Food products comprising avian stem cells

Abstract

To provide a synthetic meat product for human and animal consumption and methods for producing such food product.SOLUTION: A synthetic food product comprises or essentially consists of a cell biomass of avian cells grown in vitro in a chemically-defined serum free culture medium under controlled conditions and does not contain any hazard contaminations.SELECTED DRAWING: None

Description

Technical Field

[0001] The field of the present invention relates to the industrial production of synthetic nutritional foods for human and / or animal consumption. More specifically, the present invention relates to the use of avian cell lines, in particular chicken or duck ES cell lines derived from stem cells of embryonic origin, for producing cell biomass suitable as food or dietary supplements. The present invention encompasses a method for manufacturing such synthetic foods and the products themselves.

Background Art

[0002] World meat production has increased rapidly over the past 50 years. That is, the world's total production has increased 4 to 5 times from 1961 (Ritchie and Roser, 2018). The total meat production in 2014 was approximately 300 million tons, most of which was poultry, pork, and beef. At the same time, the total number of livestock is approximately 1.4 billion cattle, 1.2 billion sheep, 1 billion goats, and approximately 1 billion pigs, and is showing a very strong increasing trend mainly due to the increasing demand in Asia. The total meat consumption per capita has doubled over the past 50 years. That is, the meat consumption is exceeding the population growth. Furthermore, it is estimated that the world's meat consumption will increase by 25% in 2030 compared to 2015 and reach 460 million tons in 2050 (GEAS 2012).

[0003] Behind this remarkable growth, there are serious problems associated with current animal meat production, which is expected to further increase.

[0004] First, the conventional methods of producing animal meat are highly inefficient. A significant portion of all the grains produced agriculturally is used for animal consumption. Furthermore, thousands of pounds of water are required to produce one pound of meat. For example, 5988, 8768, and 15415 liters of water are needed to produce one kilogram of pork, sheep / goat, or beef meat, respectively (Mekonnen and Hoekstra, 2010). Nevertheless, current efforts focus on fixing livestock growth by using hormones and antibiotics and thus reducing the consumption of grains and water. However, this development brings another problem where livestock meat contaminated with growth hormones (especially steroid hormones such as testosterone, progesterone, estrogen, or their synthetic derivatives) and antibiotics poses a threat to public health (Galbraith, 2002; Jeong et al., 2010).

[0005] Second, the intensification of livestock production is associated with the rapid spread of pathogens and emerging diseases worldwide (Greger, 2007). Foodborne pathogens such as Salmonella, Campylobacter, and E. coli cause millions of illnesses every year, resulting in huge expenditures in human and animal health systems.

[0006] Third, the massive emissions of carbon dioxide and methane from the livestock sector are a serious environmental problem (GEAS 2012; Opio et al., 2013; Hedenus et al., 2014). According to the World Bank's estimate, 18% of the world's CO 2 emissions are caused by current inefficient meat production. The Worldwatch Institute claims that the true figure is 51% (see https: / / www.independent.co.uk / environment / climate-change / study-claims-meat-creates-half-of-all-greenhouse-gases-1812909.html).

[0007] Fourthly, current meat production methods involve animal suffering, to which many people today object.

[0008] Fifthly, a further drawback of using natural meat for consumption is its high content of harmful substances such as cholesterol and saturated fat, which poses problems threatening diet and health.

[0009] Therefore, there is a need to develop new approaches for the production of meat and / or meat-like products that can at least partially solve or reduce the above problems.

[0010] One approach is to develop non-conventional meat products generated ex vivo. So-called "synthetic meat" or "in vitro meat", also known as "cell-cultured meat", "artificial meat", "clean meat", "lab-grown meat", is produced using animal-derived cells cultured in vitro. Such synthetic meat has many advantages compared to conventional meat in terms of the efficient use of natural resources (land, energy, water), lower greenhouse gas production, and better animal welfare (Tuomisto, 2014). Furthermore, the nutritional composition of cultured meat can be thoroughly controlled, thereby preventing the incorporation of harmful components such as cholesterol, saturated fat, hormones, antibiotics, and infectious microorganisms.

[0011] Theoretically, synthetic meat can have physical properties, color, flavor, aroma, texture, palatability, and nutritional value equivalent to those of conventional animal meat, or can play a complementary role to conventional meat products as long as it is simply acceptable to humans, or can even be regarded as a substitute for meat. Although some progress has been made in recent years, the technology in the field of synthetic meat or meat-like product production is still in a very early stage of practice (reviewed in Kadim et al., 2015). Important problems remain to be solved, such as the selection of appropriate cell types, the refinement of culture conditions, and the development of a cost-effective and contaminant-free culture medium.

[0012] In particular, one of the important problems solved by the present invention is the upscaling for the production of large quantities of meat analogue products at affordable prices.

[0013] The inventors have developed avian cell lines that can grow continuously in culture and generate large amounts of cell biomass. In particular, the cell lines presented herein have all the characteristics necessary to enable high-level industrial-scale culturing.

Summary of the Invention

[0014] Surprisingly, culturing avian cell lines in suspension provides a very high-yield source of such food, at a time when there is a great need to find alternative ways to produce food that contains no antibiotics and requires less energy and water.

[0015] The present application provides a new process for manufacturing synthetic meat products that can help solve the serious environmental, health and ethical problems associated with conventional approaches and meet the rapidly growing consumer needs. The disclosed process does not involve the cumbersome procedures of tissue engineering, but is based on low-cost cell culture. Aspects of the invention particularly provide:

[0016] A1. A process / method for "in vitro" manufacturing of nutritious food for human or animal consumption, comprising culturing an avian cell line in suspension, wherein the avian cell line is i) derived from avian embryonic stem cells, ii) capable of growing in a basal culture medium in the absence of exogenous growth factors, feeder cells and / or animal serum, and iii) capable of continuous proliferation in suspension.

[0017] A2. The avian cell line is a) isolating avian embryonic stem cells from embryos at a developmental stage near the egg-laying period; and b) culturing said cells in a basal culture medium comprising at least one exogenous growth factor selected from SCF, IGF-1, bFGF, IL-6, IL-6R and / or CNTF, a layer of feeder cells, and animal serum for at least 20 passages; c) modifying said culture medium by gradually removing said growth factors, feeder cells and animal serum, and further culturing the cells for at least several passages; d) adapting the cells of step c) to a suspension, thereby obtaining an established avian cell line capable of growing in a basal culture medium for at least 50 days in the absence of exogenous growth factors, feeder cells and / or animal serum, the process / method of embodiment A1 obtained by a process comprising:

[0018] A3. An avian cell line is obtained by a process comprising: a) isolating avian embryonic stem cells from embryos at a developmental stage near the egg-laying period; b) culturing said cells in a basal culture medium comprising exogenous growth factors IGF-1 and CNTF, a layer of feeder cells, and animal serum for at least 1 passage; c) gradually removing said growth factors from the culture of step b) and growing further for at least 1 passage; d) gradually removing feeder cells from the culture of step c) and growing for at least 1 passage; e) gradually removing animal serum from the culture of step d) and growing for at least 1 passage; f) adapting the cells of step e) to a suspension, thereby obtaining a continuous avian cell line capable of growing in a basal medium in the absence of exogenous growth factors, feeder cells and / or animal serum, the process / method of embodiments A1 and A2 obtained by a process comprising:

[0019] A4. The process / method according to any one of embodiments A1 to A3, wherein the avian cell line is derived from chicken embryonic stem cells.

[0020] A5. A process / method according to any one of aspects A1 - A4, wherein the avian cell line is derived from duck embryonic stem cells.

[0021] A6. A process / method according to any one of aspects A1 - A5, wherein the avian cell line does not contain functional endogenous retroviruses or other viral particles.

[0022] A7. A process / method according to any one of aspects A1 - A6, wherein the avian cell line is derived from SPF species.

[0023] A8. A process / method according to any one of aspects A1 - A7, wherein the avian cell line is selected from the group consisting of chicken EB14, chicken EB line 0, chicken EBv13, chicken DL43, chicken DL46, duck EB24, duck EB26 and duck EB66 cell lines.

[0024] A9. A process / method according to any one of aspects A1 - A8, wherein the avian cell line is chicken DL43, chicken DL46, duck EB24, duck EB26.

[0025] A10. A process / method according to any one of aspects A1 - A9, wherein the avian cell line is chicken DL43 or duck EB26.

[0026] A11. The process / method according to any one of claims A1 - A10, wherein the cell line is grown in a culture medium which is a synthetic or known composition (CD) medium that does not contain harmful substances to humans and / or animals.

[0027] A12. The process / method of aspect A11, wherein the synthetic medium is Ex - Cell® GRO - I and / or HYQ CDM4 Avian medium.

[0028] The process / method according to embodiment A11, wherein the synthetic medium or CD medium is further supplemented with one or more components (multiple possible) selected from the group consisting of amino acids, nucleotides, vitamins, saccharides, fatty acids, beta-mercapto-ethanol, insulin, glycine, choline, pluronic acid F-68, and sodium pyruvate.

[0029] The process / method according to embodiment A13, wherein the additional component is L-glutamine used at a concentration of 0 to 12 mM or 1 to 5 mM, preferably about 2.5 mM.

[0030] The process / method according to any one of embodiments A11 to A13, wherein the culture medium further contains a hydrolyzate of plants and / or yeast.

[0031] The process / method according to any one of embodiments A11 to A15, wherein the culture medium does not contain any animal products containing serum.

[0032] The process / method according to any one of embodiments A1 to A16, wherein the cell line is cultured under fed-batch culture conditions.

[0033] The process according to any one of embodiments A1 to A16, wherein the cell line is cultured under perfusion conditions.

[0034] The process / method according to any one of embodiments A1 to A18, wherein the cells are cultured in a bioreactor with a volume of 30 liters, 50 liters, 100 liters, 1000 liters, preferably 10,000 liters or more.

[0035] The process / method according to any one of embodiments A1 to A19, wherein the cell line is cultured at about 37°C, pH 7.2, pO2 about 50%, and a stirring speed of about 40 rpm or more.

[0036] A21. The cell line is cultured until the cell density reaches about 10 7 cells / mL. The process / method according to any one of embodiments A1 to A20.

[0037] A22. The cell line is cultured until the cell density reaches about 10 8 cells / mL, a process / method of any of embodiments A1 - A21.

[0038] A23. The cell line is cultured until the cell density exceeds 10 8 cells / mL, a process / method of any of embodiments A1 - A21.

[0039] A24. The yield of the process is at least about 0.5 - 1 g of biomass per 1 g of medium, a process / method of any of embodiments A1 - A23.

[0040] A25. The process / method of any of embodiments A1 - A24 further includes the step of collecting the cell biomass by precipitation and decantation.

[0041] A26. The cell sedimentation is performed by adding a calcium salt to the cell suspension, the process / method of embodiment A25.

[0042] A27. The calcium salt is calcium chloride used at a final concentration of 10 - 500 mg / L, preferably 50 - 300 mg / L, more preferably 50 mg / L, the process / method of embodiment A26.

[0043] A28. The process / method of any of embodiments A1 - A27 further includes the step of adding one or more components (multiple possible) that increase the nutritional value of food selected from the group consisting of vitamins, co - vitamins, minerals, essential amino acids, essential fatty acids, enzymes, and antioxidants to the cell biomass.

[0044] A29. The process / method of any of embodiments A1 - A28 further includes adding one or more flavorings (multiple possible), flavor aromatic compounds (multiple possible), and / or colorants (multiple possible) to the cell biomass.

[0045] The process / method of any of Aspects A1 - A29, further comprising one or more (multiple possible) food processing steps selected from cooling, freezing, solidifying, drying, soaking, boiling, cooking, baking, frying, smoking, 3D printing, and packaging.

[0046] B1. Food produced by the process / method of any of Aspects A1 - A30.

[0047] C1. Cell biomass produced by the process / method of any of Aspects A1 - A27.

[0048] D1. Use of the cell biomass of Aspect C1 for producing synthetic food for human or animal consumption.

[0049] B2. Food containing or consisting essentially of the cell biomass of Aspect C1.

[0050] B3. Food of Aspect B1 or B2, further comprising other cells such as non - human muscle cells, fat cells or cartilage cells, or combinations thereof, that grow in vitro with avian cells or are added after collection of avian cells.

[0051] B4. Food of any of Aspects B1, B2, or B3, further comprising additional ingredients selected from the group consisting of minerals, vitamins, co - vitamins, essential fatty acids, essential amino acids, enzymes and antioxidants, or combinations thereof, to enhance nutritional value.

[0052] B5. Food of any of Aspects B1, B2 - B4, further comprising one or more (multiple possible) flavorings, flavor and aroma compounds (multiple possible) and / or colorants (multiple possible), or combinations thereof.

[0053] B6. Food of any of Aspects B1, B2 - B5, processed into any of the consumption forms selected from the group consisting of paste, puree, soup, pie, powder, granule, chip, tablet, capsule, spread and sausage.

Brief Description of Drawings

[0054] The present invention is further illustrated by the following figures, tables, and examples, from which further features, embodiments, and advantages can be obtained. Thus, the specific modifications discussed should not be construed as limitations to the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, modifications, and alterations can be made without departing from the scope of the present invention, and accordingly, it is to be understood that such equivalent embodiments are included herein.

[0055] In connection with the present invention,

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DETAILED DESCRIPTION OF THE INVENTION

[0056] The inventors have recognized that poultry meat, especially chicken and duck meat, is a major source of edible protein. It has also been recognized that conventional approaches to producing poultry meat or meat in general are not efficient and cannot produce sufficient quantities of healthy products to meet the rapidly growing consumer needs and the increasing number of meat consumers.

[0057] "Poultry" foods grown in vitro can serve as alternatives to conventionally produced poultry meat or as food supplements. Importantly, since in vitro culture is carried out under controlled aseptic conditions, it enables the production of synthetic foods without harmful contamination. Furthermore, the culture processes described herein are suitable for producing cell biomass on an industrial scale at reasonable prices.

[0058] Accordingly, an object of the present invention is to provide a food product manufactured from avian cells grown in vitro, which can be used as a supplement to conventional chicken or duck meat or any meat or synthetic meat products.

[0059] In one aspect, the present application provides a method for manufacturing a synthetic food cultured in vitro.

[0060] The term "synthetic food" refers to products for consumption produced by culturing cells isolated from non-human animals. The term "synthetic food", as used herein, is interchangeable with terms such as "meat-like products", "synthetic meat", "in vitro meat", "cultured meat", "cell-cultured meat", "clean meat", "artificial meat", and "lab-grown meat".

[0061] "In vitro" means that a process is carried out on isolated cells outside the body, particularly isolated cells grown in a synthetic culture medium.

[0062] Avian cell line In one embodiment, the method of the present invention is carried out on, but not limited to, an avian cell line. The term "avian" or "bird" refers to any species, subspecies, or race of organisms in the taxonomic class "Aves". More specifically, "bird" refers to any animal taxonomically belonging to the orders Anseriformes (ducks, geese, swans, and their relatives), Galliformes (chickens, quails, pheasants, turkeys, and their relatives), and Columbiformes (pigeons and their relatives).

[0063] In one embodiment, the bird is selected from specific pathogen free (SPF) species that do not produce infectious endogenous retrovirus particles. "Endogenous retrovirus particles" means retrovirus particles or retroviruses encoded by and / or expressed from ALV-E or EAV proviral sequences present in some avian cell genomes. For example, the ALV-E proviral sequence is known to be present in the genomes of poultry (except for line-0 chickens), pheasants, and quails. The EAV proviral sequence is known to be present in all genera of pheasants, including poultry, line-0 chickens, pheasants, blue-eared pheasants, silver pheasants, green pheasants, Ceylon pheasants, and their relatives (see Resnick et al., 1990). Thus, preferably, the bird is selected from the group including ducks, geese, swans, turkeys, quails, Japanese quails, partridges, and peacocks that do not produce infectious endogenous ALV-E and / or EAV particles.

[0064] In a preferred embodiment, the bird is a chicken, particularly a chicken of the genus Gallus. For example, the chicken line is selected from ev-0 poultry species (Gallus Gallus subspecies domesticus), particularly line ELL-0, DE, or PE11. In another preferred embodiment, the chicken is selected from SPF species screened for the absence of reticuloendotheliosis virus (REV) and avian exogenous leukemia viruses (ALV-A, ALV-B, ALV-C, ALV-D, or ALV-J), particularly from the White Leghorn line, most preferably from the Barrow line.

[0065] In another preferred embodiment, the bird is a duck, more preferably a Pekin or Rouen domestic duck, most preferably the Pekin duck line M14 or GL30.

[0066] In a further embodiment, the cell line of the present invention is derived from avian pluripotent embryonic stem (ES) cells. "Pluripotent" means that the cells are undifferentiated or that the cells can give rise to several different cell types, such as muscle cells, adipocytes, osteocytes or chondrocytes, but cannot develop into a complete organism. Preferably, the avian pluripotent ES cells are obtained from avian embryos, particularly at a very early stage of development, such as blastoderm stage avian embryos. More specifically, the ES cells are isolated from embryos near the time of egg laying, such as before egg laying, at the time of egg laying, or after egg laying. Preferably, the ES cells are isolated from embryos at the time of egg laying. Those skilled in the art can define the time frame before egg laying that allows for appropriate cell collection (see Sellier et al., 2006; Eyal-Giladi and Kochan, 1976).

[0067] Alternatively, the avian cell line can be derived from totipotent ES cells such as cells from the blastocyst stage of a fertilized egg.

[0068] Alternatively, the ES cell line can be obtained from primordial germ cells (PGCs). For example, PGCs can be isolated from the blood of embryos collected from the dorsal aorta of chicken embryos at stages 12-14 of the Hamburger & Hamilton classification (Hamburger & Hamilton, 1951). In other cases, PGCs can be collected from the embryonic crescent or from the gonads by mechanical dissection of avian embryos (see, for example, Chang et al., 1992; Yasuda et al., 1992; Naito et al., 1994).

[0069] Furthermore, the avian cell line of the present invention can be derived from avian induced pluripotent stem cells (iPSCs).

[0070] As a further alternative, the avian cell line of the present invention can be derived from avian somatic stem cells.

[0071] In another embodiment, the avian cell line of the present invention can function as a progenitor cell to obtain partially differentiated or differentiated cells. Indeed, these stem cells are pluripotent, which means that they can be induced to convert into multiple differentiation pathways, particularly into muscle cells, or adipocytes, or chondrocytes, or other suitable cells.

[0072] In a further embodiment, the avian cell line is a continuous cell line. By "continuous" it is meant that the cells can replicate in culture for a long period of time. More specifically, the cells of the present invention can grow in culture for at least 50 days, at least 75 days, at least 100 days, at least 125 days, at least 150 days, at least 175 days, at least 200 days, at least 250 days or indefinitely.

[0073] In a further embodiment, an avian cell line, such as a duck or chicken cell line, is continuous and stable. By "stable" it is meant that the cells have a stable population doubling time and controlled growth, a stable phenotype (shape, size, microstructure, nuclear-cytoplasmic ratio), a stable optimal density when maintained under defined conditions, as well as a stable cell cycle duration leading to stable expression of proteins (such as telomerase, etc.) and markers (such as SSEA1 and EMA-1, etc.). In a preferred embodiment, the avian cell line, particularly the EBx cell line, has a stable phenotype (shape, size, microstructure, nuclear-cytoplasmic ratio) characterized by a high nuclear-cytoplasmic ratio, high telomerase activity, and the expression of one or more ES cell markers, such as alkaline phosphatase and SSEA-1, EMA-1 and ENS1 epitopes, etc., and also has a stable cell cycle. These parameters can be measured by techniques well known in the art. For example, a stable phenotype can be measured by electron microscopy. The cell cycle can be measured based on monitoring of DNA content by flow cytometry using co-staining with bromodeoxyuridine (BrDU) and propidium iodide (PI). Those skilled in the art can also use other methods.

[0074] In a further embodiment, the cell line of the present invention is genetically stable, which means that all cells maintain a similar karyotype over passages.

[0075] Preferably, the avian ES cells of the present invention do not undergo genetic modifications specifically introduced for indefinite replication. Continuous cell lines can be obtained spontaneously after a multi-step process that allows the selection of stable cells that maintain some of the unique biological properties of ES cells, such as the expression of ES cell-specific markers (e.g., telomerase, SSEA-1, EMA-1), the ability to self-renew indefinitely in vitro, and long-term genetic stability (Olivier et al., 2010; Biswas and Hutchins, 2007).

[0076] Alternatively, continuous cell phenotypes can be obtained by genetic modification and / or immortalization processes. "Immortalization" means cells that normally do not proliferate indefinitely but, due to mutations, avoid normal cell aging and can continue to divide. Mutations can be intentionally induced, for example, by physical, chemical, or genetic modifications. Physical modifications can be achieved by UV, X-ray, or gamma-ray irradiation. Chemical modifications can be achieved by chemical mutagens (substances that damage DNA). Genetic modification means that cells can be transiently or stably transfected with viral or non-viral vectors, for example, proto-oncogenes, telomerase, or transcription factors such as OCT4, Klf4, Myc, Nanog, LIN28, etc., for gene overexpression. Methods for immortalizing cells are described, for example, in patent applications WO2009137146 (quail cells immortalized with UV light), WO2005042728 (duck cells immortalized by viral transfection), and WO2009004016 (duck cells transfected with non-viral vectors), the entire contents of which are incorporated herein by reference.

[0077] In one further embodiment, the avian cell line of the present invention is a non-adherent cell line, which means that the cells can grow in suspension without a support surface or matrix. The cells of the present invention can spontaneously become non-adherent during culture, or non-adherence can be obtained by removing the feeder layer. Non-adherent cells can proliferate in the culture suspension for a long time until a high cell density is reached. Thus, they are perfectly suitable for large-scale production in a bioreactor.

[0078] Furthermore, the cells of the present invention have at least one of the characteristics of a large nucleus, a high nuclear-cytoplasmic ratio, a stable number of chromosomes, elevated telomerase activity, positive alkaline phosphatase activity, and the expression of EMA1, ENS1 and SSEA-1 surface epitopes (ES-specific markers). Alternatively, these cells may be genetically engineered to produce a substance of interest, such as a protein, lipid, enzyme, vitamin, etc.

[0079] In one embodiment, the avian cell line of the present invention is obtained by the method previously described in WO2003076601, WO2005007840, or WO2008129058, which are hereby incorporated by reference in their entirety. Briefly, avian ES cells are isolated from avian embryos (s) near the egg-laying period. The cells contain all factors that assist cell growth, and further contain at least one, preferably two growth factors, such as insulin growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), stem cell factor (SCF) and / or fibroblast growth factor (FGF), animal serum and supporting layer cells, and are cultured in a basal culture medium supplemented with them. After several passages, the culture medium is gradually modified by reducing and / or completely removing the growth factors, animal serum, and feeder layer cells, and subsequently the cells are further adapted to the suspension. This gradual adaptation of the cultured cells to the basal synthetic medium results in an adherent or non-adherent avian cell line (also referred to herein as "EBx" or "EBx cell line (s)"), which can grow in culture for a long time, especially for at least 50 days, at least 250 days, preferably indefinitely. The established EBx cell line can grow in suspension in a basal culture medium without exogenous growth factors, animal serum, and feeder layer cells for at least 50 days, 100 days, 150 days, 300 days, or 600 days.

[0080] More specifically, the avian cell line is a) isolating avian embryonic stem cells from embryos (s) at a developmental stage near the egg-laying period; b) culturing the cells for at least 20 passages in a basal culture medium containing at least one exogenous growth factor SCF, IGF-1, bFGF, IL-6, IL-6R, and CNTF, a layer of feeder cells, and animal serum; c) modifying the culture medium by gradually removing the growth factors, feeder cells, and animal serum, and further culturing the cells for at least several passages; d) adapting the cells of step c) to a suspension, It can be obtained by a process comprising the step of obtaining an established cell line capable of growing in a basal culture medium for at least 50 days, preferably at least 600 days, in the absence of exogenous growth factors, feeder cells and / or animal serum.

[0081] Alternatively, an avian cell line a) isolating avian embryonic stem cells from embryos at a developmental stage near the egg-laying period; b) culturing said cells in a basal culture medium containing exogenous growth factors IGF-1 and CNTF, a layer of feeder cells, and animal serum for at least one passage; c) gradually withdrawing said growth factors from the culture of step b) and growing for at least one passage; d) gradually withdrawing feeder cells from the culture of step c) and growing for at least one passage; e) gradually withdrawing animal serum from the culture of step d) and growing for at least one passage; f) adapting the cells of step e) to a suspension, It can be obtained by a process comprising the step of obtaining an established avian cell line capable of growing in a basal medium for a long period (at least 50 days), preferably indefinitely, in the absence of exogenous growth factors, feeder cells and / or animal serum.

[0082] "Passage" means transferring cells from one culture vessel to another, with or without dilution. This term is synonymous with the term "subculture". The number of passages is the number of times the cells have been subcultured or passaged in a new vessel. This term is not synonymous with the population doubling time (PTD) or generation, which is the time required for a population of cells to double once. For example, the isolated avian ES cells of step a) above have a PDT of more than about 40 hours. The cells of an established avian cell line have a PDT of less than about 30 hours or less than about 20 hours. In the case of ES cells, subculture usually occurs once every three generations.

[0083] "Stepwise removal or discontinuation" means that any component gradually decreases until complete disappearance (complete discontinuation) that spreads over time. To establish the cell line of the present invention, a population of avian embryo-derived stem cells that can grow indefinitely in a basal medium is isolated by removing growth factors, serum, and / or feeder layers.

[0084] "Adapt to suspension" means adapting the cells to grow as non-adherent cells without a support surface, matrix, or carrier.

[0085] According to the present invention, "basal culture medium" means a culture medium having a classical medium formulation that enables at least cell survival and preferably better cell growth by itself. Preferably, the basal medium is a synthetic or known composition (CD) medium. Such a medium contains inorganic salts (e.g., 、 CaCl 2 , KCl, NaCl, NaHCO 3 , NaH 2 PO 4 , MgSO 4 ), amino acids (e.g., L-glutamine), vitamins (e.g., thiamine, riboflavin, folic acid, D-Ca pantothenate), and optionally other components such as glucose, sucrose, beta-mercapto-ethanol, and sodium pyruvate. Non-limiting examples of basal media are SAFC Excell medium, BME (basal Eagle medium), MEM (minimum Eagle medium), Medium 199, DMEM (Dulbecco's modified Eagle medium), GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 (Gibco) and Ham-F10 (Gibco), IMDM (Iscove's modified Dulbecco medium), MacCoy's 5A medium, RPMI 1640, and GTM3.

[0086] In some embodiments, the basal synthetic medium can be supplemented with at least one growth factor selected from the group consisting of IL-6, IL-6R, SCF, FGF, IGF-1, and CNTF. The final concentration of each growth factor used in step b) of the above process is preferably about 1 ng / mL.

[0087] Furthermore, in some embodiments, the basal synthetic medium may be supplemented with insulin at a concentration of 1 to 50 mg / L, particularly 1 to 10 mg / L, preferably about 10 mg / L.

[0088] Furthermore, in some embodiments, the basal synthetic medium may be supplemented with L-glutamine (L-Gln) at a concentration of 0 to 12 mM, preferably 1 to 5 mM, more preferably about 2.5 mM.

[0089] Furthermore, in some embodiments, the basal synthetic medium may be supplemented with one or more components (plural available) selected from the group consisting of amino acids, nucleotides, vitamins, saccharides, fatty acids, beta-mercapto-ethanol, glycine, choline, pluronic acid F-68, and sodium pyruvate.

[0090] Furthermore, the basal synthetic medium may be supplemented with animal serum (e.g., fetal bovine serum) at a concentration of 1% to 10%. Preferably, the animal serum concentration in step b) of the above process is about 5 to 10%. In some embodiments, a serum-free basal culture medium is used.

[0091] Instead of animal serum, the basal medium can be supplemented with a non-animal-derived protein hydrolysate. The non-animal origin protein hydrolysate is selected from the group consisting of bacterial tryptone, yeast tryptone, plant hydrolysates such as yeast or soybean hydrolysates, or mixtures thereof. In a preferred embodiment, the non-animal origin protein hydrolysate is soybean hydrolysate.

[0092] For the establishment of the avian cell line of the present invention, the preferred basal medium is DMEM-HamF12 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 1% vitamins, 0.16 mM beta-mercapto-ethanol, and optionally 1× yeast hydrolysate.

[0093] Details of the conditions used for the establishment of avian cell lines can be found in WO2003076601, WO2005007840 and WO2008129058.

[0094] In one embodiment, the cell line established according to the above method is a chicken cell line. In another embodiment, the cell line established according to the above method is a duck cell line. The cell line established according to the above method is genetically stable, continuous, and can grow in suspension in a basal synthetic medium in the absence of exogenous growth factors, feeder cells and / or animal serum. They also exhibit sustained viability and replicative ability under long-term culture conditions and are thus ideally suited for industrial-scale growth to produce high yields of biomass that can be used as food.

[0095] In another embodiment, the avian cell line of the present invention is selected from, but not limited to, avian EBx cell lines already described in patent applications WO2003076601, WO2005007840 and WO2008129058, provided that the cell line has all of the above characteristics. Thus, the cell line of the present invention may be a non-adherent avian cell line selected from the group consisting of chicken cell lines, particularly EB1, EB3, EB4, EB5, EB14, EB line 0 and EBv13 cell lines (described in WO2003076601 and WO2005007840). Preferably, the chicken cell line does not contain infectious endogenous retrovirus as EB line 0, or the chicken cell line is derived from SPF species as EBv13, both of which are described in WO2008129058. Most preferably, the chicken cell line is a cell line derived from EBv13, particularly DL43 and DL46, and is obtained by the process of aspect A3 described above in the summary of the invention.

[0096] According to a preferred embodiment, the cell line may be any duck EBx cell line described in WO2008129058. In particular, the duck cell line may be selected from, but not limited to, the group consisting of EB24, EB26 and EB66 cell lines. Most preferably, the duck cell line is EB24 (WP24) or EB26 (WP26). The cell line names EB24 and WP24, and EB26 and WP26 used in this application are interchangeable. All duck EBx cell lines have a common characteristic, namely that they are derived from duck ES cells, are stable, continuous, and can grow in a high-density suspension in synthetic medium for a long period or indefinitely in the absence of exogenous growth factors, feeder cells, and / or animal serum. Importantly, they do not contain ALV-E and / or EAV proviral sequences in their genome, and thus there are no endogenous replicable retrovirus particles.

[0097] In a further embodiment, the cell line of the present invention is a new avian cell line obtained by one of the above processes, characterized in that the cell line is stable, continuous, and free of endogenous or exogenous virus particles, and is capable of growing in a basal synthetic medium in the absence of additional growth factors (s), such as natural or synthetic hormones or their derivatives, feeder cells and / or any additional animal products (including serum). The provirus and / or oncogenic sequences capable of growing in suspension until the cell density is high and generating a high yield of biomass.

[0098] Alternatively, the avian cell line can be selected from any commercially available cell line including but not limited to the duck cell line AGE1.CR®.pIX (described in WO2005042728), the DuckCelt®-T17 cell line (described in WO2009004016) and the quail cell line QOR / 2E11 (described in WO2009137146). Briefly, AGE1.CR®.pIX is a genetically modified duck cell line derived from retinal or embryonic fibroblasts immortalized by transfection with adenoviral genes. Another recombinant duck cell line, DuckCelt®-T17, was generated from primary embryonic cells of Cairina moschata by integration of the E1A sequence into the genome. The quail QOR / 2E11 cell line was obtained as an adherent cell line from quail embryos by UV irradiation, but adaptation to growth in suspension has also been reported (see Kraus et al., 2011).

[0099] The avian cell line of the present invention can be further characterized by standard methods known in the art. For example, a method capable of characterizing the cell line and determining its specific feature(s) can be sequencing of the genome of the cell line. Once the complete genome is known, copies of the cell line can be obtained by starting with a cell line with a very similar genomic sequence and changing the sequence by gene editing such as the CRISPR-Cas9 method (see Hsu et al., 2014).

[0100] Process for producing avian cell biomass In another aspect, the present application provides a process for the scaled-up high-yield production of cell biomass derived from the above-described avian cell lines. Briefly described, this process includes, but is not limited to, the step of adapting cells from a master or working bank to a cell culture medium; the step of scaling up secondary cultures of the adapted cells in T-flasks or Erlenmeyer flasks of various sizes; the step of seeding the adapted cells into a suitable bioreactor; the step of culturing a suspension of the adapted avian cells in a synthetic medium until a high density of cells is reached; and the step of collecting the cell biomass by filtration, centrifugation or precipitation (sedimentation and decantation), or any kind of method capable of separating the cells from the medium.

[0101] It should be noted that variations of the above process that result in the production of large cell biomass are also encompassed by the present invention.

[0102] The present application also provides conditions for the large-scale production of avian cell biomass.

[0103] In particular, the present application provides a cell culture medium that is a synthetic medium free of substances harmful to humans and / or animals. More specifically, the medium can be selected from the group including, but not limited to, BME (Basal Medium Eagle), MEM (Minimum Essential Medium), Medium 199, DMEM (Dulbecco's Modified Eagle Medium), GMEM (Glasgow Modified Eagle Medium), DMEM-HamF12, Ham-F12, Ham-F10, IMDM (Iscove's Modified Dulbecco Medium), MacCoy’s 5A Medium, RPMI 1640, GTM3, Ex-Cell® EBx™ GRO-I, HYQ CDM4 PermAb and HYQ CDM4 Avian Medium (Hyclone), L-15 (Leibovitz), OptiPRO™ SFM and 293 SFM II, or combinations thereof. Alternatively, the culture medium may be a newly developed synthetic medium experimentally developed, for example, by combining or modifying commercially available media. To improve cell growth, additional components may be added to the medium. They include, but are not limited to, amino acids (non-essential or essential amino acids), especially L-glutamine, methionine, glutamate, aspartate, asparagine, nucleotides, insulin, vitamins (e.g., thiamine, riboflavin, folic acid, calcium D-pantothenate), saccharides (e.g., D-glucose, D-sucrose, D-galactose or mixtures thereof), fatty acids, beta-mercapto-ethanol, glycine, choline, pluronic acid F-68 and sodium pyruvate. The final concentration of L-glutamine (L-Gln) in the culture medium can be used in the range of 0 to 12 mM or 0 to 10 mM, especially 1 to 5, more especially 2 to 4 mM, preferably about 2.5 mM. The final concentration of insulin in the medium may be in the range of 1 to 50 mg / L, especially 1 to 10 mg / L, preferably about 10 mg / L.

[0104] Preferably, the culture medium does not contain any animal products, and in particular does not contain animal serum. "Serum-free medium" (SFM) means a ready-to-use cell culture medium that does not require animal serum. The SFM medium of the present invention contains many components including amino acids, vitamins, organic and inorganic salts, and a carbohydrate source, and each component is present in an amount that aids in the culture of cells in vitro. This medium does not necessarily have a known composition and may contain hydrolysates of various origins, for example from plants (e.g., soybeans) or yeast. In a preferred embodiment, the culture medium is a known composition SFM that does not contain components of animal or human origin ("animal-origin-free").

[0105] Preferably, the cell culture is carried out in HYQ CDM4 Avian medium or a combination thereof, particularly in HYQ CDM4 avian medium supplemented with L-Gln used at a concentration of 2.5 - 4 mM.

[0106] According to another embodiment of the present invention, the cells grow in suspension without a support or matrix. Alternatively, the cells can adhere to a substrate, a scaffold, or microcarrier beads or a gel.

[0107] According to other embodiments of the present invention, the cell culture can be carried out in batch, fed-batch, perfusion, or continuous mode.

[0108] Briefly described, fed-batch culture is defined in the broadest sense as an operating technique in a biotechnology process in which one or more nutrients are supplied to a bioreactor during culture and the product remains in the bioreactor until production is complete (Yamane & Shimizu, 1984). Fed-batch strategies are usually used in bioprocesses to achieve high cell densities in bioreactors. In most cases, the feed solution is highly concentrated to avoid dilution of the bioreactor, increases in pH and osmotic pressure. The controlled addition of nutrients directly affects the growth rate of the culture and helps to avoid nutrient depletion, overflow metabolism and oxygen limitation (Jeongseok Lee et al., 1999).

[0109] Fed-batch culture is a culture in which the supply rate of the growth-limiting substrate is constant, that is, the supply rate remains unchanged during the culture. When the supply rate of the growth-limiting substrate is increased in proportion to the exponential growth rate of the cells, an exponential fed-batch culture, which can maintain an exponential cell growth rate for a long period, is called.

[0110] Perfusion culture means maintaining cell culture in a bioreactor, and while the cells are retained in the reactor, an equal amount of medium is simultaneously added and removed. This provides a stable source of fresh nutrients and continuous removal of cell waste products.

[0111] The culture vessel of the present invention can be selected from a stirred flask, an Erlenmeyer flask, a spinner flask, and a stirred paddle or wave bioreactor, but is not limited thereto. In particular, the culture vessel can be selected from a continuous stirred tank bioreactor, a Wave (trademark) bioreactor, a Bello (trademark) bioreactor, a Mobius bioreactor, a stirred bioreactor (for example, Orbshake), a bioreactor equipped with a perfusion system, but is not limited thereto. In the case of scale-up production, a preferred culture vessel is a bioreactor. The volume of the bioreactor is 20 liters or more, 100 liters or more, 1,000 liters or more, preferably up to 10,000 liters. According to a preferred embodiment, the culture vessel is a continuous stirred tank bioreactor that enables control of temperature, aeration, pH, and other control conditions, and has a suitable inlet for introducing cells, sterile oxygen, and various media for culture, an outlet for attaching a probe, removing cells and media, and means for stirring the culture medium in the bioreactor.

[0112] Typically, cells are scaled up from master or working cell bank vials through T-flasks, Erlenmeyer flasks, roller bottles, or Wave™ bioreactors of various sizes. The resulting cell suspension is then fed into larger bioreactors for further culture. For example, approximately 16 billion cells are used to seed a 30 L bioreactor.

[0113] In a preferred embodiment of the present invention, cell culture is carried out at pH 7.2 (adjusted by CO 2 or NaOH injection), 50% pO 2 , a stirring speed of 40 rpm, and a temperature of 37°C.

[0114] The population doubling time (PDT) in fed-batch culture may be in the range of 10 to 40 hours, preferably 10 to 20 hours, more preferably 10 to 15 hours, and most preferably about 12 hours (or less).

[0115] The theoretical maximum cell concentration (cell density) obtainable from animal cells in suspension culture is considered to be about 10 9 ~10 11 cells / mL. In many of the conventional cell lines used in industrial production, the cell density is in the range of 2×10 6 ~4×10 6 cells / mL obtained in fed-batch mode and up to 3×10 7 cells / mL in perfusion mode (see Tapia et al., 2016).

[0116] The avian cell lines used in the process of the present invention have high potential for industrial-scale production, and the selection of an appropriate cell line is important. The main selection criteria are, in most cases, the ability to remain stable over several passages and the ability to safely produce as much biomass as possible in as short a time as possible. For example, the EB66 cell line, when cultured in perfusion mode, yields 1.6×10 8It can reach a cell density exceeding cells / mL (see Nikolay et al., 2018). Typically, the cell density obtained for EBx cells in fed-batch culture is in the range of 1×10 7 ~2×10 7 cells / mL. In a preferred embodiment, the cultured cell density is at least about 1×10 7 cells / mL, at least about 2×10 7 cells / mL, at least about 5×10 7 cells / mL, at least about 10 8 cells / mL.

[0117] Typically, the cell biomass is in the range of 0.5 - 1.0 mg or more per million cells, preferably 0.7 - 1.0 mg or more per million cells, more preferably about 1 mg or more per million cells. The bulk cell yield achievable by this process is expected to be more than 10 11 cells / L.

[0118] A typical process for culturing avian cell suspensions includes the following steps. 1) Thaw 10 - 20 million CD medium-compatible cells contained in a cryovial in a 37°C water bath, suspend them in about 30 mL of pre-warmed CD medium, and place them in an incubator at 37°C in a humidified atmosphere (over 80%) of 7.5% CO 2 while stirring on a 25 mm orbital shaker at 150 rpm. 2) After recovery, subculture the cells from step 1 and amplify them for three passages in a large Erlenmeyer flask seeded at a concentration of about 0.3×10 6 ~0.5×10 6 cells / mL. Between each subculture, incubate the Erlenmeyer flask at 37°C, 7.5% CO 2 and 150 rpm for 3 days. 3) After three passages, seed the cells in a volume ratio of about 1:10 in 20 L of CD medium in a 30 L bioreactor; culture the cells at 37°C, 40 rpm, 50% O 7 until a cell density of at least 10 2 cells / mL is reached for 3 days. 4) Harvest the cells by centrifugation at 3450 g for 10 minutes, or by filtration, or by precipitation.

[0119] In one embodiment, cell precipitation can be performed by adding a calcium salt to the cell suspension. The calcium salt can be selected from the group consisting of, but not limited to, calcium chloride, calcium acetate, calcium carbonate, calcium citrate, and calcium lactate. Preferably, calcium chloride is used. The final concentration of calcium chloride is in the range of 10 - 500 mg / L, preferably 50 - 300 mg / L, more preferably 50 mg / L. After the addition of calcium chloride, avian cells form large aggregates (clots) that precipitate. Calcium chloride can be added to the bioreactor at the end of the cell amplification process. As a result, the cell biomass settles to the bottom of the vessel, and the supernatant can be removed by decantation. If the collection port is located at the bottom of the vessel, a reduced volume of concentrated cell "paste" can be collected and used in the next step of the bioprocess.

[0120] Examples of perfusion cultures of avian EBx cell lines, particularly the EB66 cell line, in a bioreactor are described in Nikolay at el., 2018. Briefly, a 1 L bioreactor was operated with a scalable hollow fiber-based tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion system.

[0121] Cultures in the perfusion bioreactor were performed at a fixed cell-specific perfusion rate (CSPR) calculated as CSPR = D perf / X v (where D perf is the volume of the perfusion medium and X v is the viable cell concentration). The CSPR can vary widely between bioprocesses and is typically selected in the range of 50 - 500 pL / cell / day depending on the feed profile (Konstantinov et al., 2006). When EB66 cells were cultured in a known composition CDM4Avian medium at a CSPR of 34 pL / cell / day, the cell concentration was 1.6×10 8It became cells / mL. In another example of perfusion, in the culture of the AGE1.CR.pIX® cell line run in manual mode with a CSPR of about 60 pL / cell / day, a cell concentration of 5.0×10 7 cells / mL was achieved (Vazquez-Ramirez et al., 2018).

[0122] In a preferred embodiment of the present invention, in order to culture avian cells and prepare a final food substantially free of hazardous microorganisms such as bacteria, fungi, viruses, prions, protozoa, or any combination thereof, it is necessary to use aseptic techniques. Preferably, the production is carried out under Good Manufacturing Practice (GMP) conditions that avoid harmful contamination.

[0123] In another aspect, the present application provides a cell biomass derived from an avian cell line cultured in vitro. The cell biomass comprises or consists essentially of avian cells cultured in vitro. The cell biomass can be obtained by the processes provided herein or any modified processes. Manufacturing processes suitable for avian cell culture can be considered. High-yield cell culture carried out on an industrial scale is preferred.

[0124] In yet another aspect, the present invention relates to the use of the above avian cell lines and cell biomass for the production of synthetic food for human or animal consumption.

[0125] In yet another aspect, the present invention provides a synthetic food derived from avian cells grown in vitro, suitable for human or animal consumption.

[0126] In one embodiment, the synthetic food of the present invention comprises or consists essentially of avian cell biomass produced according to any of the above processes. In one particular embodiment, the synthetic food comprises or consists essentially of cell biomass derived from a chicken cell line, preferably selected from the group consisting of, but not limited to, the above EB1, EB3, EB4, EB5, EB14, EB line 0, and EBv13, DL43 and DL46 cell lines. In another particular embodiment, the cell line may be selected from the group consisting of, but not limited to, the duck EB24, EB26 and EB66 cell lines. Alternatively, the synthetic food may comprise or consist essentially of cell biomass derived from an avian cell line obtained by any of the processes described herein.

[0127] Preferably, the synthetic food of the present invention comprises or consists essentially of cell biomass obtained from the chicken cell line DL43 or the duck cell line EB26 (WP26).

[0128] In one embodiment, the synthetic food of the present invention does not contain additional component(s) of animal origin such as cells, proteins, polypeptides, enzymes, lipids, body fat, animal tissues, serum, etc.

[0129] In another embodiment of the present invention, the synthetic food of the present invention may further contain other cells derived from any animal tissue such as muscle, fat or chondrocytes, or a combination thereof. These cells may be primary somatic cells derived from any animal such as mammals (cows, pigs, rabbits, pigs, sheep, deer, etc.), birds (chickens, ducks, ostriches, turkeys, pheasants, etc.), fish (tunas, salmon, tuna, sea bass, trout, catfish, etc.), invertebrates (lobsters, crabs, shrimps, bivalves, oysters, mussels, sea urchins, etc.), reptiles (snakes, alligators, turtles, etc.), amphibians (e.g., the legs of Xenopus laevis, etc.). Alternatively, these cells may be cells derived from pluripotent embryonic stem cells induced into differentiated cells. For example, muscle cells may be primary muscle cells, or may be derived from pluripotent mesenchymal stem cells that give rise to muscle cells, adipocytes, osteocytes, and chondrocytes. Examples of avian cells include, but are not limited to, the ATCC cell lines DF1 (CRL-12203 chicken), QM7 (quail), DE (duck), and the chicken embryo fibroblasts described in WO2018011805. These cells can be grown in vitro together with avian cells or added after the collection of avian cells. The addition of these cells may improve the taste, aroma, and / or nutritional value of the synthetic meat. For example, meat with more fat is more delicious and can improve the taste characteristics of the product. The ratio of muscle cells to adipocytes can be adjusted in vitro to produce a food with optimal flavor and health effects. Muscle cells and chondrocytes can improve the texture (consistency) of the product. Examples of synthetic foods having muscle cells and chondrocytes include chicken breast or pork belly.

[0130] In yet another embodiment, other nutrients such as vitamins that are typically lacking in meat products of the whole animal can be added to enhance the nutritional value of the synthetic food. This can be achieved by direct addition of nutrients to the culture medium or by genetic engineering techniques. For example, genes of enzymes involved in the biosynthesis of specific vitamins such as vitamin D, A, or various vitamin B complexes can be transfected into cultured avian cells to produce specific vitamins. Other nutrients include, but are not limited to, essential trace elements, minerals, coenzymes, essential fatty acids, essential amino acids, enzymes, antioxidants, and the like.

[0131] In yet another embodiment, the process of the present invention may also include adding flavorings and / or flavor aromatics. The flavorings may be added during the mixing step or may be mixed with any of the components (e.g., cultured cells) prior to the mixing step. Examples of flavorings that create taste and sensation include artificial sweeteners, glutamate salts, glycine salts, guanylate salts, inosinate salts, ribonucleotide salts, and organic acids including acetic acid, citric acid, malic acid, tartaric acid, and polyphenols. Some representative examples of common flavor aromatics include isoamyl acetate (banana), cinnamaldehyde (cinnamon), ethyl propionate (fruity), limonene (orange), ethyl-(E,Z)-2,4-decadienoate (pear), allyl hexanoate (pineapple), ethyl maltol (sugar, cotton candy), methyl salicylate (wintergreen), and mixtures thereof.

[0132] Furthermore, the present invention provides a color enhancer (coloring agent) that can be added to cultured cells to make food more visually appealing. Furthermore, the coloring agent functions as a physiological antioxidant and can thus provide another essential nutrient. For example, colored antioxidants such as flavonoids, carotenoids, and anthocyanins from tomatoes, Physalis peruviana, grapes, blueberries, cranberries, etc. can be used. Preferably, the coloring agent is a natural product or a purified or partially purified product. For example, purified catechins, resveratrol, anthocyanins, beta-carotene, lycopene, lutein, zeaxanthin, etc. can be used as coloring agents.

[0133] In yet another embodiment, the food of the present invention can be used to produce any type of food that can contribute to taste, texture, and nutritional components. The synthetic food of the present invention can be pickled, boiled, cooked, smoked, fried, baked, dried, or frozen and can typically be eaten as a snack or as part of a meal. The final food (edible product) obtained according to the process of the present invention can be composed in any of the consumption forms including, but not limited to, soups, purees, pastes, pies, pellets, crumbles, gels, powders, granules, tablets, chips, capsules, spreads, sausages, etc. The final food can be produced by a 3D printer. 3D printed foods are being developed by companies such as Novameat, Jet-Eat, Meatech, etc. In particular, Novameat has developed 3D printed synthetic meat having the texture of beef or chicken (see https: / / www.novameat.com / ). For details on 3D food printing, see (e.g., Sun J. et al., 2015).

[0134] Each of the final foods contains a part of cultured avian cells as an essential component, but may also contain other non-toxic substances, such as plant-derived substances (including cultured plant cells).

[0135] Finally, although the above embodiments are referred to, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limited thereto. Specific practice modes can be modified or equivalently replaced, but these modifications or changes are not excluded from the protection scope of the claims of the present invention.

Example

[0136] Example 1. Production of cell biomass Materials and methods Cell bank An avian stem cell bank (Valneva, duck cell line, GMP Working Cell Bank) prepared from cells adapted to grow in Ex-Cell® EBx™ GRO-I serum-free medium (SAFC, ref. 14530C) supplemented with 2.5 mM L-glutamine (L-Gln) was used as the starting material.

[0137] The cell line was first isolated from duck blastoderms and adapted to grow in suspension in a serum-free medium without scaffolds or matrices. The cells are characterized by their ability to grow in a carrier-free suspension at 37°C, either on a small scale (in Erlenmeyer flasks) or in a larger-scale bioreactor. When maintained under constant agitation, the cells grow as aggregates.

[0138] Preparation of CD growth medium The medium used in this process was a known composition medium, HYQ CDM4 Avian medium (Hyclone, ref. SH31036.02), supplemented with 2.5 or 4 mM L-Gln (LONZA, ref. BE17-605E).

[0139] Freezing mix A 1.46 M sucrose solution was prepared by dissolving 50 g of sucrose powder (Sigma, S1888) in 100 mL of sterile water (B Braun). The solution was then sterile filtered through a 0.22 μm filter (Millipore). The freezing mix contained 20% dimethyl sulfoxide (DMSO) (Sigma, D2438), and 0.2 M sucrose diluted in fresh CD medium supplemented with 2.5 mM L-Gln. This freezing mix was prepared immediately and placed at 4 °C before use.

[0140] Thawing of the cell bank and adaptation of the CD medium before freezing Cells were thawed by placing the cryovial in a 37 °C water bath as rapidly as possible. The cells were then diluted in 30 mL of pre-warmed CD growth medium supplemented with 2.5 mM L-Gln. Cell number and viability were evaluated with a cell counter based on the trypan exclusion method (VI-Cell XR, Beckman Coulter) using a cell aliquot. To remove the freezing medium, a cell centrifugation at 1200 rpm for 10 minutes was applied. After centrifugation, the cell pellet was resuspended in complete growth medium to obtain a final seeding density between 0.5 - 1.5×10 6 cells / mL and the cell suspension was transferred to a 125 mL Erlenmeyer flask. Cells were cultured at 37 °C and 7.5% CO 2 , with a humidity of approximately 90% (Thermo Incubator, Model 311, Hepa Class 100) under constant stirring at 125 rpm (IKA stirrer, ref. KS260).

[0141] After reactivation, cell cultures were confirmed daily by microscopic observation. During this post-thaw period, cell counts were performed regularly to evaluate cell recovery. Fresh CD growth medium was added on day 2 and day 3 to avoid over-density. On day 4, cells were seeded at 0.3×10 6 cells / mL in 60 mL of CD growth medium in a 250 mL Erlenmeyer flask. The stirring speed was increased to 135 rpm.

[0142] For amplification, cells were seeded at 0.3×10 6 cells / mL in 500 mL and 1 L Erlenmeyer flasks according to the supplier's recommendations.

[0143] Freezing of the Master Cell Bank (MCB) Cells adapted to CD medium were harvested at the exponential growth phase in 500 mL tubes by centrifuging at 1200 rpm for 10 minutes. After centrifugation, the cell pellet was diluted to 40×10 6 cells / mL with spent medium, and equal volumes of cold freezing mix were added dropwise until a final cell suspension of 20×10 6 cells / mL was obtained. Finally, the cryopreservation medium consisted of DMSO (10%) (Sigma, ref D2438 - 50 mL), 0.1 M sucrose (6.5%) (Sigma, ref S188), 50% spent CD medium recovered from the culture, and 33.5% fresh CD medium supplemented with 2.5 mM L - Gln. 1 mL of the cell freezing mixture was filled into cryovials (Corning, ref 430488), placed in a freezing container (Nalgene, Mr. Frosty™) at -80 °C, and then transferred into liquid nitrogen (-196 °C) for long - term storage.

[0144] Thawing and culturing of CD medium - adapted cell lines Cryovials containing cells adapted for growth in CD medium were thawed in a 125 mL Erlenmeyer flask under 15 mL of fresh CD medium, and placed in a shaker incubator (Kuhner, ref ISF1 - XC) with a stirring speed of 150 rpm, 7.5% CO 2 and 80% humidity. 15 mL and 20 mL of medium were added on day 1 and day 2 respectively, and then the cells were sub - cultured on day 3 for further amplification steps.

[0145] Small - scale culture After thawing, under constant stirring (150 rpm (for 250, 500, or 1 L Erlenmeyer flasks) or 80 rpm (for 3 L Erlenmeyer flasks), 25 mm orbit), at 37 °C, 80% humidity and 7.5% CO 2Cells were grown in 250 mL to 3 L Erlenmeyer flasks (Corning, Ref 431144, 431147 and 431253) maintained in a shaker incubator (Kuhner, ref ISF1-XC). Cells were seeded at 0.3×10 6 cells / mL and subcultured every three days. Seeding was performed at 60 mL, 400 mL, or 1 L in 250 mL, 1 L, or 3 L Erlenmeyer flasks, respectively.

[0146] Growth kinetics Cells were seeded at 0.1 - 0.5×10 6 cells / mL in 250 mL Erlenmeyer flasks under 100 mL of CD medium supplemented with 2.5 mM L-Gln. After transfer, daily cell counts were performed to confirm cell concentration and viability after seeding.

[0147] Parameters used for large-scale production in a 30 L stirred tank bioreactor After amplification in 3 L Erlenmeyer flasks, cells were seeded at 0.8×10 6 cells / mL into 20 L of pre-warmed medium in a 30 L stainless steel bioreactor (Applikon, Ref ADI 1075). Incubation monitoring was defined as follows: pH 7.2 adjusted by CO 2 or NaOH injection, O 2 set point 50%, stirring speed 40 rpm and temperature 37°C. Consumption of carbon sources (glucose, glutamate and glutamine) and release of metabolic by-products (lactate and ammonium) were monitored daily along with cell culture (Bioprofile Flex analyzer, Nova Biomedical).

[0148] Cell harvesting and pellet preparation Three days after seeding, cells were harvested from the bioreactor into 1 L bottles and centrifuged at 3450 g for 10 minutes (Beckman Coulter, Ref AVANTI JXN-26 / rotor JL-8.1000). After removing the spent medium, the cells were resuspended and washed in 1×PBS (LONZA, Ref BE17-516F), transferred to 500 mL tubes, and subjected to a second centrifugation at 3450 g (4000 rpm) for 10 minutes (ThermoFisher Scientific, Ref Sorvall ST40). After removing the buffer, the 500 mL tubes containing the dry pellets were weighed (balance: Denver, Ref SI 4002) and stored at -80 °C (Sanyo, Ref MDF-U73V). The weight of the cell pellet was calculated by subtracting the weight of the 500 mL tube from the total weight (500 mL tube + cell pellet).

[0149] Results Adaptation of avian stem cells to a medium of known composition The first step of the process was the generation of a bank of avian stem cells adapted to growth in the medium of known composition HYQ CDM4 Avian medium.

[0150] The purpose of this step was to prepare the sole source of cells: - To avoid some adaptations - To enable possible verification / release of the master cell bank - To use the same starting material for several batches of production - To shorten the timeline assigned to a bioproduct of known composition - To minimize batch-to-batch variability

[0151] Cell adaptation and bank generation To avoid adapting the stem cells to the CD medium for each production run, one adaptation was performed as described below and shown in Figure 1, and a working bank of 165 vials was prepared.

[0152] One cryovial of avian stem cells initially grown in Ex-cell GRO-I SFM was thawed directly in 30 mL of CDM4 Avian CD medium supplemented with 2.5 mM L-Gln. After centrifugation, 7.2×10 6 cells were recovered and seeded at a concentration of 0.6×10 6 cells / mL in 12 mL of medium in a 125 mL Erlenmeyer flask. The cells were placed in an incubator on a shaker at 125 rpm. On the 2nd and 3rd days after thawing, 8 mL and 15 mL of CD medium were added respectively. On the 4th day, an aliquot was collected for cell counting and the cells were harvested by centrifugation. The cell pellet was resuspended in fresh CD medium; then, it was seeded at a concentration of 0.3×10 6 cells / mL in 60 mL in one 250 mL Erlenmeyer flask and incubated with stirring at 135+ / -15 rpm. The next two passages were performed as follows: The cells were harvested on the 7th or 10th day and transferred to three new 500 mL Erlenmeyer flasks or 3 L Erlenmeyer flasks, and diluted to 0.3×10 6 cells / mL in 200 mL or 1 L of CD medium respectively. On the 13th day, approximately 11 billion cells were collected from the 3 L Erlenmeyer flask. The final cell concentration was 9.1×106 cells / mL and the viability was 91%.

[0153] Direct adaptation to HYQ CDM4 Avian medium was very efficient. Ten days after thawing in CD medium, the cells recovered at an expected density of 5×10 6 cells / mL and with good viability (over 80%) (see Figure 2A). As a result, the population doubling time (PDT) achieved rapidly was within the expected range of 15 - 16 hours (see Figure 2B). With respect to morphology, the cells maintained the property of growing in suspension as clumps of dozens of cells that could be easily resuspended by pipetting. On the 13th day, the cell concentration was 9.1×10 6It reached cells / mL, and the cell viability reached 91%. As a result, a cell bank of 165 vials (Bank 5777) was formed. Therefore, to adapt avian stem cells in HYQ CDM4 Avian CD medium to prepare a high-quality master cell bank, only 4 passages and 13 days were sufficient.

[0154] Validation of the cell bank To ensure the quality of the avian stem cell bank after CD adaptation, the cell bank was thawed, and the robustness, viability of the cells, as well as the stability of cell density and PDT over passages were controlled.

[0155] To confirm the robustness and stability of the cells, Bank 5777 was thawed and maintained during further 4 passages of subculture. As shown in Figure 3, the viability of the bank immediately after thawing was very good at 91%. Since the total amount of cells filled in the vials was completely recovered, there was no loss of cells related to the freezing step. After 3 days of incubation, the cell density reached about 5×10 6 cells / mL, indicating rapid cell growth. In the next passage, good quality of the cell bank was confirmed at a concentration higher than 6×10 6 cells / mL.

[0156] Growth kinetics To determine the optimal density achievable by the adapted avian stem cells, different concentrations were seeded in 250 mL Erlenmeyer flasks, incubated, and cell density and viability were confirmed daily. Figure 4A shows the cell density obtained after 3 and 4 days of culture, and Figure 4B shows the corresponding cell viability. The data indicate that an increase in seeding density up to 0.4×10 6 cells / mL does not improve the optimal cell concentration after 4 days of culture. Under all conditions where the seeding amount exceeded 0.2×10 6 cells / mL, the viability tended to slightly decrease on the 4th day. Regarding viability and cell density, the appropriate compromise point to reach the optimal density with good viability in a 250 mL Erlenmeyer flask is to seed the cells at an amount of 0.3 - 0.4×10 6 cells / mL.

[0157] Scale-up for seeding of a 30 L bioreactor To produce the cell biomass required to seed a 30 L bioreactor, the adapted avian stem cell bank 5777 was thawed and the cells were amplified according to a scale-up process conducted in Erlenmeyer flasks.

[0158] The final avian cell biomass was produced in vitro using a 30 L stainless-steel bioreactor. To seed the 30 L bioreactor with 20 L of cell suspension at a concentration of 0.8×10 6 cells / mL, 16 billion cells were required. Since avian stem cells have the property of growing at high cell densities, the scale-up procedure was not cumbersome because the required amount of cells was obtained in a 2 L suspension. Figure 5 shows a typical process for the rapid amplification of cells for seeding the bioreactor.

[0159] Figure 6 shows the cell densities obtained at each passage along the scale-up process. In the final step of amplification, the achieved cell concentration was 10.3×10 6 cells / mL and a total of 20.6 billion cells could be harvested.

[0160] Batch cell growth in a 30 L bioreactor Cells harvested from both 3 L Erlenmeyer flasks were seeded into a 30 L bioreactor at a concentration of 0.8×10 6 cells / mL under 20 liters of pre-warmed CD medium supplemented with 4 mM L-Gln. The setpoints for pH and oxygen regulation were adjusted to 7.2 and 50%, respectively, and the stirring speed was 40 rpm. Since the process was conducted in batch mode, neither glucose nor glutamine was regulated. Consumption of carbon sources (glucose, glutamate, and glutamine), as well as release of metabolic by-products (lactate and ammonium), were monitored daily along with the cell culture (Bioprofile Flex analyzer, Nova Biomedical).

[0161] Using the aforementioned parameters, three runs were performed. Figure 7 shows cell growth and viability over three days of production. After seeding, no lag phase was observed and cell proliferation was very rapid, as indicated by a short population doubling time (less than 12 hours) from seeding to day 1 (see Table 1). On day 3, an increase in PDT (more than 35 hours) indicating a deceleration of growth was observed, in conjunction with a decrease in viability.

[0162] [Table 1]

[0163] Based on the average of the higher cell concentrations obtained in the three runs and the corresponding viability, it was concluded that the optimal density was reached at a concentration of approximately 14×10 6 total cells / mL between day 2 and day 3.

[0164] In the metabolite studies conducted during the three runs, high consumption of glutamine, glutamate and glucose was shown (data not shown).

[0165] Cell harvesting Centrifugation After growing cells in the bioreactor for three days, avian cells were harvested into 1 L bottles by high speed centrifugation (3450 g) (see Figure 8), rinsed with PBS, transferred to 500 mL tubes and pelleted by a second run of centrifugation (see Figure 9).

[0166] The pellet was weighed after the last run of centrifugation. 304 g, 282 g and 281 g were obtained from Run 1, Run 2 and Run 3 respectively, demonstrating the reproducibility of the process with respect to biomass production. Finally, the pellet was frozen and stored at -80 °C.

[0167] Sedimentation and decantation Harvesting cell biomass by centrifugation is a cumbersome process, and without a cooling system, a temperature increase can be observed after several centrifugation runs, posing a risk of biomaterial denaturation. Therefore, a decantation step before centrifugation (or filtration) was considered to reduce the volume of the suspension.

[0168] Since EBx cells grow as small aggregates, conditions were investigated to induce cell aggregation to promote cell sedimentation. Addition of calcium chloride to the medium causes the formation of cell clumps. Since duck and chicken cells are not sensitive to the same range of calcium concentrations, various conditions were tested. Calcium chloride at 50, 100, 150, 200, or 300 mg / L was supplemented to the cell suspension of chicken or duck at the end of the logarithmic phase, and incubated at 37 °C for 2 - 6 hours with stirring. In the EBx cell line, aggregation was already observed after 2 hours of incubation. The largest clumps were produced at the highest calcium concentration. It was found that the size of the clumps gradually increased with the calcium concentration. In duck cells, after incubation for 2 hours in the presence of 50 mg / mL calcium chloride, almost all cells aggregated, so cell aggregation was more prominent.

[0169] After incubation with calcium chloride for 6 hours and sedimentation for 20 minutes, the cell number of residual cells in the supernatant was counted to more accurately evaluate the percentage of the cell population that sedimented to the bottom of the tube. The obtained data are summarized in Tables 2 and 3. It was observed that 42.8% of the chicken cell suspension sedimented within 20 minutes without adding calcium. By the 6-hour treatment, this sedimentation percentage was improved, reaching a maximum value of 75.5% at the highest test dose of calcium chloride (300 mg / L). In duck cells, no clear sedimentation was observed after 20 minutes without calcium, but the addition of 50 mg / L calcium chloride was sufficient for 95% precipitation of the cell biomass.

[0170] Similarly, the cell sedimentation step can be applied to the bioreactor at the end of the cell amplification process. As a result, the cell biomass precipitates to the bottom of the vessel. If the collection port is located at the bottom of the vessel, a reduced volume of concentrated cell "paste" can be collected and used in the next step of the bioprocess.

Table 2

Table 3

[0171] Other calcium salts such as calcium acetate, calcium carbonate, calcium citrate, and calcium lactate may be considered as alternatives.

[0172] Productivity Runs 1, 2, and 3 produced 304 g, 282 g, and 281 g of avian stem cells, respectively. Thus, based on the amount of cells harvested from the bioreactor (see Table 2), the biomass productivity (value obtained by dividing the total weight by the total number of cells harvested) was 1.18 + / - 0.07 mg per million cells. Since 385.6 g of media powder was required to run the 20 L bioreactor, the productivity was approximately 0.75 g of biomass per g of media powder.

Table 4

[0173] Therefore, based on the kinetics and metabolite consumption data obtained in the Erlenmeyer flask, an improvement in the product yield can be achieved as follows. - Modify the initial cell seeding to extend cell growth beyond day 3; - Supplement the CD media to avoid depletion; - Apply a fed-batch or perfusion process.

[0174] List of references: Biswas and Hutchins. 2007. “Embryonic Stem Cells.” Stem cells and Development 16:213-221. Chang et al., 1992. “Simple method for isolation of primordial germ cells from chick embryos.” Cell Biol Int. Reports 16(9):853-857. Eyal-Giladi and Kochan, 1976. “From cleavage to primitive steak formation: a complementary normal table and a new look at the first stage of the development of the chick.” Developmental Biology 49:321-337. Galbraith H. 2002. “Hormones in international meat production: biological, sociological and consumer issues.” Nutrition Research Reviews 15:293-314. GEAS. October 2012. “Growing greenhouse gas emissions due to meat production.” Greger M. 2007. “The human / animal interface: emergence and resurgence of zoonotic infection diseases”. Critical Reviews in Microbiology 33:243-299. Hamburger V. and Hamilton H. 1951. “A series of normal stages in the development of the chick embryo”. Hedenus F., Wirsenius D. and Johansson J.A. 2014. “The importance of reduced meat and dairy comsimption for meeting stringent climate change targets”. Climatic Change 124:79-91. Hsu P., Lander E.S., and Zhang F. 2014. “Development and application of CRISPR_Cas9 for genome engineering”. Cell 157, June 5:1262-1278. Jeong SH. et al., 2010. “Risk assessment of growth hormones and antimicrobial residues in meat:” Toxicol.Res. 26(4):301.313. Jeongseok Lee et al. 1999. “Control of fed-batch fermentations.” Biotechnol Adv 17:29-48. Kadim I.T. et al. 2015. “Cultured meat from muscle stem cells: a review of challenges and prospects.” J.Integrative Agriculture 14(2):222-233. Konstantinov K. et al. 2006. “The “push-to-low” approach for optimization of high-density perfusion cultures of animal cells.” Adv Biochem Engin Biotechnol 101:75-98. Kraus B. et al. 2011. “Avian cell line - technology for large scale vaccine production.” BMC Proceedings 5(suppl 8):52. Mekonnen and Hoekstra. “The green, blue and gray water footprint of farm animals and animal products.” UNESCO-IHE, Research Report Series No.48. December 2010. Naito M. et al. 1994. “Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells.” Molecule Reproduction and Development 39:153 - 161. Nikolay A. et al. 2018. “Process intensification of EB66 cell cultivations leads to high-yield yellow fever and Zika virus production.” Applied Microbiol Biotechnol 102:8725 - 8737. Olivier S. et al. 2010. “EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antoibodies with enhanced ADCC activity.” MAbs 2(4):405 - 15. Opio C., Gerber P., Falcucci A., et al., 2013. “Greenhouse gas emissions from ruminant supply chains. A global life cycle assessment.” Report prepared by Food and Agriculture organization of the United Nations (FAO), Rome. Resnick R. et al. 1990. “Phylogenetic Distribution of the Novel Avian Endogenous Provirus Family EAV-0.” L Virology 64(10):4640:4653. Ritchie and Roser. 2018. “Meat and Seafood Production & Consumption.” Sellier N., Brillard J-P and Bakst M.R. 2006. “Comparative staging of embryo development in chicken, turkey, duck, goose, Guinea fowl, and Japanese quail assessed from five hours after fertilization through seventy-two hours of incubation.” J Appl Poult Res 15:219-228. Sun J.et al. 2015. “A Review on 3D Printing for Customized Food Fabrication”, Procedia Manufacturing 1: 308-319. Tapia F. et al. 2016. “Bioreactors for high cell density and continuous multi-stage cultivations: options for process intensification in cell culture-based viral vaccine production.” Appl Microbiol Biotechnol 100:2121-2132. Tuomisto H, Ellis M.J. and Haastrup P. “Environmental impacts of cultured meat: alternative production scenarios.” Proceedings of the 9th International Conference on Life Cycle Assessment in the Agri-Food Sector. 2014 Vazquez-Ramirez D. et al. 2018. “High-cell-density cultivations to increase MVA virus production.” Vaccine 36:3124-3133. Yamane & Shimizu. 1984. “Fed-batch Techniques in Microbial Processes.” Advances in Biochem Eng. Biotechnol 30:147-194. Yasuda Y. et al. “A method to obtain avian germ-line chimaeras using isolated primordial germ cells.” J Reprod.Fert. 96:521-528. 1992.

Claims

1. 1. A process for producing a nutritional food in vitro for human or animal consumption, comprising culturing in suspension an avian cell line that i) is derived from avian embryonic stem cells, ii) is capable of growing in a basal culture medium in the absence of exogenous growth factors, feeder cells and / or animal serum, and iii) is capable of continuous proliferation in suspension.

2. The avian cell line is a) isolating avian embryonic stem cells from an embryo(s) at a developmental stage near egg-laying; b) culturing said cells for at least 20 passages in a basal culture medium containing at least one exogenous growth factor SCF, IGF-1, bFGF, IL-6, IL-6R and / or CNTF, a layer of feeder cells, and animal serum; c) modifying the culture medium by stepwise removal of the growth factors, feeder cells and animal serum and further culturing the cells for at least several passages; d) adapting the cells of step c) to a suspension; and thereby obtaining an established avian cell line capable of proliferation for at least 50 days in a basic culture medium in the absence of exogenous growth factors, feeder cells and / or animal serum.

3. The avian cell line is a) isolating avian embryonic stem cells from an embryo(s) at a developmental stage near egg-laying; b) culturing the cells for at least one passage in a basal culture medium containing the exogenous growth factors IGF-1 and CNTF, a layer of feeder cells, and animal serum; c) gradually withdrawing the growth factors from the culture of step b) and further growing it for at least one passage; d) gradually withdrawing the feeder cells from the culture of step c) and growing for at least one passage; e) gradually withdrawing the animal serum from the culture of step d) and growing it for at least one passage; f) adapting the cells of step e) to a suspension; The process according to claims 1 and 2, thereby obtaining a continuous avian cell line capable of growing in a basal medium in the absence of exogenous growth factors, feeder cells and / or animal serum.

4. The process according to any one of claims 1 to 3, wherein said avian cell line is derived from chicken embryonic stem cells.

5. The process according to any one of claims 1 to 4, wherein said avian cell line is derived from duck embryonic stem cells.

6. The process according to any one of claims 1 to 5, wherein the avian cell line is free of functional endogenous retroviruses or other viral particles.

7. The process according to any one of claims 1 to 6, wherein the avian cell line is derived from an SPF species.

8. 8. The process according to any one of claims 1 to 7, wherein the avian cell line is selected from the group consisting of chicken EB14, chicken EB line 0, chicken EBv13, chicken DL43, chicken DL46, duck EB24, duck EB26 and duck EB66 cell lines.

9. The process according to any one of claims 1 to 7, wherein said avian cell line is selected from the group consisting of chicken DL43, chicken DL46, duck EB24 and duck EB26 cell lines.

10. The process according to any one of claims 1 to 7, wherein the avian cell line is chicken DL43 or duck EB26.

11. The process according to any one of claims 1 to 10, wherein the cell line is grown in a culture medium that is a synthetic or chemically defined (CD) medium that is free of substances harmful to humans and / or animals.

12. 12. The process of claim 11, wherein the synthetic medium is Ex-Cell® GRO-I and / or HYQ CDM4 Avian medium.

13. 12. The process of claim 11, wherein the synthetic medium or CD medium is further supplemented with one or more component(s) selected from the group consisting of amino acids, nucleotides, vitamins, saccharides, fatty acids, beta-mercapto-ethanol, insulin, glycine, choline, pluronic acid F-68 and sodium pyruvate.

14. The process according to any one of claims 11 to 13, wherein the additional component is L-glutamine used at a concentration of 0 to 12 mM, preferably 1 to 5 mM, more preferably about 2.5 mM.

15. The process according to any one of claims 11 to 14, wherein the culture medium further comprises a plant and / or yeast hydrolysate.

16. The process according to any one of claims 11 to 15, wherein the culture medium does not contain any animal products, including serum.

17. The process according to any one of claims 1 to 16, wherein the cell line is cultured under fed-batch conditions.

18. The process according to any one of claims 1 to 16, wherein the cell line is cultured under perfusion conditions.

19. The process according to any one of claims 1 to 18, wherein the cell line is cultivated in a bioreactor with a volume of 30 liters, 50 liters, 100 liters, 1000 liters, preferably 10,000 liters or more.

20. The cell line is maintained at about 37° C., pH 7.2, pO ２ 20. The process according to any one of claims 1 to 19, which is cultivated at about 50% and an agitation speed of about 40 rpm or more.

21. The cell line has a cell density of about 10 ７ The process according to any one of claims 1 to 20, wherein the culture is continued until a cell / mL is reached.

22. The cell line has a cell density of about 10 ８ The process according to any one of claims 1 to 20, wherein the culture is continued until a cell / mL is reached.

23. The cell line is cultured at a cell density of 10 ８ 21. The process of any one of claims 1 to 20, wherein the culture is cultured until the cell count exceeds 100 cells / mL.

24. 24. The process of any one of claims 1 to 23, wherein the yield of the process is at least about 0.5 to 1 g of biomass per gram of medium.

25. 25. The process of any one of claims 1 to 24, further comprising the step of harvesting the cellular biomass by sedimentation and decantation.

26. 26. The process of claim 25, wherein cell precipitation is carried out by addition of a calcium salt to the cell suspension.

27. 27. The process according to claim 26, wherein the calcium salt is calcium chloride used at a final concentration of 10 to 500 mg / L, preferably 50 to 300 mg / L, more preferably 50 mg / L.

28. 28. The process of any one of claims 1 to 27, further comprising the step of adding to the cell biomass one or more ingredient(s) that increase the nutritional value of the food (selected from the group comprising vitamins, covitamins, minerals, essential amino acids, essential fatty acids, enzymes and antioxidants).

29. 29. The process of any one of claims 1 to 28, further comprising adding one or more flavour(s), flavour aroma compound(s) and / or colourant(s) to the cell biomass.

30. 30. The process of any one of claims 1 to 29, further comprising one or more food processing step(s) selected from cooling, freezing, solidifying, drying, marinated, boiling, cooking, baking, frying, smoking, 3D printing, and packaging.

31. A nutritional food product produced by the process of any one of claims 1 to 30.

32. 28. Cellular biomass produced by the process of any one of claims 1 to 27.

33. 33. Use of the cell biomass of claim 32 for producing a synthetic food for human or animal consumption.

34. 33. A synthetic food product comprising or consisting essentially of the cellular biomass of claim 32.

35. 35. The synthetic food product of claim 31 or 34, further comprising other cells, such as non-human muscle cells, adipocytes or chondrocytes, or combinations thereof, grown in vitro with the avian cells or added after harvesting the avian cells.

36. 36. The synthetic food of any one of claims 31, 34 and 35, further comprising additional ingredients that enhance the nutritional value selected from the group comprising minerals, vitamins, co-vitamins, essential fatty acids, essential amino acids, enzymes and antioxidants, or combinations thereof.

37. 37. The synthetic food of any one of claims 31, 34-36, further comprising one or more flavouring(s), flavour aroma compound(s) and / or colouring(s), or combinations thereof.

38. 37. The composite food of any one of claims 31, 34 to 36, processed into any of the consumption forms selected from the group comprising paste, puree, soup, pie, powder, granule, chips, tablet, capsule, spread and sausage.

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