Cell composition

Abstract

A cell composition of non-human animal origin is described comprising a plurality of cells of non-human animal origin wherein the cells comprise at least one exogenous supplement contained in a carrier selected from the group consisting of nanocarriers, nanoparticles, micelles, liposomes or vesicles. The cellular compositions are characterized by having improved organoleptic properties, such as improved flavor, color, texture or mouthfeel and / or with a meat-like flavor. Methods of producing them and comestible products comprising them are also described.

Description

Technical Field

[0001] The present disclosure relates to the field of cultured meat. More specifically, the present disclosure relates to methods and compositions for improving the sensory properties of cultured meat, including its color, odor, taste, aroma, texture, or mouthfeel, such that it resembles meat from whole animal sources.

[0002] Background

[0003] To sustain livestock production, many valuable resources are exploited, including water, food, land, and energy, and the demands of the world's rapidly growing population will lead to even greater exploitation of these valuable resources. Therefore, the ability to produce meat and meat products in a way that will reduce our dependence on non-human animals is a highly desirable goal. Replacing animals such as cattle, sheep, pigs, and poultry as the main meat sources, as well as fish and shellfish, is also morally beneficial as it would reduce the sometimes cruel conditions associated with intensive livestock and agricultural production. Cultured meat production also provides an option for consumers who do not eat meat for ethical reasons, and for those consumers who freely consume meat today, it can offer additional health benefits as the production of cultured meat does not require the use of growth hormones and antibiotics.

[0004] However, creating some or all of the sensory properties of whole meat in cultured products such that these products resemble whole meat remains a challenging task.

[0005] There is still a need to provide cultured meat products that can offer consumers an alternative to meat consumption without compromising any of the beneficial or sensory properties of intensively farmed meat.

[0006] The development of food products containing cultured meat has shown that there are still significant gaps between food products from cultured meat and livestock meat in terms of the macro- and micro-nutritional value, flavor, color, perception, and texture of the final food product. Food products from cultured meat are completely different in properties from intensively farmed meat and its food products. WO2020 / 100143 (ALEPH Farms) took a first step in this direction by, specifically, improving the micro-nutritional value of the final food product while also providing a more authentic meat flavor for cultured meat. WO 2015 / 038988 (Modern Meadows) disclosed millimeter-sized edible microcarriers that serve as scaffolds for adhering cells to lock or adsorb thereon and avoid the need to separate the microcarriers from the cell culture medium.

[0007] The goal of the present disclosure is to incorporate molecules such as flavoring compounds, flavoring precursors, colorants, and / or nutrients into cell cultures to provide cultured raw meat products having sensory properties and nutritional content as similar as possible to livestock meat.

[0008] However, the applicant's in-depth research has confirmed that flavoring agents cannot be directly incorporated into cell cultures because the excretion and decomposition processes of cells immediately begin when exogenous (i.e., non-cellular or non-physiological) contents come into contact with cells and cell culture media. In addition, the hydrophobic and cytotoxic properties of certain compounds may also pose problems for their incorporation.

[0009] Therefore, it is necessary to immerse and encapsulate or embed non-physiological and exogenous compounds that can be directly utilized in cells into and then into the final cultured raw meat, and subsequently into the final food product, in order to provide the characteristic meat properties of raw or cooked meat, such as meat flavor, flavor precursors or color, which may provide characteristic color changes after cooking, or supplements such as nutritional products, proteins or other bioactive compounds, or supplements that provide other typical characteristics of meat (such as fat, texture and taste).

[0010] To avoid the excretion and degradation of compounds, other methods are needed to deliver exogenous compounds to cells and retain them within the cells.

[0011] Possible delivery methods include, but are not limited to, transduction, electroporation, nanoparticle delivery and liposome delivery.

[0012] Liposomes have been used in medical and pharmaceutical research, especially for the targeted delivery of drugs to different parts of the body, such as different tissues of the body (e.g., connective tissues), or to cells including, but not limited to, endothelial cells (e.g., blood vessels), for example through pH control or other mechanisms: but there are also published studies using liposomes to directly deliver active substances to cells through mitochondria ("MITO-Porter" and "Mitochondrial delivery system using liposomes as nanocarriers for targeting myoblasts").

[0013] In food science, liposomes have been used for different purposes, such as the delivery and preservation of nutrients or bioactive agents, for encapsulation and packaging, for example in "Application of Liposomes in the Food Industry, Z. Mirafzali, C. S. Thompson, K. Tallua in Microencapsulation in the Food Industry - A Practical Implementation Guide, 2014, Chapter 13, pp. 139 - 150", "Antioxidant activity of spice extracts in a liposome system and in cooked pork patties and the possible mode of action; Kong, Baohua; Zhang, Huiyun; Xiong, Youling L. Meat Science (2010), 85(4), 772 - 778"; or for emulsification, for example in "Domínguez, R.; Pateiro, M.; Munekata, P. E. S.; McClements, D. J.; Lorenzo, J. M. Encapsulation of Bioactive Phytochemicals in Plant - Based Matrices and Application as Additives in Meat and Meat Products. Molecules 2021, 26, 3984".

[0014] Liposomes have been successfully used to deliver proteins into cells, such as in "Fu et al., 'Promises and Pitfalls of Intracellular Delivery of Proteins, Bioconjugate Chem., 2014, 25, 1602 - 1608'; or 'Nahum, V.; Domb, A. J. Recent Developments in Solid Lipid Microparticles for Food Ingredients Delivery. Foods 2021, 10, 400'; or 'Sharma, S.; Mulrey, L.; Byrne, M.; Jaiswal, A. K.; Jaiswal, S. Encapsulation of Essential Oils in Nanocarriers for Active Food Packaging. Foods 2022, 11, 2337'; or 'Akbarzadeh et al., Nanoscale Research Letters 2013, 8:102'.

[0015] Overview

[0016] According to a first exemplary aspect, there is provided a cell composition comprising cells derived from a cultured non - human animal, the cell composition being enriched with an exogenous supplement that provides or is capable of providing meat - like sensory properties or meat - like coloring to the cell composition, wherein the exogenous supplement is contained within a carrier. According to certain exemplary aspects, the exogenous supplement is contained in the form of a nanocarrier, nanoparticle, micelle, liposome, or vesicle composition.

[0017] According to a second exemplary aspect, there is provided cultured meat containing the exogenous supplement composition.

[0018] According to a third exemplary aspect, there is provided an edible product containing the cultured meat composition.

[0019] According to a fourth exemplary aspect, there is provided a cell culture medium enriched with an exogenous supplement, the supplement being provided by or contained within a carrier, such as, but not limited to, a nanocarrier, nanoparticle, micelle, liposome, or vesicle composition.

[0020] According to a fifth exemplary aspect, there is provided a scaffold useful for the preparation of cultured meat, the cultured meat being rich in exogenous supplements contained in a carrier such as, but not limited to, a nanocarrier, nanoparticle, micelle, liposome or vesicle composition, which provides or is capable of providing meat-like sensory properties to the scaffold and / or cells cultured in the scaffold.

[0021] This disclosure is in part based on the surprising and unexpected finding that culturing cells in a culture medium containing exogenous supplements such as flavorants, flavorant precursors or flavorant enhancers, pigments, etc., if the flavorant or precursor is provided in a nanocarrier, nanoparticle, micelle, liposome or vesicle composition, results in the uptake, accumulation and / or adhesion of the flavorant or precursor in or on the cells, thereby providing taste, aroma or color to the cultured meat, with an effect superior to that achievable by simply injecting or coating the cultured meat product with such flavoring or coloring compounds, such that the cultured meat produced according to the methods of the present disclosure has sensory properties substantially similar to those of meat from whole organisms, such as flavor, flavor precursors, color and nutritional content. In addition, hydrophobic (e.g., CLogP value > 2) and cytotoxic exogenous supplements can be readily and efficiently taken up, accumulated and / or adhered to cells or on cells.

[0022] By controlling the content of exogenous supplements added to the culture medium and / or the scaffold used in combination therewith or generated therein by providing the exogenous supplements in the form of a nanocarrier, nanoparticle, micelle, liposome or vesicle composition as a supplement composition or a more specific exogenous supplement composition, the disclosed compositions, edible products and methods provide advantages and improvements over the prior art. The generation of flavor is achieved by administering, generating or enhancing the required amount of exogenous supplements contained in a carrier (e.g., but not limited to, in such nanocarrier, nanoparticle, micelle, liposome or vesicle composition) during the growth, proliferation and / or differentiation of the cells, thereby effectively controlling the uptake of the supplement composition into and / or on the cell surface, or into and / or on any scaffold used therewith. In addition, by judiciously selecting the exogenous supplement composition or the exogenous supplement itself added to or generated in the cell culture medium and / or scaffold, it is possible to use simple, non-artificial, non-synthetic ingredients that consumers consider to be healthy to impart, alter or improve the flavor of the cultured meat and thereby meet the so-called "clean label" requirements or expectations related to flavor.

[0023] It should be clearly understood that one or more cells containing a certain amount of one or more exogenous supplements according to the present disclosure refer to non-human animal cells or a plurality of such cells, which are combined with one or more exogenous supplement compositions according to the present invention, and the combination can be intracellular, within the cell membrane, adhered to the cell membrane or other cell parts and any combination thereof, provided that it is provided in a nanocarrier, nanoparticle, micelle, liposome or vesicle composition.

[0024] The exogenous supplements described in the present disclosure are food-grade supplements suitable for human consumption. The exogenous supplements can be of biological origin, that is, they can be extracted and / or derived from natural sources (such as plants, fungi, bacteria, algae or animal sources). They may be natural, that is, extracted without modification from their natural state, or obtained and purified from their natural state, or even chemically or biochemically modified, or they can be synthetic materials identical to the materials found in nature.

[0025] Other aspects, embodiments, features and advantages of the present disclosure will be further described in the following detailed description of the exemplary aspects and embodiments.

[0026] Detailed Description

[0027] In recent decades, the need to provide cultured meat and edible products containing it has been recognized, but there are currently no high-quality, reasonably priced products. In addition to the price of current products, consumer doubts regarding the sensory characteristics of the products and especially their taste and aroma are important limiting factors that are widely accepted. The present disclosure addresses aspects of consumer appeal by providing compositions and methods that confer, alter or improve sensory characteristics such as taste and aroma, as well as color, texture, mouthfeel and even the nutritional value of cultured meat or edible products containing them.

[0028] For convenience, certain terms used in the specification, examples and claims are described herein.

[0029] Definitions

[0030] The term "cultured meat" is used herein to describe meat grown in vitro in non-human animal cell culture, rather than meat obtained from slaughtered animals.

[0031] The term "exogenous supplement" refers to a composition of one or more ingredients that is added to a cell culture medium and / or scaffold in the form of a nanocarrier, nanoparticle, micelle, liposome or vesicle composition to form a "supplement composition" or "exogenous supplement composition", which is absorbed, attached or bound by the cell or scaffold when in contact with the cell or scaffold.

[0032] As used herein, the term "incorporated in a carrier" refers to any compound or molecule that can serve as a carrier (e.g., but not limited to, the nanocarriers, nanoparticles, micelles, liposomes, or vesicles described below).

[0033] As used herein, the term "nanocarrier" refers to a nanomaterial that serves as a means of transporting another substance (i.e., a nanocarrier is a means for carrying or transporting another substance). According to the present disclosure, the other substance is an exogenous supplement or a cell culture medium containing an exogenous supplement. Nanocarriers used in the present disclosure include, for example, micelles, polymers, carbon-based materials, liposomes, and other substances. The diameter size range of the nanocarriers used herein is from 1 to 1000 nm, preferably from 1 to 500 nm, and more preferably from 1 to 200 nm.

[0034] As used herein, the term "nanoparticle" refers to particles in the nanoscale range having different shapes or compositions, and its diameter size range is from 1 to 1000 nm, preferably from 1 to 500 nm, and most preferably from 1 to 200 nm.

[0035] Examples of nanoparticles and nanocarriers used herein include, but are not limited to, nanomaterials of food-related proteins (such as albumin such as whey protein, globulin such as lactoglobulin, gluten such as gliadin, zein such as zein, scleroprotein or protamine, glycoprotein such as avidin, lipoprotein, phosphoprotein or chromoprotein). Nanocarriers used in the present disclosure can explicitly include virus-like particles or chylomicrons. Other examples are oil-like materials, such as, but not limited to, palm oil, sunflower oil, or triglycerides, such as, but not limited to, medium-chain triglycerides.

[0036] As used herein, the term "micelle or micellar" refers to a spherical aggregate of amphiphilic molecules, in which the non-polar molecular regions form a core that is shielded by the polar molecular regions in the micelle shell.

[0037] As used herein, the term "liposome" refers to a spherical aggregate having at least one lipid bilayer that forms a cavity capable of containing a liquid. The bilayer is preferably composed of amphiphilic molecules, such as, but not limited to, phospholipids, sphingolipids, or combinations thereof. Liposomes can optionally contain additives in an aqueous core encapsulated by a monolayer or multilayer phospholipid shell.

[0038] As used herein, the term "vesicle" refers to a structure inside or outside a cell that consists of a liquid or medium (including a buffer) encapsulated by a lipid layer.

[0039] As used herein, the term "spherulite" refers to a multi-layered vesicle composed of concentric double layers, which can be used to encapsulate suitable exogenous supplements. Inside the multi-layered vesicle, several hydrophilic and lipophilic layers alternate. Spherulites suitable for the applications described herein are described in US 5908697 and WO 2023 / 111230, the disclosures of which are incorporated herein by reference.

[0040] As used herein, the term "nanocarrier, nanoparticle, micelle, liposome or vesicle composition" refers to a mixture containing one or more different nanocarrier, nanoparticle, micelle, liposome or vesicle structures that encapsulate supplements such as flavorings, flavoring precursors, pigments, pigment precursors, bioactive compounds that produce flavorings, flavoring precursors, nutrients, etc., or proteins or other additives such as cell culture media or food-related additives, including nutritional products, texture additives, and mixtures thereof.

[0041] When used alone herein or together in phrases such as "meat-like" and "meat from non-human animals", the term "meat" refers to meat derived from non-human animals, including but not limited to mammals such as farm animals like cows, sheep, and pigs, wild game such as deer, roe deer, and moose; marsupials such as kangaroos; rodents such as mice, guinea pigs, or squirrels; reptiles such as crocodiles, turtles, or snakes; oviparous animals such as poultry and ducks; aquatic animals such as shellfish and fish; and arthropods such as insects. The term "meat" as used herein can be used for, but is not limited to, products suitable for human and pet consumption.

[0042] The term "sensory properties" with respect to meat and / or cultured meat refers to organoleptic properties such as color, odor, taste, aroma, and texture, including mouthfeel.

[0043] The term "pluripotent stem cell (PSC)" refers to a cell that can proliferate indefinitely and give rise to every other cell type in the body, including muscle cells, bone cells, and fat cells.

[0044] The term "induced pluripotent stem cell (iPSC)" refers to a type of pluripotent stem cell that can be directly generated from differentiated cells through a reprogramming process well-known in the art.

[0045] The term "embryonic stem cell (ESC)" refers to a type of pluripotent stem cell derived from a blastocyst.

[0046] The term "immortalized cell line" refers to a cell that does not undergo replicative senescence due to mutations and can be maintained in culture for a long time.

[0047] The term "primary cell" refers to a cell freshly isolated from animal tissue and grown in vitro.

[0048] The terms "reprogramming" or "differentiation" refer to the conversion of one specific cell type to another specific cell type. According to certain embodiments of the present invention, reprogramming is the conversion of a somatic cell type to a pluripotent cell type known as induced pluripotent stem cells or iPSCs.

[0049] The methods of the present invention

[0050] The cell compositions and cultured meat of the present disclosure, as well as their edible products, can be prepared according to a method comprising the steps of: contacting a single type or multiple cells with at least one exogenous supplement contained in a carrier, such as, but not limited to, in a nano - carrier, nanoparticle, micelle, liposome or vesicle composition, wherein the exogenous supplement is selected from flavoring materials, flavoring precursors, flavoring enhancers, pigments and pigment precursors, and bioactive compounds and other food - related additives or mixtures thereof.

[0051] According to certain embodiments, the cells are pluripotent stem cells (PSCs) and / or cells differentiated therefrom.

[0052] According to certain embodiments, the PSCs are induced PSCs (iPSCs) reprogrammed from somatic cells of non - human animals and / or cells differentiated therefrom.

[0053] According to certain embodiments, the PSCs are non - embryonic stem cells (non - ESCs).

[0054] According to certain embodiments, the PSCs are embryonic stem cells (ESCs).

[0055] According to certain exemplary embodiments, non - genetically modified PSCs are reprogrammed cells produced by a method comprising introducing a combination of the following into at least one cell: (a) at least one reprogramming mRNA encoding a reprogramming factor; and (b) at least one double - stranded microRNA; thereby producing at least one iPSC as described in WO2020230138A1, which publication is incorporated herein by reference.

[0056] In certain embodiments, the PSCs are embryonic stem cells (ESCs) of bovine origin and are cultured according to the method described in WO2020230138A1, the disclosed method of which is incorporated herein by reference.

[0057] According to certain embodiments, the cells are pluripotent stem cells differentiated into myocytes.

[0058] According to certain embodiments, the cells are pluripotent stem cells differentiated into adipocytes (lipocytes) and / or their progenitors.

[0059] According to certain embodiments, the cells are pluripotent stem cells that differentiate into stromal cells (connective tissue) and / or their progenitor cells.

[0060] According to certain embodiments, the cells are pluripotent stem cells that differentiate into endothelial cells (blood vessels) and / or their progenitor cells.

[0061] According to certain embodiments, the cells are pluripotent stem cells that differentiate into red blood cells / erythroblasts (hemoglobin-containing cells) and / or their progenitor cells.

[0062] According to certain embodiments, the cells are myoblasts that are induced to differentiate into myotubes and are cultured according to the method described in WO201916795A1, the disclosed method of which is incorporated herein by reference.

[0063] According to certain embodiments, the cells are satellite cells that differentiate into muscle cells and / or their progenitor cells.

[0064] According to certain embodiments, the cells are selected from muscle cells, adipocytes, stromal cells, fibroblasts, pericytes, endothelial cells and / or their progenitor cells and combinations thereof.

[0065] According to certain embodiments, the non-human animals are selected from cattle, sheep, pigs, birds, poultry (such as ducks, chickens and turkeys), wild animals (such as deer, roe deer or moose), shellfish, fish, insects, reptiles (such as crocodiles, turtles or snakes), rodents (such as mice, guinea pigs or squirrels) and any combination thereof. Each possibility represents a separate embodiment of the present disclosure.

[0066] According to certain exemplary embodiments, the non-human animal is cattle, more specifically cattle of the Bos Taurus species.

[0067] According to certain exemplary embodiments, the cells comprise a combination of cells from non-human animals, including muscle cells and their progenitor cells; adipocytes and their progenitor cells; stromal cells and their progenitor cells; endothelial cells and their progenitor cells, or red blood cells / erythroblasts and their progenitor cells.

[0068] According to certain embodiments, the cells are stromal vascular fraction cells isolated from adipose tissue according to the method described in "Mehta F, Theunissen R, Post MJ. Adipogenesis from Bovine Precursors. Methods Mol Biol. 2019;1889:111-125", the disclosed method of which is incorporated herein by reference.

[0069] According to certain exemplary embodiments, the cells form organoids. An "organoid" is a self-organizing, self-renewing three-dimensional cell structure that is similar to an organ in terms of structure and function. They can be derived from adult stem cells, embryonic stem cells, or induced pluripotent stem cells or mixtures thereof. They contain most of the relevant cell types, and their topology and cell-cell interactions are similar to those in in vivo tissues.

[0070] For example, organoids can be cultured according to the method described in Kar, S.K. et al. (Kar, S.K., Wells, J.M., Ellen, E.D. et al. Organoids: a promising new in vitro platform in livestock and veterinary research. Vet Res 52, 43 (2021)), which is incorporated herein by reference.

[0071] According to certain embodiments, the cell culture of any of the above embodiments forms cultured meat. According to certain exemplary embodiments, the cultured meat according to the present disclosure comprises a combination of cells from non-human animal sources, including myocytes and their progenitors; adipocytes and their progenitors; stromal cells and their progenitors; endothelial cells and their progenitors, erythrocytes / erythroblasts and their progenitors, organoids, and combinations thereof.

[0072] According to the method of the present disclosure, the cells are contacted with at least one exogenous supplement contained in a carrier, such as, but not limited to, in a nanocarrier, nanoparticle, micelle, liposome, or vesicle composition. The cells can be grown in a culture medium.

[0073] The term "medium" as used herein refers to any liquid composed of a mixture of basal nutrients and complex nutrients that provides conditions for maintaining cell viability and / or allowing it to grow, proliferate, and / or differentiate over a desired period of time. Those nutrients can include, but are not limited to, essential and non-essential amino acids, glucose, vitamins, inorganic salts, and buffers. Additional supplements can include hormones, proteins (such as, but not limited to, albumin, fetuin, and transferrin), lipids, cholesterol, growth factors, heparin, and trace elements (minerals), such as selenium.

[0074] Examples of media that can be used in the present invention include, but are not limited to, isotonic media, ready-to-use media, or custom media. Ready-to-use media are described in detail in the literature and are suitable for certain cell types, cell lines, and cell banks. The custom media in the present invention can refer to isotonic media or ready-to-use media supplemented with other components (such as growth factors, etc.) that are suitable for providing optimal conditions for the cells maintained in the medium.

[0075] The term "isotonic medium" as used herein refers to a medium that provides the minimum requirements for maintaining cell viability. According to one exemplary embodiment, the isotonic medium does not have any one or more of the following products: growth factors (such as insulin, EGF, FGF10, noggin, R-Spondin, bioactive proteins, nutrients such as minerals and vitamins), FBS, antibiotics, etc.

[0076] In other embodiments, the medium can be a ready-to-use medium or a custom medium that provides all the nutrients, growth factors, etc. required for cell growth, proliferation, and / or differentiation. Those skilled in the art will understand that the medium (ready-to-use or custom) will be adjusted according to the cell line or cell bank employed, the cell cycle, the differentiation stage, and other variables commonly known in the art. Thus, more than one medium can subsequently be used according to the methods of the present disclosure.

[0077] According to certain embodiments, the cell culture medium can contain or not contain antibiotics.

[0078] Antimicrobial peptides (AMPs) are a variety of natural proteins present in animals, plants, insects, and bacteria. These peptides are part of the host's defense mechanism against pathogenic organisms and have been found to be alternatives to chemical preservatives.

[0079] According to certain embodiments, the medium of the present disclosure further contains AMPs that prevent contamination of the cultured cells. As described above, these peptides are natural preservatives. AMPs are produced by bacteria that have been present in a variety of foods (such as cheese, yogurt, and Portuguese fermented meat) since ancient times and have been shown to be safe for human consumption and are thus approved for use in the food industry.

[0080] A commonly used AMP is nisin, which has a molecular weight of 3.5 kDa, 34 amino acids, a positive charge, and antimicrobial activity against Gram-positive bacteria (including bacilli, micrococci, Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum) and low antimicrobial activity against Gram-negative bacteria. Nisin is used to protect and extend the shelf life of pasteurized cheese, dairy desserts, canned foods, cured meats, and seafood. Although nisin has been used in the food industry for decades, the emergence of resistant food spoilage organisms has not been detected.

[0081] The culture medium will be adapted to the cell type being cultured. Examples of different culture media specifically designed for the different cell types mentioned herein can be found in the literature, such as in the following: Barsh and Cunningham et al. (J Cell Physiol. July 1977; 92(1):115-28.; 1977); Verma et al., (Animal Biotechnology. 2020:269-29), Palm and Thompson (Nature. June 7, 2017; 546(7657):234-242)., Specht, Liz, and S. Scientist. (“An analysis of culture medium costs and production volumes for cultivated meat.” The Good Food Institute: Washington, DC, USA (2020)), Freshney, R.I., (2021. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Culture of Animal Cells), Sigma-Aldrich, E ECACC Cell Culture Laboratory Handbook, Fundamental Techniques in Cell Culture, Laboratory Handbook. ECACC Handbook, WO201914652A1, the culture media disclosed therein are incorporated herein by reference.

[0082] In addition to the choice of cell type and cell culture medium, the bioprocess (i.e., the culture conditions and bioreactor) is also important for creating the conditions for large-scale production of cultured meat. An important aspect of the bioprocess is the design of the bioreactor (or incubator) as it will control conditions such as temperature, oxygen levels, the rate of delivery of the cell culture medium to the cells, and other important parameters well known to those skilled in the art. They are also able to monitor other key parameters such as metabolite levels, pH, biomass accumulation, cell growth and morphology, metabolite profiles, etc.

[0083] Most cells of non-human animal origin (e.g., chicken embryo fibroblast (CEF) cells) are adherent and can thus grow attached to a support (such as flasks, culture vessels, and other culture supports). Cells of non-human animal origin can also grow in suspension in a suitable suspension support medium.

[0084] The scaffold can assist in cell growth, cell-cell interaction, and cell proliferation as well as adhesion to the support, and can thus be used for culturing cells according to the methods of the present disclosure. The scaffold can also facilitate the separation of cells from the culture medium (Furuhashi, M. et al., 2021. Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak. npj Science of Food, 5.).

[0085] Any edible scaffold known in the art for culturing cells can be used according to the methods of the present disclosure, including edible protein scaffolds, edible hydrogel scaffolds, edible polysaccharide scaffolds, etc. According to certain embodiments, the scaffold is derived from plants, algae, fungi, and / or microorganisms. According to certain embodiments, the scaffold can be a 2D or 3D scaffold, as described in Campuzano S. and Pelling A.E. Front. Sustain. Food Syst., May 17, 2019, the scaffolds disclosed therein are hereby incorporated by reference.

[0086] Thus, in certain embodiments of the present disclosure, the cell composition as defined above can comprise a scaffold.

[0087] There are many prior art references that describe various supports and cell suspension cultures, including WO2021102375A1, US20210106032A1, and WO2020243324A1, the supports and cell suspension cultures disclosed therein are hereby incorporated by reference.

[0088] Cells can be grown in a bioreactor, such as described in Madeline, B. et al., (2015. Culturing a duck ES-derived cell line in single-use bioreactors: A rapid, efficient, and cost-effective vaccine manufacturing system based on suspension culture. BioProcess International, 13.), or in a large-scale stirred tank bioreactor (STR), such as described in Eibl, R. et al. (2009. Cell and Tissue Reaction Engineering). Similarly, US 2011 / 0287508 discloses bioreactors and methods of using them to produce tissue engineering products or culture cells. In addition, WO2020222239A1 describes systems and methods for producing cultured food products (such as cultured meat) using multiple cell culture bioreactors, all of their disclosed bioreactors and methods of use are incorporated herein by reference.

[0089] In certain embodiments of the invention, the cell composition as defined above can be grown in a bioreactor (such as a stirred bioreactor) or on a suspended microcarrier support that provides a solid surface on which the cells can attach and proliferate.

[0090] One of the greatest challenges in suspension cell culture is to prevent the sedimentation of the microcarrier support (such as beads or disks) and, if high stirring speeds are required, not to expose the cells acutely to shear forces. The optimal stirring conditions required for the microcarrier system must be determined experimentally and addressed on a case-by-case basis, as will be understood by those skilled in the art. An operating window needs to be determined within which the stirring rate allows sufficient microcarrier suspension without damaging the cells being cultured. The level of hydrodynamic shear must be balanced with the mixing of large volumes of liquid.

[0091] In one embodiment, cell growth and morphology as well as metabolite profiles are monitored.

[0092] According to the method according to the present disclosure, cells can be contacted with the at least one exogenous supplement composition in a manner commonly known in the art. For example, by adding a required amount of the exogenous supplement composition to a culture medium for culturing cells derived from non-human animals, the cells can be contacted with the exogenous supplement composition in the cell culture medium. The supplement composition can be added in a single dose at a single time point, or portions of the supplement composition can be added sequentially over time during the cell culture process. It can also be prepared before adding the enriched medium (which is a culture medium supplemented with the exogenous supplement composition) to the cells. The culture medium thus obtained is the enriched culture medium according to the present invention.

[0093] In other embodiments, the cells can be separated from a growth medium (such as a ready-to-use medium or a custom medium) and then contacted with at least one exogenous supplement composition. Using a suitable dilution medium, such as an isotonic solution preferably containing only the exogenous supplement or the exogenous supplement composition, the exogenous supplement composition can be provided at the desired dilution. Preferably, the isotonic solution contains only food-grade products and optionally does not contain growth factors, FBS, vitamins, antibiotics, etc.

[0094] When culturing cells to grow, proliferate or differentiate in the presence of a scaffold, the scaffold can be a scaffold rich in one or more exogenous supplements, and the exogenous supplements are contained in a carrier, such as, but not limited to, a nanocarrier, nanoparticle, micelle, liposome or vesicle composition, optionally before the scaffold is used for cell culture. An enriched scaffold (i.e., a scaffold supplemented with the exogenous supplement composition) can also be prepared before adding the cells to the scaffold. The scaffold thus obtained is the enriched scaffold according to the present disclosure.

[0095] For example, but not limited to, at least one exogenous supplement contained in a carrier (such as, but not limited to, a nanocarrier, nanoparticle, micelle, liposome or vesicle composition) can be contacted with the scaffold for a suitable period of time considered suitable for enriching the scaffold with the supplement composition, such as at least 1 minute, at least 5 minutes, at least 30 minutes, at least 1 hour or at least 10 hours before contacting the scaffold with the cells.

[0096] The contact of the at least one exogenous supplement composition with cells derived from non-human animals can be carried out at any stage of cell growth, proliferation or differentiation. The exogenous supplement composition can also be added to the harvested cells at the end of the culture growth.

[0097] In certain embodiments of the present disclosure, the step of contacting the cells with one or more exogenous supplement compositions can be carried out when the cell division rate is high. Doing so can achieve a more intense flavor, aroma or other sensory effects, such as masking off-flavors.

[0098] Cell growth represents an increase in the total mass of a cell (including the volume of cytoplasm, nucleus, and organelles). Cell growth occurs when the overall rate of cell biosynthesis (production or anabolism of biomolecules) is greater than the overall rate of cell degradation.

[0099] Cell division (or cell proliferation) is the process by which a mother cell divides into two or more daughter cells. The rate of cell division varies depending on the cell line or type.

[0100] Cell growth and cell proliferation may occur simultaneously.

[0101] Cells may also differentiate. Cell differentiation is the process by which a cell changes from one cell type to another. Typically, the cell changes to a more specialized type.

[0102] In certain embodiments of the present disclosure, the step of contacting a cell with one or more exogenous supplement compositions may be carried out during cell growth, proliferation, and / or differentiation.

[0103] Any known method in the art can be used to measure the cell growth and proliferation rate, for example, using a cell division marker as described in Bernard S. et al. (Analysis of Cell Kinetics Using a Cell Division Marker: Mathematical Modelling of Experimental Data, May 2003; 84(5): 3414 - 3424), which is incorporated herein by reference.

[0104] In certain embodiments of the present disclosure, the step of contacting a cell with one or more exogenous supplement compositions may be carried out when the cell division rate is low. Doing so can achieve a more intense flavor, aroma, or other sensory effects, such as masking off - flavors.

[0105] In certain embodiments of the present disclosure, the step of contacting a cell with one or more exogenous supplement compositions may be carried out when the cell differentiation rate is low. Doing so can achieve a more intense flavor, aroma, or other sensory effects, such as masking off - flavors.

[0106] In certain embodiments of the present disclosure, the step of contacting a cell with one or more exogenous supplement compositions may be carried out when the cell differentiation rate is high. Doing so can achieve a more intense flavor, aroma, or other sensory effects, such as masking off - flavors.

[0107] In certain embodiments of the present disclosure, the step of contacting the cells with one or more exogenous supplement compositions can be carried out at a low cell growth rate. Doing so can achieve a more intense flavor, aroma, or other sensory effects, such as masking off-flavors.

[0108] In certain embodiments of the present disclosure, the step of contacting the cells with one or more exogenous supplement compositions can be carried out at a high cell growth rate. Doing so can achieve a more intense flavor, aroma, or other sensory effects, such as masking off-flavors.

[0109] According to certain exemplary embodiments, the step of contacting the cells with one or more exogenous supplement compositions is carried out on cells having a substantially constant glucose uptake rate (GUR).

[0110] According to certain embodiments, the step of contacting the cells with one or more exogenous supplement compositions can be facilitated using an additional step that promotes the incorporation of the exogenous supplement into the cells.

[0111] According to certain embodiments, the contacting of the cells with one or more exogenous supplement compositions is carried out for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, such as at least 4 days, although the exact time may vary depending on the cell type and cell line and will be determined based on the desired improvement or modification of the flavor and aroma.

[0112] According to one exemplary embodiment, the step of contacting the cells with one or more exogenous supplement compositions is carried out between 30 minutes and 2 days, more preferably between 1 and 24 hours.

[0113] In one embodiment, the cells are contacted with at least one exogenous supplement composition such that at least 30% of the cells contain at least one exogenous supplement, such as at least 40% of the cells, at least 50% of the cells, at least 60% of the cells, at least 70% of the cells, at least 80% of the cells, at least 90% of the cells, at least 95% of the cells, such as at least 99% of the cells contain at least one exogenous supplement composition.

[0114] In one embodiment, cells of non-human animal origin are contacted with at least one exogenous supplement composition for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, such as at least 4 days or at least 1 week.

[0115] According to an exemplary embodiment, a non-human animal cell is contacted with at least one or more exogenous supplement compositions for at least 30 minutes and at most 2 days, more preferably at least 1 hour and at most 24 hours.

[0116] The exogenous supplement composition used according to the present disclosure may comprise an exogenous supplement, which may be any ingredient suitable for human consumption and which may impart, modify or improve the flavor of the matrix to which it is applied.

[0117] This may be a substance that inherently has the desired flavor, or it may be a flavor precursor or a substance that enhances other flavors. It may be a specific chemical, or it may be a complex composition obtained from materials of plant, animal or microbial origin by appropriate physical, chemical, enzymatic or microbial processes. Alternatively, the supplement may be a flavor precursor, i.e., a substance that, although it does not itself have the desired flavor, can be converted into a substance having the desired flavor in response to a suitable stimulus (such as heat treatment, physical treatment) or as a result of a chemical, enzymatic or microbial process. For example, the flavor precursor may be a mixture of various materials, such as one material containing an amino functional group and another material being a reducing sugar, which can react under heat stimulation to form a heat-reaction flavor through a series of complex consecutive and / or competing reactions, such as the Maillard reaction, Schiff base formation, Strecker degradation, caramelization reaction and / or other reactions conducive to flavor and / or color generation, all of which are well known to those skilled in the art. Alternatively, metabolites formed during cell growth, proliferation or differentiation may increase the desired flavor or may be precursors of the desired flavor in response to the above-mentioned appropriate stimuli.

[0118] The amino acid / amine source may be selected from cysteine, methionine, alanine, glycine, lysine, arginine, histidine, tryptophan, proline, valine, glutamic acid, glutamine, aspartic acid, glutathione, other sulfur-containing peptides, HVP (peanut, soybean, wheat / corn gluten), other hydrolyzed proteins (such as those derivable from milk, egg, fish, blood, liver, bone, collagen), yeast extract, autolyzed yeast, meat extract, taurine, pyrrolidone carboxylic acid and combinations thereof.

[0119] Reducing sugars are those that have an aldehyde group or are capable of forming an aldehyde group in solution through isomerization. The aldehyde group enables the sugar to act as a reducing agent in the Maillard reaction, which is important for the browning of many foods. The cyclic hemiacetal form of an aldose can open to expose the aldehyde, and certain ketoses can undergo tautomerization to become aldoses. Examples of reducing sugars include, but are not limited to: glucose, fructose, xylose, glyceraldehyde, galactose, lactose, arabinose, maltose, glucose polymers such as starch, hydrolyzed starch, and starch derivatives such as glucose syrup, maltodextrin, dextrin, and combinations thereof.

[0120] Reaction flavorants can produce a rich variety of flavoring materials that can be used to prepare baked, savory, poultry, and animal or meat flavors, including, but not limited to, ketopiperazines, piperazines, pyrrolazines, pyrazines, sulfides, thiols, and maltol derivatives, and mixtures thereof.

[0121] Flavorants, flavorant precursors, or flavorant enhancers are not intended to be foods per se; they are manufactured products intended to impart, modify, or enhance the flavor of cultured meat or edible products containing cultured meat. They are essentially non-nutritive, that is, their primary purpose is to impart flavor, or enhance, modify, or improve the flavor of the matrix to which they are added, rather than to provide nutrition.

[0122] The flavoring agent can be selected from the following components: 1-octen-3-ol, 1-octen-3-one, 2,3-dimethylpyrazine, 2,3-pentanedione, 2,4-decadienal, 2,4-nonadienal, 2,4-undecadienal, 2,5-dimethyl-3-furanthiol, 2-acetylfuran, 2-acetyl-2-thiazoline, 2-acetylthiazole, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-furfurylthiol, 2-methyltetrahydrofuran-3-one (coffee furanone), 2-methyl-3-furanthiol, 2-methyl-3-tetrahydrofuranthiol, 2-octen-4-one, 2-pentylfuran, 2-tridecanone, 3-mercapto-2-butanone, 4,5-epoxy-2-decenal, 4-mercapto-4-methyl-2-pentanone, 5-methylfurfural, acetoin, acetyl-2-pyrazine, 4-methylcaprylic acid, alanine, anserine, arachidonic acid, arginine, bis(2-methyl-3-furyl) disulfide, butyric acid, methylcyclopentenolone, cysteine, damascenone, δ-decalactone, capric acid, dihydroxyacetone, dimethyldisulfide, disodium guanylate, disodium inosinate, δ-dodecalactone, ethyl humulone (Emoxyfurone), ethyl 3-methyltetracid; 4-, NAT, 1-PG (maple furanone), ethyl oleate, fructose, furaneol, furfural, furfuryl disulfide, furfurylthiol, furfuryl methyl disulfide, γ-nonalactone, glucose, glutathione, glycine, Glycogene, guaiacol, hexadecanoic acid (oleic acid), hexanal, hexanoic acid, histidine, indole, isobutylthiol, isobutyl-4-methyl-5-ethylthiazoline, isoleucine, isovaleraldehyde, leucine, linoleic acid, linolenic acid, lysine, 1-p-menthene-8-thiol (Mercapto-8Menthene-1para), mercapto-butanone, methional, methionine, methoxy-2-methylpyrazine, methyl-12-tridecenal, methyl-2-butyric acid, methyl-2-keto-3-tetrahydrothiophene, methylthiol, nonanal, phenylethyl alcohol, phenylethylthiol, phenylacetaldehyde, phenylalanine, plasmalogen, proline, ribose, skatole, sotolone, succinic acid, sulfurol, δ-tetradecalactone, thialdine, trans-2-nonenal, 6-nonenal, trans-2-octenal, trans-2-undecenal, trimethylpyrazine, 2,3,5-Nat, trithione, tryptophan, tyrosine, valeraldehyde, valine, xylose and mixtures thereof.

[0123] According to certain embodiments, the flavorant material (such as a flavorant, flavorant precursor, or flavorant enhancer) has a ClogP of at least -4, such as at least -3, such as at least -2, such as at least -1, such as at least 0, such as at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6.

[0124] According to certain embodiments, the flavorant or flavorant precursor has a ClogP between -4 and 6, preferably such as between -3 and 5, more preferably such as between -2 and 4, and most preferably such as between -1 and 3.

[0125] The calculated logP (ClogP) is a virtual partition coefficient that represents a non-experimental method for determining logP, which takes into account different correction factors and determines logP based on the fragment contributions of almost every atom of the compound. Briefly, regression techniques are used to model the experimentally verified logP values of compounds or compound fragments and added as a product of defined correction factors. ClogP can be calculated according to any method known in the art, such as the method disclosed in Klopman G. et al. (Mini-reviews in Medical Chemistry. 2005, 5, 127-133).

[0126] According to certain exemplary embodiments, the flavorant material (such as a flavorant, flavorant precursor, or flavorant enhancer) can be selected from materials having a ClogP of at least -4 to up to 6 (such as 12-methyltridecanal, 2,4-decadienal, 2,4-undecadienal, 2-acetyl-2-thiazoline, 2-pentylfuran, 2-tridecanone, bis(2-methyl-3-furyl) disulfide, damascenone, capric acid, δ-dodecalactone, ethyl oleate, furfuryl disulfide, hexadecanoic acid (oleic acid), isobutyl-4-methyl-5-ethylthiazoline, linoleic acid, linolenic acid, 1-p-menthene-8-thiol, nonanal, trithione, and combinations thereof).

[0127] In certain embodiments of the present disclosure, flavorants selected from the following can be used to produce a particularly interesting meaty flavor: thiazole sulfide, methanethiol, methylthiopropionaldehyde, isobutyl mercaptan, hexadecanoic acid (oleic acid), furanone, methylcyclopentenolone, acetoin, 2-acetylthiazole, 2-decenal, 2,6-nonadienal, and 1-octen-3-ol, and combinations thereof.

[0128] The present disclosure now unexpectedly discloses contacting cells with a culture medium and / or a scaffold, the culture medium and / or the scaffold comprising one or more flavorants contained in a carrier, the flavorants selected from Thialdine, methional, 1-p-menthene-8-thiol, indole, hexanal, furanone, acetoin, 4-mercapto-4-methyl-2-pentanone, 3-mercapto-2-butanone, 2-octen-4-one, 2-methyl-3-furanthiol, and 2,4-decadienal, 1-octen-3-ol, thereby producing cultured cells that can be used to prepare a cultured cell composition (such as cultured meat) having an improved meaty flavor.

[0129] According to certain exemplary embodiments, the flavorant material is selected from one or more or a combination of Thialdine, methional, 1-p-menthene-8-thiol, indole, hexanal, furanone, acetoin, 4-mercapto-4-methyl-2-pentanone, 3-mercapto-2-butanone, 2-octen-4-one, 2-methyl-3-furanthiol, and 2,4-decadienal and 1-octen-3-ol.

[0130] The present disclosure now unexpectedly discloses contacting cells with a culture medium and / or a scaffold, the culture medium and / or the scaffold comprising one or more of the following flavorant materials contained in a carrier: such as thiazole sulfide, 1-p-menthene-8-thiol, hexanoic acid, furfuryl mercaptan, furanone, 2,3-butanedithiol, acetoin, 2-methyltetrahydrofuran-3-one (coffee furanone), 2-methyl-3-furanthiol, and 2-acetylthiazole, or a combination thereof, thereby producing cultured cells that can be used to prepare cultured meat having an improved meaty flavor.

[0131] The present disclosure now unexpectedly discloses contacting cells with a culture medium and / or a scaffold, the culture medium and / or the scaffold comprising one or more of the following flavorant materials: myristic acid, skatole, methional, 2-undecanal, 2-undecenal, 2-octenal, 2-nonenal, 6-nonenal, 2-methyl-3-furanthiol, 2-decenal, 2,4-decadienal, 2,4-nonadienal, 12-methyltridecanal, thereby producing cultured cells that can be used to prepare cultured meat having an improved meaty flavor.

[0132] Flavoring agents or meat-like flavors and aromas used in the preparation may include a sulfur source. The sulfur source may be selected from hydrogen sulfide, cysteine, cystine, methionine, glutathione, thiamine, inorganic sulfides, organic thiols and sulfides, 2-mercaptoethanol derivatives (such as mercaptoacetaldehyde and / or its dimer 2,5-dihydroxy-1,4-dithiane), 5-hydroxy-3-mercapto-2-pentanone, 3-mercaptopropan-1-ol, 4,5-substituted thiazoles, thiocarbonates, thioamides, 2-mercaptoalkanoic acids / amides, mercaptoalkylamines, aminosulfides, S-acetylmercaptosuccinic acid, plant extracts, fermented vegetable juices, yeast extracts, autolyzed yeast, egg white, meat extracts and combinations thereof.

[0133] Taking into account certain physicochemical parameters, such as the CLogP, molecular weight and functional groups of the exogenous supplement, certain fragrances or compounds intended to be used as exogenous supplements may preferentially be transported through a specific carrier material or carrier system.

[0134] In a preferred embodiment, wherein the carrier is a liposome, the exogenous supplement may be selected from vitamin B12, succinic acid, butyric acid, lactic acid, cysteine, thiamine, 2,4-decadienal, 2-hexenal, methylcyclopentenolone and combinations thereof.

[0135] In a preferred embodiment, wherein the carrier is a nanoparticle or nanocarrier, the exogenous supplement may be selected from arachidonic acid, 12-methyltridecanal, linoleic acid, 2-pentylfuran, 4-methyloctanoic acid, 2,4-decadienal and combinations thereof.

[0136] In a preferred embodiment, wherein the carrier is a pellet as described herein, the exogenous supplement may be selected from vitamin B12, succinic acid, butyric acid, lactic acid, cysteine, thiamine, 2,4-decadienal, 2-hexenal, methylcyclopentenolone, arachidonic acid, 12-methyltridecanal, linoleic acid, 2-pentylfuran, 4-methyloctanoic acid, Thialdine, furanone, disodium guanylate, lactic acid, leucine, methionine, disodium inosinate, ribose, xylose, glucose, glutamic acid and combinations thereof.

[0137] Yeast extract can also be used to add flavor. Yeast extract may consist of natural components from yeast cells: proteins, amino acids, carbohydrates, vitamins, and minerals. To produce yeast extract, the contents of yeast cells are broken down with enzymes and the cell walls are removed. Yeast extract is a food ingredient that contains many flavor-providing components, including glutamate and its derivatives. It has various flavor-enhancing properties. Yeast extract is a common flavor source for a range of savory food products, especially when a meaty flavor is desired (Ames JM and Elmore JS. 1992. FlavorFragr.J 7:89-103).

[0138] According to certain embodiments, the enrichment medium and / or scaffold comprises yeast extract at a concentration of from about 10 pg / ml to about 5 g / ml. According to certain exemplary embodiments, the enrichment medium and / or scaffold comprises yeast extract at a concentration of from about 50 pg / ml to about 1 g / ml. According to certain exemplary embodiments, the enrichment medium and / or scaffold comprises yeast extract at a concentration of from about 500 pg / ml to 50 mg / ml.

[0139] A complete flavor composition can comprise flavor precursors, aroma volatiles, and other ingredients commonly known in the art for generating flavor, such as other synergists or enhancers, including fats or fatty acids, or their sources, herbs, spices, etc.; pH regulators; inorganic salts; flavor masking agents, taste sensors; vitamins; dyes; colorants; pigments, etc.

[0140] Examples of flavor enhancers and their sources include MSG, IMP, GMP, autolyzed yeast, HVP, 2-furfuryl-thioinosine-5'-phosphate, 2-allyloxyinosine-5'-phosphate, 2-(lower alkoxy)inosine-5'-phosphate, 2-benzylthioinosine-5'-phosphate, 4-glucosyl gluconic acid, and cyclotene.

[0141] Examples of pH regulators include mono-, di-, and tri-basic inorganic acids such as HCl, sulfuric acid, and phosphoric acid, organic acids including succinic acid, citric acid, lactic acid, malic acid, tartaric acid, acetic acid, and propionic acid; amino acids including valine, glycine, and glutamic acid.

[0142] Examples of fats include the fats of beef, chicken, coconut, other triglycerides, fatty acids, and their esters.

[0143] Examples of inorganic salts include chlorides and phosphates.

[0144] It may also be possible to employ flavor masking agents to mask any off-flavors associated with the basic contents of the cultured meat. Flavor masking agents include, but are not limited to, dihydrochalcone, nucleotides, sodium salts, hydroxyflavanone, etc.

[0145] Taste sensates may also be employed. Taste sensates include pungent flavors, substances that induce salivation, substances that cause a warming or tingling sensation, and cooling active ingredients. Examples of pungent flavors and / or substances that induce salivation and / or substances that cause a warming and / or tingling sensation on the skin or mucosa are capsaicin, dihydrocapsaicin, gingerol, shogaol, zingerone, piperine, carboxylic acid-N-vanillylamides (e.g., but not limited to, nonanoic acid-N-vanillylamide, pellitorin, or spilanthol), 2-nonanamide (e.g., but not limited to, 2-nonanoyl-N-isobutylamide, 2-nonanoyl-N-(4-hydroxy-3-methoxyphenyl)amide), alkyl ethers of 4-hydroxy-3-methoxybenzyl alcohol (e.g., but not limited to, 4-hydroxy-3-methoxybenzyl-n-butyl ether), alkyl ethers of 4-acetyloxy-3-methoxybenzyl alcohol (e.g., but not limited to, 4-acetoxy-3-methoxybenzyl-n-butyl ether and 4-acetoxy-3-methoxybenzyl-n-hexyl ether), alkyl ethers of 3-hydroxy-4-methoxybenzyl alcohol, alkyl ethers of 3,4-dimethoxybenzyl alcohol, alkyl ethers of 3-ethoxy-4-hydroxybenzyl alcohol, alkyl ethers of 3,4-methylenedioxybenzyl alcohol, (4-hydroxy-3-methoxyphenyl)acetamides (e.g., but not limited to, (4-hydroxy-3-methoxyphenyl)acetic acid-N-n-octylamide), vanillic-mandelic acid alkylamides, ferulic acid-phenethylamides, nicotinaldehyde, methyl nicotinate, propyl nicotinate, 2-butoxyethyl nicotinate, benzyl nicotinate, 1-acetoxychavicol, polygonal aldehyde, and isodrimeninol.

[0146] Natural extracts of pungent flavors and / or natural extracts that cause a warming and / or tingling sensation on the skin or mucosa and can be components of a complete flavoring composition include: paprika extract, pepper extract (e.g., capsicum extract), chili pepper extract, ginger root extract, Aframomum melegueta extract, Spilanthes acmella extract, Kaempferia galanga extract, or Alpinia galangal extract.

[0147] Depending on the flavor profile the flavorist wishes to achieve, the complete flavorant composition may additionally contain one or more of the following ingredients: dimethyl sulfide, ethyl acetate, ethyl propionate, methyl butyrate, and ethyl butyrate; flavor oils containing volatile aldehydes or esters, including, for example, cinnamyl acetate, cinnamaldehyde, citral, diethyl acetal, dihydrocarvyl acetate, eugenyl formate, and p-methylanisole.Other examples of volatile compounds that may be present in a flavor oil include oleoresins, essential oils, caraway oil, clove oil, onion oil, pepper oil, rosemary oil, spearmint oil, allium spices, garlic, leek, chive, and onion, plant extracts, proteolytic hydrolyzates, hydrolyzed vegetable proteins, meat protein hydrolyzates, milk protein hydrolyzates, and natural and artificial compound flavorings, including those disclosed in S. Heath, Source Book of Flavors, Avi Publishing Co., Westport Connecticut, 1981, pages 149 - 277; valerian oil; 3,4 - dimethoxyphenol; amyl acetate; pentyl cinnamate, butyrolactone; furfural; trimethylpyrazine; phenylacetic acid; isovaleraldehyde; ethyl maltol; ethyl vanillin; ethyl valerate; ethyl butyrate; cocoa powder extract; coffee extract; peppermint oil; spearmint oil; clove oil; anethole; cardamom oil; wintergreen oil; cinnamaldehyde; ethyl - 2 - methyl valerate; γ - hexenolactone; 2,4 - decadienal; 2,4 - heptadienal; methyl thiazolol (4 - methyl - 5 - β - hydroxyethyl thiazole); 2 - methylbutanethiol; 4 - mercapto - 2 - butanone; 3 - mercapto - 2 - pentanone; 1 - mercapto - 2 - propane; benzaldehyde; furfural; furfuryl alcohol; 2 - mercaptopropionic acid; alkylpyrazines; methylpyrazine; 2 - ethyl - 3 - methylpyrazine; tetramethylpyrazine; polysulfides; dipropyl disulfide; methyl benzyl disulfide; alkylthiophenes; 2,3 - dimethylthiophene; 5 - methylfurfural; acetylfuran; 2,4 - decadienal; guaiacol; phenylacetaldehyde; β - decalactone; D - limonene; acetoin; amyl acetate; maltol; ethyl butyrate; levulinic acid; piperonal; ethyl acetate; n - octanal; n - valeraldehyde; n - hexanal; diacetyl; monosodium glutamate; monopotassium glutamate; sulfur - containing amino acids, e.g., cysteine; hydrolyzed vegetable protein; 2 - methylfuran - 3 - thiol; 2 - methyldihydrofuran - 3 - thiol; 2,5 - dimethylfuran - 3 - thiol; hydrolyzed fish protein; tetramethylpyrazine; propyl allyl disulfide; propyl allyl trisulfide; diallyl disulfide; diallyl trisulfide; di - propenyl disulfide; di - propenyl trisulfide; 4 - methyl - 2 - [(methylthio) - ethyl] - 1,3 - dithiolane; 4,5 - dimethyl - 2 - (methylthiomethyl) - 1,3 - dithiolane; and 4 - methyl - 2 - (methylthiomethyl) - 1,3 - dithiolane.

[0148] Flavoring materials (e.g., but not limited to, those that can be used to flavor farmed poultry meat) include, but are not limited to, 2-methylbutanal, methylpyrazine, 2,5-dimethylpyrazine, 2,3-dimethylpyrazine, trimethylpyrazine, 2-methyl-3-furanthiol, 2-acetylpyrrole, furanone, nor-furaneol, thiazole, proline-valine diketopiperazine, proline-isoleucine diketopiperazine, 3-methylbutanal, 1,2-dimercaptoethane, 2-(1-mercaptoethyl)furan, 3-mercapto-2-butanone, 2-mercapto-3-pentanone, 3-mercapto-hexan-4-one, phenylacetaldehyde, 4-methylpentanoic acid, 5-hydroxy-5,6-dihydromaltol, 2-methylthiazolidine, 2-isopropylthiazolidine, 2-isobutylthiazolidine, 2-sec-butyl-thiazolidine, 4,5-dimethylthiazole, 2,5-dimethyl-3-furanthiol, 2-methyl-3-mercaptothiophene, 2-methyl-5-(1-mercaptomethyl)-thiophene, 2-mercaptopropionic acid, valine-valine diketopiperazine, valine-alanine diketopiperazine, proline-alanine diketopiperazine, and proline-leucine diketopiperazine.

[0149] Other ingredients include aldehyde and ketone sources, including acetaldehyde, propionaldehyde, butyraldehyde, methyl propionaldehyde, C3 to C5 alkanals, HVP, α-diketones and their sources, including butanedione, pentane-2,3-dione, pyruvaldehyde, pyruvic acid, glyceraldehyde, glyoxal, dihydroxyacetone, α-ketobutyric acid, heptane-3,4-dione-2,5-diacetate, HMFone, HDFone and related derivatives, ascorbic acid, 5-ketogluconic acid, cyclotene, maltol, lactic acid, glycolic acid, malic acid, tartaric acid, and protein hydrolysates.

[0150] Flavoring materials (e.g., but not limited to, those that can be used to flavor farmed red meat) include, but are not limited to, 2-methylbutanal, 2-methyl-1-butene-1-thiol, 2-methyl-3-furanthiol, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3-dimethylpyrazine, 2-methyl-4,5-dihydrofuran-3-thiol, trimethylpyrazine, furfuryl mercaptan, 2-acetylpyrrole, furanone, nor-furaneol, thiazole, proline-valine diketopiperazine, proline-isoleucine diketopiperazine, 3-methylbutanal, 3-mercapto-2-butanone, 2-ethyl-6-methylpyrazine, 2-methylthiazolidine, 2-isopropylthiazolidine, 2-isobutylthiazolidine, maltol, and proline-leucine diketopiperazine.

[0151] Other components include aldehyde and ketone sources, including acetaldehyde, propionaldehyde, butyraldehyde, methyl propionaldehyde, C3-C5 alkanals, HVP, α-diketones and their sources, including butanedione, pentane-2,3-dione, pyruvaldehyde, pyruvic acid, glyceraldehyde, glyoxal, dihydroxyacetone, α-ketobutyric acid, heptane-3,4-dione-2,5-diacetate, HMFone, HDFone and related derivatives, ascorbic acid, 5-ketogluconic acid, cyclotene, maltol, lactic acid, glycolic acid, malic acid, tartaric acid, and protein hydrolysates.

[0152] According to certain embodiments, the exogenous supplement composition is added to the culture medium and / or scaffold at a concentration of about 100 pg / ml to about 5 mg / ml, such as about 1000 pg / ml to about 100 ng / ml, such as about 10 ng / ml to about 100 ng / ml, such as about 100 ng / ml to about 5 mg / ml, such as about 100 ng / ml to about 1 mg / ml.

[0153] According to certain embodiments, the supplement composition is added to the culture medium and / or scaffold to provide a total concentration of about 5 ng / ml to about 500 ng / ml.

[0154] According to certain embodiments, the supplement composition is added to the culture medium and / or scaffold to provide a concentration of about 0.1 pM to about 500 mM, more specifically about 0.1 nM to about 200 nM, and even more specifically about 1 mM.

[0155] The values mentioned herein refer to the amount of a single supplement, or in the case of using a mixture of supplements, refer to the sum of the supplement mixture in the supplement composition.

[0156] In certain embodiments, the cells are further contacted with at least one additional exogenous supplement composition selected from vitamins, minerals, yeast extracts, bioactive compounds, bacterial extracts, pigments, nutrients, texture additives, and any combination thereof.

[0157] A vitamin is an organic molecule (or a group of chemically related molecules, i.e., provitamins) that is an essential micronutrient required by organisms in small amounts to maintain the normal functions of their metabolism. Essential nutrients cannot be synthesized in organisms (either not at all or in insufficient amounts) and must therefore be obtained through the diet. Vitamins that may come into contact with cells include: vitamin A (as all-trans-retinol, all-trans-retinyl-esters, and all-trans-β-carotene and other provitamin A carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid or folate), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (calciferol), vitamin E (tocopherols and tocotrienols), and vitamin K (phylloquinone and menaquinones) and combinations thereof.

[0158] A colorant is a substance added or applied to change the color of a material (in this case, a cell). Colorants used in the present disclosure include natural colorants and synthetic colorants. According to certain exemplary embodiments, the colorant is a food-grade colorant.

[0159] As used herein, the term “natural colorant” refers to pigments that can be found in natural sources (including plants, algae, fungi, etc.). It should be clearly understood that the natural colorants of the present invention can be derived from natural sources or can be chemically synthesized. Examples of colorants include, but are not limited to, carotenoids, caramel color (e.g., Class IV E150d), caramelized fruit and vegetable juice concentrates, anthocyanin-containing colorants, phycobilin-containing colorants, betaine-containing colorants, betacyanin-containing colorants, and any mixtures thereof.

[0160] Anthocyanins can be present as extracts obtained or obtainable from plants of the Brassicaceae family (such as Raphanus sativus L. (radish)), Rosaceae family (such as Fragaria (strawberry)), Solanaceae family (such as Solanum tuberosum (red potato)), Convolvulaceae family (such as Ipomoea batatas (purple sweet potato)), Apiaceae family (such as Daucus carota sativus subsp. atrorubens Alef var. (black carrot)) or mixtures thereof.

[0161] In one embodiment, the phycobilin may be present as an extract obtained or obtainable from cyanobacteria of the species Arthrospira platensis, A. fusiformis or A. maxima. Optionally, the phycobilin is obtained or obtainable from Arthrospira platensis (Spirulina).

[0162] In one embodiment, the betalain may be present as an extract obtained from or obtainable from plants of the family Amaranthaceae. Optionally, the plant of the family Amaranthaceae may be Beta vulgaris (beet).

[0163] Anthocyanin glycosides are glycosides of sugar-free anthocyanidins (aglycones). The sugar molecule in anthocyanin glycosides is bound via an O-glycosidic bond to one or more hydroxyl groups that are usually present in the anthocyanidin molecule. Most naturally occurring anthocyanin glycosides are 3-O-glycosides.

[0164] In one embodiment, the anthocyanin glycoside is a pigment derived from black carrots.

[0165] Phycobilins are light-harvesting pigments found in cyanobacteria, but they are not present in higher plants. The basic structure of phycobilins consists of a tetrapyrrole unit in which four pyrrole rings form an open chain. There are mainly four phycobilins in photosynthetic organisms, namely phycoerythrobilin, phycocyanobilin, phycoerythrocyanin and phycourobilin. Differences in the degree of π-electron conjugation result in different absorption spectral properties and colors of the chromophores. Phycoerythrobilin is red, phycocyanobilin is blue, phycoerythrocyanin is purple, and phycourobilin is yellow.

[0166] In one embodiment, the phycobilin is a pigment derived from the genus Spirulina. Optionally, the phycobilin in the present invention may be phycocyanobilin, which has a blue color.

[0167] Betalains are a class of red and yellow tyrosine-derived pigments present in plants of the order Caryophyllales, where they replace anthocyanin glycoside pigments. There are two types of betalains:

[0168] a) Betacyanins, which are pale red to violet. Examples of betacyanins present in plants include betanin, isobetanin, probetanin and neobetanin; and

[0169] b) Betaxanthins, which are yellow to orange. Betaxanthins present in plants include indicaxanthin, miraxanthin, portulaxanthin and phyllocactin.

[0170] Thus, the betalain used in the present disclosure may be betacyanin, such as betanin, isobetanin, probetanin, and neobetanin; and / or betaxanthin, such as indicaxanthin, miraxanthin, portulaxanthin, and phyllocactin.

[0171] In one embodiment, the betalain is a beetroot-derived pigment. Optionally, the betalain used in the present invention may be betanin.

[0172] Carotenoids (also known as tetraterpenoids) are yellow, orange, and red organic pigments produced by plants and algae, as well as several bacteria and fungi. Carotenoids give pumpkins, carrots, corn, tomatoes, canaries, flamingos, salmon, lobsters, shrimp, and daffodils their distinctive colors. In one embodiment, the carotenoid is a carrot-derived pigment. In one embodiment, the carotenoid is a Dunalliela-derived pigment.

[0173] According to an exemplary embodiment, a colorant (such as a natural colorant) provides brown, red, pink, or orange.

[0174] According to certain embodiments, the enriched medium and / or enriched scaffold contains a pigment at a concentration of about 10 pg / ml to about 5 g / ml. According to certain exemplary embodiments, the enriched medium and / or enriched scaffold contains a pigment at a concentration of about 50 pg / ml to about 1 g / ml. According to certain exemplary embodiments, the enriched medium and / or enriched scaffold contains a pigment at a concentration of about 500 pg / ml to about 50 mg / ml.

[0175] Techniques known in the art and described in the examples below can be used to measure the concentration of the exogenous supplement composition in the medium, scaffold, and cells.

[0176] According to certain embodiments, the exogenous supplement composition or cell composition and cultured meat of the present disclosure, and their edible products contain a carrier, which may be selected from nanocarriers, nanoparticles, micelles, liposomes, or vesicles, preferably selected from nanoparticles, micelles, and liposomes, more preferably the carrier is a liposome, or most preferably the carrier is a liposome composed of phospholipids.

[0177] According to certain exemplary embodiments of the present invention, the exogenous supplement composition is colored or capable of producing color, such that its uptake from the culture medium into the cells and / or its arrival at the cell surface and / or its amount within or on the cell surface can be measured by a spectrophotometer at wavelengths specific to each supplement and such supplement compositions. Minerals include, but are not limited to, calcium, phosphorus, potassium, sodium, magnesium, sulfur, iron, chlorine, cobalt, copper, zinc, manganese, molybdenum, iodine, and selenium.

[0178] Optionally, the method according to the present disclosure may further comprise the step of separating any excess supplement or supplement composition from the cells once the cells have incorporated the exogenous supplement composition.

[0179] In certain embodiments, the cells are partially separated from the enriched culture medium and / or the enriched scaffold after contacting with the exogenous supplement composition for at least 1 minute, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 2 days, or at least 1 week.

[0180] According to certain exemplary embodiments, the cells are partially separated from the enriched culture medium and / or the enriched scaffold after at least 1 minute to at least 2 days, more preferably after at least 4 hours to at least 24 hours.

[0181] The cells can be separated from the enriched culture medium and / or the enriched scaffold in whole or in part by any method known in the art, such as centrifugation, filtration, decantation, fluorescence-activated cell sorting, or isopycnic sedimentation, and other methods. The cells can be separated from discontinuous or continuous cultures.

[0182] In certain embodiments, at least one exogenous supplement composition is present in the cells at sites selected from the intracellular space of the cells, the intramembrane space, on the membrane, and any combination thereof.

[0183] In certain embodiments, the cells are separated from the enriched culture medium and / or the enriched scaffold before the exogenous supplement composition is partially or completely metabolized.

[0184] According to certain exemplary embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% of the cells have not partially or completely metabolized at least one exogenous supplement, such as at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of cells of non-human animal origin have not partially or completely metabolized at least one exogenous supplement.

[0185] According to certain exemplary embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% of the cells have not partially or completely metabolized at least one exogenous supplement composition, such as at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of cells of non-human animal origin have not partially or completely metabolized at least one exogenous supplement composition.

[0186] The term "metabolize" shall cover any type of change that a compound undergoes through a chemical reaction, e.g., but not limited to, through a chemical degradation reaction, but shall also include changes in chemical or biological activity. The term metabolite shall cover products derived from such reactions or changes in activity.

[0187] In certain embodiments, when at least 30% of the cells contain at least one exogenous supplement composition, such as at least 40% of the cells, at least 50% of the cells, at least 60% of the cells, at least 70% of the cells, at least 80% of the cells, at least 90% of the cells, at least 95% of the cells, at least 99% of the cells contain at least one exogenous supplement composition, the cells are separated from the enrichment medium and / or enrichment scaffold.

[0188] In accordance with the teachings of the present invention, any method known in the art can be used to measure the amount of exogenous supplement composition absorbed by the cells. For example, HPLC analysis can be performed on the percentage of the supplement incorporated into the cells according to the examples of the present disclosure. For example, the cells can be centrifuged to precipitate cell debris and membrane proteins. The centrifugation can be carried out at 200G to 500G, e.g., at 125G. The sample can be centrifuged for 1 to 20 minutes, e.g., 15 minutes, and the temperature can be 4°C. Then the supernatant is collected and can be dried, or the nanocarrier can be subjected to HPLC analysis after decomposition.

[0189] In certain embodiments, the method further comprises washing the cells in a water-based solution to remove the cell culture medium and the exogenous supplement composition not absorbed by the cells.

[0190] In certain embodiments, the cells can be dried with or without separating the cells from the enrichment medium and / or enrichment scaffold of the present invention.

[0191] Any method known in the art can be used, such as centrifugation, filtration, decantation, etc.

[0192] If cells grow on a scaffold, steps to disrupt the interaction between the cells and the scaffold surface may be required to separate the cultured meat from the scaffold. Various proteolytic enzymes are used to separate the cells from the scaffold surface, with trypsin being the most frequently used, which is a member of the serine protease family. Trypsin is produced from the zymogen trypsinogen secreted by pancreatic exocrine cells. Trypsin acts on the C-terminal side of lysine or arginine. Optimal activity is achieved at 37 °C, so pre-warmed trypsin can accelerate the separation (see, for example https: / / www.sigmaaldrich.com / CH / de / technical-documents / protocol / cell-culture-and-cell-culture-analysis / mammalian-cell-culture / cell-dissociation-with-trypsin ).

[0193] The cell composition thus obtained can be used directly or can be dried.

[0194] The obtained cells can be dried by any known method in the art, such as freeze-drying (Zhang et al., 2017 Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity. Scientific reports, 7(1): 6198-6198), drying by microwave treatment (Gd et al., 2013 Dry preservation of animal cells: state of the art in microwave processing. Cryo letters, 34: 203-204), oven drying, spray drying, adsorption on a solid support, or any other technique known in the art. The drying step can also represent thermal and physical methods for generating flavorants from flavorant precursors or colorants from colorant precursors.

[0195] The cell composition prepared according to the method described herein represents another aspect of the present disclosure.

[0196] The cell composition of the present disclosure comprises one or more cells, wherein at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90% of the cells contain at least one exogenous supplement contained in a carrier, and wherein the cell composition is characterized by having improved sensory properties, such as flavor, which is substantially similar to whole meat.

[0197] The cell compositions of the present disclosure are rich in one or more exogenous supplement compositions. In one embodiment, the cell composition comprises at least 10% of cells containing at least one exogenous supplement composition, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of cells containing at least one exogenous supplement composition.

[0198] The cell compositions of the present invention may comprise one or more types of cells. The cell compositions of the present invention may comprise harvested cells, cell aggregates, or structured 2D or 3D cultured meat tissues.

[0199] In certain embodiments, the exogenous supplement composition is present in the cells of the cell composition at a site selected from the intracellular space of the cell, the intramembrane space, on the membrane, and any combination thereof.

[0200] In certain embodiments, the cell compositions of the present disclosure further comprise at least one additional exogenous supplement composition, wherein the exogenous supplements are selected from vitamins, minerals, yeast extracts, bioactive compounds, bacterial extracts, pigments, texture additives, and any combination thereof. Examples of vitamins, minerals, yeast extracts, bacterial extracts, pigments, and texture additives are discussed above or below.

[0201] In certain embodiments, the cell compositions of the present disclosure further comprise at least one additional exogenous supplement composition, wherein the carrier is selected from nanocarriers, nanoparticles, micelles, liposomes, or vesicles, preferably selected from nanoparticles, micelles, and liposomes, more preferably the carrier is a liposome, and most preferably the carrier is a liposome composed of phospholipids.

[0202] According to certain embodiments, the cell composition comprises a plurality of cells containing a mineral (such as iron or its salt) in an amount of from about 0.01 mg / 100 g of cells to about 50 mg / 100 g of cells. According to certain embodiments, the mineral (such as iron or its salt) is present in the cell composition in an amount of from about 0.01 mg / 100 g of cells to about 40 mg / 100 g of cells. According to certain embodiments, the mineral (such as iron or its salt) is present in the cell composition in an amount of from about 0.05 mg / 100 g of cells to about 5 mg / 100 g of cells.

[0203] According to certain embodiments, the cell composition further comprises at least one vitamin (such as vitamin D). According to certain embodiments, the cell composition comprises a plurality of cells containing a vitamin (such as vitamin D) in an amount of from about 0.01 pg / 100 g of cells to about 150 pg / 100 g of cells. According to certain embodiments, the vitamin (such as vitamin D) is present in an amount of from about 0.1 pg / 100 g of cells to about 100 pg / 100 g of cells. According to certain embodiments, the amount of the vitamin D is from about 0.1 pg / 100 g of cells to about 15 pg / 100 g of cells.

[0204] In certain or preferred embodiments, the carrier comprising the exogenous supplement may have an average particle size of less than 500 nm, preferably less than 350 nm, more preferably less than 200 nm or between 100 nm and 200 nm, wherein the carrier is preferably selected from nanocarriers, nanoparticles, micelles, liposomes or vesicles. At these average particle sizes, the carrier comprising the exogenous supplement has a size suitable for being taken up or incorporated by cells.

[0205] As used herein, the term "(average) particle size" refers to the particle size of the exogenous supplement composition described herein. One skilled in the art can measure the particle size using known methods (such as dynamic light scattering).

[0206] In certain embodiments, the at least one exogenous supplement is a food-grade supplement (such as a food-grade flavoring, flavor precursor, food-grade coloring agent, food-grade texture additive, etc.).

[0207] In certain embodiments, the cell composition of the present disclosure contains at least one exogenous supplement in an amount of from about 0.01 mg / 100 g of cells to about 100 mg / 100 g of cells, more preferably from 0.05 mg / 100 g of cells to about 10 mg / 100 g or even more preferably from 0.1 mg / 100 g of cells to 5 mg / 100 g of cells.

[0208] In certain embodiments, the cell composition of the present disclosure contains at least one exogenous supplement composition in an amount of from about 0.05 mg / 100 g of cells to about 500 mg / 100 g of cells, more preferably from 0.1 mg / 100 g of cells to about 50 mg / 100 g or even more preferably from 0.5 mg / 100 g of cells to 25 mg / 100 g of cells.

[0209] In certain embodiments, the cell composition is selected from:

[0210] a) pluripotent stem cells (PSCs) and / or cells differentiated therefrom,

[0211] b) Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells of non-human animals and / or cells differentiated therefrom,

[0212] c) Embryonic stem cells (ESCs),

[0213] d) Satellite cells

[0214] e) Primary progenitor cells

[0215] and any combination thereof.

[0216] In certain embodiments, the cells are selected from muscle cells and their progenitors; adipocytes and their progenitors; stromal cells and their progenitors; endothelial cells and their progenitors; and any combination thereof.

[0217] In certain embodiments, the cells are derived from a non-human animal selected from cattle, sheep, pigs, poultry, reptiles, rodents, wild animals, shellfish, fish, and insects, and any combination thereof.

[0218] The one or more flavoring agent materials may be selected from 1-octen-3-ol, 1-octen-3-one, 2,3-dimethylpyrazine, 2,3-pentanedione, 2,4-decadienal, 2,4-nonadienal, 2,4-undecadienal, 2,5-dimethyl-3-furanthiol, 2-acetylfuran, 2-acetyl-2-thiazoline, 2-acetylthiazole, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-furfurylthiol, 2-methyltetrahydrofuran-3-one (cafefuranone), 2-methyl-3-furanthiol, 4-methylcaprylic acid, 2-methyl-3-tetrahydrofuranthiol, 2-octen-4-one, 2-pentylfuran, 2-tridecanone, 3-mercapto-2-butanone, 4,5-epoxy-2-decenal, 4-mercapto-4-methyl-2-pentanone, 5-methylfurfural, acetoin, acetyl-2-pyrazine, alanine, anserine, arachidonic acid, arginine, bis(2-methyl-3-furyl) disulfide, butyric acid, methylcyclopentenolone, cysteine, damascenone, δ-decalactone, capric acid, dihydroxyacetone, dimethyldisulfide, disodium guanylate, disodium inosinate, δ-dodecalactone, ethyl fenchylideneacetate, ethyl 3-methyltetracid; 4-,NAT,1-PG (maple furanone), ethyl oleate, fructose, furanone, furfural, furfuryl disulfide, furfurylthiol, furfuryl methyl disulfide, γ-nonalactone, glucose, glutathione, glycine, Glycogene, guaiacol, hexadecanoic acid (palmitic acid), hexanal, hexanoic acid, histidine, indole, isobutyl mercaptan, isobutyl-4-methyl-5-ethylthiazoline, isoleucine, isovaleraldehyde, leucine, linoleic acid, linolenic acid, lysine, 1-p-menthene-8-thiol, mercapto-butanone, methional, methionine, methoxy-2-methylpyrazine, methyl-12-tridecenal, methyl-2-butyric acid, methyl-2-keto-3-tetrahydrothiophene, methyl mercaptan, nonanal, phenylethyl alcohol, phenylethyl mercaptan, phenylacetaldehyde, phenylalanine, plasmalogen, proline, ribose, skatole, sotolon, succinic acid, thiazole, δ-tetradecalactone, thialdine, trans-2-nonenal, 6-nonenal, trans-2-octenal, trans-2-undecenal, trimethylpyrazine, 2,3,5-Nat, trithione, tryptophan, tyrosine, valeraldehyde, valine, xylose, and mixtures thereof.

[0219] According to certain embodiments, the exogenous supplement has a ClogP of at least -4, such as at least -3, such as at least -2, such as at least -1, such as at least 0, such as at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, or such as at least 6.

[0220] According to certain embodiments, the flavorant is selected from flavorants, flavorant precursors, or flavorant enhancers having a ClogP of at least -4 to up to 6 (such as 12-methyltridecanal, 2,4-decadienal, 2,4-undecadienal, 2-acetyl-2-thiazoline, 2-pentylfuran, 2-tridecanone, bis(2-methyl-3-furyl) disulfide, damascenone, decanoic acid, δ-dodecalactone, ethyl oleate, furfuryl disulfide, hexadecanoic acid (oleic acid), isobutyl-4-methyl-5-ethylthiazoline, linoleic acid, linolenic acid, 1-p-menthene-8-thiol, nonanal, trithione, or one or more thereof).

[0221] According to certain embodiments, the flavorant is selected from thiazole sulfide, methyl mercaptan, methional, isobutyl mercaptan, hexadecanoic acid (oleic acid), furanone, methylcyclopentenolone, acetoin, 2-acetylthiazole, 2-decenal, 2,6-nonadienal, 6-nonenal, 1-octen-3-ol, and combinations thereof.

[0222] According to certain embodiments, the flavorant is selected from thialdine, methional, 1-p-menthene-8-thiol, indole, hexanal, furanone, acetoin, 4-mercapto-4-methyl-2-pentanone, 3-mercapto-2-butanone, 2-octen-4-one, 2-methyl-3-furanthiol, 2,4-decadienal, 1-octen-3-ol, or one or more thereof.

[0223] According to certain embodiments, the flavorant is selected from thiazole sulfide, 1-p-menthene-8-thiol, hexanoic acid, furfuryl mercaptan, furanone, 2,3-butanedithiol, acetoin, 2-methyltetrahydrofuran-3-one (coffee furanone), 2-methyl-3-furanthiol, 2-acetylthiazole, or one or more thereof.

[0224] According to certain embodiments, the flavorant is selected from myristic acid, skatole, methional, 2-undecenal, 2-undecenal, 2-octenal, 2-nonenal, 6-nonenal, 2-methyl-3-furanthiol, 2-decenal, 2,4-decadienal, 2,4-nonadienal, 12-methyltridecanal, or one or more thereof.

[0225] In certain embodiments, at least one exogenous supplement is not metabolized in at least 60% of the cells, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells.

[0226] In certain embodiments, at least one exogenous composition is not metabolized in at least 60% of the cells, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the cells.

[0227] In certain embodiments of the present disclosure, the uptake of exogenous supplements in the cell composition is from about 0.01 mg / 100 g of cells to about 100 mg / 100 g of cells, preferably from 0.05 mg / 100 g of cells to about 10 mg / 100 g of cells.

[0228] In certain embodiments of the present disclosure, the uptake of exogenous supplement compositions in the cell composition is from about 0.05 mg / 100 g of cells to about 500 mg / 100 g of cells, preferably from 0.1 mg / 100 g of cells to about 50 mg / 100 g of cells.

[0229] In certain embodiments, the present disclosure provides cultured meat comprising a cell composition as defined herein, such as cultured poultry meat or cultured beef.

[0230] In certain embodiments, the present disclosure provides an edible composition comprising the cell composition defined herein mixed with other food-grade ingredients.

[0231] The cell compositions of the present disclosure can be used to prepare cultured meat or edible products containing cultured meat.

[0232] The cell composition in the form of cytoplasm can be further processed, for example, by drying and / or extrusion, and optionally mixed with food-grade ingredients such as meat analogs, such as plant proteins, and other food-grade excipients to form edible products such as chunks, minced meat, sausages, hamburgers, and other processed meat products. Alternatively, the cell composition can be in the form of structured 2D or 3D whole cultured meat. In addition, the cell composition can be used as a flavoring ingredient, for example, in bouillon cubes and soups, or can be used in the form of a powder or slurry, which is intended to be sprayed as a coating onto savory snacks.

[0233] In certain embodiments, the cell composition can be used in an edible product at a concentration of at least 0.1% w / w, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% w / w based on the weight of the edible product.

[0234] Food products cover the following general food categories as defined by the US Food and Drug Administration (FDA): bakery and baking mixes, including all ready-to-eat and ready-to-bake products, flours, and mixes that require preparation before consumption; beverages, alcoholic beverages, including malt beverages and cocktail mixes; beverages and beverage bases, non-alcoholic beverages, including only specialty or spiced teas, soft drinks, coffee substitutes, and fruit and vegetable flavored gelatin beverages; cheeses, including cottage and whey cheeses, creams, natural cheeses, grated cheeses, processed cheeses, spreadable cheeses, dip cheeses, and miscellaneous cheeses; chewing gums, including all forms; coffee and tea, including regular, decaffeinated, and instant types; condiments and seasonings, including regular sauces and spreads, olives, pickles, and relishes, but not including spices or herbs; confections and frostings, including candies and flavored frostings, marshmallows, baking chocolate, brown sugar, lumpsugar, rock candy, maple sugar, powdered sugar, and raw sugar; ingredients and other non-dairy products; egg products, including liquid, frozen, or dried eggs, and egg dishes made therefrom, i.e., egg rolls, egg foo young, egg salad, and frozen multi-egg meals, but not including fresh eggs; fats and oils, including margarine, salad dressings, butter, salad oil, shortening, and cooking oils; fish products, including all prepared main courses, salads, appetizers, frozen multi-course dishes, and spreads containing fish, shellfish, and other aquatic animals, but not including fresh fish; fresh eggs, including cooked eggs and egg dishes made only from fresh shell eggs; fresh fish, including only fresh and frozen fish, shellfish, and other aquatic animals; fresh meat, including only fresh or home-frozen beef or veal, pork, lamb or mutton, and home-made dishes, salads, appetizers, or sandwich spreads made therefrom; fresh poultry, including only fresh or home-frozen poultry and game birds, and home-made dishes, salads, appetizers, or sandwich spreads made therefrom; pasta products, including macaroni and noodle products, rice, and frozen multi-course dishes without meat or vegetables; gravies and sauces, including all meat sauces and gravies, and tomato, milk, butter, and specialty sauces; herbs, seeds, spices, seasonings, mixes, extracts, and flavorings, including all natural and artificial spices, mixes, and spice blends; meat products, including all meats and meat-containing dishes, salads, appetizers, frozen multi-meat meals, and sandwich spreads prepared commercially or at home using commercially processed meat; whole and skim milk, including only whole, low-fat, and skim liquid milk; dairy products, including flavored milk and milk beverages, powdered milk, ingredients, snack dips, spreads, weight control milk beverages, and other milk source products; vegetable protein products, including the "reconstituted vegetable protein" category of the National Academy of Sciences / National Research Council, and meat, poultry, and fish substitutes, analogs, and extender products made from vegetable protein;Poultry products, including all poultry and poultry-containing dishes, salads, appetizers, frozen multi-course poultry meals, and sandwich spreads prepared at home using commercially processed or commercially processed poultry; all commercially processed vegetables, vegetable dishes, frozen multi-course vegetable meals, and vegetable juices and blends; snacks, including potato chips, pretzels, and other novelty snacks; home-made soups, including meat, fish, poultry, vegetable, and home-made combination soups; soups and soup blends, including commercially prepared meat, fish, poultry, vegetable, and combination soups and soup blends;

[0235] In one embodiment, the cell compositions of the present disclosure are added to meat edible products (including fish, poultry) or plant-based edible products (such as, but not limited to, meat analogs) to enhance the sensory properties of the products (such as generating, altering, or improving the flavor, color, texture, and / or mouthfeel of the edible product). In one embodiment, the cell composition can be added to an edible product to provide a beef flavor. In certain embodiments, the cell composition can be added to an edible product to generate, alter, or improve a chicken flavor.

[0236] The following examples are provided to more fully illustrate certain embodiments of the present disclosure. However, they should not be construed as limiting the scope of the present disclosure in any way. Those skilled in the art can readily devise various variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0237] Figures 1-3 show the fluorescence intensity of C2C12 cells sourced from being cultured in complete medium and incubated with Nile Red-loaded liposomes for 1, 7, and 24 hours. Figures 4-6 similarly show the fluorescence intensity of C2C12 cells sourced from being cultured in DMEM and incubated with Nile Red-loaded liposomes for 1, 3, and 7 hours. Since the cells were thoroughly washed after incubation with the fluorescent liposomes, it can be concluded that the fluorescence signal originated from fluorescent substances adhered to or within the cells. Fluorescence measurements were always carried out under the same measurement protocol (such as exposure time, excitation wavelength, gain settings), enabling comparison between the figures. Increasing the incubation time and increasing the liposome concentration added to the cell culture both led to an increase in the measured fluorescence intensity. This indicates that uptake of the fluorescent substance is possible under both serum and serum-free conditions (CM and DMEM, respectively). Examples

[0238] 1. Liposomes

[0239] 1.1. General manufacturing procedures for obtaining liposomes

[0240] 1.2. Liposomes loaded with fluorescent markers

[0241] 1.3. Liposomes loaded with different model compounds

[0242] 1.3.1. Flavoring agents

[0243] 1.3.2. Colorants

[0244] 1.4. Liposome stability study

[0245] 2. Uptake of flavoring agents by cells - Cell preparation

[0246] 2.1. Growth conditions

[0247] 2.2.1 Liposome uptake - Serial dilution

[0248] 2.2.2 Liposome uptake - Cell imaging

[0249] 2.2.3 Liposome uptake and retention / metabolism of exogenous compounds (encapsulated in liposomes) in cells

[0250] 2.2.4 Viability of cells incorporating exogenous compounds (encapsulated in liposomes)

[0251] 3. Use of non - human animal cells derived from oviparous animals

[0252] 4. Use of non - human animal cells derived from stem cells harvested from umbilical cord, bone marrow, placenta or other tissues

[0253] 5. Nanoparticles

[0254] 5.1 Preparation of flavoring agent - loaded nanoparticles

[0255] 5.2 Stability data of flavoring agent - loaded nanoparticles

[0256] Materials:

[0257] 1,2 - Distearoyl - sn - glycero - 3 - phosphocholine (DSPC) was purchased from Avanti Lipids (Birmingham, AL, USA).

[0258] Cholesterol was purchased from Merck & Cie. (Schaffhausen, Switzerland).

[0259] Nile red was purchased from Merck & Cie. (Schaffhausen, Switzerland).

[0260] Curcumin was provided by Naturex SA (Givaudan SA, Switzerland).

[0261] Quinine was purchased from Merck & Cie. (Schaffhausen, Switzerland).

[0262] Dulbecco's Modified Eagle Medium (DMEM) containing sodium pyruvate and L-glutamine was purchased from ThermoFisher Scientific (Waltham, MA, USA).

[0263] Dulbecco's Phosphate Buffered Saline (DPBS) was purchased from ThermoFisher Scientific (Waltham, MA, USA).

[0264] DMEM plus: DMEM containing 4.5 g / L glucose, sodium pyruvate and L-glutamine was purchased from Gibco-ThermoFisher Scientific (Waltham, MA, USA) and supplemented with a 1% antibiotic-antimycotic solution (i.e., a solution of penicillin / streptomycin / amphotericin B) purchased from Merck & Cie. (Schaffhausen, Switzerland).

[0265] Complete Medium (CM): DMEM plus supplemented with 10% fetal bovine serum (FBS) purchased from Biowest (Riverside, MO, USA) meeting US standards.

[0266] Cell lines can be obtained from one of the suppliers listed in the following publication https: / / www.labome.com / method / Cell-Lines-Companies.html or initiated by one of the known methods such as Freshney's Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. A Capes-Davis, RI Freshney - 2021, Choi et al., 2021. comprehensive reviews in food science and food safety. Volume 20, Issue 1. Pages 1 - 1117. January 2021, Mehta et al., 2019, Adipogenesis from Bovine Precursors. Methods Mol Biol, 2019; 1889: 111 - 125.

[0267] C2C12 cells (a mouse myoblast cell line) were purchased from ECACC (European Collection of Authenticated Cell Cultures) through (Merck & Cie, Schaffhausen, Switzerland).

[0268] A 96-well plate with a transparent bottom and black sidewalls was purchased from ThermoFisher Scientific (Waltham, MA, USA).

[0269] Palm oil was provided by Givaudan Schweiz AG (Dubendorf, Switzerland).

[0270] Medium-chain triglycerides (MCTs) were provided by Givaudan Schweiz AG (Dubendorf, Switzerland).

[0271] Soybean lecithin was purchased from Lipoid AG (Steinhausen, Switzerland).

[0272] Polysorbate 80 was provided by Givaudan Schweiz AG (Avignon, France).

[0273] All other chemicals used were commercially available reagents or chemical grade products.

[0274] Example 1 - Liposomes

[0275] 1.1 General manufacturing procedure for obtaining liposomes with and without exogenous compounds for cell delivery.

[0276] Liposomes were constructed by the lipid film hydration method, specifically:

[0277] Step 1a : After acclimating to room temperature, phospholipids (such as lecithin or distearoyl phosphatidylcholine) and cholesterol were mixed with a solvent (such as a 70:30 v / v mixture of dichloromethane DCM and methanol MeOH).

[0278] Approximately 20 mg of total solid mass of phospholipid:cholesterol in different molar ratios of 50:50 to 100:0 was placed in a flask and mixed with 50 to 100 mL of a solvent (such as chloroform:methanol, 2:1).

[0279] Step 1b : Depending on the intended function of the liposomes, an exogenous compound solution was added to the dissolved lipids, with a weight ratio of exogenous compound to lipid of 1:500 to 1:1. The exogenous compound added at this stage was preferably a hydrophobic compound (such as cLogP > 2).

[0280] Step 1c : The mixture was stirred and agitated at room temperature until a clear solution was obtained.

[0281] Step 2:The lipid film of liposomes is formed on the flask wall by evaporating the organic solvent in a rotary evaporator at about 30 °C - 40 °C and a rotation speed of 60 - 100 rpm. First, the pressure is reduced to 300 mbar, and then the pressure is continuously and slowly reduced to about 10 mbar to avoid violent boiling. The resulting thin and opaque lipid film is flushed with nitrogen for one minute and then thoroughly dried overnight in a vacuum drying oven at 10 mbar - 100 mbar and 40 °C.

[0282] Step 3 :Then the lipid film is prepared for further use as follows: Add 5 mL of preheated water or 5 mL of the relevant buffer (such as PBS buffer, pH 7.4), and heat the flask in a water bath, pressurize it, and keep it at a temperature 5 °C higher than the lipid phase transition temperature, all at a rotation speed of 50 rpm. Hydrate the lipid layer for at least 1 hour and then store it at 4 °C for further experiments. If the encapsulated exogenous compounds have sufficient water solubility, they can be added to the hydration buffer in step 3.

[0283] Step 4 :To form unilamellar vesicles with a narrow size distribution, extrusion is utilized. Extrusion is carried out in an Avanti mini-extruder equipped with a polycarbonate membrane with a pore size of 100 nm. Actuate the extruder at a temperature 5 °C higher than the phase transition temperature of the lipid being processed (such as 65 °C) for at least 10 times.

[0284] After extrusion, the liposomes have a median diameter of less than 200 nm. Then the extruded liposomes can be stored at 4 °C until further use. If necessary, a PD Miditrap G-25 gel filtration column (Cytiva, Grens, Switzerland) can be used to remove unencapsulated exogenous compounds from the sample. Then the sample is filtered through a 220 nm sterile filter.

[0285] 1.2 Liposomes Loaded with Fluorescent Markers

[0286] Different liposomes are prepared according to the general manufacturing process listed in step 1a (including step 1b), and the fluorescent markers are:

[0287] Nile red, 108 μg, is added to the lipid / cholesterol solution as a 108 μL solution of 1 mg / mL in acetone.

[0288] Liposomes containing encapsulated fluorescent markers were purified by passing the preparation through a PD Minitrap desalting column containing Sephadex G-25 resin (Cytiva, commercially available from cytivalifesciences.com, Switzerland). Size-exclusion based separation resulted in the separation of the fluorescently labeled liposomes from the unencapsulated fluorescent markers. After column filtration, encapsulation of the fluorescent marker in the liposomes was confirmed by measuring fluorescence with a fluorescence microplate reader (Synergy H1 Hybrid Microplate Reader, BioTek, Agilent Technologies, Santa Clara, CA, USA).

[0289] 1.3 Liposomes Loaded with Different Model Compounds

[0290] 1.3.1 Liposomes Loaded with Flavor Compounds

[0291] All experiments and characterizations were done in three separate liposome preparations.

[0292] 52.2 mg of soy lecithin and 7.8 mg of cholesterol were weighed into a 50 mL round-bottom flask and dissolved in 5 mL of chloroform:methanol (70:30). A stock solution of the meat flavor 2,4-decadienal was prepared by dissolving 120 mg of 2,4-decadienal in 10 mL of pure ethanol. From the 2,4-decadienal stock solution, 83 μL was added to the soy lecithin and cholesterol solution, thereby adding 1 mg of 2,4-decadienal to the dissolved lipids. The mixture was dried by rotary evaporation under reduced pressure at 30 °C to obtain a dried lipid film. The lipid film was further dried in a vacuum drying oven at 40 °C for 4 h. Next, 5 mL of phosphate-buffered saline (PBS) preheated to 65 °C was added to the film, and the round-bottom flask was stirred in a 65 °C water bath for 1 h. During hydration, the mixture was vortexed for 1 min to accelerate hydration. The hydrated lipid mixture was extruded through a mini-extruder equipped with a 100 nm mesh polycarbonate membrane ( Extruded from Polar Lipids). The resulting liposomes were analyzed by dynamic light scattering (Zetasizer nano ZS90, Malvern), showing an average diameter of 142.1 ± 3.2 nm and a polydispersity index of 0.090 ± 0.011, indicating a narrow diameter distribution. The liposomes were filtered through a PD MiniTrap desalting column containing Sephadex G-25 resin (Cytiva, commercially available from cytivalifesciences.com, Switzerland) to remove unencapsulated flavoring compounds. The phospholipid concentration in the filtered liposome suspension was analyzed by the Stewart assay (John Charles Marshall Stewart, Colourimetric determination of phospholipids with ammonium ferrothiocyanate, Analytical Biochemistry, Volume 104, Issue 1, 1980, pp. 10-14).

[0293] Briefly, a reagent solution containing 0.1 M FeCl3 and 0.4 M NH4SCN was prepared. 50 μL of the liposome sample was added to 2 mL of the reagent solution, 0.45 mL of H2O, and 3 mL of chloroform. The mixture was vortexed and centrifuged to separate the aqueous and organic phases. The lower chloroform layer was removed with a Pasteur pipette and the absorption at 485 nm was analyzed. The liposome sample was compared with a standard curve of a soy lecithin phospholipid solution with a known concentration. The filtered liposome sample in this example contained 6.3 ± 2.4 mg / mL of phospholipids. The purified liposomes were diluted 10-fold in pure ethanol to disrupt the liposome membrane, and the flavoring concentration was measured by ultraviolet absorption at 282 nm. The liposome suspension prepared in this example contained 26.8 ± 2.5 μg / mL of 2,4-decadienal. Compared with the unfiltered liposomes (34.0 ± 3.2 μg / mL, including unencapsulated 2,4-decadienal), the encapsulation percentage was 78.7% ± 2.8%. The data for each individual batch can be seen in Table 1 below.

[0294] Table 1 - Liposomes Loaded with Flavoring Compounds

[0295]

[0296]

[0297] 1.3.2 Liposomes Loaded with Coloring Compounds

[0298] Different liposomes were prepared according to the general manufacturing process listed in step 1a (including step 1b), with different model compound markers as follows:

[0299] Curcumin, 100 μL of a 1 mg / mL solution in acetone was added to the lipid / cholesterol solution.

[0300] Quinine, 100 μL of a 1 mg / mL solution in ethanol was added to the lipid / cholesterol solution.

[0301] Quinine is a fluorescent flavorant material. Fluorescence microscopy and fluorescence microplate readers were used to quantify the intracellular quinine levels.

[0302] Liposomes containing encapsulated quinine and curcumin were purified by passing the formulation through a PD Minitrap desalting column containing Sephadex G-25 resin (Cytiva, commercially available from cytivalifesciences.com, Switzerland). Size-exclusion-based separation resulted in the separation of fluorescently labeled liposomes from unencapsulated fluorescent markers. After column filtration, encapsulation of the fluorescent marker in the liposomes was confirmed by measuring fluorescence with a fluorescence microplate reader (Synergy H1 Hybrid Microplate Reader, BioTek, Agilent Technologies, Santa Clara, CA, USA).

[0303] Liposome loading was quantified as follows: Liposomes were disrupted in ethanol (100 μL of liposome suspension in 900 μL of ethanol) and the concentration of the colored compound was measured by UV / visible absorption or HPLC analysis. The UV absorption peak of quinine is at 350 nm and the fluorescence absorption peak is at 460 nm, while the UV / visible absorption peak of curcumin is at approximately 420 nm.

[0304] 1.4 Liposome stability studies

[0305] The stability of liposomes prepared in PBS was tracked over 28 days under storage conditions (4 °C) (Table 2). Under these storage conditions, liposomes containing 20 mol% cholesterol exhibited excellent size stability, with minimal changes in the average diameter and polydispersity (size distribution). The stability of liposomes was tracked over 14 days under cell culture mimicking conditions (diluted in DMEM, 37 °C) (Table 3). The internalized aqueous phase of liposomes in DMEM contained PBS.

[0306] Table 2 - Results of average diameter measurements of liposomes with different cholesterol contents determined by dynamic light scattering (DLS) and polydispersity index (PDI)

[0307]

[0308] Table 3 - Measurement results of the average diameter of liposomes with a lipid:cholesterol molar ratio of 80:20 determined by dynamic light scattering (DLS) and polydispersity index (PDI)

[0309]

[0310] Table 3.1 describes other exemplary exogenous compounds encapsulated in liposomes prepared by the methods described herein and the related stability data determined by dynamic light scattering:

[0311] Table 3.1 - Measurement results of the average diameter of liposomes with encapsulated exogenous compounds determined by dynamic light scattering (DLS) and polydispersity index (PDI)

[0312]

[0313] N / M = not measured

[0314] Example 2 - Uptake of flavorants by cells - Cell preparation

[0315] 2.1 Growth conditions

[0316] All the following experiments were performed in triplicate.

[0317] 2.2.1 Uptake of liposomes by growing cell cultures - Serial dilutions.

[0318] C2C12 cells were seeded at 3000 cells / well (200 μl / well) in CM in a 96-well plate with a clear bottom and black sidewalls. Growth medium (DMEM) (5 mL) with 5 - 20% (v / v) FBS was added to the wells, and the cells were cultured until approximately 70% confluent and evenly distributed (cell culture stage 1). Serial dilutions of liposomes were prepared in different media under low light conditions (without hood light). Table 4 below gives the different dilutions of different media.

[0319] The cells were treated with liposome dilutions (200 μL / well). As a reference, "cell-free control" (NOC) and "untreated control" were performed. The cell-free control was a well without cells treated with the highest liposome concentration, while the untreated control (no treatment) was a well with cells treated only with growth medium (CM or DMEM).

[0320] The incubation times were 1, 3, 7, or 24 hours. At the end of the corresponding incubation time, the treatment medium was removed and the cells were washed three times with DPBS, leaving the last DPBS wash in the wells. The fluorescence intensity measurement results are shown in Figures 1 - 6.

[0321] 2.2.2 Uptake of Growing Cell Cultures by Liposomes - Cell Imaging

[0322] C2C12 cells were seeded at 5000 - 10000 cells / well (400 - 500 μL / well) in DMEM growth medium supplemented with 10% (v / v) FBS in 8-chambered glass slides (Corning Life Sciences). The cells were cultured until they reached approximately 70% confluence and were evenly distributed (cell culture stage 1). Under low light conditions (no hood light), serial dilutions of liposomes were prepared in different media. The cells were treated with 1.5% to 50% liposome dilutions (500 μL / well) and incubated for 1 to 24 hours. At the end of the corresponding incubation time, the treatment medium was aspirated, and each chamber well was washed three times with DPBS supplemented with 10% FBS (v / v), and the final DPBS-FBS supernatant was left in the well. Then, the cells were imaged using a 20 - 60X objective lens and a LionHeart FX automated microscope (Agilent BioTek). The Nile red liposome signal in the cells was detected by imaging the cells in fluorescence detection mode with excitation / emission wavelengths of 586 nm and 647 nm, respectively. At the same time, the cells were also imaged in phase contrast mode, and the images were captured / overlaid / analyzed using Gen5 Prime software (Agilent BioTek).

[0323] The results of cell imaging are as Figure 7 shown.

[0324] In another experiment, on day 0, mouse C2C12 cells were seeded at a density of 2000 - 5000 cells per well of an 8-well culture slide (BD-Falcon #354108) in growth medium (Dulbecco's Modified Eagle Medium (DMEM; Gibco; catalog number 11995-065); supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin) and grown at 37 °C for 48 hours. On day 2, the master stock solution / suspension of fluorescent liposomes (e.g., nitrobenzofurazan-labeled) was diluted 6-fold in phenol-free DMEM containing 10% fetal bovine serum. The final liposome concentration of this diluted solution was 16.67%. The growth medium was removed from the cells and replaced with 400 μl of the liposome solution, and the culture slide was returned to the cell culture incubator for 17 - 18 hours. On day 3, after the liposome incubation period, the liposome solution was removed from the cells, and the cells were washed four times with 500 - 1000 μl of serum-free, phenol-free DMEM. The liposome-bearing cells were restained with 500 μl of a solution containing 1X CellTracker Blue CMF2HC (Invitrogen #C12881), which was prepared in serum-free, phenol-free DMEM (1:1000 dilution = 1X), and incubated on the cells for 2 - 4 hours. After the incubation period, the cells were washed four times with serum-free, phenol-free DMEM. Then the cells were fixed at room temperature for 20 minutes by adding 500 μl of PBS solution containing 4% paraformaldehyde (Thermo Scientific; catalog number J61899). The slide was washed four times with 500 - 1000 μl of PBS. After washing, the chamber walls were removed from the culture slide using a chamber disassembly key. After removing the chamber, a few drops of Epredia PermaFluor Mounting Medium were placed on the slide and then the entire slide was covered with a coverslip. The mounting medium was allowed to cure at 4 °C in the dark for 24 - 48 hours. Day 4: After the mounting medium had cured, the cells were imaged by phase contrast and epifluorescence microscopy using the 4X, 20X, and 40X objectives of a LionHeart FX imager (BioTek / Agilent). The signal of the fluorescent liposomes was detected using a GFP filter set (excitation 469 nm, emission 525 nm), and the cells stained with Cell Tracker Blue were detected using a DAPI filter set (excitation 377 nm, emission 447 nm). Cell images were captured using Gen5 microplate reader and imager software (BioTek / Agilent). The cell imaging results are as Figure 9 shown.

[0325] Table 4: Dilution with different media

[0326]

[0327]

[0328] Detect the fluorescence of cells and separate treatment media in a plate reader using the following settings:

[0329] - Fluorescence endpoint, detected from the bottom

[0330] - Excitation: 554 nm, Emission: 638 nm; Gain 100 RFU (Relative Fluorescence Unit)

[0331] 2.2.3 Liposome uptake and retention / metabolism of exogenous compounds in cells

[0332] To test for potential compound retention / metabolism effects, C2C12 cells were incubated with liposomes loaded with exogenous compounds or free compounds applied at the same concentration for 17 h by the method described herein. The cells were washed with DPBS and incubated with complete medium for 3 h, 6 h, 24 h (post-incubation steps) to promote normal cell metabolism. After the post-treatment incubation steps, the treatment medium was removed and the cells were washed twice with DPBS and harvested. Viability assays and cell counting of the harvested cells were performed using a NucleoCounter NC-202 (ChemoMetec A / S, Allerod, Denmark). To determine the amount of exogenous compound absorbed and / or bound to the cells, the cells were centrifuged at 130 * g for 5 min, washed with DPBS and snap-frozen. Samples of the treatment medium and cell pellet were analyzed / quantified by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS). The samples were diluted with ethanol (for vitamin B12) and with ethanol:water (50:50) (for thiamine hydrochloride). The cell pellet was dissolved using an ultrasonic bath. The content of thiamine hydrochloride or vitamin B12 was calculated using linear regression analysis of a calibration curve generated with reference standards.

[0333] By comparing the concentration of exogenous compound in the cell pellet harvested immediately after treatment and the concentration of exogenous compound in the cell pellet after the post-treatment incubation steps, the stability of the compound bound to the cells when applied in free form or when encapsulated can be understood. Figure 8 - A and B show the results for two exogenous compounds, vitamin B12 and thiamine hydrochloride, respectively. The amount of exogenous compound detected or analyzed (encapsulated in liposomes and free) was normalized to 100%. As in Figure 8As shown in A, the vitamin B12 clearance rate of cells loaded with free vitamin B12 was higher (for all time points) compared to vitamin B12 encapsulated in liposomes. Cells treated with vitamin B12 encapsulated in liposomes retained 81% and 64% of the 0-hour concentration at 3 and 6 hours, respectively, after the post-treatment incubation step, while cells treated with free vitamin B12 alone retained 67% and 24% at the same time points. This indicates that vitamin B12 encapsulated in liposomes is not only taken up by and / or binds to cells, but also that cells can retain liposome-encapsulated vitamin B12 for a longer time. Similar observations can be made for liposome-encapsulated thiamine hydrochloride compared to free thiamine hydrochloride ( Figure 8 B). Without being bound to any theory, this effect may be attributed to liposomes protecting exogenous compounds from intracellular metabolism and thus improving retention.

[0334] 2.2.4 Viability of cells incorporating (liposome-encapsulated) exogenous compounds

[0335] The following data show cell counts and viability determinations under different conditions in the vitamin B12 and thiamine hydrochloride experiments. Cell counts of harvested cell pellets at a given concentration were compared and measured using the methods described herein. The counts and viability of cells incubated with liposomes were comparable to those of untreated cells, and the numbers of both doubled overnight, indicating that liposomes loaded with vitamin B12 or thiamine do not reduce cell viability and are not cytotoxic. Percentages were normalized to control cells (100%).

[0336] Vitamin B12

[0337] 6 h incubation Normalized cell count [%] Viability [%] Control cells (untreated) 100 100 Cells treated with free vitamin B12 97 104 Cells treated with liposomal vitamin B12 111 99 17 h incubation Normalized cell count [%] Viability [%] Control cells 100 100 Cells treated with free vitamin B12 100 99 Cells treated with liposomal vitamin B12 104 104

[0338] Thiamine hydrochloride

[0339]

[0340]

[0341] Example 3 - Use of non-human animal cells derived from oviparous animals

[0342] Culturing non-human animal cells as described in the following references: Olivier, S. et al., (2010. EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity. MAbs, 2(4):405-15), Madeline, B. et al., (2015. Culturing a duck ES-derived cell line in single-use bioreactors: A rapid, efficient, and cost-effective vaccine manufacturing system based on suspension culture. BioProcess International, 13), Silim, A., Azhary, M.A.S.Y.E., Roy, R.S., (1982. A Simple Technique for Preparation of Chicken-Embryo-Skin Cell Cultures. Avian Diseases, 26(1):182-185); Chen, Y.-C. et al., (2019. In vitro culture and characterization of duck primordial germ cells. Poultry science, 98(4):1820-1832.) Farzaneh, M., et al. (2017). The evolution of chicken stem cell culture methods. British Poultry Science, 58(6):681-686).

[0343] The addition of the supplement composition, its harvest, and analysis are carried out according to Example 2.

[0344] Example 4 - Use of non-human animal cells derived from stem cells harvested from umbilical cord, bone marrow, placenta, or other tissues

[0345] Non-human animal cells are cultured as described in the following references: Pham, P.V. et al., (2014. Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells. Journal of Translational Medicine, 12(1):56) Smith, J.R. et al., (2016. Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method. Stem Cells International, 2016:).

[0346] The supplement composition is added, harvested, and analyzed according to Example 2.

[0347] Example 5 - Nanoparticles

[0348] 5.1 Preparation of Loaded Nanoparticles

[0349] (Method M1) Lipid nanoparticles are prepared by weighing wax (e.g., palm oil) and an oil that is liquid at room temperature (e.g., medium-chain triglycerides, MCT), with the weight ratio of wax to oil being between 90:10 and 50:50. Soybean lecithin (90% PC) is added to the melt in a ratio of 98:2 wax + oil:soybean lecithin. The combined weight of wax, oil, and soybean lecithin is about 10 grams. The mixture is heated above the melting temperature of the wax (about 70 °C). Up to 1 gram of an exogenous compound (e.g., 2,4-decadienal) can be mixed with the wax melt. The mixture forms an organic phase. Additionally, a polysorbate 80 solution containing 2.4 grams of polysorbate 80 in 87.4 grams of distilled water is prepared and heated to about 70 °C. This solution forms an aqueous phase. The organic phase is slowly injected into the aqueous phase under strong magnetic stirring (1200 rpm). After complete addition of the organic phase, the resulting emulsion is sonicated (Banson digital sonicator model 250) for 5 minutes and then processed by a high-shear homogenizer (Polytron 6100D) at 12000 rpm for about 10 minutes. The resulting emulsion is cooled to room temperature under ambient conditions to solidify the emulsion and form lipid nanoparticles. If desired, unencapsulated flavorants can be removed from the sample by using a PD Miditrap G-25 gel filtration column (Cytiva, Grens, Switzerland). The sample is then subsequently filtered through a 220 nm sterile filter.

[0350] (Method M2) Prepare the organic phase by dissolving wax (e.g., palm oil) and an oil that is liquid at room temperature (e.g., medium-chain triglycerides, MCT) in a mixture of chloroform:methanol (2:1). The weight ratio of wax to oil is between 90:10 and 50:50. The total amount of lipid (wax + oil) is about 300 mg. Up to 100 mg of the desired exogenous compound (e.g., 2,4-decadienal) can be mixed with the dissolved wax and oil. Remove the organic solvent by rotary evaporation at about 65 °C under about 300 mbar vacuum. Then remove traces of the remaining organic solvent in a vacuum drying oven at about 40 °C and about 100 mbar for at least 15 hours. Next, prepare a polysorbate 80 solution by dissolving 300 - 600 mg of polysorbate 80 in about 20 mL of distilled water and heating to about 65 °C. Add the heated solution to the dried organic phase, and treat the mixture with a high-shear homogenizer (Polytron 6100D) at 12000 rpm for 3 minutes. Then, sonicate the formed dispersion with a probe sonicator (Banson digital sonicator model 250) at a 50% amplitude setting for about 20 minutes. Cool the resulting emulsion to room temperature under ambient conditions to solidify the emulsion and form lipid nanoparticles. If desired, unencapsulated flavorant can be removed from the sample by using a PD Miditrap G-25 gel filtration column (Cytiva, Grens, Switzerland). Then filter the sample subsequently through a 220 nm sterile filter.

[0351] 5.2 Stability data of flavorant-loaded nanoparticles

[0352] The stability and polydispersity index (PDI) of the size or average particle size (average diameter) were measured using the methods described previously for liposomes (e.g., dynamic light scattering). The results are listed in Table 5. The flavorant-loaded nanoparticles were analyzed by GC-MS, and the following amounts of the loaded or encapsulated flavorant (2,4-decadienal) were determined: Method M1: 4490 μg / mL, Method M2: 2426 μg / mL.

[0353] The following GC-MS method was carried out to quantify the loaded or encapsulated flavorants: 2,4-decadienal was analyzed by gas chromatography-mass spectrometry selective ion monitoring (GC-MS SIM). For GC-MS SIM sample preparation, 200 μL of the sample containing the loaded delivery system (e.g., nanoparticles) was taken, an internal GC-MS SIM standard in acetone was added, and the volume was increased to 1 mL with acetone to extract all the loaded flavorants. Additionally, a high-shear mixer (Polytron PT 1200E, Kinematica, Eschbach, Germany) could be used to homogenize the cell pellet for 1 minute. The sample was filtered through a 220 nm syringe filter. GC-MS SIM (GC = 7890B, MS = 5977B MSD, Agilent Technologies, Lautengartenstrasse 6, Basel) measured a sample injection volume of 1 μL. The content of the loaded or encapsulated flavorants was calculated using linear regression analysis of the calibration curve generated with the reference substance.

[0354] To measure the average particle size and PDI, 20 - 50 μL of the sample was diluted in the same dispersion medium until the volume in a disposable cuvette was 2 mL. The cuvette was placed inside a Zetasizer Nano ZS90 (Malvern Panalytical), and measurements were carried out by dynamic light scattering. Measurements were performed in triplicate and the average value was taken.

[0355] Table 5 - Measurement results of the average diameter of liposomes with encapsulated exogenous compounds determined by dynamic light scattering (DLS) and polydispersity index (PDI)

[0356]

[0357] N / M = Not measured.

Claims

1. A method for producing a cell composition from non-human animals, the method comprising: contacting a single type or multiple cells from non-human animals with at least one exogenous supplement selected from flavoring materials, flavoring precursors, flavoring enhancers, pigments, pigment precursors, bioactive compounds, or other food-related additives and mixtures thereof.

2. The method according to claim 1, wherein the exogenous supplement is contained in a carrier.

3. The method according to claims 1 to 2, wherein the carrier is a nanocarrier, nanoparticle, micelle, liposome, or vesicle.

4. The method according to claims 1 to 3, wherein the carrier is a nanoparticle or a particle having a lipid bilayer such as a micelle, liposome, or vesicle.

5. The method according to claims 1 to 4, wherein the carrier is a liposome preferably composed of phospholipids.

6. The method according to claim 1 or 5, wherein the flavorant material is selected from 1-octen-3-ol, 1-octen-3-one, 2,3-dimethylpyrazine, 2,3-pentanedione, 2,4-decadienal, 2,4-nonadienal, 2,4-undecadienal, 2,5-dimethyl-3-furanthiol, 2-acetylfuran, 2-acetyl-2-thiazoline, 2-acetylthiazole, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-furfurylthiol, 2-methyltetrahydrofuran-3-one (coffee furanone), 2-methyl-3-furanthiol, 2-methyl-3-tetrahydrofuranthiol, 2-octen-4-one, 2-pentylfuran, 2-tridecanone, 3-mercapto-2-butanone, 4,5-epoxy-2-decenal, 4-mercapto-4-methyl-2-pentanone, 5-methylfurfural, acetoin, acetyl-2-pyrazine, alanine, anserine, arachidonic acid, arginine, bis(2-methyl-3-furyl) disulfide, butyric acid, methylcyclopentenolone, cysteine, damascenone, δ-decalactone, capric acid, dihydroxyacetone, dimethyldisulfide, disodium guanylate, disodium inosinate, δ-dodecalactone, ethyl fenchylideneacetate, ethyl 3-methyltetronic acid; 4-, NAT, 1-PG (maple furanone), ethyl oleate, fructose, furanone, furfural, furfuryl disulfide, furfurylthiol, furfuryl methyl disulfide, γ-nonalactone, glucose, glutathione, glycine, Glycogene, guaiacol, hexadecanoic acid (palmitic acid), hexanal, hexanoic acid, histidine, indole, isobutyl mercaptan, isobutyl-4-methyl-5-ethylthiazoline, isoleucine, isovaleraldehyde, leucine, linoleic acid, linolenic acid, lysine, 1-p-menthene-8-thiol, mercapto-butanone, methional, methionine, methoxy-2-methylpyrazine, methyl-12-tridecenal, methyl-2-butyric acid, methyl-2-keto-3-tetrahydrothiophene, methyl mercaptan, nonanal, phenylethyl alcohol, phenylethyl mercaptan, phenylacetaldehyde, phenylalanine, plasmalogen, proline, ribose, skatole, sotolon, succinic acid, thiazole, δ-tetradecalactone, thiazolidine, trans-2-nonenal, trans-2-octenal, trans-2-undecenal, trimethylpyrazine, 2,3,5-Nat, trithione, tryptophan, tyrosine, valeraldehyde, valine, xylose and combinations thereof.

7. The method according to any one of claims 1 to 6, wherein the flavor precursor is a material or combination of materials that forms the desired flavorant in response to a suitable stimulus such as heat treatment, physical treatment or as a result of a chemical, enzymatic or microbial process or any combination thereof.

8. The method according to any one of the preceding claims, wherein the non-human animal-derived cells are further contacted with at least one additional exogenous supplement selected from vitamins, minerals (such as exogenous iron and / or its salts), bioactive compounds, bacterial extracts, pigments, pigment precursors, bioactive compounds, food-related additives, and any combination thereof, and wherein the exogenous supplement is contained in a carrier.

9. The method according to any one of the preceding claims, wherein the cells are contacted with the exogenous supplement contained in a carrier to obtain a non-human animal-derived cell composition having improved flavor, aroma, texture, taste, and / or color.

10. The method according to any one of the preceding claims, wherein the carrier is a liposome having a size polydispersity of less than 0.3, preferably less than 0.2, more preferably less than 0.

1.

11. The method according to any one of claims 1 to 10, wherein the average particle size is less than 500 nm, preferably less than 350 nm, more preferably less than 200 nm.

12. The method according to any one of claims 1 to 10, wherein the cells are contacted with the exogenous supplement contained in a carrier at a low cell differentiation rate.

13. The method according to any one of claims 1 to 10, wherein the cells are contacted with the exogenous supplement contained in a carrier at a high cell differentiation rate.

14. The method according to any one of claims 1 to 10, wherein the cells are contacted with the exogenous supplement contained in a carrier at a low cell division rate.

15. The method according to any one of claims 1 to 10, wherein the cells are contacted with the exogenous supplement contained in a carrier at a high cell division rate.

16. The method according to any one of the preceding claims, wherein the cells are in suspension.

17. The method according to any one of the preceding claims, wherein the non-human animal-derived cells are selected from: i. Pluripotent stem cells (PSCs) and / or cells differentiated therefrom, ii. Induced pluripotent stem cells PSCs (iPSCs) reprogrammed from non-human animal somatic cells and / or cells differentiated therefrom, iii. Non-embryonic stem cells (non-ESCs), iv. Satellite cells, v. Embryonic stem cells (ESCs), vi. Primary progenitor cells, vii. and any combination thereof.

18. The method according to any one of the preceding claims, wherein the cells of non-human animal origin are selected from muscle cells and their progenitors; adipocytes and their progenitors; stromal cells and their progenitors; endothelial cells and their progenitors; and any combination thereof.

19. The method according to any one of the preceding claims, wherein the non-human animal is selected from cattle, sheep, pigs, poultry, reptiles, rodents, wild animals, shellfish, fish, and insects or any combination thereof.

20. The method according to any one of the preceding claims, wherein the time for contacting the cells with at least one exogenous supplement contained in a carrier is sufficient to produce, alter, or improve the sensory properties of the cells, and wherein the cells are contacted with the exogenous supplement contained in the carrier for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, at least 5 days, or at least 1 week.

21. The method according to claim 20, wherein the cells are contacted with the exogenous supplement contained in the carrier for at least 30 minutes and at most 2 days.

22. The method according to claim 21, wherein the cells are contacted with the exogenous supplement contained in the carrier for at least 1 and at most 24 hours.

23. The method according to any one of the preceding claims, wherein the contact time is sufficient such that at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90% of the cells contain the exogenous supplement contained in the carrier at sites selected from intracellular space of the cells, intramembrane space, on the membrane, and any combination thereof.

24. The method according to any one of the preceding claims, the method further comprising the step of separating the non-human cells from the culture medium.

25. The method according to any one of the preceding claims, the method further comprising the step of dehydrating the cell composition by freeze-drying, spray-drying, drying, or adsorbing the cell composition onto a solid support.

26. A cell composition of non-human animal origin obtained by the method according to any one of claims 1 to 25.

27. A cell composition of non-human animal origin, comprising a plurality of cells of non-human animal origin, wherein at least 20%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90% of the cells comprise at least one exogenous supplement, wherein the cell composition is characterized by having improved sensory properties and / or having a meat-like flavor, and wherein the exogenous supplement is contained in a carrier.

28. The cell composition of non-human animal origin according to claim 27, wherein the at least one exogenous supplement is selected from flavoring materials, flavorings, flavoring precursors, flavor enhancers, pigments, pigment precursors, bioactive compounds or other food-related additives and mixtures thereof.

29. The cell composition of non-human animal origin according to claims 27 to 28, wherein the carrier is a nanocarrier, nanoparticle, micelle, liposome or vesicle.

30. The cell composition of non-human animal origin according to any one of claims 27 to 29, wherein the carrier is a nanoparticle or a particle having a lipid bilayer such as a micelle, liposome or vesicle.

31. The cell composition of non-human animal origin according to any one of claims 27 to 30, wherein the carrier is a liposome preferably composed of phospholipids.

32. The cell composition of non-human animal origin according to claim 26 or 31, wherein the flavoring material is selected from 1-octen-3-ol, 1-octen-3-one, 2,3-dimethylpyrazine, 2,3-pentanedione, 2,4-decadienal, 2,4-nonadienal, 2,4-undecadienal, 2,5-dimethyl-3-furylthiol, 2-acetylfuran, 2-acetyl-2-thiazoline, 2-acetylthiazole, 2-ethyl-3,5-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2-furfurylthiol, 2-methyltetrahydrofuran-3-one (coffee furanone), 2-methyl-3-furylthiol, 2-methyl-3-tetrahydrofurylthiol, 2-octen-4-one, 2-pentylfuran, 2-tridecanone, 3-mercapto-2-butanone, 4,5-epoxy-2-decenal, 4-mercapto-4-methyl-2-pentanone, 5-methylfurfural, acetoin, acetyl-2-pyrazine, 4-methylcaprylic acid, alanine, anserine, arachidonic acid, arginine, bis(2-methyl-3-furyl) disulfide, butyric acid, methylcyclopentenolone, cysteine, damascenone, δ-decalactone, capric acid, dihydroxyacetone, dimethyldisulfide, disodium guanylate, disodium inosinate, δ-dodecalactone, ethyl fenchylideneacetate, ethyl 3-methyltetracarboxylate; 4-, NAT, 1-PG (maple furanone), ethyl oleate, fructose, furanone, furfural, furfuryl disulfide, furfurylthiol, furfuryl methyl disulfide, γ-nonalactone, glucose, glutathione, glycine, Glycogene, guaiacol, hexadecanoic acid (palmitic acid), hexanal, hexanoic acid, histidine, indole, isobutylthiol, isobutyl-4-methyl-5-ethylthiazoline, isoleucine, isovaleraldehyde, leucine, linoleic acid, linolenic acid, lysine, 1-p-menthene-8-thiol, mercapto-butanone, methional, methionine, methoxy-2-methylpyrazine, methyl-12-tridecenal, methyl-2-butyric acid, methyl-2-keto-3-tetrahydrothiophene, methylthiol, nonanal, phenylethyl alcohol, phenylethylthiol, phenylacetaldehyde, phenylalanine, plasmalogen, proline, ribose, skatole, sotolon, succinic acid, thiazole, δ-tetradecalactone, thiazolidine, trans-2-nonenal, trans-2-octenal, trans-2-undecenal, trimethylpyrazine, 2,3,5-Nat, trithione, tryptophan, tyrosine, valeraldehyde, valine, xylose, and combinations thereof.

33. The cell composition of non-human animal origin according to any one of claims 26 to 32, wherein the exogenous supplement is selected from flavoring materials, flavoring precursors, flavoring enhancers, pigments, pigment precursors, bioactive compounds, or other food-related additives having a ClogP of at least -4, such as at least -3, such as at least -2, such as at least -1, such as at least 0, such as at least 1, such as at least 2, such as at least 3, such as at least 4, such as at least 5, or such as at least 6.

34. The cell composition of non-human animal origin according to claim 27 or 33, wherein the flavor precursor is selected from materials or combinations of materials that form the desired flavor in response to a suitable stimulus such as heat treatment, physical treatment, or as a result of a chemical, enzymatic, or microbial process or any combination thereof.

35. The cell composition of non-human animal origin according to any one of claims 26 to 34, wherein the cells of non-human animal origin further comprise at least one additional exogenous supplement selected from vitamins, minerals (such as exogenous iron and / or its salts), bioactive compounds, bacterial extracts, pigments, or other food-related additives or any combination thereof.

36. The cell composition of non-human animal origin according to any one of claims 26 to 35, wherein the cells are selected from: i. Pluripotent stem cells (PSCs) and / or cells differentiated therefrom, ii. Induced pluripotent stem cells PSCs (iPSCs) reprogrammed from non-human animal somatic cells and / or cells differentiated therefrom, iii. Non-embryonic stem cells (non-ESCs), iv. Satellite cells, v. Embryonic stem cells (ESCs), vi. Primary progenitor cells, vii. And any combination thereof.

37. The cell composition of non-human animal origin according to any one of claims 26 to 36, wherein the cells are selected from muscle cells and their progenitors; adipocytes and their progenitors; stromal cells and their progenitors; endothelial cells and their progenitors; and any combination thereof.

38. The cell composition of non-human animal origin according to any one of claims 26 to 37, wherein the non-human animal is selected from cattle, sheep, pigs, poultry, reptiles, rodents, wild animals, shellfish, fish, and insects or any combination thereof.

39. The cell composition of non-human animal origin according to any one of claims 26 to 38, wherein at least one exogenous supplement contained in the carrier is present in the cells at a site selected from the intracellular space of the cell, the intramembrane space, on the membrane, and any combination thereof.

40. The cell composition of non-human animal origin according to any one of claims 26 to 39, wherein the cell composition is dehydrated by a method selected from freeze-drying, spray-drying, drying, and adsorption on a solid support.

41. A cell composition of non-human animal origin according to any one of claims 26 to 40, wherein at least one exogenous supplement contained in the carrier is not metabolized in at least 60% of the cells of non-human animal origin, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the cells of non-human animal origin.

42. A cell composition of non-human animal origin according to any one of claims 26 to 41, wherein the plurality of cells contain an amount of at least one exogenous supplement from about 0.01 mg / 100 g of cells to about 100 mg / 100 g of cells.

43. A cell composition of non-human animal origin according to any one of claims 26 to 42, wherein the at least one exogenous supplement composition is present in an amount from about 0.05 mg / 100 g of cells to about 500 mg / 100 g of cells, more preferably from 0.1 mg / 100 g of cells to about 50 mg / 100 g of cells or even more preferably from 0.5 mg / 100 g of cells to 25 mg / 100 g of cells.

44. A cell composition of non-human animal origin according to any one of claims 26 to 43, wherein at least one exogenous supplement contained in the carrier is food-grade and the cells are characterized by enhanced sensory properties.

45. An enriched cell culture medium comprising at least one exogenous supplement, wherein the amount of the supplement is sufficient to impart a sensory effect to cells cultured in the medium according to the method of claims 1 to 25, and wherein the exogenous supplement is contained in a carrier.

46. The enriched cell culture medium according to claim 45, wherein the at least one exogenous supplement is selected from flavoring materials, flavoring precursors, flavor enhancers, pigments, pigment precursors, bioactive compounds or other food-related additives and any combination thereof.

47. An enriched scaffold comprising at least one exogenous supplement, wherein the amount of the supplement is sufficient to impart a sensory effect to cells cultured in the presence of the scaffold according to the method of claims 1 to 25, and wherein the exogenous supplement is contained in a carrier.

48. The enriched scaffold according to claim 47, wherein the at least one exogenous supplement is selected from flavoring materials, flavorings, flavoring precursors, flavor enhancers, pigments, pigment precursors, bioactive compounds or other food-related additives and any combination thereof.

49. The enriched cell culture medium according to claim 45 or 46 or the enriched scaffold according to claim 47 or 48, wherein the carrier is a nanocarrier, nanoparticle, micelle, liposome or vesicle. Use of the cell composition according to any one of claims 26 to 44 for flavoring a food or beverage edible product.

51. Use according to claim 50, wherein the cell composition is used for flavoring an edible product selected from cultured meat, meat analogues or meat products.

52. An edible product comprising a cell composition of non-human animal origin according to any one of claims 26 to 44.

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