Process for the production of cultivated fat cells

Abstract

The present invention relates to a method for the cultivation of adipose cells, which method comprises: culturing adipose cells in a culture medium; and harvesting the adipose cells, wherein prior to harvesting, the adipose cells are aggregated in the culture medium. Cells obtained from the method may be incorporated into food products.

Description

[0001] Process for the production of cultivated fat cells

[0002] Field of the invention

[0003] The present invention relates to a method for the production of cultivated fat cells.

[0004] Background of the invention

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

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

[0007] Summary of the invention

[0008] The invention relates to a method for cultivation of fat cells in which the recovery of cells in a cell culture may be improved. It is based on the finding that customary harvesting techniques often fail to recover all of the cells present in a cell culture since, for example, single cells present in the cell culture may not be recovered by settling or centrifugation. The inventors have found that aggregating adipose cells prior to harvesting results in larger recoveries of cells that would otherwise be achieved.

[0009] According to the invention, there is thus provided a method for the cultivation of adipose cells, which method comprises: cultivating adipose cells in a culture medium; and harvesting the adipose cells, wherein prior to harvesting, the adipose cells are aggregated in the culture medium.

[0010] Critically, aggregation may take place prior to harvesting.

[0011] In one embodiment, the adipose cell is selected from the group consisting of embryonic stem cells, pluripotent stem cells, such as induced pluripotent stem cells, embryonic cell lines, somatic cell lines and immortalized cell lines.

[0012] The invention further relates to: a cell obtainable by or obtained by the method of the invention; a cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method of the invention; a cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to the invention; a method according to the invention, further comprising the step of: incorporating the cultured cell into a food product for animal, preferably human, consumption; and use of a cell obtained according to a method according to any one of claims 1 to 9 fortissue engineering, optionally for the production of cultured meat.

[0013] Description of the drawings

[0014] Figure 1 demonstrates that the addition of food safe cross-linking agents such as transglutaminase can significantly improve the harvesting yield of cultivated adipose cells. The bar graph in Figure 1A demonstrates that addition of 15 g / L transglutaminase to the cell / medium suspension at the end of culture collects at least 50% more yield (yellow bars) than standard centrifugation alone (white bars, blue outline). This ensures collecting at least equal to initial weight of product inoculated at the beginning of harvest (blue bars). This method is successful regardless of variations in culture conditions, such as media recipes or bioreactor vessel. Standard harvest techniques collect a pellet of cells or aggregates via centrifugation; removing the cells from the cell / medium suspension and leaving behind the supernatant of spent culture medium. However, it was noted (Figure 1 B) that the supernatant still contained many single floating cells (nuclei stain; blue), presumably due to the high lipid content of the cells making them buoyant (neutral lipid stain; green). After incubation with transglutaminase tested from 2-24 hours at 38C or 4C it was found that the supernatant contained a negligible number of remaining cells after centrifugation (Figure 1 C. Blue; nuclei). Moreover, the pellet collected contained a high yield of cells (Figure 1 D. Blue; nuclei) and the cells were filled with lipids (Fig 1 E. Green; neutral lipids). This was confirmed at higher magnification that each cell collected was enlarged and filled with neutral lipid droplets (Figure 1 F. Green; neutral lipids).

[0015] Figure 2 demonstrates addition of a flocculant agent such as s-polylysine during harvest of cultivated adipose cells significantly improves yield and productivity. Moreover, addition of a flocculant agent such as s-polylysine at the beginning or during cell culture in suspension to improve aggregate stability. After incubation with 200mg / L s-polylysine up to 4 hours at room temperature followed by standard centrifugation harvest steps, it was found that the pellet collected contained a high yield of cells (Figure 2A Blue; nuclei) and the cells were filled with lipids (Figure 2B. Blue: nuclei, Green; neutral lipids). Whereas the supernatant did not contain any surplus of floating cells (Figure 2C. Blue; nuclei. Figure 2D. Blue: nuclei, Green; neutral lipids). Furthermore, it was noted that addition of a flocculant agent such as s-polylysine during the culture resulted in a more stable and homogeneous aggregate population (Figure 2F) compared to 3D cultures without added s- polylysine (Figure 2E).

[0016] Description of the invention

[0017] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

[0018] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0019] As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0020] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.

[0021] The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.

[0022] 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) or hepatocytes (liver cells).

[0023] The term “lipid accumulation” as used herein is defined as an increase in the level of at least one form of fatty acid in a cell or tissue. Lipid accumulation is detected, for example, by Nile red staining, by C1-BODIPY-C12 staining, by lipid extraction and gas chromatography / mass spectroscopy, or by any other method known in the art. Lipid accumulation refers to, for example, an increase in the level of total lipids or an increase in at least one type of fatty acid (e.g., very long chain fatty acids). As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo- derived stem cells, induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using, methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

[0024] As used herein, a “medium” or “cell culture medium” refers to an aqueous based solution that provides for the growth, viability, or storage of cells. A medium as contemplated herein can be supplemented with one or more nutrients to promote the desired cellular activity, such as cell viability, growth, proliferation, differentiation of the cells cultured in the medium. A medium, as used herein, includes a serum replacement, a medium supplement, a complete medium or a cryopreservation medium. The pH of a culture medium should be suitable to the microorganisms that will be grown. Most bacteria grow in pH 6.5-7.0 while most animal cells thrive in pH 7.2-7 .4.

[0025] The terms “cultured” and “cultivated”, in the context of “cultured meat” and "cultivated meat”, “cultured fat / muscle”, cultivated fat / muscle” may be used interchangeably herein.

[0026] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0027] During the culture of cells, it is typical for the culture medium to be separated from the cell mass. Conventional methods for carrying this out include settling and centrifugation. It was observed that not all cells in the case of culture of adipose cells can be recovered using such standard harvesting techniques. It appears that there are cells in a cell culture, in particular single cells, which float and are not susceptible to recovery by standard techniques.

[0028] Accordingly, the invention relates to a method in which cells, typically adipose cells, are aggregated in the culture medium prior to harvesting. In this way, harvested yields may be appreciably increased. According to the invention, there is thus provide a method for the cultivation of adipose cells, which method comprises: cultivating the adipose cells in a culture medium; and harvesting the adipose cells, wherein prior to harvesting, the adipose cells present in the culture medium are aggregated in the medium.

[0029] Accordingly, the aggregation step occurs prior to harvesting. Harvesting may be carried out using any suitable method for the separation of cells from a culture medium. Techniques customarily used include centrifugation and settling followed by removal of culture medium. Such techniques may be used in the invention. In the context of 3D suspension culture of cells, ’’aggregation” refers to the phenomenon whereby individual cells come together and adhere to each other, forming a collective structure or cluster while being suspended in a liquid medium.

[0030] In the invention, any suitable technique to promote aggregation. In particular, a crosslinking agent and / or a flocculation agent may be used. A preferred example of a cross-linking agent is a transglutaminase. A preferred example of a flocculation agent is a polylysine,

[0031] Cross-linking in the invention is the formation of cross-links which are bonds or short sequences of bonds that link one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or as is the case here natural polymers (such as proteins). A cross-linking agent is any substance that promotes cross-linking between cells, i.e. the formation of cross-links between cells, creating larger aggregates.

[0032] Flocculation in the invention is a process by which a substance added to the culture medium acts to facilitate bonding between cells through hydrophobic or hydrogen bonds. A flocculation agent is any substance that promotes flocculation between cells, i.e. the formation of hydrophobic or hydrogen bonds between cells, creating larger aggregates.

[0033] A transglutaminase suitable for use in the invention is any enzyme from the a class of enzymes that catalyze the formation of a covalent bond between the y-carboxamide group of protein- or peptide-bound glutamine (acyl donors) and the free amine group of protein- or peptide- bound lysine (acyl acceptors). The transglutaminase may be a member of EC2.3.2.13.

[0034] A polylysine suitable for use in the invention is any suitable lysine homopolymer. These may differ from each other in terms of stereochemistry (D / L; the L form is natural and usually assumed) and link position (a / s). Of these types, only s-poly-L-lysine is produced naturally, s- polylysine (s-poly-L-lysine, EPL) is typically produced as a homopolypeptide of approximately 25- 30 L-lysine residues and is suitable for use in the invention.

[0035] A cross-linking agent may alternatively be referred to as a cross-linking substance. A flocculation agent may alternatively be referred to as a flocculation substance

[0036] In order to collect cells that have not formed aggregates in a cell culture, or that have detached from the aggregates (eg. via buoyancy due to lipid accumulation), a cross-linking agent such as a transglutaminase can be added to collect the cells chemically. Alternatively or in addition, addition of an agent that promotes flocculation leverages the cells negative charge from the cell membrane and these regents, such as a polylysine, preferably s-polylysine, interact with cell surfaces and induce attractive forces between cells resulting in easier formation of cellular aggregates.

[0037] A suitable dosage rate for a cross-linking agent or a flocculation agent may be determined by the skilled person depending on the context, including the specific cells being cultured, the specific culture medium and culture conditions. A transglutaminase may be used in a culture medium at a concentration of from about 1 g / L to about 20 g / L, such as about 15g / L. A polylysine, such as s-polylysine may be used in a culture medium at a concentration of from about 50 mg / L to about 500 mg / L, such as about 200 mg / L. A cell suitable for use in the invention is a cell is an adipose cell, i.e. a cells that is capable of lipid accumulation such as a fat cell (adipocytes) or precursor cells thereof (such as fibro- adipogenic progenitor cells, lipoblasts, adipoblasts and, pre-adipocytes) or liver cells (hepatocyte) or precursor cells thereof (such as pre-adipocytes, hepatoblasts and pre-heptatocytes). Thus, the cell may be one which is partially or fully differentiated into an mature adipocyte or may be one which is capable of being differentiated into a mature adipocyte such as a pluripotent stem cell. A method of the invention may comprise differentiation of a cell, such as a pluripotent stem cell, into a mature adipocyte.

[0038] A cell used in the invention may be a “pluripotent stem cell”. As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, epiblast-derived stem cells, induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is selected from the group consisting of embryonic stem cells, embryo-derived stem cell lines, epiblast-derived stem cell lines, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSC). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of: OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using, methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

[0039] The pluripotent stem cell according to the invention can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate pluripotent stem cells, such as embryonic stem cells embryo-derived stem cells and epiblast-derived stem cells (EpiSC), from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0040] Similarly, iPS cells can be from any species. These iPS cells have been successfully generated using mouse and human cells. Furthermore, iPS cells have been successfully generated using embryonic, fetal, newborn, and adult tissue. Accordingly, one can readily generate iPS cells using a donor cell from any species. Thus, one can generate iPS cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

[0041] In certain embodiments, the pluripotent stem cell according to the invention, or for use in the invention is an animal cell. In certain embodiments the pluripotent stem cell according to the invention, or for use in the invention if from an edible animal species. In certain embodiments, the cell is from a mammal, preferably a non-human mammal. Preferably, the cell is an animal cell. In certain embodiments cell according to the invention is from an edible non-human animal species.

[0042] Accordingly, in one embodiment, the cells produced by the method as described herein are suitable for human and non-human dietary consumption.

[0043] In certain embodiments, the pluripotent stem cells are of a livestock or poultry species.

[0044] Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons. In certain embodiments, the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare.

[0045] Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits.

[0046] In certain embodiments, the cells are derived from an immortalized cell line. Exemplary cell lines include, but are not limited to, 3T3-L1 (mouse pre-adipocytes), buffalo rat liver cells (BRL 3A), chicken liver cells (LMH),

[0047] In certain embodiments, the cell is an adipose cell originating from a domestic pig or domestic cattle.

[0048] In certain embodiments, the cell is an hepatocyte originating from a duck or a goose.

[0049] In certain embodiments, the cell is a pluripotent cell. Preferably the pluripotent cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. Preferably, when a pluripotent cell is cultured, the pluripotent stem cell is a porcine or a bovine pluripotent stem cell. In certain embodiments, the stem cell according to the invention is a porcine or bovine epiblast stem cell (pEpiSCs and bEpiSCs). Preferably a pluripotent stem cell according to the invention, or for use in the invention, is not a human cell.

[0050] In certain embodiments, the cells as used in the methods as described herein are described in International patent publication number W02024 / 084082. Any cell described in this patent application may be useful in the methods of the invention for adipose culturing cells.

[0051] In one embodiment, the method according to the invention is for the proliferation (i.e. expansion) of the pluripotent cell.

[0052] In one embodiment, the method as described herein can be used for differentiation the pluripotent cell.

[0053] Aggregation may take place at any point in the cell culture prior to harvesting. Thus, a cross-linking agent or a flocculation agent may be added at the start of cell culture or immediately prior to harvesting or at any other time point in the culture process.

[0054] In certain embodiments, the culture medium is a basal medium that is supplemented with said medium components.

[0055] The term ‘basal medium’, as used herein, includes reference to a liquid medium that supports cellular growth by providing essential components for growth. A basal medium may be provided in liquid or powdered format. A basal medium that is not supplemented with any compound may enable cellular growth, but supplementation may be required for growth depending on the cell type. A basal medium may be supplemented with one or more components selected from the nonlimiting group consisting of amino acids, lipids, sugars, carbohydrates, anions, cations, buffering agents, colorants, vitamins, antioxidants, hormones, enzymes, proteins and trace elements. In some embodiments, the basal medium as disclosed herein is a commercially available basal medium such as DMEM (Dulbecco’s Modified Eagle Medium) and Ham’s F- 12. In some embodiments, the basal medium is the medium described in any of International patent publication nos. W02024 / 084082, WO2024 / 170696 and W02024 / 170702and W02024 / 252001 , which are incorporated by reference herein. Any of the media described in any one of those patent applications may be used as basal medium in the methods of the invention.

[0056] In certain embodiments, the methods as described in the various embodiments of the invention further comprises the step of:

[0057] - incorporating the cultured cell into a food product for animal, preferably human, consumption.

[0058] In a further aspect, the invention provides for a cell obtainable by or obtained by the methods as described in the various embodiments as described herein. In one embodiment, cells obtained by the method as described herein are suitable for human and non-human dietary consumption.

[0059] In yet a further aspect, the invention provides for a food product (also referred to as “foodstuff’) comprising the cells produced, obtained by the methods and / or cultured in any of the mediums as described herein. In certain embodiments, the food product is for animal, preferably human, consumption.

[0060] A food product of the present invention may comprise one or more of minerals, synthetic substances, flavors, texture enhancers, nutritional additives, preservatives, and fats. In an aspect of the invention, the flavors are selected from one or more of essential oils, oleoresin (ESO), enzymes (ENZ), natural substances and extractives (NAT), non-nutritive sweetener (NNS), nutritive sweetener (NUTRS), herbs, spices, natural seasonings & flavorings (SP), and synthetic flavors (SY / FL), fumigant (FUM), artificial sweeteners and yeast extract. In another aspect of the present invention, the texture enhancers are selected from one or more of pureed plant material, guar gum, cellulose, hemicellulose, lignin, beta glucans, soy, wheat, maize and rice isolates and beet fiber, pea fiber, bamboo fiber, plant derived fiber, plant derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate, natural polysaccharides, grain husk, calcium citrate, calcium phosphates, calcium sulfate, magnesium sulfate and salts. In another aspect of the present invention, the nutritional additives are selected from one or more of trace elements, bioactive compounds, endogenous antioxidants, A, B-complex, C, D, E vitamins, zinc, thiamin, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, protein, amino acids salt, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, Retinol, folic acid, dietary fiber and amino acids. In yet another aspect of the present invention, the fats are selected from one or more of saturated, monounsaturated, polyunsaturated fats, corn oil, canola oil, sunflower oil, safflower oil, olive oil, peanut oil, soybean, flax seed oil, sesame oil, canola oil, avocado oil, seed oils, nut oils, safflower and sunflower oils, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fat, adipose tissue, cellular agriculture derived fat essential oil and oleoresin. In another aspect of the present invention, the preservative and / or antioxidant is selected from one or more of: sodium salt, chloride salt, iodine salt. Nitrates, nitrosamines. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium benzoate, potassium benzoate and benzene ascorbic acid, citric acid, potassium, monosodium glutamate (MSG), sulphur dioxide, sulphites, antibiotics. It is noted here that any one additive, flavor, texture enhancer, nutrient additive, fat / oil and / or preservative / antioxidant may supply more than one attribute to food product of the present invention.

[0061] In certain embodiments the food product of the present invention further comprises (cultured) myocytes. In certain embodiments, the food products comprises the cultured myocytes as described in International patent publication nos. WO2024 / 170696and WO2024 / 170702. Any cell described in any one of those patent applications may be useful in a method of the invention for culturing cells.

[0062] In certain embodiments, the food product is a cultured meat product.

[0063] The cultured meat product may be a structured product. Optionally, the product is structured with scaffolding. In one embodiment the structured product is free of scaffolding and the structure is provided by the cells as obtained by the methods as described herein.

[0064] In yet a further aspect, the invention provides for a cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method as described herein.

[0065] In yet a further aspect, the invention provides for a use of the cell obtained or obtainable by the method as described herein for tissue engineering, optionally for the production of cultured meat.

[0066] Examples

[0067] The present invention is further illustrated by the following Examples which should not be construed as limiting the scope of the invention.

[0068] Example 1. Addition of cross-linking agent, such as transglutaminase, during harvest significantly improves yield and productivity

[0069] Transglutaminase (TGlut) was added to a cell / medium suspension from 3D aggregate culture of differentiated epiblast derived adipose cells at a concentration of 15 g / L. It was observed that the TGlut binds floating cells together through cross-linking and in this way it was possible to collect at least 50% more mass at harvest vs centrifugation harvest methods alone (see Figure 1A).

[0070] Using standard harvest methods, i.e. centrifugation to collect the pellet of cells or tissue, it was observed that there were many floating cells left in the supernatant, for example floating due to buoyancy from lipid content (see Figure 1 B). By incubating TGlut at a concentration of 15 g / L, with this cell / medium mix, it was then possible to collect the cells via centrifugation (See Figure 1C) into a pellet (see Figure 1 D).

[0071] All cells collected were lipid filled adipose cells or tissue demonstrated by positive neutral lipid stain in green (see Figure 1 E). Extra cells collected were to the same high quality as those cells and tissues collected from non-floating population; large cell size (Scale 10 pm) with many lipid droplets (see Figure 1 F).

[0072] Example 2: Addition of a flocculant agent such as c-polylysine (polyL) during harvest significantly improves yield and productivity or during cell culture in suspension to improve aggregate integrity / stability s-polylysine was added to a cell / medium suspension from 3D aggregate culture of differentiated epiblast derived adipose cells at a concentration of 200 mg / L. It was observed that floating cells were bound together by facilitating the bonding of cells due to their charged membranes, resulting in the collection at least 50% more mass at harvest vs centrifugation harvest methods alone.

[0073] Using standard harvest methods (centrifugation to collect the pellet of cells or tissue), it could be seen that there were many floating cells left in the supernatant (eg floating due to buoyancy from lipid content). By incubating s-polylysine with the cell / medium suspension it was then possible to collect the cells via centrifugation into a pellet (See Figure 2A) and these cells were differentiated towards lipid rich adipose cells (see Figure 2B).

[0074] All cells collected were lipid filled adipose cells or tissue demonstrated by positive neutral lipid stain in green (see Figure 2E). All samples collected were confirmed by a clear supernatant after centrifugation for cells (see Figure 2C) therefore there was no loss of lipid filled product (see Figure 2D).

[0075] Addition of a flocculant agent such as s-polylysine (polyL) at the beginning and / or during cell culture in suspension improve aggregate integrity / stability (see Figure 2F) compared to standard culture conditions in the absence of s-polylysine (see Figure 2E).

[0076] Embodiments

[0077] The present invention provides at least the following numbered statements / embodiments:

[0078] 1 . A method for the cultivation of adipose cells, which method comprises: culturing adipose cells in a culture medium; and harvesting the adipose cells, wherein prior to harvesting, the adipose cells are aggregated in the culture medium.

[0079] 2. A method according to embodiment 1 , wherein the adipose cells are aggregated by the addition to the culture medium of a cross-linking agent or a flocculation agent. 3. A method according to embodiment 2, wherein the cross-linking agent is a transglutaminase.

[0080] 4. A method according to embodiment 2, wherein the flocculation agent is a polylysine.

[0081] 5. A method according to any one of the preceding embodiments, wherein the adipose cells in the culture medium are partially aggregated.

[0082] 6. A method according to any one of the preceding embodiments, wherein the adipose cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, somatic cell lines.

[0083] 7. A method according to any one of the preceding embodiments, wherein the adipose cells produced by the method are suitable for human and non-human dietary consumption.

[0084] 8. A method according to any one of the preceding embodiments, wherein the method is for the proliferation of cells or for the differentiation of cells.

[0085] 9. A method according to any one of the preceding embodiments, wherein the method is carried out in one or more bioreactors.

[0086] 10. A cell obtainable by or obtained by the method according to any of the preceding embodiments.

[0087] 11. A cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method according to any one of embodiments 1 to 9, optionally further comprising cultivated mammalian myocytes.

[0088] 12. A cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to any one of embodiments 1 to 9.

[0089] 13. A method according to any one embodiments 1 to 9, further comprising the step of:

[0090] - incorporating the cultured cell into a food product for animal, preferably human, consumption.

[0091] 14. Use of a cell obtained according to a method according to any one of embodiments 1 to 9 fortissue engineering, optionally for the production of cultured meat.

Claims

Claims1 . A method for the cultivation of adipose cells, which method comprises: culturing adipose cells in a culture medium; and harvesting the adipose cells, wherein prior to harvesting, the adipose cells are aggregated in the culture medium.

2. A method according to claim 1 , wherein the adipose cells are aggregated by the addition to the culture medium of a cross-linking agent or a flocculation agent.

3. A method according to claim 2, wherein the cross-linking agent is a transglutaminase.

4. A method according to claim 2, wherein the flocculation agent is a polylysine.

5. A method according to any one of the preceding claims, wherein the adipose cells in the culture medium are partially aggregated.

6. A method according to any one of the preceding claims, wherein the adipose cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, somatic cell lines.

7. A method according to any one of the preceding claims, wherein the adipose cells produced by the method are suitable for human and non-human dietary consumption.

8. A method according to any one of the preceding claims, wherein the method is for the proliferation of cells or for the differentiation of cells.

9. A method according to any one of the preceding claims, wherein the method is carried out in one or more bioreactors.

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

11. A cultured meat product for animal, preferably human, consumption, comprising at least one cell obtained by the method according to any one of claims 1 to 9, optionally further comprising cultivated mammalian myocytes.

12. A cultured fat product for animal, preferably human, consumption, comprising at least one cell obtainable by a method according to any one of claims 1 to 9.

13. A method according to any one claims 1 to 9, further comprising the step of:- incorporating the cultured cell into a food product for animal, preferably human, consumption.

14. Use of a cell obtained according to a method according to any one of claims 1 to 9 for tissue engineering, optionally for the production of cultured meat.

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