.0164 mg / mL;

[0121] Butyric acid, preferably in a concentration of about 0,10 to 0,4 mg / mL, such as 0.1322 mg / mL;

[0122] Biotin, preferably in a concentration of about 0,002 to 0,008 mg / mL, such as 0.004 mg / mL;

[0123] EGF, preferably in a concentration of about 1x10-4to 1x10-8mg / mL, such as 1x10-5;chromium(lll) chloride, preferably in a concentration of about 0,0018 to 0,0035 mg / mL, such as 0.0027 mg / mL

[0124] In a second aspect, the invention provides for a cell obtainable by or obtained by the methods as described in the various embodiments as described herein.

[0125] In a third aspect, the invention provides for a medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises an mTor agonist, preferably 2 hydroxy 2 methyl butyrate and a component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEBPa) agonist as described herein; at least one growth factor, preferably wherein the growth factor is an ERK-modulator as described herein; at least one carbon source as described herein; and at least one thiol-containing antioxidant as described herein.

[0126] In an alternative embodiment of the third aspect, the invention provides for a medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises an mTor agonist, preferably 2 hydroxy 2 methyl butyrate or a suitable salt thereof, and a component selected from the group consisting of: at least one growth factor, preferably wherein the growth factor is an ERK-modulator as described herein, more preferably wherein the growth factor is FGF2; at least one carbon source as described herein; and at least one thiol-containing antioxidant as described herein.

[0127] In an alternative embodiment of the third aspect, the invention provides for a medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises an mTor agonist, preferably a calcium salt of 2 hydroxy 2 methyl butyrate, and a component selected from the group consisting of: at least one growth factor, preferably wherein the growth factor is an ERK-modulator as described herein, more preferably wherein the growth factor is FGF2; at least one carbon source as described herein; and at least one thiol-containing antioxidant as described herein. In an alternative embodiment of the third aspect of the invention, the invention provides for a medium for increasing the differentiation and / or the lipid accumulation in cells, wherein the medium comprises a thiol-containing antioxidant as described herein and a component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as described herein ; at least one growth factor, preferably wherein the growth factor is an ERK- modulator as described herein; at least one carbon source as described herein; and an mTor agonist as described herein.

[0128] In a further alternative embodiment of the third aspect of the invention, the invention provides for a medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises least (-)-catechin and magnolol and a component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as described herein ; at least one growth factor and / or cytokine, preferably wherein the growth factor is an ERK- modulator as described herein ; at least one carbon source as described herein; an mTor agonist as described herein; and at least one thiol-containing antioxidant as described herein.

[0129] In certain embodiments, the medium for increasing the differentiation and / or the lipid accumulation in cells comprises:

[0130] - (-)-catechin, preferably in a concentration of about 2.0 to 4,0 pg / mL such as for example 3,0 pg / mL;

[0131] - magnolol, preferably in a concentration of about 3.5 to 5,0 pg / mL such as for example 3,7 pg / mL;

[0132] - fructose, preferably in a concentration of about 1 ,3 to 2,3 mg / mL such as 1 ,8 mg / mL;

[0133] - citrate, preferably in a concentration of about 320 to 580 pg / mL, such as about 330 pg / mL;

[0134] - FGF2, preferably in a concentration of about 4x10-4to 6x10-4pg / mL such as 5x10-4pg / mL;

[0135] - 2 hydroxy 2 methyl butyrate preferably in a concentration of about 0,20 to 0,30 mM such as about 0,25mM; and

[0136] - acetyl cysteine preferably in a concentration of about 30 to 500 pg / mL such as about 50g / mL optionally, the method further comprises

[0137] DHA, preferably in a concentration of about 0,008 to 0,020 mg / mL, such as 0.0164 mg / mL;

[0138] Butyric acid, preferably in a concentration of about 0,10 to 0,4 mg / mL, such as 0.1322 mg / mL; Biotin, preferably in a concentration of about 0,002 to 0,008 mg / mL, such as 0.004 mg / mL;

[0139] EGF, preferably in a concentration of about 1x10-4to 1x10-8mg / mL, such as 1x10-5;chromium(lll) chloride, preferably in a concentration of about 0,0018 to 0,0035 mg / mL, such as 0.0027 mg / mL.

[0140] In yet a further aspect, the invention provides for a food product (also referred to as “foodstuff’) comprising the cells produced, obtained by the methods and / or cultured in any of the mediums as described herein. In certain embodiments, the food product is for animal, preferably human, consumption.

[0141] A food product of the present invention may comprise one or more of minerals, synthetic substances, flavors, texture enhancers, nutritional additives, preservatives, and fats. In an aspect of the invention, the flavors are selected from one or more of essential oils, oleoresin (ESO), enzymes (ENZ), natural substances and extractives (NAT), non-nutritive sweetener (NNS), nutritive sweetener (NUTRS), herbs, spices, natural seasonings & flavorings (SP), and synthetic flavors (SY / FL), fumigant (FUM), artificial sweeteners and yeast extract. In another aspect of the present invention, the texture enhancers are selected from one or more of pureed plant material, guar gum, cellulose, hemicellulose, lignin, beta glucans, soy, wheat, maize and rice isolates and beet fiber, pea fiber, bamboo fiber, plant derived fiber, plant derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate, natural polysaccharides, grain husk, calcium citrate, calcium phosphates, calcium sulfate, magnesium sulfate and salts. In another aspect of the present invention, the nutritional additives are selected from one or more of trace elements, bioactive compounds, endogenous antioxidants, A, B-complex, C, D, E vitamins, zinc, thiamin, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, protein, amino acids salt, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, Retinol, folic acid, dietary fiber and amino acids. In yet another aspect of the present invention, the fats are selected from one or more of saturated, monounsaturated, polyunsaturated fats, corn oil, canola oil, sunflower oil, safflower oil, olive oil, peanut oil, soybean, flax seed oil, sesame oil, canola oil, avocado oil, seed oils, nut oils, safflower and sunflower oils, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fat, adipose tissue, cellular agriculture derived fat essential oil and oleoresin. In another aspect of the present invention, the preservative and / or antioxidant is selected from one or more of: sodium salt, chloride salt, iodine salt. Nitrates, nitrosamines. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium benzoate, potassium benzoate and benzene ascorbic acid, citric acid, potassium, monosodium glutamate (MSG), sulphur dioxide, sulphites, antibiotics. It is noted here that any one additive, flavor, texture enhancer, nutrient additive, fat / oil and / or preservative / antioxidant may supply more than one attribute to food product of the present invention.

[0142] In certain embodiments the food product of the present invention further comprises (cultured) myocytes. In certain embodiments, the food products comprises the cultured myocytes as described in International patent application nos. WO2024 / 170696 and W02024 / 170702. Any cell described in any one of those patent applications may be useful in a method of the invention for culturing cells.

[0143] The cultured meat product may be a structured product. Optionally, the product is structured with scaffolding. In one embodiment the structured product is free of scaffolding and the structure is provided by the cells as obtained by the methods as described herein.

[0144] In certain embodiments, the food product is a liver pate. Accordingly, in certain embodiments the food product comprises hepatocytes or precursors thereof as obtained by the methods as described herein or as cultured in the medium as described herein. The hepatocytes can originate from any animal suitable for consumption e.g pigs or cattle, preferably from fowl e.g turkey, chicken and / or guinea fowl, more preferably duck or goose. In certain embodiments, the food product is a foie gras (i.e. a “faux gras”).

[0145] In yet a further aspect, the invention provides for a cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method as described herein.

[0146] In yet a further aspect, the invention provides for a use of the cell obtained or obtainable by the method as described herein or as cultured in the medium as described herein for increasing lipid accumulation in a cell.

[0147] Description of the figures

[0148] Figure 1 : 2D adipocyte differentiation over 7 days with or without booster. A) Bright-field microscopy, 10X. B) Fluorescent microscopy, 10X zoom. Oil Red O (red, neutral lipids) and DAPI (blue, nuclei).

[0149] Figure 2: 2D adipocyte differentiation over 7 days with or without speed booster. A) Bright-field microscopy, 10X or 40X. B) Fluorescent microscopy, 10X zoom. Phalloidin (red, actin filaments) and DAPI (blue, nuclei), LipidTOX (green, neutral lipids)

[0150] Figure 3: A) Triglyceride content of adipocytes cultured with or without speed booster during differentiation B) increase of fat content after 7 day differentiation in bioreactors lipid content (wet pellet) versus lipid content (dry pellet) compared to commercially available porcine shoulder.

[0151] Examples

[0152] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0153] Materials and Methods

[0154] Cultivation procedure

[0155] PPARy-CEBPa induced pEplS cells (produced as set out in W02024 / 084082) were seeded with in proliferation medium with a coating additive in 6-well plates at final concentrations of 125.000 cells / cm2per well. After 24 hours cells were transferred to a differentiation medium with doxycycline at 0.5 ug / mL. The differentiation media was supplemented with a broad range of ingredients to screen for adipogenic- and lipogenic stimulating compounds. The screening set-up was designed by fractional factorial Design of Experiments methodology.

[0156] Fresh differentiation medium was added to the cells every other day. At day 7, the cells were harvested and fixated by incubating the cells for 15 min with a 4% PFA solution. Afterthe incubation period, the PFA solution was aspirated, and the wells were washed three times with PBS to remove any residual PFA.

[0157] Sample Preparation

[0158] 1 mL of 5% Nonidet P40 was added to the dry pellet and the solution was heated to 80-100 °C, for 2-5 minutes or until the Nonidet P40 becomes cloudy. After this initial heating, samples were cooled to room temperature and the heating step was repeated. Following the second heating, samples were centrifuged at top speed (13,000xg) for 2 minutes to eliminate insoluble material.

[0159] In some experiments, commercially available porcine shoulder was used as a comparison for triglyceride content. Samples originating from porcine shoulder were lyophilized and grinded. Fat was liberated from the cells using Nonidet P40 as described above.

[0160] Staining procedure

[0161] Neutral lipid content was estimated by BODIPY according to manufacturer’s instructions. DAPI was added to the Oil Red O solution at 1 ug / mL. Cells were analyzed by capturing the stained cultures with the EVOS7000. Fluorescent pictures were made for the Oil Red O and the DAPI staining. These pictures were analyzed by software and quantified for lipid droplets per cell.

[0162] Fixation and Staining Procedure:

[0163] Following the differentiation period, cultures were fixed using 4% paraformaldehyde (PFA) for 20 minutes and subsequently washed thriced with phosphate-buffered saline (PBS) were performed to remove excess PFA. Cells were analyzed by capturing the stained cultures with an EVOS 7000 microscope. Fluorescent pictures were made for the Oil Red O and the DAPI staining. Imaging was carried out using an EVOS 7000 microscope. DAPI staining was captured using the blue channel with an excitation wavelength of 357 nm and emission at 447 nm. The Oil Red O stain, utilizing Texas Red (excitation at 585 nm, emission at 624 nm), was captured in the red channel.

[0164] Image Analysis:

[0165] Trained artificial intelligence (Al) was employed for the quantification of the fat droplet area and the DAPI-stained nuclear area. The Al utilized image analysis algorithms to provide accurate measurements for these parameters, allowing for a comprehensive understanding of adipocyte morphology and distribution. This staining and imaging protocol, combined with advanced image analysis techniques, allowed for a detailed characterization and comparison of adipocyte cultures under different conditions. The utilization of specific dyes and imaging channels provided the necessary contrast for accurate assessment of lipid droplet accumulation. Fat (triglyceride) quantification was performed with the Triglyceride (TG) Colorimetric Assay Kit (ThermoFisher) according manufacturers instructions.

[0166] Results

[0167] Example 1

[0168] In all experiments, the compound HMB was present, additional components N-Acetyl Cystein (NAC), FGF2, Fructose, Citrate, (-) Catechin and Magnolol were added and their effect on the triglyceride content of the adipocytes was measured. Results are presented in Table 1. As can be seen the addition of NAC plus at least one of the components mentioned result in a significant increase in the amount of triglycerides after 7 day differentiation.

[0169] The best results were obtained when all compounds HMB, NAC, FGF2, Fructose, Citrate, (-)Catechin and Magnolol were simultaneously added to the culture, the combination of all these compounds is referred to as “booster” in this application. Figure 1 shows lipid accumulation after 7 day differentiation as analyzed under bright-field microscopy. As can be seen, the cells cultured in combination with the booster have bigger accumulated lipid droplets.

[0170] Table 1 : Effect of compound addition to the amount of triglycerides present after 7 day differentiation.

[0171] ++ indicates an significant increase in triglycerides of at least p <0,01 + indicates an significant increase in triglycerides of at least p <0,05

[0172] Example 2

[0173] The possibility to further improve lipid accumulation was analyzed by running a further screening set-up was designed by fractional factorial Design of Experiments methodology. From this further screening, an additional four components (butyric acid; biotin; EGF; and chromium(lll) chloride) were identified that further increase lipid accumulation during differentiation (Figure 2A and 2B). Samples for triglycerides quantification were obtained on days 0, 1 , 2, 3, 4, 5, 6 and 7. From day 2 on a significant increase in triglycerides content was observed for cells treated with the booster comprising the additional components (Figure 3A). The sample taken at day 7 after differentiation in a bioreactor was further analyzed for lipid content and compared to commercially available pork shoulder (Figure 3B).

[0174] Embodiments

[0175] The present invention provides at least the following numbered statements / embodiments:

[0176] 1 . A method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises, an mTor agonist and a component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist ; at least one growth factor, preferably wherein the growth factor is an ERK-modulator; at least one carbon source; and at least one thiol-containing antioxidant.

[0177] 2. The method according to embodiment 1 , wherein the mTor agonist is selected from the group consisting of 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1 ,3,5-triazin-2-amine (MHY1485) or derivatives thereof, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO), TSC1 knockdown agents (miRNA, siRNA, shRNA for example), PDK1 activators, AKT1 activators, TSC2 knockdown agents, PTEN knockdown agents, 2 hydroxy 2 methyl butyrate or a salt thereof, preferably the mTor agonist is 2 hydroxy 2 methyl butyrate or the calcium salt of hydroxy 2 methyl butyrate (HMB-Ca).

[0178] 3. The method according to embodiment 1 or 2, wherein the thiol-containing antioxidant is selected from the group consisting of glutathione, N-acetyl-L-cysteine, alpha lipoic acid, L-cysteine, dihydrolipoic acid, thioredoxin, mercaptoethanol and derivatives thereof, preferably the thiol- containing antioxidant is N-acetyl-L-cysteine.

[0179] 4. The method according to any of the preceding embodiments, wherein the carbon source is a sugar and / or a component involved in metabolic pathway preferably the citric acid pathway.

[0180] 5. The method according to any of the preceding embodiments, wherein the carbon source is a sugar preferably a monosaccharide or a disaccharide, preferably selected from the group consisting of galactose, fructose, xylose, sucrose, lactose, 1 ,6-bisphosphate maltose, isomaltulose, trehalose glucose, glucose 6-phosphate, fructose 6-phosphate, preferably fructose.

[0181] 6. The method according to any of the preceding embodiments, wherein the carbon source is a component involved in metabolic pathway preferably the citric acid pathway selected from the group consisting of succinyl-CoA, acetyl-CoA, succinate, fumarate, L-malate, oxaloacetate preferably wherein the component is citrate.

[0182] 7. The method according to any of the preceding embodiments, wherein the growth factor is a ERK modulator selected from the group consisting of FGF2, GF-EGF, AMD3100, RLX-33 and ACA-28, preferably wherein the growth factor is FGF2, more preferably the FGF2 is of bovine origin.

[0183] 8. The method according to any of the preceding embodiments, wherein the at least one PPARy agonist and / or a CEPBa agonist is selected from the group consisting of: Indomethacin, magnolol, amorfrutins (comprising, for example, amorfrutin 1 , amorfrutin 2, amorfrutin A, amorfrutin B, amorfrutin C and amorfrutin D), honokiol, lecithine (such as L-a-lecithine from soy beans), formononetin, bixin, norbixin, catechin, A9-tetrahydrocannabinol, (9S, 13R)-12-oxo-phytodienoic acid, odoratin, hydroxy unsaturated fatty acids from Coix lacrymajobi, commipheric acid, kaempferol-3-O-B-glucopyranoside, citral, alkamides from Echinacae purpera, tocotrienols, deoxyelephantopin, acetylated flavonol glycosides, kampferol, , 5’-formulglabridin, (2R,3R)-3,4’,7- trihydroxy-3’-prenylflavane, echinatin, (3R)-2’,3’,7-trihydroxy-4’- methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, glabrone, licochalcone E, flavonoids from Glycyrrhiza uralensis, 3-arylcoumarins from Glycyrrhiza uralensis, meranzin, fatty acids from Lycium chinense, lunularin, fatty acids from Melarnpyrum pratense, cucurbitane-type triterpene glycosides, polyacetylenes from Notopterygium incisum, biochanin A, ginsenoside 20(S)-protopanaxatriol, ginsenoside Rbi, fatty acids from Pineilia ternata, oleaninic acid, pseudolaric acid B, daidzein, amorphastilbol, carnosic acid, carnosol, 12-O-methul carnosic acid, u-linolenic acid, inoleic acid, naringenin, saurufuran A, isosilybin A, gallotannins, carvacrol, isoflavones from Trifolium pratense, ellagic acid, epicatechin gallate, flavonoids from Vitis vinifera, dehydrotrametenolic acid and 6-shogaol, preferably the agonist is magnolol and / or catechin.

[0184] 9. The method according to any of the preceding embodiments, wherein the culture medium is a basal medium that is supplemented with said medium components.

[0185] 10. The method according to any of the preceding embodimens, wherein the cell is capable of lipid accumulation such as adipocytes, hepatocytes, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells mesenchymal cells, fibro-adipogenic progenitor cells, lipoblasts, adipoblasts, pre-adipocytes, hepatoblasts, pre-heptatocytes

[0186] 11 . The method according to any of the preceding embodiments, wherein the culture medium further comprises at least one of the following compounds:

[0187] - at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid;

[0188] - at least one vitamin B;

[0189] - at least one additional growth factor; and

[0190] - at least one chromium (III) salt. 12. The method according to embodiment 11 , wherein

[0191] - the at least PUFA is an omega 3 fatty acid and the at least one short chain fatty acid is butyric acid;

[0192] - the at least one vitamin B is selected from the group consisting of thiamine, riboflavin, niacin, niacinamide, nicotinamide riboside, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, Biotin, folate and cobalamins, preferably biotin ;

[0193] - the least one additional growth factor is EGF; and

[0194] - the least one chromium (III) salt is chromium(lll) chloride.

[0195] 13. A cell obtainable by or obtained by the method according to any of the preceding embodiments.

[0196] 14. A medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises an mTor agonist as defined in embodiment 2 and at least one component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARg) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as defined in embodiment 8; at least one growth factor, preferably wherein the growth factor is an ERK-modulator as defined in claim 7; at least one carbon source as defined in any one of embodiments 4-6; and at least one thiol-containing antioxidant as defined in embodiment 3; and optionally comprising:

[0197] - at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid as defined in embodiment 12;

[0198] - at least one vitamin B as defined in embodiment 12;

[0199] - at least one additional growth factor as defined in embodiment 12; and

[0200] - at least one chromium (III) salt as defined in embodiment 12.

[0201] 15. A cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method according to any one of embodiments 1-11 , optionally further comprising (cultured) mammalian myocytes.

[0202] 16. A cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to any one of embodiments 1-11 .

[0203] 17. The method according to any one embodiments 1-11 , further comprising the step of:

[0204] - incorporating the cultured cell into a food product for animal, preferably human, consumption.

Claims

Claims1 . A method for increasing the differentiation and / or lipid accumulation in cells, wherein the method comprises culturing the cells in a culture medium that comprises, an mTor agonist and a component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist ; at least one growth factor, preferably wherein the growth factor is an ERK-modulator; at least one carbon source; and at least one thiol-containing antioxidant.

2. The method according to claim 1 , wherein the mTor agonist is selected from the group consisting of 4,6-di-4-morpholinyl-N-(4-nitrophenyl)-1 ,3,5-triazin-2-amine (MHY1485) or derivatives thereof, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO), TSC1 knockdown agents (miRNA, siRNA, shRNA for example), PDK1 activators, AKT1 activators, TSC2 knockdown agents, PTEN knockdown agents, 2 hydroxy 2 methyl butyrate or a salt thereof, preferably the mTor agonist is 2 hydroxy 2 methyl butyrate or the calcium salt of hydroxy 2 methyl butyrate (HMB- Ca).

3. The method according to claim 1 or 2, wherein the thiol-containing antioxidant is selected from the group consisting of glutathione, N-acetyl-L-cysteine, alpha lipoic acid, L-cysteine, dihydrolipoic acid, thioredoxin, mercaptoethanol and derivatives thereof, preferably the thiol- containing antioxidant is N-acetyl-L-cysteine.

4. The method according to any of the preceding claims, wherein the carbon source is a sugar and / or a component involved in metabolic pathway preferably the citric acid pathway.

5. The method according to any of the preceding claims, wherein the carbon source is a sugar preferably a monosaccharide or a disaccharide, preferably selected from the group consisting of galactose, fructose, xylose, sucrose, lactose, 1 ,6-bisphosphate maltose, isomaltulose, trehalose glucose, glucose 6-phosphate, fructose 6-phosphate, preferably fructose.

6. The method according to any of the preceding claims, wherein the carbon source is a component involved in metabolic pathway preferably the citric acid pathway selected from the group consisting of succinyl-CoA, acetyl-CoA, succinate, fumarate, L-malate, oxaloacetate preferably wherein the component is citrate.

7. The method according to any of the preceding claims, wherein the growth factor is a ERK modulator selected from the group consisting of FGF2, GF-EGF, AMD3100, RLX-33 and ACA-28, preferably wherein the growth factor is FGF2, more preferably the FGF2 is of bovine origin, even .

8. The method according to any of the preceding claims, wherein the at least one PPARy agonist and / or a CEPBa agonist is selected from the group consisting of: Indomethacin, magnolol, amorfrutins (comprising, for example, amorfrutin 1 , amorfrutin 2, amorfrutin A, amorfrutin B, amorfrutin C and amorfrutin D), honokiol, lecithine (such as L-a-lecithine from soy beans), formononetin, bixin, norbixin, catechin, A9-tetrahydrocannabinol, (9S, 13R)-12-oxo-phytodienoic acid, odoratin, hydroxy unsaturated fatty acids from Coix lacrymajobi, commipheric acid, kaempferol-3-O-B-glucopyranoside, citral, alkamides from Echinacae purpera, tocotrienols, deoxyelephantopin, acetylated flavonol glycosides, kampferol, , 5’-formulglabridin, (2R,3R)-3,4’,7- trihydroxy-3’-prenylflavane, echinatin, (3R)-2’,3’,7-trihydroxy-4’- methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, glabrone, licochalcone E, flavonoids from Glycyrrhiza uralensis, 3-arylcoumarins from Glycyrrhiza uralensis, meranzin, fatty acids from Lycium chinense, lunularin, fatty acids from Melarnpyrum pratense, cucurbitane-type triterpene glycosides, polyacetylenes from Notopterygium incisum, biochanin A, ginsenoside 20(S)-protopanaxatriol, ginsenoside Rbi, fatty acids from Pineilia ternata, oleaninic acid, pseudolaric acid B, daidzein, amorphastilbol, carnosic acid, carnosol, 12-O-methul carnosic acid, u-linolenic acid, inoleic acid, naringenin, saurufuran A, isosilybin A, gallotannins, carvacrol, isoflavones from Trifolium pratense, ellagic acid, epicatechin gallate, flavonoids from Vitis vinifera, dehydrotrametenolic acid and 6-shogaol, preferably the agonist is magnolol and / or catechin.

9. The method according to any of the preceding claims, wherein the culture medium is a basal medium that is supplemented with said medium components.

10. The method according to any of the preceding claims, wherein the cell is capable of lipid accumulation such as adipocytes, hepatocytes, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells mesenchymal cells, fibro-adipogenic progenitor cells, lipoblasts, adipoblasts, pre-adipocytes, hepatoblasts, pre-heptatocytes11. The method according to any of the preceding claims, wherein the culture medium further comprises at least one of the following compounds:- at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid;- at least one vitamin B;- at least one additional growth factor; and- at least one chromium (III) salt.

12. The method according to claim 11 , wherein- the at least PUFA is an omega 3 fatty acid and the at least one short chain fatty acid is butyric acid;- the at least one vitamin B is selected from the group consisting of thiamine, riboflavin, niacin, niacinamide, nicotinamide riboside, pantothenic acid, pyridoxine, pyridoxal, pyridoxamine, Biotin, folate and cobalamins, preferably biotin ;- the least one additional growth factor is EGF; and- the least one chromium (III) salt is chromium(lll) chloride.

13. A cell obtainable by or obtained by the method according to any of the preceding claims.

14. A medium for boosting the differentiation and / or the lipid accumulation in cells, wherein the medium comprises an mTor agonist as defined in claim 2 and at least one component selected from the group consisting of: at least one peroxisome proliferator-activated receptor gamma (PPARg) agonist and / or a CCAAT / enhancer-binding protein alpha (CEPBa) agonist as defined in claim 8; at least one growth factor, preferably wherein the growth factor is an ERK-modulator as defined in claim 7; at least one carbon source as defined in any one of claims 4-6; and at least one thiol-containing antioxidant as defined in claim 3; and optionally comprising:- at least one polyunsaturated fatty acid (PUFA) and / or at least one short chain fatty acid as defined in claim 12;- at least one vitamin B as defined in claim 12;- at least one additional growth factor as defined in claim 12; and- at least one chromium (III) salt as defined in claim 12.

15. A cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method according to any one of claims 1-11 , optionally further comprising (cultured) mammalian myocytes.

16. A cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to any one of claims 1-11.

17. The method according to any one claims 1-11 , further comprising the step of:- incorporating the cultured cell into a food product for animal, preferably human, consumption.

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