Large scale fish cell production

Abstract

Provided are a growth medium and a method for growing cells.

Description

[0001] Applicant: Bluu GmbH

[0002] Our reference: P80988WO

[0003] LARGE SCALE FISH CELL PRODUCTION

[0004] BACKGROUND

[0005] Fish has been a part of the human diet for ten thousands of years. Global demand for seafood will continue to grow as the population steadily increases. At the same time, the per capita protein consumption is also projected to grow. However, industrial fishing has resulted in over 90% of the global fish stocks being overfished or fished to a maximum sustainable level. Therefore, it will be difficult to meet the future demand for fish products. While fish is generally a healthy food, microplastics and heavy metals in fish products are more and more common. Industrial aquaculture - another form of mass animal farming - often pollutes their surroundings and are hotbeds for fish diseases.

[0006] Cultured fish products have the potential to: (1) substantially reduce reliance on fish from the oceans or fresh water for food use, (2) lessen the environmental burden of raising animals for food supply, and (3) provide a reliable source of protein that is both safe and has consistent quality.

[0007] Therefore, there is a need for new fish products and methods for producing those products.

[0008] SUMMARY

[0009] The invention is defined by the appended claims.

[0010] DETAILED DESCRIPTION

[0011] The following description is presented to enable one of ordinary skill in the art to make and use the disclosed subject matter and to incorporate it in the context of applications.

[0012] Definitions As used herein, the term “edible food product” refers to a food product safe for human consumption. For example, this includes, but is not limited to a food product that is generally recognized as safe per a government or regulatory body (such as the United States Food and Drug Administration and the European Union). In certain aspects, the food product is considered safe to consume by a person of skill. Any edible food product suitable for a human consumption should also be suitable for consumption by another animal and such an aspect is intended to be within the scope herein.

[0013] As used herein, “growth medium” refers to a medium or culture medium that supports the growth of microorganisms or cells or small plants. A growth medium may be, without limitation, liquid or semi-solid. Growth medium shall also be synonymous with “growth media” or “cultivation medium.”

[0014] As used herein, “in vitro” refers to a process performed or taking place in a test tube, culture dish, bioreactor, or elsewhere outside a living organism. In the body of this disclosure, a product may also be referred to as an in vitro product, in which case in vitro shall be an adjective and the meaning shall be that the product has been produced with a method or process that is outside a living organism.

[0015] As used herein, “suspension culture” refers to a type of culture in which single cells or small aggregates of cells multiply while suspended in agitated liquid medium. It also refers to a cell culture or a cell suspension culture.

[0016] As used herein, “fibroblasts” refers to mesenchymal-derived cells that are responsible for the extracellular matrix, epithelial differentiation, and regulation of inflammation and wound healing. In addition, fibroblasts are also responsible for the secretion of growth factors and work as scaffolds for other cell types. Fibroblasts are one cell type found in conventional fish.

[0017] As used herein, “substantially pure” refers to cells that are at least 80% cells by dry weight. Substantially pure cells are between 80%-85% cells by dry weight, between 85%- 90% cells by dry weight, between 90%-92% cells by dry weight, between 92%-94% cells by dry weight, between 94%-96% cells by dry weight, between 96%-98% cells by dry weight, between 98%-99% cells by dry weight. As used herein, “primary fish fibroblast cells” refers to cells from a parental animal that survive in a suitable growth medium, for instance under controlled environmental conditions. Cells in primary culture have the same karyotype (number and appearance of chromosomes in the nucleus of a eukaryotic cell) as those cells in the original tissue.

[0018] As used herein a “immortalized fish fibroblast cells” or simply “fish fibroblast cells” are fish fibroblast cells which can proliferate ex vivo, i.e. separated from the parental animal, indefinitely. “Immortalized fish fibroblast cells” are obtainable from “primary fish fibroblast cells”.

[0019] As used herein, “proliferation” refers to a process that results in an increase in the number of cells. It is characterized by a balance between cell division and cell loss through cell death or differentiation

[0020] As used herein, and unless otherwise indicated, percentage (%) refers to total % by weight typically on a dry weight basis unless otherwise indicated.

[0021] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain aspects, the term “about” indicates the designated value± 10%, ±5%, or ±1%. In certain aspects, the term “about” indicates the designated value ± one standard deviation of that value.

[0022] Disclosures

[0023] Disclosed is a cultivation medium and a production method.

[0024] Provided is a cultivation medium for cultivating cells in suspension comprising methylcellulose.

[0025] Cells grown in suspension can be prone to adhere to each other and forming spheroids of ever increasing size. Cells within the core of such large spheroids tend to die. This especially applies to cells isolated from multicellular tissue from multicellular organisms. It has been surprisingly found that methylcellulose is capable of preventing the cell spheroids becoming too large and thereby the cultivation medium prevents the dying of cells.

[0026] Methylcellulose is a chemical compound derived from cellulose; chemically it is a cellulose ether. It is a hydrophilic white powder and dissolves in cold (but not hot) water, forming a low-viscosity or very viscous solution until it even gives the impression of a gel.

[0027] Methylcellulose does not occur naturally and is synthetically produced by heating cellulose with caustic solution (e.g. a solution of sodium hydroxide) and treating it e.g. with methyl chloride. In the substitution reaction that follows, the hydroxyl residues (-OH functional groups) are replaced by methoxide (-OCH3 groups, also known as methoxyl).

[0028] Different kinds of methyl cellulose can be prepared depending on the number of hydroxyl groups substituted. Cellulose is a polymer consisting of numerous linked glucose molecules, each of which exposes three hydroxyl groups. The Degree of Substitution (DS) of a given form of methyl cellulose is defined as the average number of substituted hydroxyl groups per glucose. The theoretical maximum is thus a DS of 3.0 which corresponds to a degree of substitution of 100% for methoxyl.

[0029] The degree of substitution of the methylcellulose of this disclosure can be 20-40%, 25- 35%, 27-32%, for methoxyl.

[0030] It has been found that particularly these degrees of substitution provide a methylcellulose which is useful for cultivating cells and preventing an unlimited size increase of cell spheroids.

[0031] The methyl cellulose can be a modified cellulose which does not contain any other modifications, but methoxyl, in particular, a modified cellulose which does not contain (any) modifications with hydroxypropoxyl.

[0032] Methylcellulose lacking hydroxypropoxyl has been found to be especially useful for growing cells in culture. The apparent viscosity of the methylcellulose via Brookfield can be 50,000 — 200,000 mPa 70,000 — 150,000 mPa, 100,000 — 120,000 mPa, or about 110,000 mPas at 2% in water and 20°C.

[0033] It has been found that particularly methylcellulose exhibiting the above viscosity properties provides a methylcellulose which is useful for cultivating cells and preventing an unlimited size increase of cell spheroids.

[0034] The cultivation medium can further comprise poloxamer.

[0035] It has been surprisingly found that poloxamer can improve the viability of cultivated cells in suspension even when the medium already contains methylcellulose.

[0036] Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).

[0037] For the generic term poloxamer, these copolymers are commonly named with the letter P (for poloxamer) followed by three digits: the first two digits multiplied by 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage polyoxyethylene content (e.g. P407 = poloxamer with a polyoxypropylene molecular mass of 4000 g / mol and a 70% poloxamer).

[0038] The poloxamer can have a (e.g. relative) molecular weight of 7000-10000 g / mol, 8000- 9000 g / mol, 8200-8600 g / mol, or 8400 g / mol. The polyoxyethylene content of the poloxamer can be 60%-95%, 70%-90%, 75%-85%, or about 80 %.

[0039] The cultivation medium can comprise 40 - 400 mg / L, methylcellulose 50 - 300 mg / L, 60 - 200 mg / L, 70 - 150 mg / L, 80 - 120 mg / L, 90 - 110 mg / L, or about 100 mg / L methyl cellulose. The cultivation medium can comprise 0.04 -- 0,4 % (w / v), 0.05 -- 0,3 % (w / v), 0.06 -- 0,2 % (w / v), 0.07 -- 0, 15 % (w / v), 0,075 -- 0,5 % (w / v), 0.09 -- 0,11 % (w / v), or about 0.1 % (w / v) methyl cellulose.

[0040] The cultivation medium can comprise 0.1 -- 1.0 % (w / v), 0.1 -- 0,8 % (w / v), 0.3 -- 0,7 % (w / v), 0.4 -- 0,6 % (w / v), or about 0.5 % (w / v) poloxamer.

[0041] In particular, the cultivation medium can comprise 0.06 -- 0,2 % (w / v) methyl cellulose and 0.1 -- 1.0 % (w / v) poloxamer, 0,05 -- 0,15 % (w / v) methyl cellulose and 0.1 -- 1.0 % (w / v) poloxamer, or 0,05 -- 0,15 % (w / v) methyl cellulose and 0.3 -- 0,7 % (w / v) poloxamer.

[0042] It has been found that these concentrations suited above are especially suited for cultivating cells.

[0043] The cultivation medium may comprise any compounds that are required to maintain or grow cells, especially, in suspension.

[0044] Proliferation can occur in suspension or adherent conditions, with or without feeder-cells and / or in serum-containing or serum-free media conditions. Media for proliferation can contain one or more of amino acids, peptides, proteins, carbohydrates, essential metals, minerals, vitamins, buffering agents, anti-microbial agents, growth factors, and / or additional components.

[0045] Proliferation can be measured by any method known to one skilled in the art. In some aspects, proliferation is measured through direct cell counts. In certain aspects, proliferation is measured by a haemocytometer. In some aspects, proliferation is measured by automated cell imaging. In certain aspects, proliferation is measured by a Coulter counter.

[0046] Proliferation, especially, viability can be measured by using viability stains. In certain aspects, the stains used comprise trypan blue.

[0047] The fish fibroblast cells can be grown attached to a substrate. The fish fibroblast cells can be grown in a suspension culture system. In some aspects, the fish fibroblast cells are grown in a batch, fed-batch, semi continuous (fill and draw) or perfusion culture system or some combination thereof. When grown in suspension culture, the suspension culture can be performed in a vessel (fermentation tank, bioreactor)) of a desired size. The vessel is a size that is suitable for growth of fish cells without unacceptable rupture of the cells. In some aspects, the suspension culture system can be performed in vessel that is at least 25 liters (L), 50 L, 100 L, 200 L, 250 L, 350 L, 500 liters (L), 1000 L, 2,500 L, 5,000 L, 10,000 L, 25,000 L, 50,000 L, 100,000 L, 200,000 L, 250,000 L, or 500,000 L. For smaller suspension cultures, the cultivation of the cells can be performed in a shaking or spinner flask that is least 125 mL, 250 mL, 500 mL, 1 L, 1.5 L, 2 L, 2.5 L, 3 L, 5 L, 10 L, or larger.

[0048] For example, the cultivation medium can comprise:

[0049] - carbohydrates (monosaccharides);

[0050] - proteins;

[0051] - isolated amino acids comprising L-glutamine;

[0052] - fetal bovine serum;

[0053] - antibiotics (penicillin and streptomycin);

[0054] - omega- 3 fatty acids comprising eicosapentaenoic acid (EP A) and docosahex-aenoic acid (DHA);

[0055] - vitamin A; and / or

[0056] - vitamin D.

[0057] The cultivation medium may not include growth factors (e.g. Fibroblast growth factor 2, FGF2, insulin-like growth factor 1, IGF-1) except for those growth factors included in FBS, if FPS is present in the cultivation medium. Thus, no growth factors are added to the cultivation medium except for those contained in FBS, if FBS is present.

[0058] In particular, the cultivation medium can comprise less than 4, less than 3 % (v / v) or 0-5, 1-4, or 2-3 % (v / v) fetal bovine serum, FBS. These low FBS values are especially useful for growing cells in suspension. It is also disclosed that the cultivation medium does not contain any FBS. The cultivation medium can also comprise a pH indicator wherein the indicator causes the color of the solution in the range visible to the human eye to change depending on the pH, e.g. phenol red.

[0059] The cultivation medium can also comprise a pH buffer, e.g. a carbonated and / or a phosphate buffer system.

[0060] Disclosed is also a bottle, container, or any other suitable vessel comprising the cultivation medium described above. The bottle, container, or any other suitable vessel may comprise any value between 10 ml and 500 liter.

[0061] Disclosed is also a kit comprising the cultivation medium without methylcellulose and (separated therefrom) methylcellulose.

[0062] Disclosed is also a method comprising:

[0063] - providing a cultivation medium comprising methylcellulose, wherein the cultivation medium is the above described cultivation medium;

[0064] - adding cells to the cultivation medium;

[0065] - proliferating the cells in the cultivation medium in suspension.

[0066] In some aspects, the cells are fish cells. In some aspects, the fish cells are selected from stem cells, induced pluripotent stem cells (iPSC), fibroblast cells, myoblast cells, myocyte cells, adipocyte cells, osteoblast cells, mesenchymal cells, epidermal cells, epithelial cells, or precursor cells of the before mentioned . In some aspects, the cells comprise fish fibroblast cells.

[0067] The fish cell lines can have a spontaneously immortalized fibroblast phenotype. The fish cell lines can have high proliferation rates. The cells can have both an immortalized fibroblast phenotype and high proliferation rates.

[0068] The cells can be not recombinant or engineered in any way (i.e., non-GMO). In some aspects, the cells have not been exposed to any viruses and / or viral DNA. The cells can be both not recombinant or have not been exposed to any viruses and / or viral DNA and / or RNA. The cells can be preferentially fish cells.

[0069] It has been surprisingly found that fish cells can be successively grown in suspension using the cultivation medium described above.

[0070] The fish cells can be fish cells of a fish of the Salmonidae family. The fish cells of a fish of the of the Salmonidae family can be of the Salmoninae subfamily. The Salmoninae cells can be trout or salmon cells.

[0071] The cells can be fibroblasts. The cells can be fish fibroblast cells.

[0072] It has been surprisingly found that fibroblast cells and especially fish fibroblast cells can be successively grown in suspension using the cultivation medium described above.

[0073] The proliferated cells can be in the form of spheroids comprising multiple cells. The diameter of the spheroids can be 10 - 300 micrometer, 20 - 250 micrometer, 20 - 200 micrometer, 20 - 150 micrometer, 20 - 100 micrometer, 20 - 75 micrometer, or 20 -50 micrometer.

[0074] It has been found that cell spheroids of the preferred diameters allow the cells in the core of the spheroids to survive while at the same time the cells can proliferate.

[0075] The cells can be grown in shaking flasks, spinner flasks, or stirred fermenters. The stirred fermenter can be stirred tank bioreactors.

[0076] The cells can be grown in suspension under shaking, stirring or moving of the medium.

[0077] The method can involve maintaining the CO2concentration at 1-4 %(v / v), 1-3 %(v / v), 1.5-2.5 %(v / v), or about 2%(v / v).

[0078] The method can involve maintaining the temperature at 15-25°C, 17.5-22.5°C, 19-21C or about 20°C. These conditions have been found to be particularly advantageous for growing fish cells.

[0079] The method can further comprise:

[0080] - harvesting the proliferated cells; and

[0081] - manufacturing a food product from the harvested cells.

[0082] The cells can be recovered / harvested by any technique apparent to those of skill. In some aspects the fish fibroblast cells are separated from the growth media or are removed from a bioreactor or a scaffold. In certain aspects, the fish fibroblast cells are separated by centrifugation, a mechanical / filter press, filtration, flocculation or coagulation or gravity settling or drying or some combination thereof. In certain aspects, the filtration method comprises tangential flow filtration, vacuum filtration, rotary vacuum filtration and similar methods. In certain aspects the drying can be accomplished by flash drying, bed drying, tray drying and / or fluidized bed drying and similar methods. In certain aspects, the fish fibroblasts are separated enzymatically. In certain aspects, the fish fibroblasts are separated mechanically.

[0083] Disclosed is also a method for preparing immortalized fish fibroblasts from fish skin tissue.

[0084] Thus, a method is provided comprising:

[0085] - isolating explants of skin tissue from a skin biopsy of a fish;

[0086] - incubating explants in a cell cultivation medium (a), which comprises 15-25 % (v / v) fetal bovine serum, for 5-9 days, removing the supernatant, detaching the cells adhered to the substrate by incubating in a serum-free cell cultivation medium comprising a proteolytic enzyme for 15-25 minutes at 18-22°C; removing the supernatant and seeding resuspended cells;

[0087] - passaging the cells for 8-12 passages in cell cultivation medium a), the passaging involving during each passage the use of the above described detaching step with a proteolytic enzyme;

[0088] - passaging the cells for 8-12 passages in cell cultivation medium b), which comprises 8- 12 % (v / v) fetal bovine serum, the passaging involving during each passage the use of the above described detaching step with a proteolytic enzyme; - passaging cells for 8-12 passages in cell cultivation medium c), which comprises 1-3 % (v / v) fetal bovine serum, the passaging involving during each passage the use of the above described detaching step with a proteolytic enzyme;

[0089] - thereby obtaining immortalized fibroblast fish cells.

[0090] It has been surprisingly found that the above method starting from fish skin tissue is particularly useful for providing immortalized fibroblast fish cells which can also grow in suspension in subsequent procedures.

[0091] When the step of isolating explants of skin tissue from a skin biopsy of a fish is performed the non-skin tissue is (e.g. at least substantially) removed. For example, subcutaneous and muscle tissue is removed.

[0092] The explants can be approximately cube-shaped. Each side of the cube shaped explant can have a length of 0.5-1.5 mm, 0.75-1.25 mm, or 0.9-1 mm.

[0093] These sizes resulted in high number of cells useful for subsequent cultivation steps.

[0094] The proteolytic enzyme used for detaching the cells can be a proteolytic enzyme which at the end of the incubation period with the proteolytic enzyme does not require the use of a proteinaceous proteolytic enzyme inhibitor, like e.g. trypsin inhibitor to deactivate the enzymatic activity of the proteolytic enzyme. For example, the enzymatic activity of the proteolytic enzyme used for detaching the cells can be deactivated by being diluted merely by the addition of medium or buffer. These enzymes are designated as mild proteolytic enzyme (e.g. for detaching adherently growing cells from a substrate).

[0095] For example, the proteolytic enzyme used for detaching can be a fungal proteolytic enzyme. These enzymes do not require the use of a proteinaceous proteolytic enzyme inhibitor. These enzymes may comprise a non-mammalian, non-bacterial enzyme mixture with proteolytic and collagenolytic enzyme activity (e.g. accutase),

[0096] - a recombinant fungal trypsin-like serine endo-protease cleaving peptide chains mainly or only at the carboxyl side of the amino acids lysine or arginine (e.g. TrypLE), or an enzyme mixture obtainable from Aspergillus oryzae comprising two aminopeptidases, two dipeptidyl peptidases, three endopeptidases, and one a-amylase (e.g. Flavourzyme).

[0097] It has been surprisingly found that immortalized (fish) fibroblast can be successfully prepared with mild proteolytic enzyme, but not with non-mild proteolytic enzymes like pancreatic trypsin, which require the use of a proteinaceous proteolytic enzyme inhibitor.

[0098] Also disclosed is the use of methylcellulose for growing cells in suspension. As explained above, methylcellulose comprised in a culture medium prevents the formation of oversized cell spheroids when the cells are grown in suspension. All the features described above in relation to the methylcellulose to be used in the cultivation medium apply corresponding to the methylcellulose for growing cells in suspension.

[0099] In particular, the use of methylcellulose is disclosed to prevent cells growing in suspension from forming spheroids comprising multiple cells having a diameter of above 300 micrometer, 250 micrometer, 200 micrometer, 150 micrometer, 100 micrometer, 75 micrometer, or 50 micrometer.

[0100] Disclosed are also fibroblast fish cells and food products comprising the same.

[0101] Thus, fibroblast fish cells are disclosed, wherein the fibroblast fish cells are immortalized. The cells can also be isolated. This means they are separated from the parental animal and the parental tissue.

[0102] The cells can be capable of growing in suspension culture.

[0103] The immortalized fish fibroblasts can be obtainable by the methods described above, e.g. by growing the immortalized fish fibroblasts obtained from fish skin biopsies in suspension cultures using the method for growing immortalized fish fibroblasts described above.

[0104] Surprisingly, it was possible to provide fibroblast fish cells, wherein the cells are isolated and immortalized. In addition, the cells were not only capable of growing adherently, but also in suspension.

[0105] The cells have a doubling time of less than 70 hours, 60 hours, or 54 hours; or wherein the have a doubling time of be-tween 20 and 70 hours, 30 and 60 hours, 42 and 54 hours, or about 48 hours.

[0106] These doubling times are surprising since primary fish fibroblast (which are the cells directly obtained from tissue explants) have doubling times of about 168 hours. The fibroblast fish cells disclosed herein exhibit doubling times that allow to grow the cells to large quantities within a short time frame.

[0107] The cells are capable of expressing cy-tokeratin- 18 and collagen type 1 and do not express Pax7 or myosin heavy chain.

[0108] Thus, the cells are identifiable as fibroblast and can be distinguished from other cell types.

[0109] The cells are capable of proliferating without the addition of growth factors (e.g. Fibroblast growth factor 2, FGF2, insulin-like growth factor 1, IGF-1), not taking into account. FBS, if FBS is included in the medium.

[0110] The cells can exhibit an elongated and spindle-shaped appearance.

[0111] Thus, the appearance of the cells can be used to identify the cells as fibroblast and distinguish them from other cell types.

[0112] The cells can be capable of proliferating in a medium comprising less than 5, 4, or 3 % (v / v) or 0-5, 1-4, or 2-3 % (v / v) fetal bovine serum.

[0113] Thus, it has been advantageously possible to provide a fish fibroblasts, i.e. a fish fibroblast cell line, that replicate fast and grow under serum-depleted conditions.

[0114] The cells can be in the form of spheroids comprising multiple fibroblast fish cells.

[0115] Thus, the cells can adhere to each other in suspension. The spheroids cam have a diameter of 10 - 300 micrometer, 20 - 250 micrometer, 20 - 200 micrometer, 20 - 150 micrometer, 20 - 100 micrometer, 20 - 75 micrometer, or 20 -50 micrometer.

[0116] These diameters are especially useful for preventing the formation of dead cells in the core of the spheroids.

[0117] The fish fibroblast can be substantially pure.

[0118] Tests can be administered at one or more steps of cell culturing to determine whether the fish fibroblast cells are substantially pure.

[0119] The fish fibroblast cells can be tested for the presence or absence of bacteria. In certain aspects, the types of bacteria tested include, but are not limited to: Salmonella enteritidis, Staphylococcus aureus, Campylobacter jejunim, Listeria monocytogenes, Fecal streptococcus, Mycoplasma genus, Mycoplasma pulmonis, Coliforms , and Escherichia coli.

[0120] Components of the cell media can be tested for the presence or absence of viruses. In certain aspects, the viruses include, but are not limited to: Bluetongue, Bovine Adenovirus, Bovine Parvovirus, Bovine Respiratory Syncytial Virus, Bovine Viral Diarrhea Virus, Rabies, Reovirus, Adeno-associated virus, BK virus, Epstein-Barr virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Herpes Simplex 1, Herpes Simplex 2, Herpes virus type 6, Herpes virus type 7, Herpes virus type 8, HIV1, HIV-2, HPV-16, HPV 18, Human cytomegalovirus, Human Foamy virus, Human T-lymphotropic virus, John Cunningham virus, and Parvovirus Bl 9.

[0121] The tests can be conducted for the presence or absence of yeast and / or molds.

[0122] The tests can be for metal concentrations by mass spectrometry, for example inductively coupled plasma mass spectrometry (ICP-MS). In certain aspects, metals tested include, but are not limited to: arsenic, lead, mercury, cadmium, and chromium. The cells comprise substantially no heavy metals. The heavy metals can be selected from the group consisting of arsenic, lead, mercury, cadmium, and chromium. In particular the cells contain less 0.3, 0.1 or 0.01 ppm of heavy metal, i.e. of each heavy metal element or / and of the total of all heavy metal elements.

[0123] It is a particular advantage of the cells disclosed herein that they contain substantially no heavy metals. Especially fish cells form other sources, like fishes from natural sources or fishes grown in farms often contain substantial amounts of heavy metal.

[0124] The cells can be monitored by any technique known to a person of skill in the art. In some aspects, differentiation is measured and / or confirmed using transcriptional markers of differentiation after total RNA extraction using RT-qPCR and then comparing levels of transcribed genes of interest to reference, e.g. housekeeping genes.

[0125] Also disclosed is a food product comprising the fibroblast fish cells described above.

[0126] Advantageously, a food product similar to traditional fish food products can be provided that does not require to fish or kill large amounts of animals.

[0127] The food product can comprises gelling agents, texturing agents, thickening agents, flavoring agents, vegetable oil or fat, algal oils, algal protein, fungal proteins, or / and vegetable protein.

[0128] In this way, the food product can be adapted to imitate traditional fish food products.

[0129] The food product can be in the shape of a bar, a paste, fish fillet, fish eggs (roe, caviar) or a ball.

[0130] Disclosed is also a method of producing a first food product comprising: providing a composition comprising gelling agents, texturing agents, thickening agents, flavoring agents, vegetable oil or fat, or / and vegetable protein; providing the fibroblast fish cells described above; mixing the fibroblast fish cells with the composition to obtain a mixture; shaping a food product from the mixture to obtain a shaped food product. The method of producing a first food product can further comprise freezing the shaped food product at temperatures below -15°C.

[0131] The method of producing a first food product may further comprise a step of coating the obtained shaped food product with a coating.

[0132] The coating step may comprise covering the shaped food product with a batter and subsequently covering the food product with crumbs.

[0133] Disclosed is also a method of producing a second food product comprising: providing the fibro-blast fish cells described above mixing the fibroblast fish cells with crosslinking agents suitable for crosslinking fibroblast fish cells to obtain a mixture; incubating the mixture in a mold for a predetermined time; thereby obtaining a shaped food product.

[0134] Provided herein are food compositions or food products comprising fish fibroblast cells. As described above, the fish fibroblast cells are combined with other substances or ingredients to make a composition that is an fish food product composition. The fish fibroblast cells can be used alone to make a composition that is an fish food product composition. The fish food product composition is a product that resembles: fish nuggets, fish fillet, fish dumplings, fish balls, fish cakes, fish paste, fish sausage, fish eggs, or fish skin.

[0135] The recovered fish fibroblast cells can be prepared into a composition with other ingredients. In certain aspects, the composition comprises fish fibroblast cells, protein, fat, carbohydrates and water.

[0136] The food composition or food product can have a wet cell paste content of at least 100%, 90%, 80%, 75%, 70%, 65%, 60%, 50%, 40%, 30%, 35%, 25%, 15%, 10%, 5% or 1% by weight. The food composition or food product can have a wet cell paste content by weight of between 10%-20%, 20%-30%, 30%-40%, 40%-50%, 60%-70%, 80%-90%, or 90%- 100%. The food composition or food product can comprise a fat content of at least 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% by weight. The food composition or food product can have a fat content by weight of between 10%-20%, 20%-30%, 30%-40%, 40%-50%, 60%-70%, 80%-90%, or 90%-95%. The food composition or food product can comprise a water content of at least 50%, 40%, 30%, 25%, 20%, 15%, 10% or 5% by weight. The food composition or food can have a water content by weight of between 10%-20%, 20%-30%, 30%-40%, 40%-50%, 60%-70%, 80%-90%, or 90-95%. The food composition or food product can comprise a wet cell paste content of between 2%-5%,

[0137] 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%,

[0138] 45%-50%, 50%-55%, 55%-60%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, or 90%-95%.

[0139] In certain aspects, the food composition or food product comprises peptide cross-linking enzyme. Exemplary peptide cross-linking enzymes are selected from the group consisting of transglutaminase, sortase, subtilisin, tyrosinase, laccase, peroxidase, and lysyl oxidase. In certain aspects, the composition comprises a cross-linking enzyme of between 0.0001%-0.025%, 0.0001%-0.020%, 0.0001%-0.0175%, 0.0001%-0.0150%, 0.0001%- 0.0125%, 0.0001%-0.01%, 0.0001%-0.0075%, 0.0001%-0.005%, 0.0001%-0.0025%, 0.0001%-0.002%, 0.0001%-0.0015%, 0.0001%-0.001%, 0.0001%-0.00015% by weight. In certain aspects, the food composition or food product comprises a transglutaminase content between 0.0001%-0.025%, 0.0001%-0.020%, 0.0001%-0.0175%, 0.0001%- 0.0150%, 0.0001%-0.0125%, 0.0001%-0.01%, 0.0001%-0.0075%, 0.0001%-0.005%, 0.0001%-0.0025%, 0.0001%-0.002%, 0.0001%-0.0015%, 0.0001%-0.001%, 0.0001%- 0.00015% by weight. Without being bound by theory, the peptide cross-linking enzyme is believed to cross-link the pulse or vetch proteins and the peptide cross-linking enzyme is believed to cross-link the pulse or vetch proteins to the fish cells.

[0140] In one aspect, the food composition or food product comprises 0.0001% to 0.0125% transglutaminase, and exhibits reduced or significantly reduced lipoxygenase activity or other enzymes which oxidize lipids, as expressed on a volumetric basis relative to cell paste without the transglutaminase. More preferably, the food composition or food product is essentially free of lipoxygenase or enzymes that can oxidize lipids. In some aspects, a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% reduction in oxidative enzymatic activity relative to a composition is observed. Lipoxygenases catalyze the oxidation of lipids that contribute to the formation of compounds that impart undesirable flavors to compositions.

[0141] In some aspects, the addition of additional edible ingredients can be used to prepare the food composition of food product. Edible food ingredients comprise texture modifying ingredients such as starches, modified starches, gums and other hydrocolloids. Other food ingredients comprise pH regulators, anti-caking agents, colors, emulsifiers, flavors, flavor enhancers, foaming agents, anti-foaming agents, humectants, sweeteners, and other edible ingredients.

[0142] In certain aspects, the methods and food composition or food product comprise an effective amount of an added preservative in combination with the food combination.

[0143] Preservatives prevent food spoilage from bacteria, molds, fungi, or yeast (antimicrobials); slow or prevent changes in color, flavor, or texture and delay rancidity (antioxidants); maintain freshness. In certain aspects, the preservative is one or more of the following: ascorbic acid, citric acid, sodium benzoate, calcium propionate, sodium erythorbate, sodium nitrite, calcium sorbate, potassium sorbate, BHA, BHT, EDTA, tocopherols (Vitamin E) and antioxidants, which prevent fats and oils and the foods containing them from becoming rancid or developing an off-flavor.

[0144] EXAMPLES

[0145] Example 1: Preparation / Propagation of adherent trout fibroblast cell culture

[0146] 1. Tissue Procurement and Preparation:

[0147] A skin biopsy measuring 2-3 cm in length is obtained from a Rainbow Trout that was surface sterilized with 70% alcohol. Biopsy is washed with sterile Phosphate Buffered Saline (PBS) to remove contaminants and debris. Subcutaneous and muscle tissue are meticulously removed to isolate the skin tissue. The skin tissue is dissected into explants of approximately 1 mm2in size.

[0148] 2. Primary Culture Initiation (Passage 0):

[0149] Explants are transferred into tissue culture plates and supplemented with growth medium (DMEM high glucose, 20% FBS, 1% P / S). Incubation is performed at 20 °C with a 2% CO2 environment for 7 days. Medium is carefully changed every 3 days.

[0150] 3. Harvest of primary cells (Passage 1):

[0151] When outgrowth of cells from the explants is observed (about 7 days), growth medium is removed, and the plate is carefully rinsed once with sterile Phosphate Buffered Saline. The outgrown cells are detached by adding Trypsin (in DMEM high glucose, 0% FBS, 1% P / S) to the culture plates to a final concentration of 0.05%. Incubation for 20 minutes at 20 °C is followed by aspiration of all liquid and centrifugation at 650 ref for 10 min. The supernatant is discarded. The cells from each plate are suspended in growth medium and all cells from one plate are reseeded into a fresh tissue culture plate.

[0152] 4. Subsequent Passaging:

[0153] Cells are seeded in tissue culture plates at 5000 cells per cm2. Incubation is performed at 20 °C with a 2% CO2 environment. Media is changed every 2 days. Passaging is performed based on confluency: P0-P6: 60-70% confluency; from P6: 100% confluency; splitting ratio is 1 :2. Cells can be maintained for many passages under these conditions.

[0154] 5. Development of cell lines:

[0155] Cells are seeded in tissue culture plates at 5000 cells per cm2. Incubation is performed at 20 °C with a 2% CO2 environment. Media is changed every 2 days. After P10 the growth medium is changed from Medium 1 with 20% FBS to Medium2 with 10% FBS. After an additional 10 Passages the growth medium is changed from Medium2 to Medium3 with only 2.5% FBS.

[0156] Medium 1 :

[0157] DMEM high glucose, 20% FBS (Gibco #26140), 1% P / S (Penicillin 10,000U / mL / Streptomycin 10 mg / mL, PAN Biotech)

[0158] Medium2:

[0159] ADF12 high glucose (advanced DEMEM / F12, Gibco), 10% FBS, 1% P / S

[0160] Medium3 :

[0161] ADF12 high glucose, 2.5% FBS, 1% P / S, Panexin basic 7.5% (complex media supplement, PAN Biotech), Cod liver oil 0.1% (cod lipid mixture lOOOx, Sigma) L-Glutamine 1% (200mM, PAN Biotech)

[0162] At this stage a robust fibroblast fish cell line with excellent growth characteristics is established.

[0163] The fibroblast fish cells from Rainbow Trout skin explants exhibit a fibroblastic morphology, characterized by their elongated, spindle-shaped appearance. This morphology is essential for adhering to surfaces, facilitating cell attachment, and supporting tissue development. The fibroblast fish cells express marker proteins specific to skin cells, such as cytokeratin-18 and collagen type 1 but do not express Pax7 or myosin heavy chain.

[0164] Tissue Procurement and Preparation (Salmon):

[0165] The tissue procurement and preparation procedure that had been used for trout fibroblast cells has also been applied to a skin biopsy from salmon (Atlantic salmon, Salmo salar). It was possible to establish a robust cell line with excellent growth characteristics. Similar to the Rainbow Trout skin fibroblast cell line the salmon skin fibroblast cell line exhibits a fibroblastic morphology, characterized by their elongated, spindle-shaped appearance. This morphology is essential for adhering to surfaces, facilitating cell attachment, and supporting tissue development. The fibroblast fish cells of salmon express marker pro- teins specific to skin cells, such as cytokeratin-18 and collagen type 1 but do not express Pax7 or myosin heavy chain.

[0166] Example 2: Preparation of fish (trout) fibroblast suspension cell culture in flasks from adherently growing fish cells.

[0167] A T75 flask of adherently growing fish cells is cultured in medium3 (as described above) for 5 days at 20° C in a humidified incubator with 2% CO2.

[0168] After aspiration and washing with PBS the cells are dissociated from the substrate by treatment with a protease (e.g. a trypsin, e.g. TrypLE Select Enyzme, Thermofisher, USA).

[0169] The dissociated cells are centrifuged at 20°C and 650 ref for 10 min. The supernatant is discarded. The cells are washed once in PBS and are centrifuged at 20°C and 650 ref for 10 min. PBS is discarded. The cell pellet is carefully resuspended in medium3 by agitation and pipetting if necessary. The resuspended cells were transferred to a shaker flask, which is placed in a shaking incubator at 80 rpm, 20°C and 2% CO2for cultivation.

[0170] After 5 and 10 days in culture samples are removed from the cultures, the cells are counted and their viability is determined.

[0171] Example 3: Comparison of different media types

[0172] It was observed that fish cells (trout) grown in suspension form spheroids and these are getting larger and larger during culture. Increasing stirring speed does not prevent the formation of large spheroids. Moreover, the cells in the center of large spheroids are observed to die. Therefore, factors which might be responsible for limiting the growth of the spheroids were analysed.

[0173] Different media compositions were tested. The tested media contained - 0 mg / L (0.00%), 500 mg / L (0.05%), 750 mg / L (0.075%), 1000 mg / L (0.1%), 5000 mg / L (0.5%), and 10000 mg / L (1.0%) of methylcellulose (Tylopur MCE-100 TS, SE Tylose GmbH)

[0174] 0.1%, 0.5%, 0.8% poloxamer (Pluronic F68, Gibo, USA).

[0175] The following table shows the observations regarding spheroid size and viability of fish cells (trout) after 10 days of culture: Addition of methyl cellulose (MC) did not prevent the formation of spheroids but limited the size of them during culture over several days. This enables continuous growth of the cell cultures. At 0.05% there is no effect, at 1% the culture gets too viscous. It was observed that at surprisingly low concentrations around 0.075% the concentration provides spheroids of limited size consisting of cells with high viability.

[0176] Of note, other methyl celluloses (Sigma # M6385, #M0262, #M0512, CAS #9004-67-5) exhibiting similar chemical and physico-chemical properties as Tylopur MCE- 100 TS have also been tested and found to achieve substantially the same effect.

[0177] In addition, it was observed that the viability can be further increased by using about 0.5% of poloxamer (Pluronic F68, Gibo, USA).

[0178] The experiments were repeated with 0 mg / L (0.00%), 100 mg / L (0.01%), 500 mg / L (0.05%), and 750 mg / L (0.075%) of dextran sulfate (Merck, USA). Dextran sulfate has been used by other labs in mammalian cell cultures to prevent cell aggregation. However, dextran sulfate decreased the viability of the fish cells compared to media lacking dextran sulfate, without a positive effect on cell aggregation.

[0179] The above experiments were also repeated with salmon fibroblast fish cells and confirmed the above observations.

[0180] It is also assumed that the observed positive effects on cells grown in suspension cell culture can be extended to other cells (different cell types, different species including mammalian cells) grown in suspension.

[0181] Example 4: Preparation of fish (trout) fibroblast long-term suspension cell culture in bioreactors

[0182] A 250 ml flask of fish cells growing in shaking flasks is cultured in medium3 for 5 days at 20° C in a humidified incubator with 2% CO2. The cell suspension is centrifuged at 20°C and 650 ref for 10 min. The supernatant is discarded. The cells are washed once in PBS and are centrifuged at 20°C and 650 ref for 10 min. PBS is discarded. Peptidase (10 U / ml, Flavourzyme, Novozymes, Denmark) is added. The cells are incubated in a shaking incubator at 20°C for a total of 30 min. The solution is carefully resuspended by pipetting after 15 min to ensure proper mixing during the enzymatic dissociation. Next, gentle pipetting (1 mL Eppendorf pipette) of the cell suspension is performed to obtain good dissociation until no or only few aggregates are left.

[0183] The dissociation is stopped by adding medium. A small sample is removed from the cultures and the cells are counted.

[0184] The cell pellet was resuspended thoroughly in complete suspension medium (containing 0.5% poloxamer (Pluronic F68, Gibo, USA; i.e. Poloxamer 188) and 0.1 % methylcellulose (Tylopur MCE-100 TS).

[0185] Resuspended cells are seeded at 300,000 / ml into a spinner flask comprising 1000 ml of various tested suspension culture media. The flasks are placed on a stirrer plate in an incubator at 120 rpm, 20°C and 2 % CO2. Alternatively, cells are seeded at 100,000 / ml into a bioreactor comprising 3000 ml of various tested suspension culture media. The bioreactor is set to 150 rpm, 20°C and 2 % CO2.

[0186] In this way it is possible to propagate fish suspension cultures continuously.

[0187] Example 5: Production of food products comprising fish cells

[0188] Fish balls:

[0189] 1. Dissolve required amount of salt in water

[0190] 2. Add dry ingredients: Konjac flower, potato protein, potato starch, soy protein, Tara gum, Gellan gum, yeast extract

[0191] 3. Blend in a mixer until homogeneous

[0192] 4. Add vegetable oil while blending

[0193] 5. Add fish cells to reach desired amount (5% - 50% w / v)

[0194] 6. Mix until the dough appears homogeneous 7. Fill aliquots of the dough into molds

[0195] 8. Freeze at -20C° until firm or store for use in cooking

[0196] If breading is required: Pry fish balls out of the mold, dip in batter of choice, roll in Panko or bread crumps until covered. Store at -20C°

[0197] FURTHER EXAMPLES

[0198] 1. A cultivation medium for cultivating cells in suspension comprising methylcellulose.

[0199] 2. The cultivation medium of example 1, wherein the degree of substitution of the methylcellulose is 20-40%, 25-35%, 27-32%, for methoxyl.

[0200] 3. The cultivation medium of examples 1 or 2, wherein the methyl cellulose is a modified cellulose which does not contain any other modification, but methoxyl, in particular, does not contain modifications with hydroxypropoxyl.

[0201] 4. The cultivation medium of any of the above examples, wherein the apparent viscosity of the methylcellulose via Brookfield is 50,000 — 200,000 mPa 70,000 — 150,000 mPa, 100,000 — 120,000 mPa, or about 110,000 mPas at 2% in water and 20°C.

[0202] 5. The cultivation medium of any of the above examples; wherein the cultivation medium further comprises poloxamer, wherein optionally the poloxamer has a molecular weight of 7000-10000 g / mol, 8000-9000 g / mol, 8200-8600 g / mol, or 8400 g / mol and 60%-95%, 70%-90%, 75%-85%, or about 80 % polyoxyethylene.

[0203] 6. The cultivation medium of any of the above examples, wherein the cultivation medium comprises 40 - 400 mg / L, methylcellulose 50 - 300 mg / L, 60 - 200 mg / L, 70 - 150 mg / L, 80 - 120 mg / L, 90 - 110 mg / L, or about 100 mg / L methyl cellulose.

[0204] 7. The cultivation medium of any of the above examples, wherein the medium comprises: carbohydrates; proteins; isolated amino acids comprising L-glutamine;

[0205] 4, or 3 % (v / v) or 0-5, 1-4, or 2-3 % (v / v) fetal bovine serum; antibiotics; omega- 3 fatty acids comprising eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA);

[0206] - vitamin A; and / or vitamin D.

[0207] 8. A method comprising: providing a cultivation medium comprising methylcellulose, wherein the first cultivation medium is the cultivation medium of any of examples 1-7; adding cells to the cultivation medium; proliferating the cells in the cultivation medium in suspension. The method of any of example 8, wherein the cells are fish cells, wherein the fish cells are fish cells of a fish of the Salmonidae family, wherein optionally the fish cells are fish cells of a fish of the Salmoninae subfamily, wherein optionally the Salmoninae cells are trout or salmon cells. The method of examples 8 or 9, wherein the cells are fibroblasts. The method of examples 8 - 10, wherein the proliferated cells are in the form of spheroids comprising multiple cells, wherein optionally the diameter of the spheroids is 10 - 300 micrometer, 20 - 250 micrometer, 20 - 200 micrometer, 20 - 150 micrometer, 20 - 100 micrometer, 20 - 75 micrometer, or 20 -50 micrometer. The method of examples 8 - 11, wherein the cells are grown in shaking flasks, spinner flasks, or stirred fermenters, wherein optionally the stirred fermenter are stirred tank bioreactors. The method of examples 8 - 12, wherein the cells are grown in suspension under shaking, stirring or moving of the medium. The method of any of examples 8 - 13, further comprising: harvesting the proliferated cells; manufacturing a food product from the harvested cells. A method compri sing : isolating explants of skin tissue from a skin biopsy of a fish; incubating explants in a cell cultivation medium (a), which comprises 15-25 % (v / v) fetal bovine serum, for 5-9 days, removing the supernatant, detaching the cells adhered to the substrate by incubating in a serum-free cell cultivation medium comprising a proteolytic enzyme for 15-25 minutes at 18-22°C; removing the supernatant and seeding resuspended cells; passaging the cells for 8-12 passages in cell cultivation medium a); passaging the cells for 8-12 passages in cell cultivation medium b), which comprises 8-12 % (v / v) fetal bovine serum; passaging cells for 8-12 passages in cell cultivation medium c), which comprises 1-3 % (v / v) fetal bovine serum;

[0208] - thereby obtaining immortalized fibroblast fish cells. The method of example 15, wherein the proteolytic enzyme used for detaching is a fungal proteolytic enzyme. Use of methylcellulose to grow cells in suspension. The use of claim 17, further comprising the use of methylcellulose to prevent cells growing in suspension from forming spheroids comprising multiple cells having a diameter of above 300 micrometer, 250 micrometer, 200 micrometer, 150 microm- eter, 100 micrometer, 75 micrometer, or 50 micrometer.

Claims

CLAIMS1. A cultivation medium for cultivating cells in suspension comprising methylcellulose.

2. The cultivation medium of claim 1, wherein the degree of substitution of the methylcellulose is 20-40%, 25-35%, 27-32%, for methoxyl.

3. The cultivation medium of claims 1 or 2, wherein the methyl cellulose is a modified cellulose which does not contain any other modification, but methoxyl, in particular, does not contain modifications with hydroxypropoxyl.

4. The cultivation medium of any of the above claims, wherein the apparent viscosity of the methylcellulose is 50,000 — 200,000 mPa 70,000 — 150,000 mPa, 100,000 — 120,000 mPa, or about 110,000 mPas (Brookfield viscosity at 2%, 20 rpm, in water at 20°C.

5. The cultivation medium of any of the above claims, wherein the cultivation medium comprises 0.04 - 0.4 % (w / v), 0.05 - 0.3 % (w / v), 0.06 - 0.2 % (w / v), 0.07 - 0.15 % (w / v), 0.075 - 0.5 % (w / v), 0.09 - 0.11 % (w / v), or about 0.1 % (w / v) methyl cellulose.

6. The cultivation medium of any of the above claims; wherein the cultivation medium further comprises poloxamer, wherein optionally the poloxamer has a molecular weight of 7000-10000 g / mol, 8000-9000 g / mol, 8200-8600 g / mol, or 8400 g / mol and 60%-95%, 70%-90%, 75%-85%, or about 80 % polyoxyethylene.

7. The cultivation medium of any of the above claims, wherein the cultivation medium comprises 0.1 - 1.0 % (w / v), 0.1 - 0.8 % (w / v), 0.3 - 0.7 % (w / v), 0.4 - 0.6 % (w / v), or about 0.5 % (w / v) poloxamer.

8. The cultivation medium of any of the above claims, wherein the cultivation medium comprises 0.06 - 0.2 % (w / v) methyl cellulose and 0.1 - 1.0 % (w / v) poloxamer, 0.05 - 0.15 % (w / v) methyl cellulose and 0.1 - 1.0 % (w / v) poloxamer, or 0.05 - 0.15 % (w / v) methyl cellulose and 0.3 - 0.7 % (w / v) poloxamer.

9. The cultivation medium of any of the above claims, wherein the medium comprises: carbohydrates; proteins; isolated amino acids comprising L-glutamine;4, or 3 % (v / v) or 0-5, 1-4, or 2-3 % (v / v) fetal bovine serum; antibiotics;omega- 3 fatty acids comprising eicosapentaenoic acid (EP A) and docosahexaenoic acid (DHA);- vitamin A; and / or vitamin D.

10. A method comprising: providing a cultivation medium comprising methylcellulose, wherein the first cultivation medium is the cultivation medium of any of claims 1-7; adding cells to the cultivation medium; proliferating the cells in the cultivation medium in suspension.

11. The method of any of claim 10, wherein the cells are fish cells, wherein the fish cells are fish cells of a fish of the Salmonidae family, wherein optionally the fish cells are fish cells of a fish of the Salmoninae subfamily, wherein optionally the Salmoninae cells are trout or salmon cells.

12. The method of claims 10 or 11, wherein the cells are fibroblasts.

13. The method of claims 10 - 12, wherein the proliferated cells are in the form of spheroids comprising multiple cells, wherein optionally the diameter of the spheroids is 10 - 300 micrometer, 20 - 250 micrometer, 20 - 200 micrometer, 20 - 150 micrometer, 20 - 100 micrometer, 20 - 75 micrometer, or 20 -50 micrometer.

14. The method of claims 10 - 13, wherein the cells are grown in shaking flasks, spinner flasks, or stirred fermenters, wherein optionally the stirred fermenter are stirred tank bioreactors.

15. The method of claims 10 - 14, wherein the cells are grown in suspension under shaking, stirring or moving of the medium.

16. The method of any of claims 10 - 15, further comprising: harvesting the proliferated cells; manufacturing a food product from the harvested cells.

17. A method comprising: isolating explants of skin tissue from a skin biopsy of a fish; incubating explants in a cell cultivation medium (a), which comprises 15-25 % (v / v) fetal bovine serum, for 5-9 days, removing the supernatant, detaching the cells adhered to the substrate by incubating in a serum-free cell cultivation medium comprising a proteolytic enzyme for 15-25 minutes at 18-22°C; removing the supernatant and seeding resuspended cells; passaging the cells for 8-12 passages in cell cultivation medium a);passaging the cells for 8-12 passages in cell cultivation medium b), which comprises 8-12 % (v / v) fetal bovine serum; passaging cells for 8-12 passages in cell cultivation medium c), which comprises 1-3 % (v / v) fetal bovine serum;- thereby obtaining immortalized fibroblast fish cells.

18. The method of claim 17, wherein the proteolytic enzyme used for detaching is a fungal proteolytic enzyme.

19. The method of claims 17 or 18, further comprising performing the method of any of claims 10 - 16 with the immortalized fibroblast fish cells.

20. Use of methylcellulose to grow cells in suspension.

21. The use of claim 20 comprising the use of a combination of methylcellulose and poloxamer.

22. The use of claims 20 or 21, further comprising the use of methylcellulose to prevent cells growing in suspension from forming spheroids comprising multiple cells having a diameter of above 300 micrometer, 250 micrometer, 200 micrometer, 150 micrometer, 100 micrometer, 75 micrometer, or 50 micrometer.

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