Patent Application
20240344028
The invention is in the field of serum-free, chemically defined media that allow for the differentiation of adipogenic progenitor (precursor) cells into adipocytes for animal, preferably human, consumption. The serum-free media of the invention allow for the differentiation of mammalian adipogenic progenitor cells, such as bovine, ovine or porcine adipogenic progenitor cells, into adipocytes and do not require the use of conventional food-incompatible differentiation inducers. The cultured adipocytes obtained by the methods of the invention can be used in the manufacturing of cultured fat products and cultured meat products for human consumption.
For the culturing and differentiation of animal cells, serum is traditionally used as part of a medium. Serum provides important nutrients, vitamins, growth factors, proteins and electrolytes. An especially widely used serum is fetal bovine serum (FBS), which is rich in growth factors. Although serum is beneficial in the proliferation and differentiation of animal cells, there are some clear disadvantages to the use of serum.
The use of serum in products for human consumption may introduce a health risk, as serum may contain bacteria, viruses and prions. In addition, the composition of serum depends on the source from which it is obtained, different batches of serum may lead to different or non-reproducible results in relation to cell culture. An additional ethical consideration is the well-being of animals used for obtaining serum. Also, the use of serum is expensive and introduces a cost burden when used on a large scale. Therefore, demand has arisen for alternatives to serum.
However, it remains a challenge to provide for serum-free and chemically defined media that at least achieve similar results in terms of adipogenic differentiation potential as compared to conventional media. Serum-free media for the production of cell culture-based fat or meat products do not only need to allow for sufficient differentiation levels compared to traditional serum-based media, but should also not include components that are not safe for human consumption and therefore incompatible with human food regulations. Culture media for differentiating adipogenic progenitor cells often include as differentiation inducers the compounds 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and rosiglitazone, which are a hazard to human health, and therefore not suitable and acceptable in human food manufacturing processes.
There is a need in the art for serum-free adipogenic differentiation media that provide for advantageous differentiation of adipogenic precursor cells and which can be applied in a cell-culture based human food manufacturing method.
The present inventors discovered amongst others a serum-free, chemically defined adipogenic differentiation medium that can advantageously be used in the culture and differentiation of mammalian adipogenic progenitor cells and which can be applied in a cell-culture based human food manufacturing methods. It was further discovered that such a serum-free medium outperforms traditional and conventional serum-based differentiation media that employ adipogenic differentiation inducers such as the often used 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and rosiglitazone, in terms of differentiation potential. It was also discovered that adipogenic progenitor cells of different mammalian species can be successfully differentiated.
Therefore, the invention provides in a first aspect a method for differentiating an adipogenic progenitor cell, comprising the step of:—culturing an adipogenic progenitor cell in a serum-free medium for differentiating an adipogenic progenitor cell, wherein the serum-free medium comprises:—at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist;—at least one hormone selected from the group consisting of insulin and hydrocortisone;—at least one cytokine and/or growth factor selected from the group consisting of bone morphogenetic protein 4 (BMP4) and epidermal growth factor (EGF); and-ascorbic acid or a derivative thereof.
In a preferred embodiment of said method for differentiating, said serum-free medium comprises bone morphogenetic protein 4 (BMP4) and epidermal growth factor (EGF) as said at least one cytokine and/or growth factor; and wherein said serum-free medium optionally further comprises fibroblast growth factor (FGF).
In another preferred embodiment of said method for differentiating, said serum-free medium comprises insulin and hydrocortisone as said at least one hormone.
In another preferred embodiment of said method for differentiating, said serum-free medium comprises a basal medium that is supplemented with said medium components.
In another preferred embodiment of said method for differentiating, said serum-free medium further comprises at least one biogenic amine such as putrescine.
In another preferred embodiment of said method for differentiating, said serum-free medium further comprises a source of lipids such as a source of saturated and/or unsaturated fatty acids, preferably supplemented to said basal medium in the form of a concentrate.
In another preferred embodiment of said method for differentiating, said basal medium is DMEM, Ham's F-12 or a mixture thereof.
In another preferred embodiment of said method for differentiating, said serum-free medium does not comprise a differentiation inducer selected from the group consisting of isobutyl-methyl-xantane (IBMX), dexamethasone and/or a thiazolidinedione such as rosiglitazone, pioglitazone, lobeglitazone, cigilitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone and/or balaglitazone. Preferably, said serum-free medium does not comprise differentiation inducers rosiglitazone, isobutyl-methyl-xantane (IBMX) and dexamethasone.
In another preferred embodiment of said method for differentiating, said at least one PPARy agonist is a food-compatible PPARy agonist.
In another preferred embodiment of said method for differentiating, said at least one PPARy agonist is selected from the group consisting of indomethacin, amorfrutin B, magnolol and honokiol, preferably indomethacin or magnolol, more preferably indomethacin.
In another preferred embodiment of said method for differentiating, said serum-free medium comprises a source of energy that allows for differentiation of said adipogenic progenitor cell. In another preferred embodiment of said method for differentiating, said source of energy is an (energy-containing) substrate involved in at least one energy metabolism pathway such as glycolysis, mitochondrial respiration and/or pentose phosphate pathway.
In another preferred embodiment of said method for differentiating, said source of energy is at least one sugar, preferably a monosaccharide or a disaccharide, such as glucose or galactose, optionally in combination with a source of glutamine, a pyruvate and/or acetate.
In another preferred embodiment of said method for differentiating, said source of energy is (i) glucose and glutamine, (ii) galactose and pyruvate, (iii) glucose and pyruvate, (iv) acetate, galactose and pyruvate, (v) glucose, galactose and pyruvate, (vi) glucose and alpha-ketoglutarate (aKG) and/or (vii) glucose, alpha-ketoglutarate (aKG) and pyruvate.
It was surprisingly established that, instead of using glucose and glutamine to support adipogenic differentiation, it was also found to be possible to use other sources of energy for that purpose. For instance, a combination of galactose and pyruvate was found to be beneficial for that purpose. An advantage of such a combination, as compared to glucose and glutamine, is that it reduces the amount of waste such as ammonia that the cells produce, which means that less medium is required, and the cells differentiated better in general.
Thus, in a preferred embodiment of a method for differentiating of the invention, said serum-free medium comprises galactose and pyruvate. In another preferred embodiment of said method for differentiating, said serum-free medium for differentiating further comprises:—progesterone.
In another preferred embodiment of said method for differentiating, said serum-free medium for differentiating further comprises:—sodium bicarbonate.
In another preferred embodiment of said method for differentiating, said serum-free medium for differentiating further comprises:—a source of glutamine and/or glucose.
In another preferred embodiment of said method for differentiating, said serum-free medium for differentiating comprises: at least one peroxisome proliferator-activated receptor gamma (PPARy) agonist, preferably indomethacin or magnolol;—hydrocortisone;—insulin;—bone morphogenetic protein 4 (BMP4);—epidermal growth factor (EGF);—ascorbic acid or a derivative thereof; and—a basal medium, preferably DMEM/F12; and wherein said serum-free medium for differentiating optionally further comprises—at least one biogenic amine such as putrescine,—a fibroblast growth factor (FGF),—a source of lipids, preferably wherein said source of lipids is a source of saturated and unsaturated fatty acids,—progesterone; and/or-HEPES.
In another preferred embodiment of said method for differentiating, (i) the at least one PPARy agonist is present in said serum-free medium in a final concentration of 0.05-5000 μM, (ii) the at least one hormone that is a hydrocortisone is present in said serum-free medium in a final concentration of 0.1-1000 nM, (iii) the at least hormone that is an insulin is present in said serum-free medium in a final concentration of 0.01-200 μM, (iv) the at least one cytokine and/or growth factor that is EGF is present in said serum-free medium in a final concentration of 3-30,000 pM, (v) the at least one cytokine and/or growth factor that is BMP4 is present in said serum-free medium in a final concentration of 0.03-60,000 nM, and/or (vi) the ascorbic acid or a derivative thereof is present in said serum-free medium in a final concentration of 0.01-10,000 μM; and optionally wherein (vii) said source of lipids is present in said serum-free medium in a final concentration of 0.0001%-1% (vol. % (v/v)), (viii) said at least one biogenic amine is present in said serum-free medium in a final concentration of 0.01-1000 μM and/or (ix) said FGF is present in said serum-free medium in a final concentration of 1-10,000 pM.
In another preferred embodiment of said method for differentiating, said serum-free medium comprises: a DMEM/F12 basal medium, HEPES (e.g. at about 4.9 mM), hydrocortisone (e.g. at about 0.1 μM), insulin (e.g. at about 1-2 μM), a lipid concentrate (e.g. at about 0.001%), a biogenic amine such as putrescine (e.g. at about 56 μM), EGF (e.g. at about 322 pM), FGF (e.g. at about 115 pM), progesterone (e.g. at about 17.8 nM), ascorbic acid or a derivative thereof (e.g. at about 227 μM), indomethacin (e.g. at about 50 μM) and BMP4 (e.g. at about 300 nM).
In another preferred embodiment of said method for differentiating, said serum-free medium is entirely free of animal-derived components.
In another preferred embodiment of said method for differentiating, said serum-free medium is a (chemically) defined serum-free medium.
In another preferred embodiment of said method for differentiating, said method for differentiating an adipogenic progenitor cell is a method for producing a cultured adipocyte for animal, preferably human, consumption by differentiating said adipogenic progenitor cell into an adipocyte.
In another preferred embodiment of said method for differentiating, said method is a method for proliferating an adipogenic progenitor cell followed by differentiating proliferated adipogenic progenitor cells, wherein said method further comprises, prior to differentiating said adipogenic progenitor cell, a step of:—culturing an adipogenic progenitor cell in a serum-free medium for proliferating adipogenic progenitor cells, to thereby provide proliferated adipogenic progenitor cells.
In another preferred embodiment of said method for differentiating, said method for differentiating and/or said method for proliferating of an adipogenic progenitor cell followed by differentiating proliferated adipogenic progenitor cells, is entirely free of animal-derived components and preferably (only) employs (chemically) defined serum-free media.
In another preferred embodiment of said method for differentiating, said adipogenic progenitor cell is a mammalian adipogenic progenitor cell, preferably a bovine adipogenic progenitor cell such as a muscle-derived bovine adipogenic progenitor cell such as a fibro-adipogenic progenitor (FAP) cell.
In another preferred embodiment of said method for differentiating, said culturing of said adipogenic progenitor cell in said serum-free medium for differentiating is performed under conditions that allow for differentiation of said adipogenic progenitor cell into an adipocyte.
In another preferred embodiment of said method for differentiating, said culturing of said adipogenic progenitor cell in said serum-free medium for proliferating is in the form of, or is performed in (or as), a two-dimensional or three-dimensional cell culture, such as a microcarrier-based cell culture.
In another preferred embodiment of said method for differentiating, said culturing of said adipogenic progenitor cell in said serum-free medium for differentiating is in the form of, or is performed in (or as), a two-dimensional or three-dimensional cell culture such as a hydrogel, aggregate and/or scaffold, preferably in a hydrogel such as a hydrogel comprising alginate.
In another preferred embodiment of said method for differentiating, said method further comprises the step of:—incorporating said cultured adipocyte into a food product for animal, preferably human, consumption.
In another preferred embodiment of said method for differentiating, said food product is (i) a cell-culture based fat product or (ii) a cell-culture based meat product that comprises myocytes, myotubes and/or myofibers.
In another aspect, the invention provides a serum-free medium for differentiating an adipogenic progenitor cell, wherein said medium is as defined in any one of the preceding aspects and/or embodiments relating to a method for differentiating an adipogenic progenitor cell.
In another aspect, the invention provides a composition comprising a serum-free medium for differentiating of the invention and an adipogenic progenitor cell and/or a partially or terminally differentiated cell obtained therefrom.
In a preferred embodiment of said method for differentiating, said serum-free medium of the invention or said composition of the invention, said adipogenic progenitor cell is a mammalian adipogenic progenitor cell, preferably a bovine, ovine, porcine or murine adipogenic progenitor cell. In another aspect, the invention provides a culture of adipocytes obtainable by a method for differentiating according to the invention.
In another aspect, the invention provides a cultured fat product for animal, preferably human, consumption, comprising adipocytes obtainable by a method for differentiating according to the invention.
In another aspect, the invention provides a cultured meat product for animal, preferably human, consumption, comprising adipocytes obtainable by a method for differentiating according to the invention and (cultured) mammalian myocytes, myotubes and/or myofibers, preferably bovine, ovine, porcine or murine myocytes, myotubes and/or myofibers.
In a preferred embodiment of said cultured fat product or cultured meat product of the invention, said fat product or said meat product:—comprises cultured fat with a different triglyceride composition compared to the triglyceride composition of a (preferably bovine) subcutaneous fat tissue, preferably (i) wherein the relative contribution of unsaturated triglycerides, preferably triglycerides with a single unsaturation, to the total amount of triglycerides is higher in cultured fat as compared to the relative contribution of unsaturated triglycerides, preferably triglycerides with a single unsaturation, to the total amount of triglycerides in said (preferably bovine) subcutaneous fat tissue or (ii) wherein the relative contribution of saturated triglycerides to the total amount of triglycerides is lower in said cultured fat as compared to the relative contribution of saturated triglycerides to the total amount of triglycerides in said (preferably bovine) subcutaneous fat tissue;—does not comprise antibiotics and/or antibiotic residues;—does not comprise red blood cells;—comprises lower levels of microbial contamination as compared to meat products obtained by animal slaughter; and/or—does not comprise cartilage tissue.
In another aspect, the invention provides a method for differentiating an adipogenic progenitor cell, comprising the step of:—culturing an adipogenic progenitor cell in a serum-free medium for differentiating an adipogenic progenitor cell; wherein the serum-free medium is food-compatible, preferably wherein said serum-free medium comprises (and is food-compatible by virtue of) a PPARΥ agonist selected from the group of indomethacin, magnolol, amorfrutin B and honokiol, more preferably wherein the PPARΥ agonist is indomethacin or magnolol, even more preferably wherein the PPARΥ agonist is indomethacin.
In another aspect, the invention provides a method for differentiating an adipogenic progenitor cell, comprising the step of:—culturing an adipogenic progenitor cell in a serum-free medium for differentiating an adipogenic progenitor cell; wherein the serum-free medium does not comprise a differentiation inducer selected from the group consisting of isobutyl-methyl-xantane (IBMX), dexamethasone and/or a thiazolidinedione such as rosiglitazone, pioglitazone, lobeglitazone, cigilitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone and/or balaglitazone.
In another aspect, the invention provides a method for differentiating an adipogenic progenitor cell, comprising the step of:—culturing an adipogenic progenitor cell in a serum-free medium for differentiating an adipogenic progenitor cell; wherein the serum-free medium comprises as a source of energy an (energy-containing) substrate involved in at least one energy metabolism pathway such as glycolysis, mitochondrial respiration and/or pentose phosphate pathway. In some embodiments, said source of energy is at least one sugar, preferably a monosaccharide or a disaccharide, such as glucose or galactose, optionally in combination with a source of glutamine, a pyruvate, acetate and/or alpha-ketoglutarate (aKG). In a more preferred embodiment of said method for differentiating, said source of energy is (i) glucose and glutamine, (ii) galactose and pyruvate, (iii) glucose and pyruvate, (iv) acetate, galactose and pyruvate, (v) glucose, galactose and pyruvate, (vi) glucose and alpha-ketoglutarate (aKG) and/or (vii) glucose, alpha-ketoglutarate (aKG) and pyruvate.
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, i.e. the process wherein adipogenic progenitor cells ultimately differentiate into adipocytes (fat cells).
The term ‘mammalian’, as used herein, includes reference to any animal of the class Mammalia. Non-limiting examples of animals belonging to the class Mammalia are cattle, pigs, sheep, deer, and mice. A mammalian cell includes cells isolated from, derived from, differentiated from, expanded or originally found in an animal of the class Mammalia, and also includes cultured cells. A preferred mammal is a Bos taurus.
The term ‘adipocyte’, as used herein, can be used interchangeably with the term ‘fat cell’ and includes reference to a cultured fat cell or adipocyte or cultured fat tissue. Adipocytes may be categorized as forming white adipose tissue or brown adipose tissue. Adipocytes are found throughout the body. Adipocytes synthesize and store fat, including but not limited to lipids and triglycerides.
The term ‘progenitor cell’ as used herein, includes reference to a cell that is able to differentiate into a more specialized cell. The term ‘progenitor cell’ can be used interchangeably with the term ‘precursor cell’. Progenitor cells may for example be stem cells, satellite cells, intermediate progenitor cells, radial glial cells, bone marrow stromal cells, periosteum, pancreatic progenitor cells, angioblasts, blast cells or adipogenic progenitor cells. No limitation to the stage of development is intended.
The term ‘adipogenic progenitor cell’, as used herein, includes reference to a cell that is able to differentiate into an adipocyte. Adipogenic progenitor cells may for example be pluripotent stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells, fibro-adipogenic progenitor (FAP) cells, lipoblasts, adipoblasts and preadipocytes. The adipogenic progenitor cell can for instance be derived from muscle or fat, and is in embodiments a muscle-derived adipogenic progenitor cell such as a (skeletal) muscle-derived fibro-adipogenic progenitor (FAP) cell. Preferably, the adipogenic progenitor cell is a mammalian adipogenic progenitor cell, for instance a bovine, ovine, porcine or murine, preferably bovine, adipogenic progenitor cell. FAP cells can be purified from muscle tissue samples by methods involving antigen-based cell sorting such as FACS. Cell surface markers expressed by bovine FAPs are CD9, CD14, CD49e (ITGA5), CD61 (ITGB3), CD140a (PDGFRA), and ITGA9. Bovine FAPs lack expression of hematopoietic marker CD45, endothelial marker CD321 (F11R), and the myogenic progenitor markers CD56 (NCAM1) and ITGA7. Preferably, the FAP cell is a bovine FAP cell.
The term ‘culturing’, as used herein, includes reference to the cell cultures of the cells disclosed herein and may refer, depending on the type of medium used, to propagation and/or proliferation (expansion) of adipogenic progenitor cells such as mammalian adipogenic progenitor cells or to differentiation of mammalian adipogenic progenitor cells into adipocytes. In the context of the invention, this term preferably includes reference to the differentiation of bovine adipogenic progenitor cells into bovine adipocytes. It should however be understood that a serum-free medium of the invention can also be employed to differentiate other non-human, mammalian adipogenic progenitor cells such as ovine, porcine or murine adipogenic progenitor cells. Therefore, any embodiment described herein in relation to bovine adipogenic progenitor cells, is also applicable to ovine (such as sheep), porcine (such as pig) or murine (such as mouse) adipogenic progenitor cells, i.e. progenitor cells of ovine, porcine or murine origin. Thus, in aspects and/or embodiments of a method for differentiation of the invention, a serum-free medium of the invention or a composition of the invention, instead of a bovine adipogenic progenitor cell, an ovine adipogenic progenitor, a porcine adipogenic progenitor or a murine adipogenic progenitor cell can be employed. In embodiments, the adipogenic progenitor cell is a muscle-derived or fat-derived adipogenic progenitor cell, such as a muscle-derived adipogenic progenitor cell e.g. a fibro-adipogenic progenitor (FAP) cell. The adipogenic progenitor cell can be derived from bovine skeletal muscle for instance by taking a biopsy. The term “culturing”, as used herein in relation to a method for differentiating an adipogenic progenitor cell, includes reference to cell culture conditions that allow for adipogenic differentiation of a progenitor cell into a partially or terminally differentiated cell, preferably an adipocyte. The skilled person is well aware of suitable cell culture conditions that allow for differentiation of adipogenic progenitor cells, especially in light of the present disclosure. The term ‘animal-derived’, as used herein, includes reference to components that are produced by an animal. Non-limiting examples of animal-derived components are fetal bovine serum, and components isolated from fetal bovine serum. Not animal-derived are for instance recombinantly produced animal proteins or peptides and any other component not produced by an animal but synthesized e.g. in the laboratory. If a medium as disclosed herein does not comprise any components or ingredients that are animal-derived, then the medium is animal component-free. Preferably, a serum-free medium as disclosed herein does not comprise components that are derived (obtained) from an animal.
The term “serum-free”, as used herein, includes reference to a culture medium that is formulated in the absence of serum such as human serum or bovine serum or cell lysates of cells in said serum. A serum-free medium may contain serum proteins by way of supplementation of said serum protein such as serum albumin to said medium. However, preferably, all components of said medium are animal-free, i.e. that the components are not obtained from an animal but are for instance recombinantly produced. Preferably, a serum-free medium as disclosed herein is a chemically defined medium, i.e. a medium that is defined by the presence (and/or absence) of specific components. The concentrations of the components of a serum-free medium as disclosed herein can routinely be adjusted and optimized for the culturing and differentiation of adipogenic progenitor cells such as mammalian adipogenic progenitor cells, e.g. bovine, ovine or porcine adipogenic progenitor cells. The components in a serum-free medium of the invention are present in effective amounts that allow for differentiation of an adipogenic progenitor cell in culture, preferably wherein said adipogenic progenitor cell in a previous culturing step has been proliferated or expanded (or originates or is derived from a culture of progenitor cells that was previously proliferated or expanded) under serum-free conditions such as with a serum-free medium for proliferation of progenitor cells. A non-limiting example of a serum-free medium for proliferation or expansion of adipogenic progenitor cells is provided in Example 2 (“SF-PM” medium). A non-limiting example of a serum-free medium for differentiation of an adipogenic progenitor cell of the invention is provided in Example 1 (“DMAD” medium) and in other Examples.
The term ‘hormone’, as used herein, includes reference to members of a class of signal molecules from multicellular organisms that have an effect on physiology and/or behavior. Hormones bind to specific receptors in order to activate a signal transduction pathway. Examples of types of hormones are eicosanoids, steroids, amino acid derivatives, proteins, peptides and gases. Examples of hormones in the context of media for differentiation as disclosed herein are hydrocortisone and insulin.
The term ‘cytokine’, as used herein, includes reference to proteins that are involved in cell signaling, more specifically cell signaling related to or associated with the immune system and/or morphogenic pathways. Cytokines may be constituents of media, for example for the induction of expansion and/or differentiation. Examples of cytokines in the context of media for differentiation are molecules belonging to the group of bone morphogenic proteins (BMP).
The term ‘growth factor’, as used herein, includes reference to members of a class of signal molecules that generally have an effect on cell proliferation, growth and/or death. Growth factors may be constituents of media, for example for the induction of differentiation. Examples of cytokines in the context of media for differentiation are epidermal growth factor (EGF) and molecules belonging to the group of fibroblast growth factors (FGF).
The terms ‘acceptable or suitable for animal consumption’ and ‘for animal consumption’, as used herein, includes reference to components that are not harmful to healthy, non-allergic animals when consumed under normal circumstances and normal use. The term ‘animal’ may refer to any member of the kingdom Animalia, including humans and cats. Components (or ingredients) that are acceptable for human consumption can, amongst others, for instance be found in the Food Chemicals Codex, ISO 22000, or ingredients approved by the European Food Safety Authority or United States Food and Drug Administration. Compounds that are explicitly not suitable for animal consumption within the context of the invention, are rosiglitazone, isobutyl-methyl-xantane (IBMX) and dexamethasone.
The term “food-compatible”, as used herein, includes reference to materials, ingredients or components that are non-toxic and safe for animal consumption, preferably for human consumption. The term “food-compatible” can be used interchangeably with the term “food-grade”.
The terms ‘peroxisome proliferator-activated receptor gamma’, ‘PPARG’ and ‘PPARΥ’, as used herein, include reference to a nuclear receptor that is present in cells of different tissue types such as adipose tissue, colon, rumen and placenta, and in macrophages. Alternative names of PPARΥ include glitazone receptor and NR1C3.
The term ‘PPARΥ agonist’, as used herein, includes reference to any chemical that binds to or interacts with PPARΥ, and functions as an agonist. ‘PPARΥ agonist’, as used herein, includes reference to endogenous agonists, full agonists, co-agonists, selective agonists, partial agonists, inverse agonists, superagonists, irreversible agonists and biased agonists. Non-limiting examples of the at least one PPARΥ agonist, which can be comprised in a serum-free medium for differentiating of the invention, include indomethacin, magnolol, amorfrutins (comprising, for example, amorfrutin 1, amorfrutin 2, amorfrutin A, amorfrutin B, amorfrutin C and amorfrutin D), honokiol, lecithine (such as L-α-lecithine from soy beans), formononetin, bixin, norbixin, catechin, Δ9-tetrahydrocannabinol, (9S, 13R)-12-oxo-phytodienoic acid, odoratin, hydroxy unsaturated fatty acids from Coix lacrymajobi, commipheric acid, kaempferol-3-O-β-glucopyranoside, citral, alkamides from Echinacae purpera, tocotrienols, deoxyelephantopin, acetylated flavonol glycosides, kampferol, quercetin, genistein, 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 Melampyrum pratense, cucurbitane-type triterpene glycosides, polyacetylenes from Notopterygium incisum, biochanin A, ginsenoside 20 (S)-protopanaxatriol, ginsenoside Rb1, fatty acids from Pinellia ternata, oleaninic acid, pseudolaric acid B, daidzein, amorphastilbol, carnosic acid, carnosol, 12-O-methul carnosic acid, α-linolenic acid, linoleic 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
The term “biogenic amine”, as used herein, includes reference to a molecule containing one or more amine groups. Included in this group of biogenic amines are monoamines and polyamines. Within the group of monoamines are included ethanolamine, and within the group of polyamines are included agmatine, cadaverine, putrescine, spermine and spermidine. Preferably, the medium as disclosed herein comprises a at least biogenic amine that is a polyamine. Examples of the group of polyamines are putrescine, agmatine, cadaverine, spermine and spermidine. Preferably, the medium as disclosed herein comprises at least putrescine.
The term ‘putrescine’, as used herein, includes reference to a compound represented by the formula NH2(CH2)NH2. Putrescine is otherwise referred to as tetramethylenediamine, butane-1,4-diamine or 1,4-diaminobutane. Putrescine may be produced chemically or biochemically.
The terms ‘proliferating’ and ‘proliferation’, as used herein, includes reference to expansion of adipogenic progenitor cells, i.e. to increase their cell number. During proliferation, cells are propagated.
The term ‘source of’, as used herein, includes reference to a medium component that can be provided as a precursor or derivative of said medium component, or is provided as the medium component as such. The skilled person is well aware of suitable precursors for medium components as described herein. For instance, L-alanyl-L-glutamine or glutamine can be used as a source of glutamine and glucose can be used as a source of glucose. Further, as an example, glucose can be provided as a source of energy.
The term ‘bovine’, as used herein, includes reference to any member of the subfamily Bovinae. The subfamily Bovinae includes the tribes Boselaphini, Bovini and Tragelaphini. Preferably, bovine as disclosed herein refers to the members of the subfamily Bovinae that are used for animal, preferably human, consumption. Non-limiting examples of such members include domestic cattle (Bos taurus and subspecies Bos taurus taurus; Bos taurus indicus), banteng (Bos javanicus), gayal or mithun (Bos frontalis), gaur (Bos gaurus), yak (Bos grunniens; Bos mutus), water buffalo (Bubalus arnee; Bubalus bubalis), American bison (Bison bison), kudu (Tragelaphus strepsiceros; Tragelaphus imberbis), common eland (Taurotragus oryx), giant eland (Taurotragus derbianus), and nilgai (Boselaphus tragocamelus). Especially preferred bovine species as disclosed herein are Bos taurus and its subspecies.
The term ‘ovine’, as used herein, includes reference to any member of the genus Ovis. Preferably, ovine as disclosed herein refers to the members of the genus Ovis that are used for animal, preferably human, consumption. A non-limiting example of such members includes domestic sheep (Ovis aries).
The term ‘porcine’, as used herein, includes reference to any member of the genus Sus. Preferably, porcine as disclosed herein refers to the members of the genus Sus that are used for animal, preferably human, consumption. Non-limiting examples of such members include domestic pig (Sus scrofa domesticus) and wild boar (several Sus scrofa subspecies).
The term “murine”, as used herein, includes reference to any member of the family Muridae. Preferably, murine as disclosed herein refers to the members of the family Muridae that are used for animal consumption. Non-limiting examples of such members include mice and rats.
The term ‘suspension culture’, as used herein, includes reference to different types of suspension culture such as microcarrier-based cell culture. Preferably, the expansion or proliferation of progenitor cells as described herein is performed using a microcarrier-based cell culture. A microcarrier-based cell culture preferably involves growing progenitor cells on the surface of microcarriers in suspension cultures. Alternatively, suspension culture refers to suspension cell culture, where cells are cultured in suspensions or aggregates and are not attached to a surface such as a microcarrier surface. In embodiments, expansion or proliferation of progenitor cells as described herein is performed by suspension cell culture.
The term ‘hydrogel’, as used herein, includes reference to crosslinked polymers with hydrophilic properties. Hydrogels may be formed by a variety of compounds, such as alginate, agarose, methylcellulose, hyaluronan, elastin-like polypeptides, collagen, chitosan, gelatin and starch. In embodiments, the hydrogel as disclosed herein comprises alginate.
The term ‘fat product’, as used herein, includes reference to cultured fat that is suitable for animal, preferably human, consumption. In embodiments, a cultured fat product can be distinguished from a natural animal-derived fat product, amongst others, by the absence of for instance immune cells such as antigen-presenting cells (APCs) such as monocytes or macrophages, T cells and B cells. Other distinguishing characteristics can be the absence of immune cell effector molecules such as antibodies, or red blood cells. Other distinguishing characteristics can be the absence of cartilage tissue, lower levels of fibrous tissue and/or absence of antibiotics and/or antibiotic residues.
The term ‘meat product’, as used herein, includes reference to cultured (artificial) meat that is suitable for animal, preferably human, consumption. A meat product generally comprises muscle tissue and preferably also fat tissue. In embodiments, a cultured meat product can, amongst others, be distinguished from a natural animal-derived meat product by the absence of for instance immune cells such as antigen-presenting cells (APCs) such as monocytes or macrophages, T cells and B cells. Other distinguishing characteristics can be the absence of immune cell effector molecules such as antibodies, or red blood cells. Other distinguishing characteristics can be the absence of cartilage tissue, lower levels of fibrous tissue and/or absence of antibiotics and/or antibiotic residues.
The phrase ‘incorporating into a food product’, as used herein, relates to the production of a food product using cultured fat cells optionally in combination with (cultured) muscle cells.
The term ‘myocyte’, as used herein, includes reference to a differentiated muscle cell, preferably a cultured muscle cell.
The term ‘myotube’, as used herein, includes reference to a multinuclear myocyte. Myotubes are generally formed by the fusion of myoblasts or myotube progenitor cells.
The terms ‘myofiber’ and ‘muscle fiber’, as used herein, include reference to a muscle cell in elongated or cylindrical form that comprises several myofibrils. A myofiber comprises one or more nuclei.
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 non-limiting group consisting of amino acids, lipids, sugars, carbohydrates, anions, cations, buffering agents, colorants, vitamins, antioxidants, hormones, enzymes, proteins and trace elements. Preferably, the basal medium as disclosed herein is a commercially available basal medium and may in itself already comprise a source of energy as disclosed herein.
The term ‘DMEM’, as used herein, includes reference to Dulbecco's Modified Eagle Medium, a basal medium. Without wishing to be exhaustive, DMEM may comprise amino acids, choline chloride, D-caldium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, i-inositol, calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate monobasic, glucose and phenol red. Preferably, DMEM as disclosed herein is provided in combination with Ham's F-12 medium in a 1:1 mixture (referred to as DMEM/F-12).
The term ‘Ham's F-12’, as used herein, refers to Ham's F-12 nutrient mixture, a basal medium. Without wishing to be exhaustive, Ham's F-12 may comprise amino acids, D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, d-pantothenic acid, pyrodoxin, riboflavin, thiamine, vitamin B12, glucose, hypoxanthine, linoleic acid, phenol red, putrescine, pyruvic acid, thioctic acid, thymidine, sodium bicarbonate, calcium chloride, cupric sulfate, ferrous sulfate, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic and zinc sulfate.
The terms ‘epidermal growth factor’ and ‘EGF’, as used herein, include reference to proteins that can stimulate cell growth and differentiation of animal cells, amongst other functions. The proteins belong to an epidermal growth factor family. EGFs bind to epidermal growth factor receptor (EGFR). EGFs are preferably recombinantly produced. Preferably, the EGF as disclosed herein is a recombinantly produced EGF, and can be a human EGF. It is to be understood that the terms ‘epidermal growth factor’ and ‘EGF’, as used herein, may also include reference to fragments of EGF that retain the biological function of EGF, such as EGF replacement peptides.
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, and can be a human 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.
The term ‘insulin’, as used herein, includes reference to peptide hormones that promote the uptake of glucose by cells, amongst other functions. Insulin is preferably recombinantly produced. Preferably, insulin as disclosed herein is a recombinant protein, and can be a human insulin.
The term ‘hydrocortisone’, as used herein, includes reference to a steroid hormone that is produced in animals. Hydrocortisone is involved in the fat metabolism of animals, amongst other functions. Hydrocortisone may otherwise be referred to as cortisol. Hydrocortisone may be obtained from animals, plants or microorganisms or may be chemically synthesized. In embodiments, hydrocortisone as disclosed herein is chemically synthesized or synthesized from plant sources. Preferably, hydrocortisone as disclosed herein is not derived from animals or animal material.
The term ‘progesterone’, as used herein, includes reference to a steroid hormone that is produced in animals. Progesterone may be obtained from animals, plants or microorganisms or may be chemically synthesized. In embodiments, progesterone as disclosed herein is chemically synthesized or synthesized from plant sources. Preferably, progesterone as disclosed herein is not derived from animals or animal material.
The term ‘putrescine’, as used herein, includes reference to a compound represented by the formula NH2(CH2)NH2. Putrescine is otherwise referred to as tetramethylenediamine, butane-1,4-diamine and 1,4-diaminobutane. Putrescine may be produced chemically or biochemically.
The term ‘ascorbic acid’, as used herein, includes reference to a compound represented by the formula CeH&Os. Ascorbic acid is otherwise referred to as vitamin C. Ascorbic acid may be produced chemically or biochemically. Also envisaged herein are derivates of ascorbic acid such as L-ascorbic acid 2-phosphate or salt forms thereof (e.g. L-Ascorbic acid 2-phosphate sesquimagnesium salt), which may be supplemented to a basal medium.
The terms ‘bone morphogenetic protein’ and ‘BMP’, as used herein, include reference to a group of growth factors that can be found in the animal body. This group comprises the BMP proteins BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11 and BMP15. Preferably, the BMP as disclosed herein is BMP4. Preferably, the BMP as disclosed herein is recombinantly produced, and can be a human BMP. It is to be understood that the terms ‘bone morphogenetic protein 4’ and ‘BMP4’, as used herein, may also include reference to fragments of BMP4 that retain the biological function of BMP4, such as BMP4 replacement peptides.
The term ‘lipid’, as used herein in relation to a source of lipids, includes reference to hydrophobic or partially hydrophobic hydrocarbon molecules such as fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, saccharolipids and polyketides. Preferably, a source of lipids is provided as a (chemically defined) lipid concentrate, which can be a mixture of lipids in emulsion. Without wishing to be exhaustive, a chemically defined lipid concentrate as disclosed herein may comprise one or more of arachidonic acid, cholesterol, dl-alpha-tocopherol acetate, ethyl alcohol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid and/or Tween 80. In embodiments, a chemically defined lipid concentrate as disclosed herein comprises a combination of some or all the above-listed compounds.
The term ‘thiazolidinedione’, as used herein, can be used interchangeably with ‘a compound of the thiazolidinedione family’ and includes reference to a class of compounds comprising pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, troglitazone and balaglitazone. Members of the thiazolidinedione family may also collectively be referred to as glitazones or TZDs. Preferably, compounds of the thiazolidinedione family are not comprised in a serum-free adipogenic differentiation medium as disclosed herein.
The term ‘HEPES’, as used herein, includes reference to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. HEPES is often used as a buffering agent. HEPES may be produced chemically or biochemically.
The term ‘
The term ‘chemically defined lipid concentrate’, as used herein, may include reference to a lipid emulsion. A chemically defined lipid concentrate as disclosed herein is an example of a lipid source as disclosed herein. Without wishing to be exhaustive, a chemically defined lipid concentrate as disclosed herein may comprise arachidonic acid, cholesterol, dl-alpha-tocopherol acetate, ethyl alcohol, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid and/or Tween 80. In embodiments, a chemically defined lipid concentrate as disclosed herein comprises a combination of some or all the above-listed compounds.
The expression ‘partially differentiated’, as used herein, includes reference to cells that are in the process of differentiation.
The expression ‘terminally differentiated’, as used herein, includes reference to specialized cells that are under normal conditions no longer able to further differentiate and/or proliferate.
The term ‘source of energy, as used herein, includes reference to a molecule that is involved as a substrate in a metabolism pathway of a mammalian cell that provides energy (such as energy in the form of ATP). The conversion of the energy source directly or indirectly leads to the generation of energy, for example in the form of ATP. In general, metabolites that are energy sources to a cell comprise carbon atoms. Examples of energy sources are sugars, proteins and fats.
The term ‘energy metabolism pathway’, as used herein, includes reference to any pathway of the mammalian cell that can be employed to generate energy from substrates involved in said pathway. Examples of energy metabolism pathways are glycolysis, glutaminolysis, mitochondrial respiration (also known as tricarboxylic acid cycle, citric acid cycle and Krebs cycle), pyruvate decarboxylation, ketosis and oxidative phosphorylation. An energy metabolism pathway leads, optionally in combination with other metabolism pathways, to the generation of ATP, which provides energy to multiple cellular processes.
The term ‘triglyceride’, as used herein, includes reference to a lipid that is derived from glycerol and three fatty acids. As a consequence, a triglyceride is an ester with three covalently linked fatty acids. These fatty acids may be saturated, characterized by one or more C═C double bond, or unsaturated, characterized by no C═C double bonds.
The term ‘unsaturated fatty acid’, as used herein, includes reference to fatty acids with at least one C=C double bond. Unsaturated fatty acids in animal fat and cultured fat may for example comprise 16, 18, 20 or 22 carbon atoms, and may for example comprise 1, 2, 3, 4, 5, or 6 C═C double bonds. Saturated fatty acids are fatty acids with no C═C double bond. Unsaturated and/or saturated fatty acids may be constituents of lipids, such as triglycerides. In general, unsaturated fatty acids are more beneficial to animal health than saturated fatty acids.
The term ‘inflammatory cells’, as used herein, includes reference to cells that are part of the immune system of animals. Examples include macrophages, neutrophils, dendritic cells, innate lymphoid cells, mast cells, eosinophils, basophils, natural killer cells, B cells, T cells and granulocytes.
The term ‘antibiotics’, as used herein, includes reference to antimicrobial substances that are active against bacteria, for example by killing bacteria or by inhibiting growth of bacteria. Antibiotics may be used in livestock, such as cattle. Antibiotics, or residues thereof, may be passed on to meat products after slaughtering of the animal. Examples of antibiotics used in cattle include bacitracin, bambermycin, laidlomycin, lasalocid, monensin, neomycin, and virginiamycin.
The term ‘blood residues’, as used herein, includes reference to components typically found in blood, such as serum, serum proteins, erythrocytes, leukocytes and thrombocytes.
The term ‘microbial contamination’, as used herein, includes reference to undesired presence of one or more microbe, such as bacteria, viruses, fungi and archaea. It also includes reference to pathogenic contamination of food products in general.
The term ‘cartilage’, as used herein, includes reference to elastic animal tissue that is for example found at the end of bones at joints, in the rib cage, in the ear and in the nose. The term ‘cartilage’ includes reference to the three types of cartilage, i.e. elastic cartilage, hyaline cartilage and fibrocartilage. Cartilage in meat products is not desirable for consumption.
The term ‘fibrous tissue’, as used herein, also referred to as fibrous connective tissue, includes reference to a tissue type with a high amount of fibers, such as elastic and collagenous fibers. In meat products, fibrous tissue may render the meat product tough and therefore less desirable for consumption.
The invention provides a method for differentiating an adipogenic progenitor cell, comprising the step of:-culturing an adipogenic progenitor cell in a serum-free medium for differentiating an adipogenic progenitor cell as disclosed herein.
An adipogenic progenitor cell as disclosed herein is generally provided prior to differentiation. Providing an adipogenic progenitor cell as disclosed herein may be done in any form. The adipogenic progenitor cell as disclosed herein is in embodiments provided in the form of a tissue sample comprising said adipogenic progenitor cell. Said tissue sample may be taken from anywhere in the non-human animal body. In embodiments, said tissue sample is a muscle tissue or fat tissue sample, preferably a bovine muscle tissue sample.
In embodiments, the tissue sample comprising adipogenic progenitor cells herein is obtained via biopsy on an animal.
The obtaining of the tissue sample, for example via biopsy, may be done using local anesthetic, for example at the biopsy site. Local anesthetic may for example be applied via subcutaneous injection, and may be done using procamidor.
In embodiments, the biopsy as disclosed herein is taken via skin incision in order to expose muscle. From said muscle, a bovine muscle sample as disclosed herein can be taken of e.g. about one gram. The creation of the incision as disclosed herein and/or the obtaining of the bovine muscle sample as disclosed herein may for example be routinely made using a scalpel. Said bovine muscle sample can subsequently be collected on ice.
In embodiments, the adipogenic progenitor cell as disclosed herein is obtained from a sample of semitendinosus muscle. In embodiments, the adipogenic progenitor cell is obtained from a (skeletal) muscle sample that is derived from a cadaver, or from a living animal e.g. by taking a biopsy.
In embodiments, the tissue sample as disclosed herein is subjected to enzymatic digestion such as with a collagenase to achieve dissociation of muscle fibers. For example, AFC A (Worthington, CLS-1, 2000 U/ml) may be used. Exemplary conditions for dissociation using collagenase are an incubation time of 45 minutes and an incubation temperature of 37° C.
Alternatively, or in combination with said step of enzymatic digestion, the tissue sample as disclosed herein may be incubated with erythrocyte lysis buffer. For example, 1× ACK erythrocyte lysis buffer may be used. Exemplary incubation conditions for erythrocyte lysis buffer are an incubation time of 1 minute and an incubation temperature of 37° C.
In embodiments, after said step of isolating adipogenic progenitor cells from the tissue sample as disclosed herein, cells obtained from said tissue sample are resuspended and incubated in a serum-free proliferation medium such as the proliferation media described herein (e.g. medium SF-PM, see Example 2).
In embodiments, cells obtained from a muscle tissue or fat tissue sample as disclosed herein after said enzymatic digestion treatment (e.g. with a collagenase) and/or said treatment that allows for erythrocyte lysis, can be precultured in a serum-free proliferation medium as disclosed herein and subsequently subjected to antigen-based cell sorting such as FACS in order to purify adipogenic progenitor cells.
In embodiments, adipogenic progenitor cells that are MSCs can be provided in accordance with Example 1.
In embodiments, adipogenic progenitor cells that are FAPs can be provided in accordance with Example 3.
An adipogenic progenitor cell, especially a FAP cell, as disclosed herein may be characterized and purified using antigen-based cell sorting, such as FACS, after isolation and optionally preculture. FAPs can be purified by antigen-based cell sorting, such as FACS, on the basis of (i) the presence of cell surface markers CD9, CD14, CD49e, CD61, CD140a and/or ITGA9, and/or (ii) the absence of cell surface markers CD45, CD321, CD56 and/or ITGA7. In embodiments, at least 1, at least 2, at least 3, or at least 4 of said cell surface markers are selected from the lists of both (i) and (ii). In embodiments, FAPs are purified by FACS on the basis of the presence of cell surface markers CD14, CD49e, CD61, CD140a and/or ITGA9, and/or (ii) the absence of cell surface markers CD56 and/or ITGA7. In embodiments, FAPs are characterized by the presence of cell surface marker CD140a.
The term ‘antigen-based cell sorting’, as used herein, includes reference to any protocol that allows for sorting of cell types on the basis of the presence or absence of cell surface markers. Non-limiting examples of antigen-based cell sorting protocols are protocols that employ fluorescently labelled antibodies such as FACS, or protocols that are based on magnetic labelling or isotope labelling of the cell surface marker-binding antibodies. Preferably, the antigen-based cell sorting is fluorescence-activated cell sorting.
The term ‘presence’ or ‘absence’, as used herein in relation to antigen-based cell sorting such as FACS, includes reference to the discrimination between cell types on the basis of differential expression of cell surface marker proteins (antigens). The skilled person directly understands that the term ‘absence’ does not mean that protein expression of a cell surface marker protein is necessarily fully absent (i.e. zero), but that its expression is lower as compared to another cell type and is lower to such an extent that it allows for a negative selection criterion in cell sorting. Thus, antigen-based cell sorting such as FACS allows for discrimination between cell types on the basis of (i) cell surface markers that are expressed on a cell to such an extent that they can be used as a positive selection criterion (i.e. presence; “+”) or (ii) cell surface markers that have a lower or reduced expression on a cell to such an extent that they can be used as a negative selection criterion (i.e. absence, “−”).
A method of the invention may further comprise the step of:-culturing an adipogenic progenitor cell in a serum-free medium for proliferating adipogenic progenitor cells, to thereby provide proliferated adipogenic progenitor cells. In embodiments, proliferation takes place after isolation and purification of an adipogenic progenitor cell and prior to differentiation of an adipogenic progenitor cell.
Culturing resulting in the expansion of adipogenic progenitor cells may be performed two-dimensionally or three-dimensionally, i.e. as a two-dimensional or three-dimensional cell culture. For two-dimensional culturing, adipogenic progenitor cells as disclosed herein may be propagated in tissue culture flasks in a serum-free medium for proliferating adipogenic progenitor cells as disclosed herein. For three-dimensional culturing, adipogenic progenitor cells as disclosed herein may be propagated in any suitable cell culture vessel such as spinner flasks or bioreactors. Preferably, in a method of the invention, culturing of adipogenic progenitor cells is performed in a way that leads to three-dimensional expansion. Preferably, microcarrier-based cell culturing is performed in order to expand progenitor cells.
An exemplary, but non-limiting, serum-free medium for proliferating adipogenic progenitor cells is described in Example 2 (medium “SF-PM”). In embodiments, a serum-free medium for proliferating an adipogenic progenitor cell as disclosed herein may comprise an albumin and a fibroblast growth factor (FGF, such as FGF2). In embodiments, said serum-free medium for proliferating an adipocyte progenitor cell as disclosed herein may further comprises one or more vitamins and/or hormones selected from the group consisting of ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone, and one or more cytokines and/or growth factors selected from the group consisting of a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin 6 (IL-6). In embodiments, said one or more cytokines and/or growth factors is selected from the group of combinations comprising i) an IL-6; ii) an IL-6 and an IGF; iii) an IL-6, an IGF and an HGF; iv) an IL-6, an IGF, an HGF and a PDGF; v) an IL-6, an IGF, and a VEGF; vi) an IL-6, an IGF and a PDGF; vii) an IL-6, a PDGF and a VEGF; viii) an IL-6, an IGF, a PDGF and a VEGF; and ix) an IL-6, an IGF, an HGF, a PDGF and a VEGF. In embodiments, said serum-free medium for proliferating an adipogenic progenitor cells comprises a PDGF, a VEGF, an HGF, an IGF and an IL-6. Exemplary, but non-limiting, serum-free proliferation media, and expansion culture conditions, are for instance disclosed in PCT/NL2021/050066, the contents of which are incorporated herein by reference.
Adipogenic differentiation of an adipogenic progenitor cells as disclosed herein can occur two-dimensionally or three-dimensionally, i.e. as a two-dimensional or three-dimensional cell culture. Three-dimensional differentiation of adipogenic progenitor cells may result in a structure comprising adipocytes that from a macroscopic perspective mimics subcutaneous fat in texture and appearance. This is a desirable effect, as it may be used to create meat products that mimic non-cultured meat products in terms of texture and appearance.
In a method of the invention, a cell culture vessel that allows for three-dimensional expansion is typically used. An example of a suitable culture vessel for three-dimensional expansion is a spinning flask (Corning), which may be coated, for example by siliconization. Siliconization may for example be done using Sigmacote (Sigma Aldrich), for example at a concentration of 10 cm2/mL.
Preferably, in order to achieve three-dimensional differentiation of adipogenic progenitor cells as disclosed herein, a three-dimensional system is be provided that is preferably edible and scalable.
Preferably, adipogenic differentiation is performed as a three-dimensional cell culture, for instance in a protein matrix, scaffold or cell aggregate. In embodiments, a hydrogel is used for three-dimensional cell culturing. In embodiments, said hydrogel is a hydrogel comprising alginate.
In embodiments, the step of culturing an adipogenic progenitor cell in a serum-free medium for differentiating an adipogenic progenitor cell is carried out in and/or on microfibres. These microfibres may for example be made using alginate (i.e. alginate-based microfibers). Thus, in embodiments, the adipogenic progenitor cell is provided in the form of a three-dimensional cell culture (e.g. a microfiber, such as an alginate-based microfiber).
Exemplary conditions for three-dimensional differentiation of adipogenic progenitor cells as disclosed herein are as follows. Adipogenic progenitor cells are resuspended in 0.5% high viscosity alginate solution (Sigma, W201502) at a concentration of 3×107 cells/mL. Cell-alginate suspension is injected into 66 mM CaCl2, 10 mM HEPES. Resultant microfibres are subsequently washed and transferred to a 12-well tissue culture plate containing a serum-free medium for differentiating an adipogenic progenitor cell as disclosed herein.
Incorporating Cultured Adipocytes into a Food Product
A method of the invention may further comprise the step of:-incorporating cultured adipocytes into a food product for animal, preferably human, consumption.
Preferably, said food product is i) a cell-culture based fat product or ii) a cell-culture based meat product that comprises myocytes, myotubes and/or myofibers.
Said myocytes, myotubes and/or myofibers may be obtained by differentiating muscle progenitor cells. Preferably, said method for differentiating muscle progenitor cells into myocytes, myotubes and/or myofibers is entirely serum-free, more preferably entirely animal-free. The process of differentiating muscle progenitor cells into myocytes, myotubes and/or myofibers may be preceded by one or more of the steps of:—providing a muscle progenitor cell;—isolating a muscle progenitor cell; and—culturing/proliferating a muscle progenitor cell. The process of differentiating muscle progenitor cells into myocytes, myotubes and/or myofibers may be succeeded by the step of:—incorporating a differentiated muscle progenitor cell into a food product, preferably a food product that also comprises differentiated adipogenic progenitor cells.
The invention also provides a serum-free medium for differentiating an adipogenic progenitor cell into an adipocyte for animal, preferably human, consumption. Preferably, the serum-free medium is used for differentiating a mammalian adipogenic progenitor cell, such as a bovine, ovine, porcine or murine adipogenic progenitor cell.
Preferably, a serum-free medium of the invention comprises at least one PPARΥ agonist selected from the group formed by indomethacin, magnolol, amorfrutins (comprising, for example, amorfrutin 1, amorfrutin 2, amorfrutin A, amorfrutin B, amorfrutin C and amorfrutin D), honokiol, lecithine (such as L-α-lecithine from soy beans), formononetin, bixin, norbixin, catechin, Δ9-tetrahydrocannabinol, (9S, 13R)-12-oxo-phytodienoic acid, odoratin, hydroxy unsaturated fatty acids from Coix lacrymajobi, commipheric acid, kaempferol-3-O-β-glucopyranoside, citral, alkamides from Echinacae purpera, tocotrienols, deoxyelephantopin, acetylated flavonol glycosides, kampferol, quercetin, genistein, 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 Melampyrum pratense, cucurbitane-type triterpene glycosides, polyacetylenes from Notopterygium incisum, biochanin A, ginsenoside 20(S)-protopanaxatriol, ginsenoside Rb1, fatty acids from Pinellia ternata, oleaninic acid, pseudolaric acid B, daidzein, amorphastilbol, carnosic acid, carnosol, 12-O-methul carnosic acid, α-linolenic acid, linoleic 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. More preferably, the at least one PPARΥ agonist is selected from the group consisting of indomethacin, magnolol, an amorfrutin (preferably amorfrutin B), honokiol and L-α-lecithine. In embodiments, the at least one PPARΥ agonist is indomethacin or magnolol. In embodiments, the at least one PPARΥ agonist is indomethacin. Indomethacin, magnolol, amorfrutin B, and honokiol perform very beneficially as PPARΥ agonists in a medium for differentiating an adipogenic progenitor cell as disclosed herein.
The at least one PPARΥ agonist can be present in the medium in a concentration range of 0.05-5000 μM, more preferably 0.05-500 μM, most preferably 5-100 μM such as about 50 μM.
Preferably, a serum-free medium of the invention further comprises at least one hormone such as hydrocortisone or insulin. Preferably, the hydrocortisone is an animal hydrocortisone, more preferably a mammalian hydrocortisone, such as human hydrocortisone (e.g. H0135 from Sigma Aldrich). The at least one hormone that is a hydrocortisone can be present in the medium in a concentration of 0.01-1000 nM, preferably 1-500 nM, more preferably about 100 nM. The hydrocortisone as disclosed herein is preferably present in a serum-free medium as disclosed herein (or “of the invention”, which terms can be used interchangeably herein) in combination with said at least one PPARΥ agonist as disclosed herein.
Preferably, a serum-free medium of the invention further comprises insulin as the at least one hormone. Preferably, the insulin as disclosed herein is animal insulin, more preferably mammalian insulin, such as recombinant human insulin (e.g. 10-365 from Peprotech). Insulin can be present in the medium in a concentration of 0.01-200 μM, preferably 0.1-20 μM, more preferably 1-2 μM. The insulin as disclosed herein is preferably present in a serum-free medium of the invention in combination with said at least one PPARΥ agonist and said at least one hydrocortisone as disclosed herein.
Preferably, a serum-free medium of the invention further comprises at least one cytokine and/or growth factor selected from the group consisting of bone morphogenetic protein 4 (BMP4) and epidermal growth factor (EGF). Preferably, the BMP4 as disclosed herein is animal BMP4, more preferably mammalian BMP4, such as E. coli derived recombinant human BMP4 (e.g. 120-05ET from Peprotech). The BMP4 can be present in the medium in a concentration of 0.03-60000 nM, preferably 0.3-6000 nM, more preferably 3-600 nM or about 300 nM. The BMP4 as disclosed herein is preferably present in a serum-free medium of the invention in combination with said at least one PPARΥ agonist and said at least one hormone as disclosed herein. Preferably, the EGF as disclosed herein is animal EGF, more preferably mammalian EGF, such as recombinant human EGF (e.g. AF-100-15 from Peprotech). The EGF can be present in the medium in a concentration of 3-30000 pM, preferably 30-3000 pM, more preferably about 322 pM. In embodiments the EGF can be present in the medium in a concentration of at least 161 pM. The EGF as disclosed herein is preferably present in a serum-free medium of the invention in combination with said at least one PPARΥ agonist and said at least one hormone as disclosed herein.
In a preferred embodiment, a serum-free medium of the invention comprises BMP4 and EGF as disclosed herein.
In embodiments, a serum-free medium of the invention comprises BMP4, EGF and fibroblast growth factor (FGF). The FGF as disclosed herein is preferably an animal FGF, more preferably mammalian FGF, such as recombinant human FGF2 (e.g. 100-18B from Peprotech). The FGF as disclosed herein can be present in the medium in a concentration of 1-10000 pM, preferably 10-1000 pM, more preferably 20-800 pM, more preferably 50-500 pM, more preferably 75-250 pM, more preferably 100-150 pM, more preferably about 115 pM. The BMP4, EGF and fibroblast growth factor (FGF) as disclosed herein are preferably present in a serum-free medium of the invention in combination with said at least one PPARΥ agonist and said at least one hormone as disclosed herein.
Preferably, a serum-free medium of the invention further comprises an ascorbic acid or a derivative thereof such as L-ascorbic acid 2-phosphate. Ascorbic acid or a derivative thereof may be provided as a salt, such as L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (e.g. A8960-5G from Sigma Aldrich). Ascorbic acid or a derivative thereof such as L-ascorbic acid 2-phosphate can be present in the medium in a concentration of 0.01-10000 μM, preferably 1-500 μM, more preferably about 227 μM. The ascorbic acid or a derivative thereof such as L-ascorbic acid 2-phosphate as disclosed herein is preferably present in a medium of the invention in combination with said at least one PPARΥ agonist, said at least one hormone, said FGF, said EGF and said BMP4 as disclosed herein.
In embodiments, a serum-free medium of the invention further comprises at least one biogenic amine such as a monoamine or a polyamine, e.g. a putrescine, ethanolamine, spermidine and/or spermine. Preferably, the biogenic amine is putrescine (e.g. 51799 from Sigma Aldrich). The biogenic amine can be present in the medium in a concentration of 0.01-1000 μM, more preferably 0.1 to 500 μM or 1-100 μM or 20-80 pM or 50-60 μM, most preferably about 56 μM. The biogenic amine as disclosed herein is preferably present in a serum-free medium of the invention in combination with said at least one PPARΥ agonist, said at least one hormone, said FGF, said EGF, said BMP4 and said ascorbic acid or a derivative thereof as disclosed herein.
In embodiments, a serum-free medium of the invention further comprises a source of lipids, for example a source of lipids comprising one or more of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, saccharolipids and polyketides. Said source of lipids is preferably provided by a (chemically defined) lipid mixture such as a lipid concentrate or lipid concentrate CD (e.g. 11548846 from ThermoFisher Scientific). Preferably, the source of lipids, or chemically defined lipid concentrate, as disclosed herein comprises at least one selected from the group of linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid, or, in embodiments, a combination of linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid. In embodiments, these lipids are present in a final concentration of approximately 10 μg/L each. In embodiments, the source of lipids, e.g. chemically defined lipid concentrate, as used herein comprises arachidonic acid. In embodiments, arachidonic acid is present in a final concentration of approximately 2 μg/L. In embodiments, the chemically defined lipid concentrate as used herein comprises cholesterol. In embodiments, cholesterol is present in a final concentration of approximately 220 μg/L. In embodiments, the source of lipids, e.g. chemically defined lipid concentrate, as disclosed herein comprises DL-alpha-tocopherol acetate. in embodiments, DL-alpha-tocopherol acetate is present in a final concentration of approximately 70 μg/L. In embodiments, the source of lipids, e.g. chemically defined lipid concentrate, as disclosed herein comprises tween 80® (also referred to as polysorbate 80). In embodiments, tween 80® is present in a final concentration of approximately 2.2 μg/mL. The lipid source, e.g. lipid concentrate as disclosed herein is preferably present in a serum-free medium of the invention in combination with said at least one PPARΥ agonist, said at least hormone, said at least one growth factor and/or cytokine and said ascorbic acid or a derivative thereof as disclosed herein, and optionally said biogenic amine as disclosed herein.
In embodiments, a serum-free medium of the invention further comprises one or more basal media. Preferably, a serum-free medium of the invention further comprises one or more basal media selected from the group comprising DMEM and Ham's F-12; more preferably a combination of DMEM and Ham's F-12. In embodiments, the combination of DMEM and Ham's F-12 is in a ratio of 1:10 to 10:1 v/v, more preferably in a ratio of about 1:1 v/v. The at least one basal medium as disclosed herein is preferably present in a medium of the invention in combination with said at least one PPARΥ agonist, said at least one hydrocortisone, said at least hormone, said at least one growth factor and/or cytokine and said ascorbic acid or a derivative thereof such as L-ascorbic acid 2-phosphate as disclosed herein, and optionally said biogenic amine and/or said lipid source as disclosed herein.
In embodiments, the serum-free medium of the invention may further comprise one or more additional vitamins and/or hormones, such as progesterone. Progesterone can be present in the medium in a concentration of 0.01-400 nM, preferably 0.1-40 nM, more preferably about 18 nM.
In embodiments, a serum-free medium of the invention may further comprise one or more buffering agent, such as one or more of sodium bicarbonate (e.g. S5761 from Sigma Aldrich) and/or HEPES (e.g. H3375 from Sigma Aldrich). The sodium bicarbonate can be present in the medium in a concentration of 0.2-2000 mM, preferably 2-200 mM, more preferably about 20 mM. The HEPES can be present in the medium in a concentration of 0.05-500 mM, preferably 0.5-50 mM, more preferably about 5 mM. The one or more buffering agent as disclosed herein is preferably present in a medium of the invention in combination with said at least one PPARΥ agonist, said at least one hormone, said at least one growth factor and/or cytokine and said ascorbic acid or a derivative thereof such as L-ascorbic acid 2-phosphate as disclosed herein, and optionally said biogenic amine, said lipid source and/or said basal medium as disclosed herein.
A serum-free medium of the invention may further comprises one or more source of glutamine, preferably one or more of L-glutamine (e.g. G8540 from Sigma Aldrich) and
A serum-free medium of the invention may further comprise one or more source of energy or carbohydrates such as glucose. The one or more source of carbohydrates can be present in the medium in a concentration of 0.02-2000 mM, such as 0.2-200 mM or 2-20 mM.
The skilled person can routinely calculate and adjust the osmolality of a solution in general and of a medium in particular if needed. Osmolality is typically expressed in milliosmoles per kilogram of water (mOsm/kg). Osmolality may be measured using an osmometer. In embodiments, the osmolality of a medium of the invention is within the range of 180-380 mOsm/kg, preferably within the range of 275-299 mOsm/kg.
In an embodiment, a serum-free medium of the invention comprises at least one source of energy that is selected from substrates involved in at least one energy metabolism pathway. Examples of energy metabolism pathways are glycolysis, glutaminolysis, mitochondrial respiration (also known as tricarboxylic acid cycle, citric acid cycle and Krebs cycle), pyruvate decarboxylation, ketosis, oxidative phosphorylation and pentose phosphate pathway. Examples of metabolites involved in energy metabolism pathways, which can function as energy sources, are monosaccharides, glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, glutamine, glutamate, aspartate, lactate, alanine, citrate, α-ketoglutarate, oxaloacetate, acetyl-CoA, citrate, cis-aconitate, isocitrate, oxalosuccinate, succinyl-CoA, succinate, fumarate, L-malate, galactose, fructose, xylose, sucrose, lactose, maltose, isomaltulose, trehalose and acetate.
In embodiments, said at least one energy source is an energy source selected from the group consisting of glucose, glutamine, galactose, pyruvate, acetate and alpha-ketoglutarate, or any combination thereof. In embodiments, said at least one energy source is a combination selected from the group of glucose and glutamine; glucose and galactose; glucose and pyruvate; glucose and acetate; glutamine and galactose; glutamine and pyruvate; glutamine and acetate; galactose and pyruvate; galactose and acetate; pyruvate and acetate; glucose, glutamine and galactose; glucose, glutamine and pyruvate; glucose, glutamine and acetate; glucose, galactose and pyruvate; glucose, galactose and acetate; glucose, pyruvate and acetate; glutamine, galactose and pyruvate; glutamine, galactose and acetate;
glutamine, pyruvate and acetate; galactose, pyruvate and acetate; glucose, glutamine, galactose and pyruvate; glucose, glutamine, galactose and acetate; glucose, glutamine, pyruvate and acetate; glucose, galactose, pyruvate and acetate; glutamine, galactose, pyruvate and acetate; and glucose, glutamine, galactose, pyruvate and acetate. In embodiments, said at least one energy source is a combination of glucose and glutamine; a combination of galactose and pyruvate; a combination of glucose and pyruvate; a combination of acetate, pyruvate and galactose; a combination of glucose, galactose and pyruvate; a combination of glucose and alpha-ketoglutarate (aKG); and/or a combination of glucose, alpha-ketoglutarate (aKG) and pyruvate. In embodiments, said at least one energy source is a combination of galactose and pyruvate.
Said at least one energy source may substitute the energy source or energy sources that are present in a basal medium as disclosed herein.
In embodiments, said at least one energy source is present in a concentration of 10 μM to 10 M, more preferably between 100 μM to 1 M, such as between 1 mM and 100 mM or between 2 mM and 21 mM. In embodiments, the total concentration of all energy sources selected from glucose, glutamine, galactose, pyruvate and acetate combined is between 2 and 200 mM, more preferably around 20 mM.
The invention also provides a composition comprising a serum-free medium for differentiating as disclosed herein and an adipogenic progenitor cell as disclosed herein and/or a partially or terminally differentiated cell obtained therefrom. Preferably, said adipogenic progenitor cell is a mammalian adipogenic progenitor cell, preferably a bovine, ovine, porcine or murine adipogenic progenitor cell.
The invention also provides a cultured fat product for animal, preferably human, consumption, comprising adipocytes obtainable by a method for differentiating an adipogenic progenitor cell of the invention.
The invention also provides a cultured meat product for animal, preferably human, consumption, comprising adipocytes obtainable by a method for differentiating an adipogenic progenitor cell of the invention and (cultured) mammalian myocytes, myotubes and/or myofibers, preferably bovine, ovine or porcine myocytes, myotubes and myofibers.
The cultured fat product for animal, preferably human, consumption as disclosed herein and/or the meat product for animal, preferably human, consumption as disclosed herein preferably corresponds to non-cultured fat products and/or meat products. However, the cultured fat and/or meat product for animal, preferably human, consumption, comprising adipocytes obtainable by a method for differentiating an adipogenic progenitor cell of the invention may be different from respective non-cultured fat and non-cultured meat products. Preferably, these differences have a beneficial effect on the fat or meat product, and/or on animal, preferably human, health.
Preferably, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention comprises no immune cells such as inflammatory cells. Existing traditional meat products are made from fat tissue and muscle tissue from the slaughtered animal body, and therefore comprise immune cells such as inflammatory cells.
Preferably, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention comprises fewer, preferably no, antibiotics and/or antibiotics residues compared to non-cultured fat and/or meat products. Antibiotics in fat and/or meat products are a burden to animal health when consumed, as they may kill part of the consumers gut microbiome. Also, the presence of antibiotics in food may allow for the promotion of antibiotic resistance. Furthermore, antibiotics may lead to tissue damage, for example in the animal gut.
Preferably, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention comprises no blood residues such as red blood cells. Blood components may lead to lipid oxidation and may decrease the shelf life of the food products. Non-cultured fat and/or non-cultured meat products may contain blood components.
Preferably, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention comprises lower levels of microbial contamination compared to non-cultured fat and/or non-cultured meat products. A cultured fat product for animal, preferably human, consumption of the invention and/or a cultured meat product for animal, preferably human, consumption of the invention is produced in controlled environments that aim to prevent contamination with microbials. Also, since significantly less animal tissue is used in the production of the fat product and/or the meat product as disclosed herein compared to non-cultured fat and/or meat products. Potential microbials present in or on animal tissue are incorporated in a cultured fat and/or meat product as disclosed herein in much lower levels compared to non-cultured fat and/or meat products.
Preferably, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention comprises no cartilage. Cartilage may have a negative effect on the consumption experience of the consumer, as it is much tougher than (artificial) muscle tissue or (artificial) fat tissue. Absence of cartilage in a food product is generally associated with a higher quality.
Preferably, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention comprises lower levels of fibrous tissue compared to non-cultured fat and/or meat products. Fibrous tissue, otherwise referred to as connective tissue, comprises proteins such as collagen and elastin that render meat tough and therefore less beneficial for consumption. Fibrous tissue is associated with lower quality food products. Lower levels of fibrous tissue are also beneficial in the preparation of food products, as it generally takes less time to cook food products with low levels if fibrous tissue.
A cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention may be produced or processed to resemble known food products. Non-limiting examples of food products as disclosed herein are a hamburger, a sausage, a steak, minced meat, a meatball, corned beef, a charcuterie product, jerky or stewed meat. Food products also covers the combination of several types of food products. The adipocytes obtainable by a method of the invention as disclosed herein and optionally the (cultured) mammalian myocytes, myotubes and/or myofibers as disclosed herein may be processed prior to or following incorporation into a food product. Non-limiting examples of processing as disclosed herein are boiling, grilling, freezing, pressing, salting, curing, fermenting, smoking, drying, canning, cutting, grinding, mixing, seasoning, tubing in casing and marinating. The cultured adipocytes as disclosed herein and the optional (cultured) mammalian myocytes, myotubes and/or myofibers as disclosed herein may be arranged in a specific manner in the food product, for example in order to create optical similarity with non-cultured food products and/or to improve texture.
In embodiments, a cultured fat product for animal, preferably human, consumption of the invention and/or a meat product for animal, preferably human, consumption of the invention contains between 0.01% and 70% cultured adipocytes as disclosed herein, more preferably between 1% and 30% cultured adipocytes as disclosed herein, more preferably between 5% and 20% cultured adipocytes as disclosed herein. Additionally to cultured adipocytes as disclosed herein and optionally (cultured) mammalian myocytes, myotubes and/or myofibers as disclosed herein, a cultured fat product as disclosed herein and/or a cultured meat product as disclosed herein may comprise water, one or more salt, one or more fiber, one or more carbohydrate, one or more protein, one or more starch, one or more spice, one or more herb, one or more yeast extract, one or more casing ingredient, one or more vitamin, one or more oil, one or more hydrocolloid, one or more thickening agent, one or more preservative, one or more colorant, one or more antioxidant, one or more acidity regulator, one or more stabilizer, one or more emulsifier, one or more flavor enhancer and/or one or more sweetener. Preferably, all constituents of the fat and/or meat product are animal-free.
For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the disclosure includes embodiments having combinations of all or some of the features described.
The content of the documents referred to herein is incorporated by reference.
An exemplary chemically defined, serum-free medium for adipogenic differentiation (also referred to as “DMAD”) of the invention was prepared as follows:
DMEM/F12 medium was supplemented with PSA (17-745E from Lonza) at 1%, HEPES (H3375 from Sigma Aldrich) at 4.9 mM, hydrocortisone (H0135 from Sigma Aldrich) at 0.1 μM, insulin (recombinant human insulin, 10-365 from Peprotech) at 1-2 μM, lipid concentrate (chemically defined lipid concentrate, 11548846 from Thermo Fisher) at 0.001% (v/v), putrescine (51799 from Sigma Aldrich) at 56 μM, EGF (recombinant human EGF, AF-100-15 from Peprotech) at 322 pM, FGF2 (recombinant human FGF2, 100-18B from Peprotech) at 115 pM, progesterone (P8783 from Sigma Aldrich) at 17.8 nM, ascorbic acid (L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate, A8960-5G from Sigma Aldrich) at 227 μM, indomethacin (17378 from Sigma Aldrich) at 50 μM and BMP4 (Recombinant Human BMP-4 (E. coli derived), 120-05ET from Peprotech) at 300 nM.
Further, concentration ranges around the concentration values of the components mentioned above still providing for effective adipogenic differentiation were routinely established.
Bovine mesenchymal stem cells (MSC) were isolated from bovine adipose tissue (Bos taurus) using a standard enzymatic method (Mehta et al., in Myogenesis (ed. Rønning, S. B.) vol. 1889 111-125 (Springer New York, 2019), and plated onto plastic tissue culture flasks to allow adherent cells to attach. MSCs were propagated in a serum-free proliferation medium (“SF-PM”) for at least 10 population doublings prior to differentiation. Briefly, the cells were seeded at a density of 3000 cells/cm2 in SF-PM in a collagen-coated cell culture flask and passaged every time they reach 80-90% confluence. The serum-free proliferation medium referred to as “SF-PM” contains: albumin (5 mg/ml), somatotropin (2 ng/ml), L-Ascorbic acid 2-phosphate (50 μg/ml), hydrocortisone (36 ng/ml), α-linolenic acid (1 μg/ml), insulin (10 μg/ml), transferrin (5.5 μg/ml), sodium selenite (0.0067 μg/ml), ethanolamine (2 μg/ml), L-alanyl-L-glutamine or glutamine (2 mM), IL-6 (5 ng/ml), FGF2 also referred to as bFGF (10 ng/ml), IGF1 (100 ng/ml), VEGF (10 ng/ml), HGF (5 ng/ml), PDGF-BB (10 ng/ml) and DMEM/F12 basal medium.
At 90% confluence, proliferated MSCs were trypsinized and seeded in a 96-well collagen coated cell culture plate at 30,000 cells/cm2 in SF-PM. After 24 hours, adipogenesis was induced by changing SF-PM to DMAD (the serum-free differentiation medium as depicted in Example 1), which was maintained on the cells for a period of 12 days with media changes every 3-4 days. As a control differentiation medium (ctrl), DMEM/F12 with 3% FBS was supplemented with 4 conventional adipogenic inducers (IBMX (Sigma Aldrich-15879; 0.5 mM), dexamethasone (Sigma Aldrich-D4902; 10 μM), rosiglitazone (Sigma Aldrich-R2408; 5 μM) and insulin (Peprotech-10-365; 10 μM)). All 4 inducers were used for the first 3 days of culture and only insulin and rosiglitazone were used for the remaining 9 days.
Cells were fixed on days 6 and 12 with 4% PFA for 20 minutes at room temperature and washed twice with PBS. Cells were then stained with Hoechst (ThermoFisher 493/503, 1:2000) and BODIPY (Sigma Aldrich 345880, 1:1000) in PBS for 30 minutes. Adipogenic differentiation was quantified using the ImageXPress Pico High Content Analyser (HCA; Molecular devices). The parameters measured were total area of lipid, lipid droplet average area and percentage of positive cells. For the percentage of positive cells, the HCA software assigned BODIPY immunoreactivity to the nearest nuclei, (i.e. a positive cell) and the number of BODIPY positive nuclei was then divided by the total number of nuclei.
To assess if DMAD is able to induce adipogenic differentiation to a similar level as said conventional control differentiation medium (ctrl), MSCs were cultured in 2D for 12 days in DMAD and said 3% FBS control medium. Culturing cells in DMAD and said conventional control medium demonstrated that adipogenic differentiation could be achieved to a greater extent with DMAD than the control (
Fresh bovine skeletal muscle samples were obtained from a registered abattoir according to national guidelines on animal tissue handling. Muscle-derived cells were isolated as previously described (Ding et al., Sci Rep. 8, 10808 (2018)). Briefly, bovine semitendinosus muscle was minced and dissociated with collagenase (CLSAFA, Worthington; 1 h, 37° C.). Cell slurries were filtered through a 100 μm cell strainer and incubated in ammonium-chloride-potassium (ACK) erythrocyte lysis buffer (1 min, RT). Cells were resuspended in serum-free proliferation medium “SF-PM” as defined in Example 2, and filtered through a 40 μm strainer prior to culture.
A similar procedure was applied for porcine and ovine adipogenic progenitor cells.
Prior to FACS, FAP cells were cultured for 72 h on bovine collagen type I (2.5 μg/cm2; Sigma-Aldrich, C2124) coated flasks and sorted using antigen-based sorting on the basis of the absence of expression of JAM1, CD45 and integrin alpha 7 (ITGA7), and the positive expression of integrin alpha 5 (ITGA5) or platelet derived growth factor alpha (PDGFRα; also known as CD140a). Unstained cells were used to define gating parameters, and sorting purities routinely checked by reanalysing the sorted fractions.
MSCs were obtained as described in Example 2.
Adipogenic progenitor cells (FAPs and MSCs) were resuspended in 0.5% high viscosity alginate solution (Sigma-Aldrich, W201502) at a concentration of 3×107 cells/ml. The cell-alginate suspension was injected into 66 mM CaCle in a 10 mM HEPES buffer. The formed microfibres were cultured in 12-well tissue culture plates for 28 days with the serum-free adipogenic differentiation media described in Example 1 (DMAD). As a control, DMEM/F12 with 3% FBS supplemented with 4 conventional adipogenic inducers (IBMX, dexamethasone, rosiglitazone, insulin) was used for 3 days, followed by DMEM/F12 with 3% FBS supplemented with only rosiglitazone and insulin for 25 days. The media was replaced every 3-4 days for 28 days. All fibers were incubated on a shaking platform at 75 RPM at 37° C. and 5% CO2.
The differentiated microfibres were fixed using 4% FA in 66 mM CaCl2 buffered with 10 mM HEPES for 1 h. Once fixed, the samples were washed in buffer solution I (66 mM CaCl buffered with 10 mM HEPES) and stained overnight at 4° C. (1:125 BODIPY, 1:1000 Hoechst). For immunocytochemistry, microfibres were then blocked/permeabilised in blocking solution (buffer solution I, 10% goat serum, 0.1% Triton X) for 1 h. Followed by incubation with acetyl-CoA carboxylase antibody (ACC; 1:200; Cell Signalling, #3676) overnight at 4° C. and donkey anti-rabbit Alexa 594 secondary antibody (1:200, Thermo Fisher, A32754) for 2 h. Following 2 washes with buffer solution I, the microfibres were imaged on a confocal microscope (TCS SP8, Leica Microsystems) using a 25×/1.00 objective lens and 5 μm-Z steps.
In our 3D cell model, it is clear that bovine MSCs differentiated in DMAD demonstrate earlier onset of adipogenesis compared to the control medium, as well as a higher number of differentiated cells (
Adipogenic progenitor cells were cultured as described in Example 2, with the exception that single components (FGF, EGF, insulin, vitamin C, hydrocortisone, BMP4, lipid concentrate, HEPES, putrescine, progesterone) of the DMAD medium (the serum-free differentiation medium as depicted in Example 1) were removed. In addition, different concentrations (50% and 200%) were tested for EGF, FGF, vitamin C, hydrocortisone and progesterone.
To assess if a range of concentrations of DMAD components can induce adipogenesis, 50% and 200% of the standard concentration (i.e. the concentration of indicated medium components as depicted in Example 1) was tested for EGF, FGF, vitamin C, hydrocortisone and progesterone.
Decreasing the concentration of EGF resulted in significantly decreased adipogenesis (
Removing FGF, lipid concentrate, HEPES, putrescine or progesterone from DMAD did not have significant effects on either the percentage of positive cells or the total lipid area. These components are thus not required for adipogenic differentiation (
Adipogenic differentiation
MSC cells were cultured as described in Example 2. With the exception that indomethacin (50 μM; 17378 from Sigma Aldrich) was replaced with one of the following three PPARΥ agonists to test their efficiency: magnolol (M3445 from Sigma Aldrich) at 10 μM; honokiol (H4914 from Sigma Aldrich) at 10 μM and amorfrutin B (SMB00532 from Sigma Aldrich) at 10 μM.
Adipogenic precursor cells cultured in DMAD (the serum-free differentiation medium as depicted in Example 1) were able to undergo adipogenic differentiation with a range of PPARΥ agonists, including indomethacin, magnolol, honokiol and amorfrutin B (
FAP cells were cultured as described in Example 3. With the exception that in the sugar/glutamine-free basal medium employed for the medium as depicted in Example 1, glucose (17 mM) and/or glutamine (2 mM) were replaced or combined with different combinations of monosaccharides such as galactose (17 mM), and substrates involved in energy metabolism such as glycolysis, glutaminolysis, and mitochondrial respiration including pyruvate (10 mM), acetate (10 mM), a-ketoglutarate (aKG; 5 mM).
To assess if different energy substrates can be used in DMAD to support differentiation, bovine FAPs were cultured with different combinations of glucose, glutamine, galactose, pyruvate, acetate and aKG. Our data demonstrates that DMAD can be successfully supplemented with various sugars and/or glutamine-sources to achieve robust adipogenic differentiation (
For the lipidomics sample preparation, 10-30 mg of animal-derived fat tissue, as well as cultured fat tissue from both MSCs and FAPs as obtained in accordance with the previous examples were washed in PBS and stored at −80° C. Lipids were extracted using a modified Bligh-Dyer protocol (as described in Barniol-Xicota et al., Communications biology, 4:218 (2021)) and triglycerides were analysed by hydrophilic interaction liquid chromatography mass spectrometry (HILIC LC-MS/MS). To quantify the relative amount of each fatty acid within the triglyceride class, each fatty acid was normalised to the total amount of triglycerides present per sample and the total amount of protein.
Lipid profiles were analyzed in conventional fat tissue, and cultured fat from bovine FAP and MSC cells, from four donors each. Based on this lipidomics analysis, it was observed that a relatively similar fatty acid profile was found within the triglyceride class in all samples (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2028813B1 | Jul 2021 | NL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/NL2022/050431 | 7/22/2022 | WO |
