Cell lines adapted and / or genetically modified for recombinant protein production and food applications

Abstract

The present invention is related to the field of cell cultures. In particular, the invention relates to genetically modified and / or adapted non-human metazoan cell lines to grow in conditions appropriate for cell cultivation on a large scale suitable for industrial applications, in serum-free and protein-free media. The invention further relates to the use of these non-human metazoan cell lines as expression systems for recombinant protein production and / or as a consumable product for human and / or animal use.

Description

[0001] Cell Lines Adapted and / or Genetically Modified for Recombinant Protein Production and Food Applications

[0002] FIELD OF THE INVENTION:

[0003] [1] The present invention relates to the field of cell cultures. In particular, the invention relates to genetically modified and / or adapted non-human metazoan cell lines to grow in conditions appropriate for cell cultivation on a large scale suitable for industrial applications and / or to grow in serum-free and protein-free media. The invention further relates to the use of these adapted and / or genetically modified non-human metazoan cell lines, comprising metazoan cells (i.e., non-human metazoan cells), as expression systems for recombinant protein production and / or as a consumable product for human and / or animal use, for example for preparation of cultured meat.

[0004] BACKGROUND OF THE INVENTION:

[0005] [2] Cell cultures have been established and used to study animal cell behavior in vitro since the early 20thcentury. Over the past century, cell culturing techniques have undergone significant advancements, leading to the production and isolation of biologically relevant molecules. These developments have enabled application in diverse fields, including biosimilar drug production, regenerative medicine, cell-based therapies, recombinant protein production, vaccine and antibody manufacturing, and more recently the production of cultured meat. These applications rely on highly precise and controlled cell culturing processes to ensure reproducibility and efficiency.

[0006] [3] One of the key factors determining the success of large-scale cell culture is the composition of the culture medium providing essential nutrients such as amino acids, vitamins, and growth factors necessary for cell survival and proliferation. Traditionally, fetal bovine serum (FBS) has been widely used as a medium supplement due to its high growth-promoting activity, presence of attachment factors, micronutrients, and protective elements. However, the use of serum introduces several critical drawbacks, including batch-to-batch variability, high protein content that complicates downstream purification, risk of contamination (e.g., by viruses, prions, or bacteria), high cost, and ethical concerns regarding animal sourcing.

[0007] [4] Serum-free media (SFM) have been developed to replace undefined serum components with chemically defined ingredients, offering improved control and reproducibility. These formulations range from protein-free and animal-component-free options to fully chemically defined media, which, while eliminating variability, may initially reduce growth efficiency. Various cost-effective alternatives have been explored, including protein hydrolysates and recombinant growth factors to replace serumbased media.

[0008] [5] Despite the significant progress in culture media development, the cultivation of eukaryotic cell lines remains challenging. Eukaryotic cells generally have longer doubling times compared to prokaryotic cells, requiring prolonged cultivation periods. However, eukaryotes are widely used as expression systems because, unlike prokaryotes, they can perform essential post-translational modifications of proteins resulting in their various additions and alterations, including proper protein folding, amino acid modifications, disulfide bridge formation or protein modifications that facilitate binding functions (e.g., glycosylation and acetylation) or regulate a protein activity (e.g.,phosphorylation and nitrosylation). These modifications are crucial for producing biologically active proteins, making eukaryotic systems superior for many biopharmaceutical applications.

[0009] [6] However, this complexity comes at a cost, as eukaryotic cells require substantial amounts of essential nutrients, particularly recombinant growth factors, which significantly increase production expenses. The large-scale expansion of eukaryotic cell cultures presents an economic challenge due to the high costs of the required essential media components. Therefore, it is critical to minimize the use of these expensive materials to enhance the efficiency and economic feasibility of large-scale production while still maintaining optimal growth conditions.

[0010] [7] Despite genetic modifications of cells to produce growth factors (e.g. albumin, fibronectin, transferrin, insulin, epidermal growth factor, fibroblast growth factor, platelet derived growth factor, transforming growth factor beta, or vascular endothelial growth factor) to enable growth in a SFM, challenges remain in maintaining stable and scalable cultivation without the use of FBS.

[0011] [8] The present invention overcomes these challenges by introducing genetically modified and / or adapted non-human metazoan cells that can be cultivated in a serum-free, protein-free medium by activation of the phosphoinositide 3 -kinase (PI3K-Akt) signaling pathway and / or by activation of the transforming growth factor-beta (TGF-beta) signaling pathway in those cells. These cells can serve as an expression system for recombinant protein production and / or as a consumable product for human and / or animal use.

[0012] [9] Another goal of the invention is to prepare a biologically safe food product comprising non-human metazoan cells that may be cultivated on a large industrial scale with high productivity. In one aspect, the invention provides genetically modified non-human metazoan cells engineered for resistance to prion infections, including transmissible spongiform encephalopathies (TSEs) such as bovine spongiform encephalopathy (BSE). Such prion resistance may be achieved through genetic modifications that inhibit prion protein misfolding and propagation, thereby enhancing the safety of bovine -derived cellular products for use in food applications. In another aspect, the invention provides genetically modified bovine cells engineered for resistance to endogenous retroviruses (ERVs).

[0013]

[0010] Another goal of the invention is to prepare a stable non-human metazoan cell line used as an expression system for the production of recombinant proteins that may also contribute to the development of food products, for example CHO cells or CHO-derived cells.

[0014] BRIEF SUMMARY OF THE INVENTION:

[0015]

[0011] The present invention relates to methods that provide adapted and / or genetically modified metazoan cell lines capable of growing in serum-free and / or protein-free media and / or in a large scale suitable for industrial applications.

[0016]

[0012] The present invention overcomes limitations of traditional cell culture, for example CHO cell or CHO-derived cell culture, reducing reliance on animal-derived components, improving scalability, and ensuring cost-effective production of recombinant proteins, vaccines and / or cultured food products.

[0017]

[0013] The non-human metazoan cells according to the present invention may be used for many industrial applications, for example for the above mentioned production of recombinant proteins and for production of other pharmaceutical active ingredients. The cells may be used for preparing biologically safe food products for human and / or animal consumption, for example for preparing cultured meat.

[0014] The present invention relates to methods that provide a genetically modified metazoan cell with activated PI3K-Akt signaling pathway, capable of growing in serum-free and / or protein-free media with enhanced growth, stability and / or proliferation and further provide an additive effect by expression of at least one additional gene.

[0018]

[0015] The present invention relates to methods that provide a genetically modified metazoan cell with activated TGF-beta signaling pathway, capable of growing in media without supplementation of TGF-beta, characterized by enhanced growth, stability and / or proliferation and further provide an additive effect by expression of at least one additional gene.

[0019]

[0016] The present invention relates to methods that provide a genetically modified metazoan cell with activated PI3K-Akt signaling pathway and TGF-beta signaling pathway, or any other appropriate signaling pathways or combinations thereof, capable of growing in serum-free and / or protein-free media with enhanced growth, stability and / or proliferation and further provide an additive effect by expression of at least one additional gene.

[0020]

[0017] In another aspect of the invention, the methods comprise utilization of Cre-Lox recombination and / or FLP-FRT recombination for generation of non-genetically modified metazoan cells from previously genetically modified metazoan cells.

[0021]

[0018] In another aspect of the invention, the methods comprise utilization of Cre-Lox recombination and / or FLP-FRT recombination as an exchange system of insertion cassettes within the genome of metazoan cells.

[0022]

[0019] The present invention relates to methods for providing an improved cryo-medium, referred to as M030 cryo-medium, for the cryopreservation of cells.

[0023]

[0020] All of the aforementioned aspects of the invention are described in the detailed description of the present application.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS:

[0025]

[0021] Fig. 1: illustrates proline production by cells.

[0026]

[0022] Fig. 2: illustrates cell cultivation and growth in thymidine free medium with low concentration of glutamine, w / o hypoxanthine, linoleic acid, lipoic acid, with hydrolyzate at concentration 9 g / L of protein (100%).

[0027]

[0023] Fig. 3: illustrates a cell growth curve and concentration of L-proline from batch culture.

[0028]

[0024] Fig.4: illustrate s lower lactate production or lactate consumption - better glucose conversion efficiency.

[0029]

[0025] Fig. 5: illustrates cell growth in a medium with low glutamine concentration, glutamine momentary production, and their connection with momentary ammonia consumption from the culture medium.

[0030]

[0026] Fig. 6: illustrates the cell cultivation system according to the invention.

[0031]

[0027] Fig. 7: illustrates the Pet Food product - tofu chunks.

[0028] Fig. 8: illustrates a schematic of the preparation of genetically modified metazoan cells and their use as an expression system and food product for animal and / or human use.

[0032]

[0029] Fig. 9: illustrates graph charts of doubling time of the non-modified bovine fibroblasts and genetically modified bovine fibroblasts.

[0033]

[0030] Fig. 10: illustrates the expression analysis of selected genes participating in the PI3K-Akt signaling pathway of non-modified bovine fibroblasts and genetically modified bovine fibroblasts.

[0034]

[0031] Fig. 11: illustrates a food product, which comprises chunks made from genetically modified bovine fibroblasts.

[0035]

[0032] Fig. 12: illustrates the formula for the calculation of cell recovery (%).

[0036]

[0033] Fig. 13A and Fig. 13B: illustrate a comparison between cells preserved in the M030 cryomedium and those preserved in a commercially available cryo-medium, following thawing. The comparison includes (A) the cell density (cells / μL), and (B) the percentage of cell viability in the post-thaw samples.

[0037]

[0034] Fig. 14: illustrates the adaptation process of bovine cells to a serum-free culture medium, presented in terms of the cell density (cells / μL), in comparison to bovine cells subcultured in DMEM supplemented with fetal bovine serum (FBS).

[0038]

[0035] Fig. 15A and Fig. 15B: illustrate bovine cells adapted to suspension culture conditions, resulting in (A) a single cell suspension and (B) size-defined cell aggregates.

[0039]

[0036] Fig. 16A and Fig. 16B: illustrate the growth curve and metabolic parameters of bovine cells cultivated in a protein hydrolysate-supplemented medium, shown in terms of (A) the cell density (cells / μL) over several days and (B) glucose consumption and lactate production.

[0040]

[0037] Fig. 17: illustrates exemplary local and global alignments of two amino acid sequences.

[0041] CROSS REFERENCE TO RELATED APPLICATIONS:

[0042]

[0038] This application claims priority to U. S. Provisional Application No. 63 / 760,147, filed February 19, 2025, which is hereby incorporated by reference in its entirety. The PCT application PCT / IB2024 / 053805 filed 18 April 2024, and PCT application PCT / IB2024 / 059990 filed 11 October 2024, and U. S. Provisional Patent Application No. 63 / 589,661 filed October 12, 2023, and U. S. Provisional Patent Application No. 63 / 555,543 filed February 20, 2024, and U. S. Provisional Patent Application No. 63 / 570,973 filed March 28, 2024, and U. S. Provisional Patent Application No.

[0043] 63 / 654,493 filed May 31, 2024, and U. S. Non-Provisional Patent Application No. 18 / 731,896 filed on June 3, 2024, and U. S. Non-Provisional Patent Application No. 18 / 763,199 filed on July 3, 2024, and U. S. Provisional Patent Application No. 63 / 698,265 filed September 24, 2024, U. S. Provisional Patent Application No. 63 / 497,051 filed April 19 2023, U. S. Provisional Patent Application No. 63 / 737,932 filed December 23, 2024, U. S. Non-provisional Patent Application No. 18 / 927,171 filed October 25, 2024, U. S. Non-provisional Patent Application No. 19 / 028,830 filed January 17, 2025, U. S. Non-provisional Patent Application No. 19 / 028,930 filed January 17, 2025, U. S. Non-provisional Patent Application No. 19 / 029,060 filed January 17, 2025, U. S. Provisional Patent Application No.

[0044] 63 / 765,920, U. S. Provisional Application No. 63 / 785,064 filed April 8, 2025, U. S. Provisional Application No. 63 / 785,075 filed April 8, 2025, U. S. Provisional Application No. 63 / 786,408 filed April 10, 2025, U. S. Non-Provisional Patent Application No. 19 / 175,419 filed April 10, 2025, U. S.Non-Provisional Patent Application No. 19 / 223,759 filed May 30, 2025, U. S. Provisional Patent Application No. 63 / 874,983 filed September 3, 2025, U. S. Provisional Patent Application No.

[0045] 63 / 898,215 filed October 13, 2025, the U. S. Provisional Application No. 63 / 943,891 filed December 18, 2025, the PCT publication PCT / IB2025 / 063239 filed December 19, 2025 are all incorporated herein by reference in their entirety.

[0046] DETAILED DESCRIPTION OF THE INVENTION:

[0047]

[0039] The present invention overcomes the disadvantages of CHO (Chinese Hamster Ovary) cell lines according to the state of the art, some of which are commercially available. The cell lines according to the invention have improved cultivation properties, are adapted to various conditions and are able to grow on a large scale for use in industrial applications.

[0048]

[0040] The cells according to the present invention are cells derived from CHO cells that are widely used in industry, for example in the production of recombinant proteins in the pharmaceutical industry. The cells according to the invention may be derived, for example, from CHO-K1, CHO-S, CHO-DG44 or CHO-DXB11 cell lines, or any other appropriate CHO cell lines. The cells according to the present invention may be used for many industrial applications, for example for recombinant protein production as mentioned above and the production of other pharmaceutical active ingredients. The cells may be used for preparing cosmetic ingredients or products. The cells may be used for preparing food products for human or animal consumption, for example for preparing cultured meat.

[0049]

[0041] The cell lines according to the invention may be adapted to at least one of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline, or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids, altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0050]

[0042] In one aspect of the invention the cell line may be adapted to at least one specific condition, wherein the specific conditions may be from the group of low or zero concentration of signaling proteins (protein-free conditions), e.g. growth factors in culture medium, low concentration of glutamine, proline, or thymidine in culture medium, resilience to a mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids, altered oxygen concentration, e.g. lowered oxygen concentration or increased oxygen concentration, serum-free conditions, any other appropriate conditions, or a combination thereof.

[0051]

[0043] The cell lines according to the invention may be adapted to one or more of the above mentioned conditions and may obtain one or more new modified properties. The cell lines according to the invention may be adapted independently to any one of the above mentioned conditions, or may be gradually adapted to more than one of the above mentioned conditions and may obtain a combination of more than one new modified properties.

[0052]

[0044] The cell lines according to the invention may have altered, e.g. lowered, oxygen consumption, which is advantageous for large-scale cultivation.

[0045] The cell lines according to the invention may be adapted to growth in alternative cultivation environments such as, for example, culture media comprising protein hydrolysate as the main source of amino acids.

[0053]

[0046] The cell lines according to the invention may be adapted to grow in alternative cultivation environments such as, for example, culture media comprising protein hydrolysate as the main source of amino acids.

[0054]

[0047] For the purposes of the present patent application, the term “in the range of...to... ” means the minimal and maximal values (endpoints) of the interval are included. For example “in the range of 0 to 1,000” means the values 0 and 1,000 are included in the described interval.

[0055]

[0048] The cell lines according to the invention may be adapted to at least two of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids, or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0056]

[0049] The cell lines according to the invention may be adapted to at least three of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0057]

[0050] The cell lines according to the invention may be adapted to at least four of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0058]

[0051] The cell lines according to the invention may be adapted to at least five of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0059]

[0052] The cell lines according to the invention may be adapted to at least six of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, lowconcentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0060]

[0053] The cell lines according to the invention may be adapted to at least seven of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0061]

[0054] The cell lines according to the invention may be adapted to at least eight of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0062]

[0055] The cell lines according to the invention may be adapted to nine of the following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0063]

[0056] The cell lines according to the invention may comprise:

[0064] (i) adaptation to at least one of: suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, or low or zero concentration of signaling proteins, e.g. growth factors, in culture medium; and

[0065] (ii) adaptation to at least one of: low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0066]

[0057] The cell lines according to the invention may comprise:(i) adaptation to at least two of: suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, or low or zero concentration of signaling proteins, e.g. growth factors, in culture medium; and

[0067] (ii) adaptation to at least one of: low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0068]

[0058] The cell lines according to the invention may comprise:

[0069] (i) adaptation to suspension cultivation conditions, adaptation to culture medium comprising protein hydrolysate as a source of amino acids, and adaptation to low or zero concentration of signaling proteins, e.g. growth factors, in culture medium; and

[0070] (ii) adaptation to at least one of: low concentration of glutamine, proline or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, high concentration of metabolites, for example lactate or ammonium ions, in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, or altered, e.g. lowered, oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0071]

[0059] The cell lines according to the invention may be adapted to or may have characteristics according to the description below.

[0072]

[0060] The cell lines according to the invention are suitable for industrial scale cultivation and may be used for the production of cultivated mammalian cell biomass for preparing food products for animal or human consumption, for example, for preparing cultivated meat.

[0073]

[0061] The cell lines according to the invention are suitable for industrial scale cultivation and may be used for the production of cultivated mammalian cell biomass for preparing food products for animal and / or human consumption, for example, for preparing cultivated meat.

[0074]

[0062] Phenotypic and metabolic characteristics of the cell lines according to the invention, genetic and gene expression characteristics of the cell lines, and the processes of derivation of the final cell lines from precursor cell lines are also provided herein.

[0075]

[0063] The cells according to the invention may be adapted to protein-free conditions, or conditions with low concentration of protein.

[0076]

[0064] As used herein, “protein-free” may refer to culture conditions in which no exogenously added intact proteins are intentionally included in the basal medium and / or feed, excluding proteins that may be present as trace impurities and / or protein hydrolysates and / or hydrolysate -derived peptides and amino acids. In further aspects,, “protein -free” excludes the addition of albumin, transferrin, insulin, and growth factors and cytokines, including but not limited to fibroblast growth factors (FGFs, e.g., FGF2), epidermal growth factor (EGF), insulin-like growth factors (IGF-1 and IGF-2), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), colony-stimulating factors (e.g., G-CSF, GM-CSF), and interleukins (e.g., IL-2, IL-6).

[0065] As used herein, “low concentration of protein” may refer to culture conditions in which the total concentration of exogenously added proteins in the culture medium is in the range of 0 mg / L to 0.2 mg / L, or in the range of 0 mg / L to 10 mg / L, or in the range of 0 mg / L to 200 mg / L, excluding proteins that may be present as trace impurities and / or protein hydrolysates and / or hydrolysate-derived peptides and amino acids.

[0077]

[0066] In another aspect of the invention, the terms “low concentration of protein” and “low concentration of signaling proteins” may be interchangeable.

[0078]

[0067] The protein may comprise for example signaling protein, such as a growth factor. The signaling protein may comprise at least one of: FGF family growth factor (e.g. FGF-1, FGF-2), insulin, insulin like growth factor 1 (IGF-1), a TGF family ligand, or transferrin, or any other appropriate signaling compound.

[0079]

[0068] The cells used for adaptation may be CHO cells, for example commercially available CHO cell lines, such as CHO-K1, CHO-S, CHO-DG44 or CHO-DXB 11, or any other appropriate CHO cell lines.

[0080]

[0069] The cells may be initially cultivated in adherent culture, for example using tissue culture flasks. Examples of culture media that may be used are Dulbecco's Modified Eagle Medium (DMEM) with 10 % Fetal Bovine Serum (FBS), or DMEM with 15 % FBS, or any other appropriate culture medium. The medium used may be a culture medium commonly used for the cultivation of serum-dependent mammalian cell lines, and / or it may be the culture medium recommended by the provider of the cell line.

[0081]

[0070] Cells may then be transferred, for example, to Essential 8 medium, or any other appropriate serum-free culture medium, and cultivated in adherent culture for a number of passages in the range of 1 to 10, or in the range of 2 to 8, or in the range of 3 to 6, in order to verify that cells are able to grow in the absence of serum. At this stage, cells may start to exhibit a partial suspension phenotype due to the lack of attachment factors from serum. Passaging may be accomplished by transferring a part of the culture medium containing the detached fraction of the cells, to a new cultivation vessel.

[0082]

[0071] As used herein, “serum-free” may refer to culture conditions in which no exogenously added animal serum is intentionally included in the basal medium and / or feed, including but not limited to FBS, bovine calf serum, horse serum, goat serum, and / or human serum, while allowing serum-derived components that may be present as trace impurities.

[0083]

[0072] Cells may be subsequently transferred to a medium composed, for example, of 5 parts Iscove's Modified Dulbecco’s Medium (IMDM) to 5 parts Ham's F12 to 1 part NCTC 135, or, for example, 4 parts IMDM to 4 parts Ham's F12 to 1 part NCTC 135, or, for example DMEM / F12, or any other suitable medium. The medium may be supplemented with at least one of sodium selenite, ethanolamine, or ferric citrate, or with any other appropriate supplement. This step may serve as confirmation that cells are able to grow in the absence of exogenous signaling proteins.

[0084]

[0073] The medium composition may comprise, for example, DMEM / F12 supplemented with sodium selenite, ethanolamine, and ferric citrate.

[0085]

[0074] The concentration of protein, e.g. signaling protein, in the culture medium for cell adaptation to low or protein- free conditions may be in the range of 0.001 mg / L to 2,000 mg / L, or in the range of 0.01 mg / L to 250 mg / L or in the range of 0.1 to 50 mg / L.

[0075] The concentration of protein, e.g. signaling protein, in the culture medium for cell cultivation may be in the range of 0 mg / L to 2,000 mg / L, or in the range of 0.01 mg / L to 250 mg / L, or in the range of 0.1 mg / L to 50 mg / L. The culture medium for cell cultivation may be free of protein, e.g. signaling protein.

[0086]

[0076] The concentration of TGF beta 1 may be in the range of 0 mg / L to 0.002 mg / L. The concentration of transferrin in the culture medium according to the invention may be in the range of 0 mg / L to 10 mg / L, or in the range of 0.1 mg / L to 8 mg / L, or in the range of 0.5 mg / L to 5 mg / L. In one aspect of the invention, the reduced amount of transferrin may be in the range of 0 mg / L to 0.01 mg / L.

[0087]

[0077] The concentration of insulin in the culture medium may be in the range of 0 mg / L to 2 g / L, or in the range of 0.1 mg / L to 1 g / L, or 0.5 mg / L to 500 mg / L. In one aspect of the invention, the reduced amount of insulin may be in the range of 0 mg / L to 0.1 mg / L.

[0088]

[0078] The concentration of FGF-2 in the culture medium may be in the range of 0 mg / L to 1 mg / L, or in the range of 0.1 mg / L to 0.8 mg / L, or 0.2 to 0.5 mg / L. In one aspect of the invention, the reduced amount of FGF-2 may be in the range of 0 to 0. 01 mg / L.

[0089]

[0079] The concentration of TGF beta 1 in the culture medium may be in the range of 0 to 0.2 mg / L, or in the range of 0.01 mg / L to 0.15 mg / L, or 0.05 mg / L to 0.1 mg / L. In one aspect of the invention, the reduced amount of TGF beta 1 may be in the range of 0 to 0.001 mg / L.

[0090]

[0080] In one aspect of the invention, the culture medium may be without content of any signaling compounds, for example growth factors. The culture medium according to the invention may be serum free and / or protein free.

[0091]

[0081] CHO cells may be unable to synthesize the non-essential amino acid L-proline (proline auxotrophy). This may be disadvantageous for the scale-up of cultivation processes of the cells. To solve this problem, the cells may be adapted to regain the ability to synthesize L-proline in culture medium without L-proline or in a culture medium with a low concentration of L-proline. The culture medium may be free of L-proline. The concentration of L-proline in culture medium may be in the range of 0 mg / L to 10 mg / L, or in the range of 0.001 mg / L to 5 mg / L, or in the range of 0.01 mg / L to 2 mg / L, or in the range of 0.1 mg / L to 1 mg / L. The cells may be cultivated in these conditions for a number of passages in the range of 1 to 10, or in the range of 2 to 8, or in the range of 3 to 6. Under these conditions, cells which exhibit a reversion to the common phenotype for mammalian cells, meaning the ability to synthesize proline (proline prototrophy), may be selected. The cells may be further cultivated in culture medium comprising, for example, DMEM / F12 supplemented with sodium selenite, ethanolamine, and ferric citrate, or in any other appropriate culture medium.

[0092]

[0082] In one aspect of the invention, 5-azacytidine or another suitable chemical agent may be used to increase the frequency of cells reverting to proline prototrophy.

[0093]

[0083] In one aspect of the invention, the CHO cells may be adapted to synthesize L-proline and therefore may be proline-prototrophic.

[0094]

[0084] Cells adapted to proline prototrophy using the disclosed methodology may exhibit a capacity for specific proline production in the range of 1 fg to 500 fg L-proline / cell / hour, or in the range of 50 fg to 450 fg L-proline / cell / hour, or in the range of 100 to 400 fg L-proline / cell / hour, or in the range of 180 to 350 fg L-proline / cell / hour, or in the range of 250 fg to 300 fg L-proline / cell / hour.

[0085] The proof of proline production by CHO-derived cells according to the invention (the curve of proline concentration versus time) is depicted in Fig. 1.

[0095]

[0086] Fig.3 shows the growth curve (GC) of cell density and proline production both together in one chart (the curve of proline concentration versus time and the curve of cell density versus time).

[0096]

[0087] Metazoan cells are commonly cultivated in adherent culture as a cell layer upon a suitable cultivation surface, such as plasma-treated polystyrene, gelatin or matrigel. Without a suitable cultivation surface, most metazoan cell types will fail to proliferate or will die from lack of attachment (anoikis). In order to produce a sufficient biomass of cells for cultivated meat applications, the cultivation surface would need to be impractically large. Therefore, it is necessary to adapt the cells so that they are able to proliferate without attachment to a surface.

[0097]

[0088] The cells according to the invention may be adapted to suspension cultivation conditions. Under these conditions, cells may be able to grow with minimal adhesion to the culture vessel as a suspension of single cells or small cell aggregates, and may be passaged with a simple transfer of cell suspension to a new culture vessel.

[0098]

[0089] As used herein, the terms “passage”, “passaged” or “passaging” may be interchangeable with the terms “subculture”, “subcultured” or “subculturing”.

[0099]

[0090] Suspension culture adaptation may enable scalability of cell cultivation in bioreactors, unlike adherent culture methods. The process may start with adherent cells or with a mixed culture of adherent and suspension cells. Agitation may be used in order to encourage suspension growth, for example in spinner flasks or bioreactors. The culture medium may be supplemented with a shear protectant, e.g. Pluronic F-68, or with any other appropriate shear protectant.

[0100]

[0091] The shear protectant may comprise at least one of: polyethylene glycol (PEG), Pluronic F68, Pluronic F127, methyl cellulose (MC), (hydroxypropyl)methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), or dextran sulfate, or a combination thereof.

[0101]

[0092] The total input of the shear protectant to culture medium may be in the range of 0.1 g / E to 5 g / E, or in the range of 0.5 g / L to 4 g / L, or in the range of 1 g / L to 3 g / L.

[0102]

[0093] Cell clumping may be prevented by optimizing agitation rates and adding anti-clumping agents.

[0103]

[0094] The cells according to the invention may start to exhibit a partial suspension phenotype during adaptation to serum-free media as a result of the depletion of attachment factors from the serum. In this stage of adaptation, individual cells or cell clumps may transiently detach from the culture surface and float freely in the medium, however, the culture is not capable of long-term survival in the absence of an attachment surface when the cells exhibit a partial suspension phenotype.

[0104]

[0095] To promote an increase in the suspension tendencies in the cell population, a method of passaging may be used where the detached fraction of cells is aspirated with a pipette and transferred to fresh culture medium. Unlike common passaging methods, which usually involve detaching the cell layer with trypsin, or other methods, this leads to the gradual enrichment of the suspension-prone population.

[0105]

[0096] After several passages of the detached fraction, the cells may be sufficiently robust to be transferred to a mechanically agitated culture environment, such as a shaking Erlenmeyer flask. The number of passages may be for example in the range of 1 to 20, or in the range of 3 to 15, or in therange of 5 to 10 passages. Mechanical agitation prevents cell attachment to the surface of the vessel. To further minimize surface attachment, the agitated vessel may have a low-attachment surface, for example Teflon or glass treated with a siliconizing reagent (such as Sigmacote).

[0106]

[0097] Mechanical agitation applied to the cell culture may be increased over time, leading to dissociation of cell aggregates into predominantly single cells. Advantageously, single cells may exhibit faster overall growth than cell aggregates, because oxygen and nutrient diffusion may limit the growth of cells at the center of an aggregate. Additionally, thanks to their smaller size, single cells have a lower sedimentation speed and a higher resistance to shear forces than cell aggregates.

[0107]

[0098] After cells are able to grow as a single-cell suspension with a >90 % viability, they may be considered fully adapted to suspension. With a suitable shear protectant, they may be able to survive even relatively intense stirring and sparging conditions, for example stirring with an impeller with a tip speed of 2 m / s and sparging at 0.125 vvm (volume per volume per minute).

[0108]

[0099] In one aspect of the invention, the cells may be adapted to a suitable range of pH and / or osmolality.

[0109]

[0100] The pH of culture media may affect multiple cellular processes, including growth, lactate and ammonia metabolism, and others. It may also impact cell viability, and secretion of recombinant proteins in the case that the cells are used for the purpose of producing recombinant proteins.

[0110]

[0101] The pH of commercially available or commonly used culture media is usually around 7.4, which mimics the environment of the body. In some aspects, a lower medium pH may be advantageous in an industrial setting, because less buffer and / or base is required to balance the pH of other medium components, which in sum tend to be acidic. Cells may also exhibit a more favorable metabolism, for example lower lactate production, at lower pH levels.

[0111]

[0102] The osmolality of commercially available or commonly used culture media is usually around 300 mOsm / kg. In some aspects, it may be advantageous for medium osmolality to be higher. At a higher osmolality, more nutrients may be dissolved in the culture medium, which may allow a higher cell density to be achieved. Additionally, culture medium osmolality may tend to decrease as the nutrients are consumed from medium by the cells, therefore, it may be beneficial to make the initial osmolality of the culture medium near the upper limit, and in some cases above the upper limit of the osmolality optimum (in terms of cell growth).

[0112]

[0103] Adaptation to a suitable pH and osmolality range may be achieved by a gradual exposure to culture media with the target parameters. This may include, for example, exposure to culture media with a lower pH, and / or culture media with a higher osmolality.

[0113]

[0104] Cells may be adapted, for example, so that optimal growth is achieved in the pH in the range of 6.9 - 7.3, or in the range of 7.0 to 7.2. The osmolality may be in the range of 280 mOsm / kg to 360 mOsm / kg, or in the range of 290 mOsm / kg to 350 mOsm / kg, or in the range of 300 mOsm / kg to 340 mOsm / kg, or in the range of 310 mOsm / kg to 330 mOsm / kg.

[0114]

[0105] The cells according to the invention may be adapted to low or zero concentration of thymidine. The cells according to the invention may be adapted to low or zero concentration of L-glutamine, where a low concentration of L-glutamine is understood to be lower than 365 mg / L (which is found for example in the commercial medium DMEM / F12). In one aspect of the invention, the cells may be adapted to a culture medium comprising a concentration of glutamine in the range of 10 mg / L to 100mg / L, or in the range of 20 to 90 mg / L, or in the range of 30 mg / L to 80 mg / L, or in the range of 40 mg / L to 70 mg / L, where the source of L-glutamine is a protein hydrolysate.

[0115]

[0106] Fig. 2 shows the outcome of cultivation and growth of CHO-derived cells according to the invention in thymidine free medium with low concentration of glutamine, without hypoxanthine, linoleic acid, or lipoic acid, with hydrolysate at concentration 9 g / L of protein (100%). The curve shows a cell density versus time.

[0116]

[0107] L-glutamine and thymidine are important molecules in metabolism of cells including CHO cells, and play crucial roles in cellular nutrition, growth and division.

[0117]

[0108] L-glutamine is a non-essential amino acid that is essential for dividing cells. It plays multiple roles in cellular metabolism. L-glutamine serves as an energy source, especially when glucose availability is limited. It undergoes deamination by glutaminase, producing glutamate, which enters the Krebs cycle as alpha-ketoglutarate, generating adenosine triphosphate (ATP).

[0118]

[0109] L-glutamine is a critical nitrogen donor in the biosynthesis of nucleotides and amino acids. It supplies nitrogen for the formation of carbamoyl phosphate, a precursor in the pyrimidine synthesis pathway. L-glutamine contributes to the biosynthesis of proteins, lipids, and nucleic acids. It is also involved in the production of NADPH, which is crucial for maintaining redox balance and biosynthetic reactions. Thymidine is a pyrimidine nucleoside consisting of thymine and deoxyribose. Thymidine is important for DNA synthesis and repair. Thymidine is a precursor for deoxythymidine triphosphate (dTTP), a nucleotide required for DNA replication. Thymidine availability is crucial for dividing cells, its deficiency may lead to stopping DNA synthesis and cell cycle disruption. Supplementing cell culture media with thymidine may enhance cell growth and division in certain conditions.

[0119]

[0110] Both molecules L-glutamine and thymidine are crucial for maintaining DNA replication and repair, especially in high-demand conditions like cell culture, where cells divide rapidly.

[0120]

[0111] L-glutamine and thymidine may be added to cell culture media to support cell growth and metabolic activities. L-glutamine may be supplied as part of a peptide, for example L-alanyl-L-glutamine, which is more stable than free L-glutamine.

[0121]

[0112] In order to adapt CHO cells to thymidine independence, the cells may be cultivated in the culture medium without thymidine, for example in the modified DMEM / F12 culture medium without thymidine and supplemented with sodium selenite, ethanolamine and ferric citrate, or in any other appropriate medium. The cells may be cultivated for a number of passages in the range of 1 to 10, or in the range of 2 to 8, or in the range of 3 to 6.

[0122]

[0113] The concentration of glutamine in the culture medium may be gradually decreased to a concentration in the range of 200 mg / L to 350 mg / L, or in the range of 100 mg / L to 200 mg / L, or in the range of 10 mg / L to 100 mg / L.

[0123]

[0114] Additionally, saturated and unsaturated fatty acids, for example lipoic acid or linoleic acid, short organic acids, for example pyruvate, nucleotide precursors, for example hypoxanthine, or any other appropriate organic compounds may be omitted from the culture medium. This may not lead to noticeable changes in cell growth or viability and may not require a dedicated adaptation step.

[0124]

[0115] Additionally, compounds in the culture medium may be replaced with alternative compounds that can fulfill similar requirements in cell metabolism. For example, putrescine may be replaced withornithine. Cells may be able to convert ornithine into putrescine, therefore fulfilling their metabolic requirements without the need to supplement the culture medium with putrescine.

[0125]

[0116] The cells according to the invention may be adapted so that they exhibit a low net production, or exhibit net consumption of lactate.

[0126]

[0117] The cells according to the invention may be adapted so that they exhibit a low net production, or exhibit net consumption of lactate and / or ammonia.

[0127]

[0118] As used herein, “net consumption” may refer to a decrease in the concentration of a metabolite in the culture medium over time such that the concentration measured in a waste medium is lower than the concentration present in the culture medium at the start of a cultivation period. In some embodiments, the adapted CHO cell line exhibits net consumption of ammonia and / or lactate, thereby reducing accumulation of ammonia and / or lactate in the culture and improving culture robustness and / or product yield.

[0128]

[0119] Lactate may be produced by the cells as a waste metabolite of glycolysis. Excess lactate may acidify the culture medium and negatively affect cell growth and viability.

[0129]

[0120] In one aspect of the invention, cells may be adapted to be resistant to the negative effects of lactate on growth and viability by being exposed to lactate in the culture medium over a prolonged period of cultivation, for example in the range of 1 to 100, or 2 to 50, or 3 to 30 passages. Most culture media contain no or minimal lactate initially, usually less than 1 mM. Lactate may form as a side product during the process of protein hydrolysis, and it may thus be introduced into culture media comprising a protein hydrolysate.

[0130]

[0121] In one aspect of the invention, culture medium comprising a protein hydrolysate may comprise lactate in concentration in the range of 5 mg to 150 mg lactate per gram of protein from the hydrolysate per liter, or in the range of 10 mg to 100 mg lactate per gram of protein from the hydrolysate per liter, or in the range of 15 mg to 50 mg lactate per gram of protein from the hydrolysate per liter.

[0131]

[0122] Culture medium for adaptation of cells to resist the negative effects of lactate may comprise lactate in a concentration in the range of 10 mg / L to 500 mg / L, or in the range of 50 mg / L to 450 mg / L, or in the range of 150 mg / L to 400 mg / L, or in the range of 200 mg / L to 350 mg / L, or in the range of 250 mg / L to 300 mg / L of culture medium.

[0132]

[0123] Culture medium for adaptation of cells to lactate presence may comprise protein hydrolysate, or another appropriate source of amino acids.

[0133]

[0124] Culture medium for adaptation of cells to lactate presence may further comprise at least one of: sugars, vitamins and organic micronutrients, mineral compounds, iron supplementation compounds, organic amines or shear protectants, or a combination thereof. The culture medium may further comprise other compounds, like fatty acids, phospholipids, or nucleic acids, for example.

[0134]

[0125] Adaptation of cells to higher concentration of hydrolysate may also gradually expose cells to higher concentrations of lactate, therefore promoting cell resistance to the negative effects of lactate.

[0135]

[0126] Under conditions where sources of energy and carbon (such as glucose) may be limited, cells may be adapted to produce a low amount of lactate, as directing pyruvate into the Krebs cycle is more energy efficient that directing it into lactate production. Furthermore, if the culture contains lactate,cells may be adapted to be able to utilize lactate as an energy and carbon source. Such adaptation may manifest in the net consumption of lactate from the culture medium by the cells.

[0136]

[0127] Adaptation of cells to lactate presence may result in cell lactate metabolism according to the description below. Fig. 4 depicts low lactate production or its consumption by cells corresponding to better glucose conversion efficiency. The curves show glucose concentration versus time and lactate concentration versus time.

[0137]

[0128] Preparation of culture media comprising individual free amino acids is expensive and problematic for large scale cultivation processes. All or most of the amino acids may be supplied to the culture medium in the form of a protein hydrolysate. Additionally, hydrolysates may contain peptides which may also be consumed by the cells or have other beneficial effects.

[0138]

[0129] The CHO cells according to the invention may be adapted to culture media comprising protein hydrolysate as a source of amino acids. The culture medium according to the invention may comprise soy protein hydrolysate, or any other appropriate protein hydrolysate. Examples of suitable industrially scalable protein sources for hydrolysis may include soy, pea, rice, wheat, corn, fava beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, water lentil, mung bean, flax, or baker's yeast, or any other appropriate protein source. The present invention is not limited to the listed exemplary protein sources.

[0139]

[0130] In one aspect of the invention, the composition of the culture medium may be defined in terms of the total input of medium components into the cultivation process. In this aspect of the invention, summary amounts of components introduced into the cultivation process at any time point over its entire duration are provided. Furthermore, in this aspect of the invention, the provided concentration ranges for the individual medium components describe the total amount of the given component introduced into the cultivation process at any time point during the cultivation process in relation to the volume of spent culture medium which exits the process. The spent culture medium may exit the cultivation process together with the cultivated cells (harvesting), or separately from the cultivated cells (perfusion). The cultivation process may further have the characteristics of a batch process, where all of the components are introduced into the cultivation process at a single time point and the harvest is performed at a single time point, a fed-batch process, where some components may be introduced after the start of the process and the harvest is done at a single time point, a continuous process, where components may be introduced during the whole duration of cultivation and harvesting may be performed during the whole duration of cultivation, or a combination of the described characteristics. For brevity, this aspect of the invention will be referred to herein as “total input”.

[0140]

[0131] The concentration of protein hydrolysate in the culture medium may be in the range of 1 g / L to 200 g / L, or in the range of 3 g / L to 100 g / L, or in the range of 10 g / L to 60 g / L, or in the range of 8 g / L to 50 g / L, where the concentration of the hydrolysate in the medium is expressed as grams of protein in the protein substrate per liter of the hydrolysis reaction mixture, multiplied by the volumetric percentage of the liquid hydrolysate in the culture medium. The concentration of protein hydrolysate is expressed as the total input, meaning that the amount of protein hydrolysate added to the culture medium before cultivation and at any point during cultivation is summed together and divided by sum of the volume of waste culture medium leaving the cultivation device.

[0141]

[0132] The total input of amino acids from hydrolysate, including amino acids in the form of short peptides or suitable bioavailable derivatives, for example phosphoesters, such as phosphoserine, orother derivatives, such as methylglycine, is at least 75 %, 80 %, 85 %, 90 %, or 95 % by weight of the total input of all amino acids into the culture medium.

[0142]

[0133] The culture medium according to the invention may comprise amino acids added separately from the hydrolysate, for example L-methionine, L-cysteine or L-ornithine. The total input of amino acids added separately from hydrolysate may be in the range of 0.02 g / L to 30 g / L, or in the range of 0.05 g / L to 15 g / L, or in the range of 0.1 g / L to 10 g / L.

[0143]

[0134] The total amount of L-cysteine in culture medium may be in the range of 0.1 % to 10 %, or 0.5 % to 7 %, or 1 % to 5 % by weight with respect to the total amount of hydrolysate protein in the culture medium.

[0144]

[0135] The total amount of L-ornithine in culture medium may be in the range of 0 % to 5 %, or 0.0001 % to 3 %, or 0.001 % to 0.5 % with respect to the total amount of hydrolysate protein in the culture medium.

[0145]

[0136] The total amount of L-methionine in culture medium may be in the range of 0.05 % to 6 %, or 0.1 % to 3 %, or 0.2 % to 2 % with respect to the total amount of hydrolysate protein in the culture medium.

[0146]

[0137] The total amount of L-tryptophan in the culture medium may be in the range of 0.05 % to 6 %, or 0.1 % to 3 %, or 0.2 % to 2 % with respect to the total amount of hydrolysate protein in the culture medium.

[0147]

[0138] The total amount of L-histidine in culture medium may be in the range of 0.03 % to 4 %, or 0.07 % to 2 %, or 0.15 % to 1.5 % with respect to the total amount of hydrolysate protein in the culture medium.

[0148]

[0139] The total amount of L-threonine in culture medium may be in the range of 0.1 % to 7 %, or 0.2 % to 5 %, or 0.3 % to 3 % with respect to the total amount of hydrolysate protein in the culture medium.

[0149]

[0140] The total input of amino acids added to the culture medium separately from the hydrolysate may be in the range of 0.2 % to 25 %, or in the range of 0.5 % to 15 %, or in the range of 1 % to 10 %, expressed as a percentage of the total input of hydrolysate protein into the culture medium.

[0150]

[0141] The culture medium according to the invention may further comprise at least one of: sugars, vitamins, organic micronutrients, mineral compounds, iron supplementation compounds, organic amines, and shear protectants, or a combination thereof. The media may also comprise other compounds, for example fatty acids, phospholipids, or nucleic acids, or other appropriate compounds.

[0151]

[0142] The cells may be adapted to hydrolysate gradually in several steps in order to avoid drastic change in cell cultivation conditions. The number of steps of hydrolysate concentration change or change of concentration of free amino acids added to the medium separately may be in the range of 1 to 10, or in the range of 2 to 8, or in the range of 3 to 6.

[0152]

[0143] For example, the concentration of soy protein hydrolysate in the culture medium may be increased in three steps, wherein the concentration of soy protein hydrolysate in the culture medium in the first step may be in the range of 0.2 g / L to 1.5 g / L, or in the range of 0.4 g / L to 1.2 g / L, or in the range of 0.6 g / L to 1 g / L. The concentration in the second step may be in the range of 1 g / L to 3 g / L, or in the range of 1.5 g / L to 2.5 g / L, or in the range of 1.7 g / L to 2.2 g / L. The concentration in the third step may be in the range of 2 g / L to 5 g / L, or in the range of 2.5 g / L to 4.5 g / L, or in the range of 3 g / Lto 4 g / L, wherein the concentration of the hydrolysate in the medium is expressed as grams of protein in the protein substrate per liter of the hydrolysis reaction mixture, multiplied by the volumetric percentage of the liquid hydrolysate in the culture medium.

[0153]

[0144] Any other appropriate protocol of gradual increase of protein hydrolysate concentration in the culture medium may be used.

[0154]

[0145] Examples of amino acids and their derivatives that may be supplied to the media are: glycine, L-alanine, L-arginine, L-asparagine L-aspartic acid, L-cystine L-glutamic acid, L-glutamine, L-histidine, L-hydroxyproline, L-ornithine, L-citrulline, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-pyroglutamic acid, L-phosphoserine, L-tryptophan, L-tyrosine or L-valine. For the preparation of the culture medium, the given amino acid may be added in the pure form, or as part of a complex mixture of compounds (for example a hydrolysate), or the hydrates or salts (for example hydrochlorides or sodium salts) of amino acids may be used.

[0155]

[0146] During the process of adaptation of cells to the gradually increased concentration of hydrolysate, the composition of the culture medium may be modified in order to improve scalability, prevent precipitation of medium components, and ensure sugar and micronutrient availability is not limiting to the cells.

[0156]

[0147] After the end of the cell adaptation phase, the production cell bank may be prepared, and further characterization of these cells may be performed, to determine their properties relevant to an industrial scale cultivation process.

[0157]

[0148] The cells according to the invention may have characteristics according to the description below.

[0158]

[0149] The cells may be able to grow with little to no addition of exogenous signaling proteins into the culture medium. In such culture medium, they may retain high viability, for example above 90 %, and a short doubling time, for example below 24 hours (commonly, metazoan cells require exogenous signaling proteins, for example in the form of fetal bovine serum or recombinant growth factors, in the culture medium; without such signaling proteins, they will die or fail to proliferate).

[0159]

[0150] The cells may be able to grow in suspension culture in the form of single cells or small aggregates. In this culture mode, they may retain high viability, for example above 90 %, and a short doubling time, for example below 24 hours (commonly, metazoan cells are not able to grow in suspension, a state known as anchorage dependence; such cells will die or fail to proliferate in the absence of a suitable attachment surface).

[0160]

[0151] The weight of wet cells (average per one cell) may be in the range of 0.5 ng to 3.0 ng, or in the range of 0.7 ng to 2.8 ng, or in the range of 0.9 ng to 2.5 ng, or in the range of 1.0 ng to 2.0 ng, or in the range of 1.2 ng to 1.8 ng.

[0161]

[0152] The weight of dry cells (average per one cell) may be in the range of 0.05 ng to 0.5 ng, or in the range of 0.07 ng to 0.45 ng, or in the range of 0.1 ng to 0.4 ng, or in the range of 0.15 ng to 0.35 ng, or in the range of 0.2 ng to 0.3 ng.

[0162]

[0153] The cell doubling time in exponential culture may be in the range of 10 hours to 100 hours, or in the range of 12 hours to 90 hours, or in the range of 14 hours to 80 hours, or in the range of 15 hoursto 60 hours, or in the range of 16 hours to 50 hours, or in the range of 17 hours to 40 hours, or in the range of 18 hours to 30 hours.

[0163]

[0154] The cell doubling time in continuous culture may be in the range of 15 hours to 150 hours, or in the range of 18 hours to 120 hours, or in the range of 20 hours to 100 hours, or in the range of 25 hours to 80 hours, or in the range of 28 hours to 60 hours, or in the range of 30 hours to 50 hours.

[0164]

[0155] In one aspect of the invention, cells may be characterized by their specific capacity for the consumption or production of a compound, where the maximum speeds of consumption or production of the compound observed, regardless of conditions are reported. In another aspect of the invention, cells may be characterized by their specific consumption or production of a compound, where the average values observed in continuous culture are reported. All specific capacity for consumption / production and specific consumption / production values (in the description, examples and claims) are reported as net values (averaged across the cell population) and are calculated from the change of concentration of the compound over time.

[0165]

[0156] The cell specific oxygen consumption in continuous culture may be in the range of 0.9 to 5 pmol / cell / day, or in the range of 1.0 to 4.0 pmol / cell / day, or in the range of 1.1 to 3.5 pmol / cell / day, or in the range of 1.2 to 3.0 pmol / cell / day, or in the range of 1.3 to 2.8 pmol / cell / day, or in the range of 1.4 to 2.6 pmol / cell / day.

[0166]

[0157] The cell specific carbon dioxide (CO2) production in continuous culture may be in the range of 0.9 to 5 pmol / cell / day, or in the range of 1.0 to 4.0 pmol / cell / day, or in the range of 1.1 to 3.5 pmol / cell / day, or in the range of 1.2 to 3.0 pmol / cell / day, or in the range of 1.3 to 2.8 pmol / cell / day, or in the range of 1.4 to 2.6 pmol / cell / day.

[0167]

[0158] The cells may show equal or better culture performance in terms of cell growth and viability in comparison to baseline (at around 90 to 95 % dissolved oxygen partial pressure) at a dissolved oxygen partial pressure in the culture medium (expressed in % of the partial pressure of oxygen in air at sea level) in the range of 3 % to 200 %, or in the range of 5 % to 100 %, or in the range of 10 % to 50 %, or in the range of 15 % to 25 %. Cell specific oxygen consumption, specific glucose consumption and specific lactate production may be equal or similar at the provided oxygen partial pressure compared to baseline.

[0168]

[0159] The glucose conversion efficiency (expressed as grams of glucose consumed per 1 g of biomass produced) may be in the range of 0.1 to 0.4, or in the range of 0.15 to 0.38, or in the range of 0.18 to 0.36, or in the range of 0.22 to 0.35 g of glucose consumed per 1 g of biomass produced. High glucose conversion efficiency may be facilitated by a low net lactate production; a higher production of ATP per molecule of glucose is achieved if pyruvate obtained from glucose enters the Krebs cycle and is used to make reduced coenzymes for the respiration chain instead of being converted to lactate; alternatively, glucose can be converted into cell biomass building blocks by various metabolic pathways such as the pentose phosphate pathway (PPP), whereas glucose converted to lactate does not directly contribute to formation of cell biomass.

[0169]

[0160] The protein conversion efficiency (expressed as total content of crude protein in cell biomass divided by total input of protein into the medium) may be in the range of 15 % to 70 %, or in the range of 20 % to 60 %, or in the range of 25 % to 50 %, or in the range of 30 % to 40 %.

[0170]

[0161] The cells in continuous culture may have a specific net glucose consumption from culture medium in the range of 1 to 50 pg / cell / hour, or in the range of 2 to 30 pg / cell / hour, or in the range of 3to 20 pg / cell / hour, or in the range of 4 to 15 pg / cell / hour, or in the range of 5 to 10 pg / cell / hour, or in the range of 6 to 8 pg / cell / hour.

[0171]

[0162] In continuous culture, the specific net production of lactate by the cells may be in the range of 1to 300 fg / cell / hour, or in the range of 5 to 150 fg / cell / hour, or in the range of 10 to 120 fg / cell / hour, or in the range of 30 to 80 fg / cell / hour. In favorable conditions, the net production of lactate may be close to zero (an equilibrium between lactate production and consumption may be achieved), or the lactate consumption may outpace lactate production, resulting in net lactate consumption. Specific net lactate consumption may be in the range of 1 to 500 fg / cell / hour, or in the range of 5 to 300 fg / cell / hour, or in the range of 10 to 200 fg / cell / hour, or in the range of 20 to 170 fg / cell / hour. The balance between lactate production and consumption may depend on the concentration of lactate in the culture. Lactate consumption may be favored at a high concentration of lactate, for example higher than 500 mg / L, higher than 1000 mg / L, or higher than 1500 mg / L. Advantageously, the concentration of lactate at which net lactate consumption is achieved may be lower than the concentration where cell inhibition by lactate is observed - this may allow the culture to never be inhibited by lactate under realistic conditions.

[0172]

[0163] The specific capacity for L-proline production may be in the range of 1 to 500 fg L-proline / cell / hour, or in the range of 50 to 450 fg L-proline / cell / hour, or in the range of 100 to 400 fg L-proline / cell / hour, or in the range of 200 to 350 fg L-proline / cell / hour, or in the range of 250 to 300 fg L-proline / cell / hour.

[0173]

[0164] The specific L-proline production may be in the range of 50 to 300 fg L-proline / cell / hour, or in the range of 60 to 250 fg L-proline / cell / hour, or in the range of 80 to 220 fg L-proline / cell / hour, or in the range of 100 to 200 fg L-proline / cell / hour, or in the range of 120 to 160 fg L-proline / cell / hour.

[0174]

[0165] The capacity for L-glutamine production may be in the range of 1 to 400 fg glutamine / cell / hour, or in the range of 20 to 300 fg glutamine / cell / hour, or in the range of 50 to 200 fg glutamine / cell / hour, or in the range of 100 to 250 fg glutamine / cell / hour, or in the range of 150 to 220 fg glutamine / cell / hour.

[0175]

[0166] The specific L-glutamine production may be in the range of 0.1 to 50 fg L-glutamine / cell / hour, or in the range of 1.0 to 40 fg L-glutamine / cell / hour, or in the range of 2.0 to 35 fg L-glutamine / cell / hour, or in the range of 3.0 to 30 fg L-glutamine / cell / hour, or in the range of 4.0 to 20 fg L-glutamine / cell / hour, or in the range of 5.0 to 10 fg L-glutamine / cell / hour.

[0176]

[0167] The specific capacity for ammonia consumption may be in the range of 1 to 300 fg ammonia / cell / day, or in the range of 10 to 250 fg ammonia / cell / day, or in the range of 25 to 220 fg ammonia / cell / day, or in the range of 50 to 200 fg ammonia / cell / day, or in the range of 80 to 150 fg ammonia / cell / day.

[0177]

[0168] The specific ammonia consumption may be in the range of 2 to 250 fg ammonia / cell / day, or in the range of 3 to 150 fg ammonia / cell / day, or in the range of 5 to 100 fg ammonia / cell / day, or in the range of 10 to 40 fg ammonia / cell / day.

[0178]

[0169] The mechanical resistance (average power input) may be in the range of 900 W / m3to 1850 W / m3, or in the range of 1000 W / m3to 1750 W / m3, or in the range of 1100 W / m3to 1700 W / m3, or in the range of 1200 W / m3to 1600 W / m3, or in the range of 1300 W / m3to 1500 W / m3.

[0170] The mechanical resistance - impeller tip speed may be in the range of 1.57 m / s to 2.72 m / s, or in the range of 1.7 m / s to 2.6 m / s, or in the range of 1.8 m / s to 2.5 m / s, or in the range of 1.9 m / s to 2.4 m / s, or in the range of 2.0 m / s to 2.3 m / s.

[0179]

[0171] The aeration resistance - relative gas flow rate may be in the range of 0.125 to 0.2 / min, or in the range of 0.13 to 0.19 / min, or in the range of 0.14 to 0.18 / min, or in the range of 0.15 to 0.17 / min.

[0180]

[0172] The aeration resistance - superficial gas velocity may be in the range of 0.9 mm / s to 2.11 mm / s, or in the range of 1.0 mm / s to 2.0 mm / s, or in the range of 1.2 mm / s to 1.9 mm / s, or in the range of 1.3 mm / s to 1.8 mm / s, or in the range of 1.4 mm / s to 1.7 mm / s.

[0181]

[0173] In one aspect of the invention, the cell line may be derived from the CH0-K1 cell line with the following characteristics.

[0182]

[0174] The cell wet weight is 1.2 to 1.6 nanograms per cell. The water content of cell biomass is 78 % to 85 %. The cells are able to proliferate with an average doubling time of 20 hours in the exponential phase of the culture, and 37 hours in continuous culture.

[0183]

[0175] The specific oxygen consumption of cells is 1.7 to 2.4 pmol O2 / cell / day. The value of specific oxygen consumption is lower than values of reported mammalian cells in vitro according to the state of the art, which is an advantage for large-scale culture, as less extreme sparging and stirring of the culture is required to meet oxygen demands of the cells.

[0184]

[0176] The cell consumption of glucose from the culture medium is 0.25 to 0.35 grams per one gram of wet biomass produced in continuous culture. The cell protein conversion efficiency (expressed as total content of crude protein in cell biomass divided by total input of protein into the medium) is 30 to 40 % in continuous culture.

[0185]

[0177] The cells are able to synthesize L-proline, L-glutamine and thymidine. The specific capacity for L-proline production is about 275 fg L-proline / cell / hour, and the specific capacity for L-glutamine production is about 176 fg L-glutamine / cell / hour.

[0186]

[0178] The cells are able to consume ammonia from the culture medium. The observed specific net ammonia consumption is 10 to 40 fg / cell / hour in continuous culture. The capacity for ammonia consumption is 110 fg / cell / hour.

[0187]

[0179] Fig. 5 depicts cell growth in a medium with low glutamine concentration and the influence of momentary production of glutamine and its connection with momentary ammonia consumption from the culture medium. The curves show cell density versus ammonia concentration and glutamine concentration in the culture medium.

[0188]

[0180] The specific net glucose consumption from culture medium is 6 to 6.5 pg / cell / hour in continuous culture.

[0189]

[0181] The cells may resist the following parameters when cultivated in a stirred tank bioreactor (with a suitable shear protectant, e.g. Pluronic F-68, PEG or methylcellulose): at least 900 W / m3volumetric power input (calculated as the total power input of the mixing shaft divided by the total volume of cultivation medium) or 1.57 m / s impeller tip speed. In terms of aeration, the cells may resist 0.125 vvm or 0.9 mm / s superficial gas velocity.

[0182] The processes of adaptation of cells according to the invention may be performed in systems, equipment or vessels commonly used, well-known, and used by persons skilled in the art.

[0190]

[0183] The processes of cultivation of cells according to the invention may be performed in a cultivation system. In one aspect of the invention, the cultivation system (1) is as depicted in Fig. 6.

[0191] The cultivation system (1) may comprise a seeding tank (2), a cultivation device (3), a harvesting device (4), a control unit (5), and sensors and analytical instruments (6) as depicted in Fig. 6. Optionally, the cultivation system (1) may further comprise a device for preparing food products (not depicted in Fig.

[0192] 6).

[0193]

[0184] The control unit may control and / or regulate every process taking place within the cultivation system. The control unit may be operated using at least one printed circuit board (PCB) and / or microprocessor with software capable of controlling the cultivation device, regardless of the extensions and scale of the system. The control unit may be connected to at least one central data storage. The cultivation system may comprise one or more subcontrol units.

[0194]

[0185] The culture medium may be prepared in a culture medium tank. The culture medium tank may comprise at least one of: a mixing tank, hydrolysis tank, storage tank, loading tank or waste medium tank, or any other appropriate device. The media components may be mixed in a mixing tank, which may be made from stainless steel, glass, or any other suitable material.

[0195]

[0186] The mixing tank may be equipped with a stirring unit comprising, for example, a shaft with one or more impellers. The mixing tank may be equipped with a heating system. The temperature may be in the range of 10 °C to 40 °C, or in the range of 15 °C to 38 °C, or in the range of 18 °C to 35 °C. The mixing tank may be connected to one or more storage tanks. The mixing tank may be connected to one or more cultivation devices, formed for example by a bioreactor. The culture medium components may be mixed directly in the cultivation device. The volume of the mixing tank may be in the range of 500 mL to 100 m3, or in the range of 1 L to 10 m3, or in the range of 2 L to 5 m3, or in the range of 500 L to 3 m3. The storage tanks may be made from stainless steel, glass or any other suitable material. The volume of the storage tank may be in the range of 500 mL to 100 m3, or in the range of 1 L to L to 5 m3, or in the range of 2 L to 3 m3, or in the range of 500 L to 1 m3. The media components may be dosed into the mixing tank through a sterilization filter, may be sterilized prior to the placement to the mixing tank or may be sterilized in the mixing tank. The mixing tank may be equipped with different types of sensors, such as, for example, a thermal sensor, pH probe, conductometer, or any other type of appropriate sensor according to the needs of the process.

[0196]

[0187] The culture medium may be prepared in a culture medium tank. The culture medium tank may comprise at least one of: a mixing tank, hydrolysis tank, storage tank, loading tank or waste medium tank, or any other appropriate device. The media components may be mixed in a mixing tank, which may be made from stainless steel, glass, or any other suitable material. The mixing tank may be equipped with a stirring unit comprising, for example, a shaft with one or more impellers. The mixing tank may be equipped with a heating system. The temperature of the mixing tank may be in the range of 10 °C to 40 °C, or in the range of 15 °C to 38 °C, or in the range of 18 °C to 35 °C. The mixing tank may be connected to one or more storage tanks. The mixing tank may be connected to one or more cultivation devices, formed for example by a bioreactor. The culture medium components may be mixed directly in the cultivation device. The volume of the mixing tank may be in the range of 500 mL to 100 m3, or in the range of 1 L to 10 m3, or in the range of 2 L to 5 m3, or in the range of 500 L to 3 m3. The storage tanks may be made from stainless steel, glass or any other suitable material. The volume of the storage tank may be in the range of 500 mL to 100 m3, or in the range of 1 L to 5 m3, or in the range of2 L to 3 m3, or in the range of 500 L to 1 m3. The media components may be dosed into the mixing tank through a sterilization filter, or may be sterilized prior to the placement into the mixing tank or may be sterilized in the mixing tank. The mixing tank may be equipped with different types of sensors, such as, for example, thermal sensor, pH probe, conductometer, or any other type of appropriate sensor according to the needs of the process.

[0197]

[0188] The culture media according to present invention may comprise protein hydrolysate as source of amino acids. The process of medium preparation may have the characteristics of a batch process, a continuous process or a combination thereof.

[0198]

[0189] A more detailed description of the cultivation system, the culture medium, the protein hydrolysate, the cell cultivation methods, the products from the non-human metazoan cells, including specific methodologies, formulations, and system parameters, is available in patent application PCT / IB2024 / 059990, which is hereby incorporated by reference and in its entirety. The present application provides a summary of key aspects relevant to the current invention, while additional details regarding the inventions can be found in the referenced document.

[0199]

[0190] The culture media according to present invention may comprise protein hydrolysate as source of amino acids. The process of medium preparation may further have the characteristics of a batch process, a continuous process or a combination thereof as described below.

[0200]

[0191] The cell cultivation processes according to the invention may comprise the step of cell cultivation in a cultivation device, for example, in a cultivation device formed by a bioreactor. The processes may further comprise at least one step of obtaining the metazoan cells; adaptation or modification of cells; inoculation of cells to the cultivation device; harvesting the cultured cells; processing harvested cells into the final product; or any other appropriate step, and / or combination thereof.

[0201]

[0192] The cell cultivation processes according to the invention may comprise at least one step of: obtaining and processing of the metazoan cells;

[0202] adaptation or modification of cells;

[0203] inoculation of cells to the cultivation device;

[0204] cultivation of cells in the cultivation device;

[0205] harvesting the cultured cells;

[0206] processing harvested cells into the food product;

[0207] or a combination thereof.

[0208]

[0193] The step of obtaining and processing the metazoan cells may optionally comprise cell isolation, separation, purification or any other similar processes, preparing primary cell bank, preparing a production cell bank, and / or any other appropriate processes. The cells used for cultivation may be cells derived from CHO cells, for example commercially available CHO cell lines, such as CHO-K1, CHO-S, CHO-DG44 or CHO-DXB11, or any other appropriate CHO cell lines.

[0194] The cells may be selected on adherent surfaces (passage 1) and expanded. The cells may be then collected (tissue based) and sorted. The sorted cell types may be expanded (passage 2). The cell stocks may be frozen, for example, at -75 °C to -196 °C, in order to obtain a primary cell bank.

[0209]

[0195] The thawed cells from the primary cell bank may be used in the production cell bank. The suitable cells for production cell bank may be thawed and expanded.

[0210]

[0196] Expansion or multiplication is caused by cell division under controlled circumstances. Cells may be maintained in incubators where the temperature, humidity and carbon dioxide levels are regulated to mimic the physiological environment. Cells may be passaged regularly to prevent overconfluence and to maintain the health of the culture. Passaging may involve detaching the cells from the surface of the vessel used, counting them, and seeding a new culture with a defined cell density.

[0211]

[0197] The processes according to the invention may optionally comprise other steps, such as the step of mixing different cell lines before or after the harvesting.

[0212]

[0198] The processes according to the invention may optionally comprise the step of preparing a food product for human or animal consumption. The food product may be, for example, in the form of pet food or a cultured meat product for human consumption, with the desired shape and sensory properties.

[0213]

[0199] The processes according to the invention may optionally comprise the step of preparing a food product for human and / or animal consumption. The food product may be, for example, in the form of pet food or a cultured meat product for human consumption, with the desired shape and sensory properties.

[0214]

[0200] In one aspect of the invention, the cell cultivation processes may comprise the steps of:

[0215] obtaining and processing the metazoan cells;

[0216] preparing the primary cell bank;

[0217] adaptation or modification of cells;

[0218] preparing the production cell bank;

[0219] inoculation of cells to the seeding tank or to the cultivation device;

[0220] cultivation of cells in the cultivation device;

[0221] harvesting the cultured cells; and / or

[0222] preparing the food product.

[0223]

[0201] In one aspect of the invention, the cells may be used for preparing a food product for human or animal consumption.

[0224]

[0202] In one aspect of the invention, the cells may be used for preparing a food product for human and / or animal consumption.

[0225]

[0203] The final food product may comprise one or more cultivated cell types or one or more cultivated cell types with other non-cellular compounds. Non-cellular compounds may be edible and may bring additional sensory and structural properties as well as additional nutritional values.

[0204] The food product according to the invention comprises non-human metazoan cells. The cells may be CHO-derived cell lines according to the description of this document. The cells may be in the form of a cell biomass - the primary component. The food product may further comprise a secondary component. The secondary component of the food product may comprise a source of saccharides and / or source of fats. The food product may further comprise a tertiary component. A tertiary component may comprise at least one auxiliary compound from the group of vitamins, minerals, binders, palatants, antioxidants, colorants and / or preservatives. The primary, secondary or tertiary component may comprise protein.

[0226]

[0205] In one aspect of the invention, the protein source may comprise plant-originated protein to enhance nutrition and rheologic properties of the food product. The plant -originated protein may comprise pea protein, peanut protein, soy protein, rice protein, potato protein, chickpea protein and / or any other plant-originated protein.

[0227]

[0206] In one aspect of the invention, the food product may comprise a palatant to enhance aroma and palatability of the food product.

[0228]

[0207] The step of preparing the food product may optionally comprise mixing of the cultured cells with other non-cellular additional compounds (for example, compounds for making scaffold structure). The food product may comprise one or more cell types, one or more scaffold type material, and / or other additional materials and substances, such as sources of fat, proteins, saccharides, derivatives of crop plants, food grade ingredients, or any other appropriate additional compounds according to the description below. Cells may be co-cultivated with each other and use scaffold type material and / or any other additional materials and substances. The cultivation time may be for a time period, for example, in the range of 1 hour to 7 days, in the range of 2 hours to 3 days, or in the range of 10 hours to 48 hours. Cells may or may not continue to grow, multiply, or differentiate when in the form of the food product. The processes of preparing the food product may comprise homogenization, chopping of the tissue from cultivated cells, formation of cell comprising aggregates, or filtering of the cells through a net with a size limit, a formation of blocks, or any other appropriate process according to the description below. Formation of blocks of the food product may comprise 3D print formation of requested shape including layering of various mixtures of cells with additional components. The food product may be defined as a mixture of cells and additives with a desired structure, cohesion, moisture, and nutritional parameters able to be formed into the final shape (block) which may be then passed to product packing.

[0229]

[0208] In one aspect of the invention, the food product may be cultivated meat.

[0230]

[0209] The food product or a cell biomass comprising cells according to the invention may be subjected to in vitro digestibility test. The test may mimic the animal's digestive process in a laboratory setting and simulate the gastrointestinal environment. The digestibility percentage obtained may help to assess the feed’s quality. The higher digestibility indicates a greater percentage of nutrients is available to the animal, signifying a more efficient or higher-quality feed.

[0231]

[0210] The test may be performed for example based on the disclosure in the articles:

[0232] 1. Boisen and Fernandez (1997): Animal Feed Science Technology, 68 (1997) 277-286, 2. Wild et al. (2018): J Anim Physiol Anim Nutr., 2018; 102:1306-1319.

[0233]

[0211] The food products according to the invention may have high digestibility results, for example a protein digestibility from 80% upwards that suggests that the protein is of a high quality and is readily available for the consumer's metabolism.

[0212] The protein content on a dry matter basis may be determined.

[0234]

[0213] The results of the cell biomass and the food products comprising the cells according to the present invention indicate high protein digestibility and a favorable sensory profile.

[0235]

[0214] In one aspect for the invention, adapted CHO cell lines may be used as an expression system for the production of at least one recombinant protein.

[0236]

[0215] In one aspect of the invention the term “adapted CHO cell line” may refer to the CHO cell line that was adapted to at least one following conditions: low or zero concentration of signaling proteins, e.g. growth factors, in culture medium, low concentration of glutamine, proline, or thymidine in culture medium, mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids, altered, e.g. lowered oxygen concentration, or any other appropriate conditions, or a combination thereof.

[0237]

[0216] In another aspect of the invention, adapted CHO cell lines may be used as an expression system for the production of at least one recombinant protein, wherein the adapted CHO cell lines may be capable of growing in a culture medium comprising protein hydrolysate.

[0238]

[0217] In another aspect of the invention, adapted CHO cell lines may be used as an expression system for the production of at least one recombinant protein, wherein the adapted CHO cell lines may be capable of growing in a culture medium comprising amino acids from the protein hydrolysate, wherein a total input of amino acids derived from the protein hydrolysate may be at least 75 % by weight.

[0239]

[0218] In another aspect of the invention, adapted CHO cell lines may be used as an expression system for the production of at least one recombinant protein, wherein the adapted CHO cell lines may be capable of growing in a culture medium comprising amino acids from the protein hydrolysate, wherein hydrolysate-derived amino acids may provide a substantial fraction of the nutritional input required for growth and / or production, thereby substantially reducing the use of expensive defined supplements, including supplemented amino acids, and reducing manufacturing costs associated with the culture medium.

[0240]

[0219] The adapted CHO cell line may be utilized as an expression system for the production of at least one recombinant protein encoded by at least one gene, wherein said recombinant protein may be suitable for applications in at least one industry selected from the group of pharmaceutical, food, medical, cosmetic or any other relevant industry.

[0241]

[0220] The at least one gene encoding recombinant protein may be present on a mobile genetic element (MGE) and / or in an insertion cassette located in a safe harbor site. Non-limiting examples of recombinant proteins may include monoclonal antibodies, antibody fragments, multispecific antibodies, nanobodies, cytokines, chemokines, growth factors, hormones, coagulation factors, clotting inhibitors, serum proteins, complement proteins, receptors or receptor ectodomains, enzymes, membrane proteins or soluble fragments thereof, and engineered binding proteins.

[0242]

[0221] In another aspect of the invention, the at least one gene encoding the recombinant protein may be stably integrated into the genome of the adapted CHO cell line, and the adapted CHO cell line may maintain recombinant protein expression over at least 30 population doublings.

[0222] In another aspect of the invention, at least one gene encoding the recombinant protein of interest may be operably linked to one or more genes encoding selectable markers. The selectable marker may be selected from the group of a detectable reporter (e.g., a fluorescent protein), a selection marker conferring resistance to a selection agent (e.g., an antibiotic or other chemical selection agent), a metabolic selection marker enabling synthesis or utilization of an essential nutrient, or another selectable marker suitable for selectable phenotype. The selectable marker may facilitate the isolation of clones that integrated the gene encoding recombinant protein of interest, or maintain the expression of the gene encoding recombinant protein of interest over prolonged culture periods.

[0243]

[0223] In another aspect of the invention, the at least one recombinant protein may be secreted into the culture medium, wherein the recombinant protein may be recovered from a clarified harvest comprising the supernatant obtained after removing cells and / or cell debris, for example by centrifugation and / or filtration. In another aspect of the invention, the at least one recombinant protein may be retained within the adapted CHO cell line biomass. In further aspects, the recombinant protein may be recovered from harvested cell biomass, for example from a cell pellet, following cell lysis and one or more clarification steps to remove insoluble material.

[0244]

[0224] In one aspect of the invention, the recombinant protein may be recovered from the culture by a clarification step comprising centrifugation and / or depth filtration, followed by one or more chromatography steps performed on a column to purify the recombinant protein. In further aspects, the chromatography may comprise affinity chromatography, ion-exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, and / or size -exclusion chromatography.

[0245]

[0225] In one aspect of the invention, the clarified harvest obtained from cultures grown in a medium comprising a protein hydrolysate may be compatible with column-based purification, optionally after one or more conditioning steps from the group of dilution, diafiltration, buffer exchange, pH adjustment, conductivity adjustment, precipitation, flocculation, and / or addition of filter aids. In some embodiments, the purified product meets one or more predefined quality attributes, including reduced host cell protein (HCP) and / or host cell DNA content, acceptable aggregate levels, and / or compliance with a specified purity threshold.

[0246]

[0226] Adaptation of cells to new culture conditions may be facilitated by changes in the genome of the cells. These changes may comprise, but are not limited to at least one of:

[0247] • missense mutations resulting in the change of amino acid sequence of an encoded protein, • nonsense mutations resulting in creation of a premature stop codon in the protein coding sequence, or

[0248] • insertion or deletion of nucleotides with disruption (frameshift) or without disruption of reading frame (in-frame).

[0249]

[0227] In another aspect of the invention, cells may exhibit mutations where a low impact on cell phenotype is predicted, which may include, but are not limited to at least one of:

[0250] • synonymous mutations in coding regions, which do not change the amino acid sequence, or • mutations in intergenic regions.

[0251]

[0228] The allele frequency of any particular mutation may be 1, where all the mutation in question is present in all copies of a particular gene in a cell population. The allele frequency of any particular mutation may be less than 1, in cases where the cells are heterozygous for the particular mutation, and / or the cell population is heterogenous.

[0229] The allele frequency of any particular mutation may be in the range of 0.1 to 1, or in the range of 0.2 to 0.9, or in the range of 0.3 to 0.8, or in the range of 0.4 to 0.7, or in the range of 0.15 to 0.99, or in the range of 0.25 to 0.90, or in the range of 0.35 to 0.85, or in the range of 0.5 to 0.80.

[0252]

[0230] All types of mutations may be used to characterize and identify the cell lines according to the invention. Mutations may also influence cell phenotypic characteristics, including but not limited to the phenotypic characteristics related to the adaptations described in the invention.

[0253]

[0231] To analyze mutations present in the genome of cells according to the invention, long read whole genome sequencing using the Nanopore technology may be performed.

[0254]

[0232] In one aspect of the invention, mutations described below may be detected by one or more of the following methods:

[0255] • sequencing methods, such as Sanger sequencing, Illumina sequencing or Nanopore sequencing • amplification-based methods, such as allele-specific PCR

[0256] • hybridization-based methods, such as Southern blotting.

[0257]

[0233] Mutations according to the invention are described using the CriGri-PICRH-1.0 (GCA_003668045.2) genome as a reference.

[0258] Small mutations

[0259]

[0234] For the purpose of the present application, the term “small mutations” describes transitions, trans versions, and short insertions and deletions where at most 50 nucleotides are inserted / deleted.

[0260]

[0235] In one aspect of the invention, cells according to the invention may comprise at least one of 2282 small mutations with an allele frequency in the range of 0.5 to 1, or in the range of 0.7 to 1, or in the range of 0.9 to 1, or in the range of 0.55 to 0.99, or in the range of 0.6 to 0.98, or in the range of 0.8 to 0.96, or in the range of 0.85 to 0.95.

[0261]

[0236] The list of possible small mutations is provided in Table 1. Tables 1 to 4 are attached at the end of the specification. The small mutations listed in Table 1 may have frequency equal to or higher than 0.9. In population genetics, a mutation is usually referred to as “fixed” when the corresponding allele has a frequency of 1. For the purpose of the present invention, we consider mutations fixed in the population of CHO-derived cells according to the invention if their frequency is equal to or higher than 0.9.

[0262]

[0237] The provided list of small mutations is filtered, so that only alleles fulfilling the following criteria are included:

[0263] • the number of reads aligned to the genomic locus (depth of sequencing) is at least 10

[0264] • the identified mutant allele was not detected in our long-read sequencing of CHO-K1 cells.

[0265]

[0238] In one aspect of the invention, the term “small mutations” may refer to nucleotide-level sequence variations present in an adapted cell allele relative to a corresponding reference genome allele, wherein the variations comprise single -nucleotide substitutions and / or short insertions and / or deletions, and wherein each insertion or deletion affects no more than 50 contiguous nucleotides on a single strand of DNA.

[0239] In one aspect of the invention, the term “small mutations” may refer to nucleotide -level sequence variations present in an adapted cell allele relative to a corresponding reference genome allele, including single -nucleotide substitutions and short insertions and / or deletions of no more than 50 contiguous nucleotides on a single strand of DNA, as exemplified by the small mutations listed in Table 1 or Table 2.

[0266]

[0240] In one aspect of the invention, the genome of the derived CHO cell is assembled and compared with a reference genome defined with respect to a CHO-K1 genome assembly having accession number GCA_003668045.2.

[0267]

[0241] In one aspect of the invention, the provided alleles may not be present in the genome GCA_003668045.2 nor in CHO-K1 cells, and may be considered unique to CHO-derived cells according to the invention.

[0268]

[0242] The list of selected 1219 small mutations in gene regions is provided in Table 2. The small mutations listed in Table 2 have frequency equal to or higher than 0.9.

[0269]

[0243] The list of selected 1219 small mutations within coding and / or non-coding gene regions is provided in Table 2. The small mutations listed in Table 2 have frequency equal to or higher than 0.9.

[0270]

[0244] In one aspect of the invention, the cells are CHO-derived cells having small mutations listed in Table 1.

[0271]

[0245] In one aspect of the invention, the cells may be CHO-derived cells having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 98%, or at least 99% of small mutations listed in Table 1.

[0272]

[0246] The CHO-derived cells may comprise at least one, or at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 30, or at least 50, or at least 80, or at least 100, or at least 150, or at least 200, or at least 300, or at least 500, or at least 1000, or at least 1500, or at least 2000 mutations listed in Table 1.

[0273]

[0247] In one aspect of the invention, the CHO-derived cells may comprise at least one mutation from mutations listed in Table 1.

[0274]

[0248] In one aspect of the invention, the cells are CHO-derived cells having small mutations listed in Table 2.

[0275]

[0249] In one aspect of the invention, the cells are CHO-derived cells having small mutations within coding and / or non-coding gene regions listed in Table 2.

[0276]

[0250] In one aspect of the invention, the cells may be CHO-derived cells having at least 80 %, or at least 85 %, or at least 90 %, or at least 92 %, or at least 95 %, or at least 97 %, or at least 98 %, or at least 99 % of small mutations listed in Table 2.

[0277]

[0251] The CHO-derived cells may comprise at least one, or at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 30, or at least 50, or at least 80, or at least 100, or at least 150, or at least 200, or at least 300, or at least 500, or at least 1,000 mutations listed in Table 2.

[0278]

[0252] The CHO-derived cells may comprise at least one, or at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 30, or at least 50, or at least 80, or at least 100, or at least 150, or atleast 200, or at least 300, or at least 500, or at least 1,000 small mutations within coding and / or noncoding gene regions listed in Table 2.

[0279]

[0253] In one aspect of the inventions, the CHO-derived cells may comprise at least one mutation from mutations listed in Table 2.

[0280]

[0254] For the purpose of the present patent application, the term “large mutations” describes insertions and deletions where more than 50 nucleotides are inserted / deleted.

[0281]

[0255] In one aspect of the invention, cells according to the invention may contain one more of 150 large mutations with an allele frequency in the range of 0.5 to 1, or in the range of 0.7 to 1, or in the range of 0.9 to 1, or in the range of 0.55 to 0.99, or in the range of 0.6 to 0.98, or in the range of 0.8 to 0.96, or in the range of 0.85 to 0.95.

[0282]

[0256] The list of possible large mutations is provided in Table 3. The large mutations listed in Table 3 have frequency equal to or higher than 0.9.

[0283]

[0257] In one aspect of the invention, the cells may be CHO-derived cells having large mutations listed in Table 3.

[0284]

[0258] In one aspect of the invention, the term “large mutations” refers to nucleotide -level sequence variations present in an adapted cell allele relative to a corresponding reference genome allele, wherein the variations comprise insertions and / or deletions having a length of at least 50 nucleotides on a single strand of DNA, as exemplified by the large mutations listed in Table 3 or Table 4.

[0285]

[0259] For the purposes of the present invention, references to substitutions and / or deletions and / or insertions defined in terms of a number of nucleotides on a single strand of DNA are considered equivalent to references defined in terms of the same number of base pairs in double-stranded DNA. Accordingly, a substitution, an insertion or deletion of, for example, 50 nucleotides on a single DNA strand may correspond to a substitution, an insertion or deletion spanning 50 base pairs in doublestranded DNA.

[0286]

[0260] In one aspect of the invention, the cells may be CHO-derived cells having at least 80 %, or at least 85 %, or at least 90 %, or at least 92 %, or at least 95 %, or at least 97 %, or at least 98 %, or at least 99 % of large mutations listed in Table 3.

[0287]

[0261] The CHO-derived cells according to the invention may comprise at least one large mutation present in at least one of the following genes: Riok2, Cmya5, Gzmk, Ranbp31, Retreg1, Nrp2, Scg2, Dgkd, C2cd3, Pomt2, Pou2f2, Def6, Gal3st1, Bmp2k, Pde4d, Dnajc1, Znf609, Socs5, Samd15, Smg6, Sox5, Cacna1c, Pparg, Dysf, Ctnna2, Mkln1, Ppard, Evi5, Zmiz1, Micu1, Ank2 or Rbms3.

[0288]

[0262] The large mutations may comprise at least one of: insertion or deletion causing a frameshift, gain of a premature stop codon, or deletion of one or more exons.

[0289]

[0263] The CHO-derived cells according to the invention may comprise at least one mutation in at least one of the genes: Riok2, Cmya5, Gzmk, Ranbp31, Retreg1, Nrp2, Scg2, Dgkd, C2cd3, Pomt2, Pou2f2, Def6, Gal3st1, Bmp2k, Pde4d, Dnajc1, Znf609, Socs5, Samd15, Smg6, Sox5, Cacna1c, Pparg, Dysf, Ctnna2, Mkln1, Ppard, Evi5, Zmiz1, Micu1, Ank2 or Rbms3.

[0290]

[0264] The mutation may comprise at least one of: a deletion, insertion, or substitution.

[0265] The mutations and the impacted genes are described in Table 4. The large mutations listed in Table 4 have a frequency equal to or higher than 0.9.

[0291]

[0266] In one aspect of the invention, a non-genetically modified CHO-derived cells comprising a deletion and / or insertion of 50 to 500,000 base pairs, wherein said deletion and / or insertion overlaps with the coding sequence of at least one gene from Riok2, Cmya5, Gzmk, Ranbp31, Retreg1, Nrp2, Scg2, Dgkd, C2cd3, Pomt2, Pou2f2, Def6, Gal3st1, Bmp2k, Pde4d, Dnajc1, Znf609, Socs5, Samd15, Smg6, Sox5, Cacna1c, Pparg, Dysf, Ctnna2, Mkln1, Ppard, Evi5, Zmiz1, Micu1, Ank2, or Rbms3, wherein the deletion and / or insertion is detectable by sequencing, PCR, Southern blotting, or any other suitable technique.

[0292]

[0267] In one aspect of the invention, a non-genetically modified CHO-derived cells comprising a deletion and / or insertion of 50 to 500,000 nucleotides on a single strand of DNA, wherein said deletion and / or insertion overlaps with the coding sequence of at least one gene from Riok2, Cmya5, Gzmk, Ranbp31, Retreg1, Nrp2, Scg2, Dgkd, C2cd3, Pomt2, Pou2f2, Def6, Gal3st1, Bmp2k, Pde4d, Dnajc1, Znf609, Socs5, Samd15, Smg6, Sox5, Cacna1c, Pparg, Dysf, Ctnna2, Mkln1, Ppard, Evi5, Zmiz1, Micu1, Ank2, or Rbms3, wherein the deletion and / or insertion is detectable by sequencing, PCR, Southern blotting, or any other suitable technique.

[0293]

[0268] In one aspect of the invention, the cells are CHO-derived cells having large mutations listed in Table 4.

[0294]

[0269] In one aspect of the invention, the cells may be CHO-derived cells having at least 80 %, or at least 85 %, or at least 90 %, or at least 92 %, or at least 95 %, or at least 97 %, or at least 98 %, or at least 99 % of large mutations listed in Table 4.

[0295]

[0270] The allele frequency of the mutations may be in the range of 0.5 to 1, or in the range of 0.7 to 1, or in the range of 0.9 to 1, or in the range of 0.55 to 0.99, or in the range of 0.6 to 0.98, or in the range of 0.8 to 0.96, or in the range of 0.85 to 0.95. The mutations described above may have a very high impact on the structure of the encoded protein, where it may completely lose its original function as the results of the mutation.

[0296]

[0271] All of the mentioned genes may be dispensable for cell cultivation for the purpose of human or animal nutrition.

[0297]

[0272] Micu1 may participate in Ca2+transport into the mitochondria. As mitochondrial Ca2+overload has been shown to lead to mitochondrial membrane permeabilization and cell death, this mutation may lead to a higher cell resistance against death caused by mitochondrial Ca2+overload.

[0298]

[0273] Adaptation of cells to new culture conditions may be facilitated by changes in the expression profile of the cells, which may happen through mechanisms at the genetic level, or other mechanisms, for example epigenetic regulation.

[0299]

[0274] For the purpose of the following description, internal transcriptomic characterization of the cell line CHO-K1 (ATCC cell line CCL-61) was used as the baseline to evaluate up / down regulation of expression. To analyze the transcriptome of CHO-K1 and CHO-derived cells according to the invention, long read multiplex RNA sequencing using the Nanopore technology was performed. RNA for the analysis was isolated from exponentially growing cells, either growing adherently in DMEM with 10 % (CHO-K1 cells) or growing in suspension in proprietary protein-free medium M PF 6.0 (CHO-derived cells according to the invention).

[0275] In one aspect of the invention, the cells may be characterized according to the difference in expression of transcripts corresponding to one or multiple genes in comparison to the original cell population. For the purpose of all following descriptions and examples, all data about gene expression upregulation or downregulation are reported in the linear scale.

[0300]

[0276] In another aspect of the invention, genes identified by the differential expression analysis may be connected into functional groups that may correspond to one or more signaling pathways, metabolic pathways or cell functions. To identify functional groups of genes, the database, for example Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), WikiPathways, Reactome, or any other suitable database may be used.

[0301]

[0277] In one aspect of the invention, the cells may comprise up or down regulation of genes associated with one or more GO terms. The upregulation of genes associated with one or more of the following GO terms:

[0302] GO:1905286, G0:0004601, G0:0170055, G0:0055093, G0:0005324, G0:0015245, G0:0017134, G0:0016684, G0:0043394, G0:0140354, GO:1902001, G0:0008299, G0:0010884, G0:0032994, G0:0016209, G0:0019730, GO:0016411, G0:0046112, G0:0015909, G0:0044539, G0:0022625, G0:0050820, G0:0042953, G0:0030194, GO: 1900048, G0:0044872, G0:0010878, G0:0016790, G0:0097242, G0:0098869, GO:0006721, GO:1990748, G0:0022627, G0:0010883, G0:0036296, G0:0001523, G0:0015908, G0:0050922, G0:0097237, G0:0098754, G0:0008028, G0:0016101, GO: 1905954, G0:0015718, G0:0030867, G0:0006695, GO: 1902653, G0:0008374, G0:0006720, G0:0097006, GO:0006641, G0:0002526, G0:0061844, GO: 1905953, G0:0071622, G0:0016667, GO: 1905952, G0:0032370, G0:0009167, G0:0009126, G0:0004866, G0:0022626, G0:0016126, GO:1901568, G0:0019915, G0:0030414, G0:0016765, G0:0001676, G0:0006637, G0:0035383, G0:0033559, G0:0006959, G0:0032760, GO:0002181, G0:0001540, GO: 1903557, G0:0008203, GO:0071621, G0:0032368, GO: 1902652, GO:0009161, G0:0006638, G0:0006639, G0:0016125, G0:0009123, G0:0046165, G0:0003735, G0:0061135, G0:0009636, G0:0006694, G0:0032640, G0:0032680, G0:0097530, G0:0015918, G0:0043202, G0:0071706, GO: 1903555, G0:0061134, G0:0015934, G0:0033218, G0:0015935, GO:0044391, G0:0005777, G0:0042579, G0:0072329, G0:0006066, G0:0006633, GO:0006631, G0:0008202, G0:2001235, G0:0046390, G0:0005840, GO:1905039, GO:1903825, G0:0015849, G0:0046942, GO:1901617, G0:0034284, G0:0042277, GO:1901615, G0:0009260, G0:0010876, G0:0072523, G0:0042274, G0:0006839, G0:0006869, G0:0009743, G0:0005788, G0:0032787, G0:0009165, G0:0016853, G0:0062023, G0:0015711, G0:0046434, GO:0072522, G0:0044283, G0:0006790, GO:0016491, G0:0044282, G0:0030312, G0:0031012, G0:0006457, G0:0032496, GO:1901293, G0:0002237, GO:0072521, G0:0031968, G0:0019867, G0:0034774, G0:0031983, G0:0060205, G0:0019752, G0:0055086, G0:0019693, G0:0006753, G0:0043436, G0:0006082, G0:0044255, G0:0006954, G0:0009617, G0:0006091, G0:0006163, G0:0009117, G0:0008610, G0:0006412, G0:0090407, G0:0005740, G0:0007005, G0:0043603, G0:0051604, G0:0005198, G0:0005743, G0:0019866, G0:0031966, G0:0030141, G0:0140546, or GO:1990904

[0303] and / or downregulation of genes associated with one or more of the following GO terms:

[0304] G0:0000902, G0:0098590, G0:0098794, G0:0034330, G0:0098609, G0:0007264, G0:0044089, G0:0120031, G0:0030031, G0:0061061, G0:0070161, G0:0031344, G0:0097435, G0:0051493, G0:0120035, G0:0090066, G0:0050808, G0:0005769, GO:1902903, G0:0031253, G0:0015629, G0:0005911, G0:0007517, G0:0003779, G0:0097060, G0:0030029, G0:0030036, G0:0150034, G0:0030027, G0:0031252, G0:0051495, G0:0032970, G0:0031032, G0:0099173, G0:0032535,G0:0032956, G0:0007015, G0:0031256, GO:0110053, G0:0051017, G0:0005938, GO:0061025, G0:0098858, G0:0061572, G0:0008154, G0:0051261, G0:0001726, GO:0032412, G0:0008064, G0:0030832, G0:0030426, G0:0030427, G0:0030041, G0:0032587, GO:0005884, GO:0030833, G0:0140056, G0:0030863, G0:0030864, G0:0001704, G0:0022406, GO:0043197, GO:0044309, G0:0005200, G0:0030175, G0:0043271, G0:0048278, GO:1904063, G0:0005484, G0:0022617, G0:0005902, G0:0048644, G0:0006904, G0:0060076, GO:0031941, G0:0031528, G0:0032410, G0:0032413, or G0:0048333

[0305] may be present in the CHO-derived cells according to the invention.

[0306]

[0278] The GO term description was based on the 2024-11-03 release of the GO database.

[0307]

[0279] In the CHO-derived cells according to the invention, the genes associated with GO terms listed in Table 5 may be upregulated, wherein the names of upregulated genes corresponding to each GO terms are provided.

[0308] List of upregulated genes contributing to GO term GO term upregulation

[0309] Regulation Of cGMP-mediated Signaling (G0:0010752) ['PDE2A', 'THBS1']

[0310] Regulation Of Adiponectin Secretion (G0:0070163) ['HCAR2', 'PPARG']

[0311] cGMP-mediated Signaling (G0:0019934) ['NPPB', 'PDE2A', 'CD36'] Neuron Remodeling (G0:0016322) ['C3', 'FARP2']

[0312] cGMP Metabolic Process (G0:0046068) [’NPPB’, 'PDE2A']

[0313] Lipoprotein Transport (G0:0042953) [’PPARG, 'CD36']

[0314] Cellular Response To Low-Density Lipoprotein Particle

[0315] Stimulus (GO:0071404) ['CDH13', 'PPARG', 'CD36'] Long-Chain Fatty Acid Transport (GO:0015909) ['FABP4', 'PPARG', 'CD36'] Lipoprotein Localization (G0:0044872) [’PPARG, 'CD36']

[0316] Positive Regulation Of Macrophage Chemotaxis

[0317] (GO:0010759) ['RARRES2', 'THBS1'] Regulation Of Smooth Muscle Cell Proliferation ['CDH13', 'PPARG, THBS1’, (G0:0048660) 'EREG']

[0318] Rac Protein Signal Transduction (GO:0016601) ['FARP2', 'CDH13']

[0319] Neuron Maturation (G0:0042551) ['FARP2', ’C3’]

[0320] Positive Regulation Of Macrophage Migration

[0321] (GO: 1905523) ['RARRES2', 'THBS1'] Regulation Of Granulocyte Chemotaxis (GO:0071622) ['RARRES2', 'THBS1']

[0322] Positive Regulation Of Hormone Secretion (GO:0046887) ['HCAR2', 'PPARG']

[0323] Fatty Acid Transport (GO:0015908) ['FABP4', 'PPARG', 'CD36'] Regulation Of Receptor-Mediated Endocytosis

[0324] (GO:0048259) ['C3', 'CLU', 'F3']

[0325] Regulation Of Macrophage Chemotaxis (GO:0010758) ['RARRES2', 'THBS1']

[0326] Cyclic-Nucleotide-Mediated Signaling (GO:0019935) ['NPPB', 'PDE2A', 'CD36']

[0327]

[0328] Regulation Of Lipid Storage (GO:0010883) ['C3', 'PPARG']Positive Regulation Of Receptor-Mediated Endocytosis

[0329] (G0:0048260) ['C3', 'CLU', 'F3'] Regulation Of Mononuclear Cell Migration (G0:0071675) ['RARRES2', ’THBST] Positive Regulation Of Cholesterol Efflux (GO:0010875) ['EEPD1', 'PPARG'] Negative Regulation Of Axonogenesis (G0:0050771) ['RUFY3', 'TRIM46'] Positive Regulation Of Chemotaxis (G0:0050921) ['RARRES2', 'CDH13', ’THBST] Regulation Of Lipid Catabolic Process (GO:0050994) ['HCAR2', 'RARRES2'] Mononuclear Cell Differentiation (GO: 1903131) [’PPARG, 'CLU']

[0330] Positive Regulation Of Smooth Muscle Cell Proliferation

[0331] (G0:0048661) ['CDH13', ’THBST, ’EREG] Osteoclast Differentiation (GO:0030316) ['FARP2', 'SNX10']

[0332] Cell-Cell Adhesion Mediated By Cadherin (GO:0044331) ['CDH13', 'CDH7']

[0333] Positive Regulation Of Nitric Oxide Biosynthetic Process

[0334] (GO:0045429) ['CD36', 'CLU']

[0335] Regulation Of Chemotaxis (G0:0050920) ['RARRES2', ’THBST] Positive Regulation Of Nitric Oxide Metabolic Process

[0336] (GO:1904407) ['CD36', 'CLU']

[0337] Regulation Of Macrophage Derived Foam Cell

[0338] Differentiation (G0:0010743) [’PPARG, 'CD36'] Phagocytosis, Engulfment (G0:0006911) ['CD36', ’THBST]

[0339] Positive Regulation Of Locomotion (GO: 0040017) ['RARRES2', ’THBST] Positive Regulation Of Phosphate Metabolic Process

[0340] (G0:0045937) [’THBST, 'EREG']

[0341] Positive Regulation Of Cholesterol Transport (G0:0032376) [’EEPDT, 'PPARG'] Regulation Of Cholesterol Efflux (GO:0010874) ['EEPD1', 'PPARG'] Negative Regulation Of Osteoblast Differentiation

[0342] (GO:0045668) ['TNFAIP6', 'PPARG']

[0343] ['SLPI', 'RARRES2', 'CXCL3', Antimicrobial Humoral Response (GO:0019730) 'F3']

[0344] Negative Regulation Of Blood Vessel Endothelial Cell

[0345] Migration (G0:0043537) [’PPARG, ’THBST] Regulation Of Cell-Matrix Adhesion (G0:0001952) ['CDH13', 'CD36', ’THBST] Myeloid Leukocyte Differentiation (GO:0002573) ['FARP2', 'SNX10', 'PPARG'] Negative Regulation Of Cellular Response To Growth

[0346] Factor Stimulus (G0:0090288) ['TNFAIP6', 'PPARG', ’THBST] Negative Regulation Of Blood Vessel Morphogenesis

[0347] (G0:2000181) ['NPPB', 'PPARG, ’THBST] Positive Regulation Of Endocytosis (GO:0045807) ['C3', 'F3', 'CLU'] Regulation Of Osteoblast Differentiation (GO:0045667) ['DNAI3', 'TNFAIP6', 'PPARG'] Negative Regulation Of Angiogenesis (G0:0016525) ['NPPB', 'PPARG, ’THBST] Positive Regulation Of Response To External Stimulus ['RARRES2', 'PDE2A', 'THBS1',(GO:0032103) 'EREG']

[0348] Response To Lipid (GO:0033993) ['SLPI', 'PPARG', 'CD36'] Negative Regulation Of Inflammatory Response

[0349]

[0350] (G0:0050728) ['TNFAIP6', 'PTPN6', ’PPARG]['ACTA2', 'PDE2A', 'PPARG', Positive Regulation Of Gene Expression (GO: 0010628) 'CD36', 'THBS1', 'CLU', ’EREG] Negative Regulation Of Cell Population Proliferation ['CDH13', 'PTPN6', 'IGFBP7', (G0:0008285) 'PPARG, 'THBS1', ’EREG]

[0351] ['NPPB', 'PPARG', 'ANGPTL4', Regulation Of Angiogenesis (GO:0045765) 'THBS1']

[0352] Positive Regulation Of Multicellular Organismal Process ['HCAR2', 'NPPB', 'FABP4', (GO:0051240) 'PPARG', 'CD36', 'EREG']

[0353] ['NPPB', ’CDH13’, 'PTPN6', 'IGFBP7', THBS1’, 'CLU', Negative Regulation Of Cellular Process (G0:0048523) 'EREG']

[0354] ['C3', 'RARRES2', 'THBS1', Positive Regulation Of Phosphorylation (GO:0042327) 'EREG']

[0355] Positive Regulation Of Macromolecule Metabolic Process ['ACTA2', 'PDE2A', 'PPARG', (GO:0010604)

[0356]

[0357] 'CD36', 'CLU']

[0358] Table 5 - upregulated genes associated with GO terms

[0359]

[0280] In the CHO-derived cells according to the invention, the genes associated with GO terms listed in Table 6 may be down-regulated, wherein the names of down-regulated genes corresponding to each GO terms are provided.

[0360] list of downregulated genes contributing GO term to GO term downregulation

[0361] Positive Regulation Of Ion Transmembrane Transporter

[0362] Activity (GO:0032414) ['ACTN2', 'WNK2', 'ATP1B1', 'PIRT']

[0363] ['DOCK10', 'PREX1', 'F2RL1', 'CCR7', Positive Regulation Of GTPase Activity (GO:0043547) 'ARHGAP27', 'RAPGEF3', 'NGEF']

[0364] ['DOCK10', 'PREX1', 'F2RL1', Regulation Of GTPase Activity (GO:0043087) 'ARHGAP27', 'RAPGEF3', 'NGEF'] Cell-Cell Junction Organization (G0:0045216) [’CDHT, 'FSCNT, 'F2RLT, 'PKP3'] Cell-Cell Junction Assembly (G0:0007043) [’CDHT, 'FSCNT, ’CD9’, 'PKP3'] Positive Regulation Of Rho Protein Signal Transduction

[0365] (G0:0035025) [’COL3AT, 'F2RLT, 'F2RL2'] Marginal Zone B Cell Differentiation (GO:0002315) ['DOCK10', 'PTK2B']

[0366] Positive Regulation Of Extracellular Matrix Disassembly

[0367] (G0:0090091) ['PDPN', ’FSCNT]

[0368] ['DOCK10', 'PREX1', 'F2RL1', Positive Regulation Of Hydrolase Activity (GO:0051345) 'ARHGAP27', 'RAPGEF3']

[0369] Positive Regulation Of Sodium Ion Transmembrane

[0370] Transport (GO: 1902307) ['WNK2', ’ATPIBT]

[0371] Regulation Of Intracellular Signal Transduction [’PREXT, 'F2RLT, 'GRAP', 'ARHGAP27', (GO: 1902531) 'RAPGEF3', 'NGEF']

[0372] Small GTPase Mediated Signal Transduction

[0373] (G0:0007264)

[0374]

[0375] ['SHTN1', 'RRAD', 'GRAP', 'RAPGEF3']Positive Regulation Of Pseudopodium Assembly

[0376] (G0:0031274) ['F2RL1', 'CCR7']

[0377] Regulation Of Pseudopodium Assembly (G0:0031272) ['F2RL1', 'CCR7']

[0378] Positive Regulation Of Ras Protein Signal Transduction

[0379] (G0:0046579) ['COL3A1', 'F2RL1', 'F2RL2'] Granulocyte Activation (GO:0036230) ['PREX1', 'F2RL1']

[0380] Positive Regulation Of Sodium Ion Transmembrane

[0381] Transporter Activity (G0:2000651) ['WNK2', 'ATP1B1']

[0382] Regulation Of Humoral Immune Response (G0:0002920) ['SUSD4', 'CCR7']

[0383] Mature B Cell Differentiation Involved In Immune

[0384] Response (GO:0002313) ['DOCK10', 'PTK2B']

[0385] Regulation Of Extracellular Matrix Disassembly

[0386] (G0:0010715) ['PDPN', 'FSCN1']

[0387] Protein Localization To Plasma Membrane (G0:0072659) ['CDH1', 'ACTN2', 'PKP3', 'ATP1B1'] Protein Localization To Cell Periphery (GO: 1990778) ['CDH1', 'ACTN2', 'PKP3', 'ATP1B1'] Ras Protein Signal Transduction (G0:0007265) ['SHTN1', 'PDPN', 'GRAP', 'RAPGEF3'] Regulation Of Establishment Of Cell Polarity

[0388] (G0:2000114) ['SHTN1', 'PTK2B']

[0389] Regulation Of Leukocyte Chemotaxis (G0:0002688) ['F2RL1', 'PTK2B']

[0390] Cellular Response To Organic Cyclic Compound

[0391] (G0:0071407) ['CDH1', 'SPPl', 'RAPGEF3', 'AQP1'] Regulation Of Establishment Or Maintenance Of Cell

[0392] Polarity (G0:0032878) ['SHTN1', 'PTK2B']

[0393] Neutrophil Activation (GO:0042119) ['PREX1', 'F2RL1']

[0394] Positive Regulation Of Extracellular Matrix Organization

[0395] (GO: 1903055) ['PDPN', 'FSCN1']

[0396] Myeloid Leukocyte Differentiation (G0:0002573) ['IL1RL1', 'F2RL1', 'CCR7'] Positive Regulation Of Cell-Substrate Adhesion

[0397] (G0:0010811) ['ECM2', 'PTK2B', 'CCR7'] Regulation Of Rho Protein Signal Transduction

[0398] (G0:0035023) ['COL3A1', 'F2RL1', 'F2RL2'] Endothelial Cell Development (G0:0001885) ['F2RL1', 'RAPGEF3']

[0399] Positive Regulation Of JNK Cascade (G0:0046330) ['PTK2B', 'F2RL1', 'CCR7'] Positive Regulation Of Cytoskeleton Organization

[0400] (G0:0051495) ['PTK2B', 'F2RL1', 'CCR7'] Positive Regulation Of Immune Response (G0:0050778) ['F2RL1', 'CCR7', 'CFP']

[0401]

[0402] Dendritic Cell Differentiation (G0:0097028) ['F2RL1', 'CCR7']

[0403] Table 6 - downregulated genes associated with GO terms

[0404]

[0281] The CHO-derived cells according to the invention may comprise at least one up-regulated gene, wherein the gene may be at least one of: Slpi, Cd36, Fabp4, Metazoa_SRP, Trim54, Cdh7, Acta2, Fgd3, Arhgef25, Cxcl3, Enpp6, Atp6vlcl, Marchfl, Rarres2, Faml3c, Ptpn6, Apobr, Ereg, Hcar2, Rab26, Plagl, Tnfaip6, Angptl4, Zmat4, Syt16, Thbsl, C3, Selenbpl, Arhgefl6, Cep97, F3, Rufy3, Pstpipl,Nppb, RNase_MRP, Fam43a, Trim46, Znf597, Cdhl3, U3, Kiaal217, Farp2, Pde2a, Eid3, SnxlO, Igfbp7, Pparg, Eepdl, Clu.

[0405]

[0282] The functions of the above-mentioned genes are summarized in Table 7.

[0406]

[0283] The genes may be upregulated in the range of 3x to 10,000x, or in the range of 4 to 8,000, or in the range of 5x to 7,000x, or in the range of 7x to 5,000x, or in the range of 8x to 3,000x.

[0407]

[0284] In one aspect of the invention, the genes may be up-regulated relative to a control, such as a non-adapted CHO-derived cell line, by about at least 3 -fold. In another aspect of the invention, the genes may be up-regulated in the range of 3-fold to 10,000-fold, or in the range of 4-fold to 8,000-fold, or in the range of 5-fold to 7,000-fold, or in the range of 7-fold to 5,000-fold, or in the range of 8-fold to 3,000-fold.

[0408] Function GenefsJ

[0409] Stress Response 3 Get? Survival Slpi, Trim54, TnfaipG, Angpt!4. Selenbpl, Clu, Trim46, EidfJ

[0410] Cell Proliferation & Growth Famt3c, Ereg, Plagl, Ptpnfi, F3, Pparg

[0411] Lipid Metabolism & Membrane Stability 0036, Fabp4, Enppf; Apobr. Hcar2, Rab26

[0412] Ceil Adhesion & Migration Cdh7, Cdivi 3, Thbsl, Acta2, Fgd3

[0413] Protein Folding, Secretion, & Translation Metazoa_SRP, Atp6v1c1, Zmat4, Rab26

[0414] inttaceiiolar Signaling & Regulation Arhgef25. Marchfl, Rarres2. Pde2a, SnxlO, Zaf597, Cep97, Arhgef16 immune Response & Inflammation Cxcl3. C3, Pstpipl, lgfbp7, Riify3, Nppb

[0415] Ribosome Biogenesis & RNA Precessing RNase_MRP, U3

[0416] Vesicle Trafficking & Transport Syt16, SnxlO

[0417]

[0418] Other Functions Kiaa1217. Znf597, Farp2, Eepdl, F3

[0419] Table 7

[0420]

[0285] The CHO-derived cells may comprise at least one, or at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50 up-regulated genes from the group of Slpi, Cd36, Fabp4, Metazoa_SRP, Trim54, Cdh7, Acta2, Fgd3, Arhgef25, Cxcl3, Enpp6, Atp6vlcl, Marchfl, Rarres2, Faml3c, Ptpn6, Apobr, Ereg, Hcar2, Rab26, Plagl, Tnfaip6, Angptl4, Zmat4, Syt16, Thbsl, C3, Selenbpl, Arhgefl6, Cep97, F3, Rufy3, Pstpipl, Nppb, RNase_MRP, Fam43a, Trim46, Znf597, Cdhl3, U3, Kiaal217, Farp2, Pde2a, Eid3, SnxlO, Igfbp7, Pparg, Eepdl or Clu.

[0421]

[0286] In one aspect of the invention, the CHO-derived cells may comprise up-regulation of at least one gene from the group of Slpi, Cd36, Fabp4, Metazoa_SRP, Trim54, Cdh7, Acta2, Fgd3, Arhgef25, Cxcl3, Enpp6, Atp6vlcl, Marchfl, Rarres2, Faml3c, Ptpn6, Apobr, Ereg, Hcar2, Rab26, Plagl, Tnfaip6, Angptl4, Zmat4, Syt16, Thbsl, C3, Selenbpl, Arhgefl6, Cep97, F3, Rufy3, Pstpipl, Nppb, RNase_MRP, Fam43a, Trim46, Znf597, Cdhl3, U3, Kiaal217, Farp2, Pde2a, Eid3, SnxlO, Igfbp7, Pparg, Eepdl, or Clu, wherein the genes may be associated with a GO terms selected from Table 5, wherein said up-regulation is at least in the range of 3x to 10,000x, or in the range of 4x to 8,000x, or in the range of 5x to 7,000x, or in the range of 7x to 5,000x, or in the range of 8x to 3,000x higher in linear scale and wherein said up-regulation is detectable by RNA sequencing or qPCR.

[0422]

[0287] In one aspect of the invention, the CHO-derived cells may comprise up-regulation of at least one gene from the group of Slpi, Cd36, Fabp4, Metazoa_SRP, Trim54, Cdh7, Acta2, Fgd3, Arhgef25, Cxcl3, Enpp6, Atp6vlcl, Marchfl, Rarres2, Faml3c, Ptpn6, Apobr, Ereg, Hcar2, Rab26, Plagl,Tnfaip6, Angptl4, Zmat4, Syt16, Thbsl, C3, Selenbpl, Arhgefl6, Cep97, F3, Rufy3, Pstpipl, Nppb, RNase_MRP, Fam43a, Trim46, Znf597, Cdhl3, U3, Kiaal217, Farp2, Pde2a, Eid3, SnxlO, Igfbp7, Pparg, Eepdl, or Clu, wherein the genes may be associated with a GO terms selected from Table 5, wherein said up-regulation relative to control, such as non-adapted CHO-derived cell line is at least in the range of 3-fold to 10,000-fold, or in the range of 4-fold to 8,000-fold, or in the range of 5-fold to 7,000-fold, or in the range of 7-fold to 5,000-fold, or in the range of 8-fold to 3,000-fold higher in linear scale and wherein said up-regulation is detectable by RNA sequencing or qPCR.

[0423]

[0288] The up-regulation of Cdh7, Cdhl3, Thbsl, Acta2, and Fgd3 may support suspension growth by modulating cell adhesion, cytoskeletal remodeling, and extracellular matrix interactions. Cdh7 and Cdhl3 influence dynamic cell-cell adhesion, balancing detachment and aggregation. Thbsl regulates integrin signaling and ECM interactions, promoting survival in low-attachment environments.

[0424] Acta2 enhances cytoskeletal flexibility, helping cells withstand shear forces in suspension. Fgd3 remodels the actin cytoskeleton, facilitating anchorage -independent growth. Together, these changes reduce strong substrate adhesion, improve cellular adaptability, and enhance proliferation in suspension culture, optimizing cells for bioprocessing applications.

[0425]

[0289] The up-regulation of genes encoding Cdh7, Cdhl3, Thbsl, Acta2, and Fgd3 may result in increased abundance of these proteins that may support suspension growth by modulating cell adhesion, cytoskeletal remodeling, and extracellular matrix interactions. Cdh7 and Cdhl3 influence dynamic cellcell adhesion, balancing detachment and aggregation. Thbsl regulates integrin signaling and ECM interactions, promoting survival in low-attachment environments. Acta2 enhances cytoskeletal flexibility, helping cells withstand shear forces in suspension. Fgd3 remodels the actin cytoskeleton, facilitating anchorage-independent growth. Together, these changes reduce strong substrate adhesion, improve cellular adaptability, and enhance proliferation in suspension culture, optimizing cells for bioprocessing applications.

[0426]

[0290] Up-regulation of Slpi, a protease inhibitor, may protect cells from residual protease activity when they are cultivated in the culture medium containing an enzymatic protein hydrolysate.

[0427]

[0291] Up-regulation of a gene encoding Slpi, a protease inhibitor, may result in increased abundance of this protein that may protect cells from residual protease activity when they are cultivated in the culture medium containing an enzymatic protein hydrolysate.

[0428]

[0292] The cells may show a higher expression of Enpp6 in the above-mentioned ranges, e.g. approximately 80x higher in comparison with CHO-K1 cells. Ennp6 may hydrolyze esters of choline, thus making choline available to the cell. Because choline is an essential nutrient which is not usually found in high surplus in the culture medium, the ability to recover choline from its esters may be advantageous to the cells.

[0429]

[0293] Enpp6 is a choline-specific phosphodiesterase essential for lipid metabolism and choline homeostasis. In CHO-derived cells according to the present invention, its upregulation may enhance choline availability by hydrolyzing glycerophosphocholine and lysophosphatidylcholine, supporting phosphatidylcholine synthesis and influencing cell membrane stability and function. In CHO-derived cells according to the invention, upregulation may improve their response to environmental stresses like shear stress and nutrient fluctuations in bioreactors by enhancing lipid metabolism and membrane composition, boosting cell resilience and performance. Additionally, Enpp6 modulation may impact intracellular signaling pathways, influencing growth and apoptosis, optimizing cell cultures for industrial applications. Promoting efficient choline metabolism and phospholipid synthesis throughEnpp6 could enhance cell growth, proliferation, and biomass accumulation, improving culture productivity.

[0430]

[0294] EREG may improve stress adaptation in bioreactors by enhancing responses to shear stress, nutrient limitations, and hypoxia. Furthermore, EREG may influence cell adhesion and morphology by inducing an EMT-like response, which may impact suspension culture performance by either downregulating E-cadherin and upregulating N-cadherin or vimentin, which reduces adhesion and promotes a more migratory phenotype. This EMT-like shift may enhance suspension cell growth by preventing excessive cell clumping and supporting single-cell proliferation.

[0431]

[0295] Up-regulation of the Plagl gene in CHO-derived cells according to the invention may lead to enhanced cell growth and proliferation, resulting in faster division rates and improved biomass accumulation. It may also alter metabolic pathways, optimizing energy production and nutrient utilization for high-density cultures. Additionally, Plagl upregulation may influence cellular differentiation, morphology, and stress response mechanisms, helping cells better withstand environmental challenges like shear stress, pH shifts and nutrient fluctuations. Furthermore, it may modulate intracellular signaling pathways, affecting processes such as apoptosis and cell cycle progression, ultimately enhancing cell resilience, stability, and performance in bioprocessing environments.

[0432]

[0296] Up-regulation of the Clu gene in CHO cells may enhance cell survival and resistance to stress by protecting against oxidative stress, apoptosis, and protein misfolding. It may help CHO cells resist apoptosis during prolonged culture or unfavorable conditions, promoting cell longevity and reducing cell death. Additionally, Clu upregulation may influence lipid metabolism, supporting better membrane stability and cellular integrity. It may also modulate inflammatory responses, contributing to a more favorable environment for growth and productivity, while aiding in detoxification and maintaining cellular homeostasis, ultimately improving overall cell viability and culture performance.

[0433]

[0297] Up-regulation of these genes may enhance survival, growth, and adaptability in bioprocessing environments in suspension culture of CHO-derived cells according to the invention.

[0434]

[0298] Up-regulation of above-mentioned genes may result in increased abundance of the proteins they encode that may enhance survival, growth, and adaptability in bioprocessing environments in suspension culture of CHO-derived cells according to the invention.

[0435]

[0299] According to the invention, the CHO-derived cells may comprise at least one down-regulated gene, wherein the gene may be at least one of: Tmem40, Scg5, Cdhl, Armhl, Tnfrsf4, Atp8a2, Cnksrl, Rrad, Actn2, Mmp28, Lama3, Atplbl, Castorl, F2rl2, Bik, Illrll, F2rll, Rapgef3, Id3, Des, Ip6k3, Phex, Cablesl, Neu2, U2, Cfp, Znf385b, Prexl, Ngef, Susd4, Ca3, Sppl, Ecm2, Mfsd4a, Sulf2, Mdhlb, Dkkll, Aqpl, Wnk2, Adcy7, Ppplr3g, Shtnl, Ostn, Speg, Abca9, Add2, Ackr4, Svip, Pdpn, Zfhx3, Col3al, Cd9 or Pirt.

[0436]

[0300] The genes may be down-regulated in the range of 3x to lOOOOx, or in the range of 5x to 7000x, or in the range of 7x to 5000x, or in the range of 8x to 3000x.

[0437]

[0301] In one aspect of the invention, the genes may be down -regulated relative to a control, such as a non-adapted CHO-derived cell line, by about at least 3 -fold. In another aspect of the invention, the genes may be down-regulated in the range of 3-fold to 10,000-fold, or in the range of 4-fold to 8,000-fold, or in the range of 5-fold to 7,000-fold, or in the range of 7-fold to 5,000-fold, or in the range of 8-fold to 3,000-fold.

[0302] The CHO-derived cells may comprise at least one, or at least 2, or at least 5, or at least 10, or at least 15, or at least 20, or at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50 down-regulated genes from: Tmem40, Scg5, Cdhl, Armhl, Tnfrsf4, Atp8a2, Cnksrl, Rrad, Actn2, Mmp28, Lama3, Atplbl, Castorl, F2rl2, Bik, Illrll, F2rll, Rapgef3, Id3, Des, Ip6k3, Phex, Cablesl, Neu2, U2, Cfp, Znf385b, Prexl, Ngef, Susd4, Ca3, Sppl, Ecm2, Mfsd4a, Sulf2, Mdhlb, Dkkll, Aqpl, Wnk2, Adcy7, Ppplr3g, Shtnl, Ostn, Speg, Abca9, Add2, Ackr4, Svip, Pdpn, Zfhx3, Col3al, Cd9 or Pirt.

[0438]

[0303] In one aspect of the invention, the CHO-derived cells may comprise down-regulation of at least one gene from: Tmem40, Scg5, Cdhl, Armhl, Tnfrsf4, Atp8a2, Cnksrl, Rrad, Actn2, Mmp28, Lama3, Atplbl, Castorl, F2rl2, Bik, Illrll, F2rll, Rapgef3, Id3, Des, Ip6k3, Phex, Cablesl, Neu2, U2, Cfp, Znf385b, Prexl, Ngef, Susd4, Ca3, Sppl, Ecm2, Mfsd4a, Sulf2, Mdhlb, Dkkll, Aqpl, Wnk2, Adcy7, Ppplr3g, Shtnl, Ostn, Speg, Abca9, Add2, Ackr4, Svip, Pdpn, Zfhx3, Col3al, Cd9 or Pirt, wherein the genes may be associated with a GO terms selected from Table 6, wherein said upregulation is at least in the range of 3x to 10,000x, or in the range of 4 to 8,000x, or in the range of 5x to 7,000x, or in the range of 7x to 5,000x, or in the range of 8x to 3,000x higher in linear scale and wherein said down-regulation is detectable by RNA sequencing or qPCR.

[0439]

[0304] In one aspect of the invention, the CHO-derived cells may comprise down-regulation of at least one gene from: Tmem40, Scg5, Cdhl, Armhl, Tnfrsf4, Atp8a2, Cnksrl, Rrad, Actn2, Mmp28, Lama3, Atplbl, Castorl, F2rl2, Bik, Illrll, F2rll, Rapgef3, Id3, Des, Ip6k3, Phex, Cablesl, Neu2, U2, Cfp, Znf385b, Prexl, Ngef, Susd4, Ca3, Sppl, Ecm2, Mfsd4a, Sulf2, Mdhlb, Dkkll, Aqpl, Wnk2, Adcy7, Ppplr3g, Shtnl, Ostn, Speg, Abca9, Add2, Ackr4, Svip, Pdpn, Zfhx3, Col3al, Cd9 or Pirt, wherein the genes may be associated with a GO terms selected from Table 6, wherein said down-regulation relative to a control, such as a non-adapted CHO-derived cell line is at least in the range of 3 -fold to 10,000-fold, or in the range of 4-fold to 8,000-fold, or in the range of 5-fold to 7,000-fold, or in the range of 7-fold to 5,000-fold, or in the range of 8-fold to 3,000-fold lower in linear scale and wherein said downregulation is detectable by RNA sequencing or qPCR.

[0440]

[0305] Lama3, Sppl, Ecm2 and Col3al are proteins of the extracellular matrix, and may promote cell adhesion and / or clumping. Cdhl and Cd9 are cell surface proteins that may promote cell adhesion. Actn2, Des, Shtnl and Pdpn may contribute to the formation of cytoskeletal structures which may serve as anchor points for cell adhesion. The decrease in expression of these genes may promote a suspension phenotype.

[0441]

[0306] Bik may promote apoptosis through interactions with antiapoptotic proteins, therefore, the reduction in its expression may confer to the cells a higher resistance to apoptosis.

[0442]

[0307] Cablesl may inhibit the activity of cyclin -dependent kinases and suppress cell growth, therefore, its downregulation may lead to a higher rate of proliferation in the cells.

[0443]

[0308] In one aspect of the invention, the cell according to the invention may exhibit overexpression of genes related to oxidative phosphorylation, which may facilitate shuttling pyruvate into the Krebs cycle and away from lactate formation.

[0444]

[0309] The expression of lactate dehydrogenase A (Ldha) may be decreased, for example by about 30 %, which may reduce the maximal rate of production of lactate from pyruvate.

[0310] The expression of mitochondrial pyruvate carriers 1 and 2 (Mpcl and Mpc2) may be increased about 3 times for Mpcl and about 1.5 times for Mpc2. Mitochondrial pyruvate carriers transport pyruvate into mitochondria, where it may enter the Krebs cycle.

[0445]

[0311] The expression of pyruvate carboxylase (Pc) may be increased by about 20 %, which may facilitate the conversion of pyruvate to oxaloacetate to replenish Krebs cycle intermediates.

[0446]

[0312] The gene Gotl, which catalyzes the conversion between aspartate and glutamate, may be overexpressed, for example by about 35 %. Gls2, which catalyzes the conversion of glutamine to glutamate, may be overexpressed for example by about 40 %. Cbs, which metabolizes converts homocysteine into cystathionine (part of metabolic pathway for conversion of methionine into cysteine) may be overexpressed, for example by about 3x.

[0447]

[0313] The gene encoding Gotl, which catalyzes the conversion between aspartate and glutamate, may be overexpressed, for example by about 35 % relative to a control, such as a non-adapted CHO-derived cell line. Gls2, which catalyzes the conversion of glutamine to glutamate, may be overexpressed for example by about 40 % relative to a control, such as a non-adapted CHO-derived cell line. Cbs, which metabolizes converts homocysteine into cystathionine (part of metabolic pathway for conversion of methionine into cysteine) may be overexpressed, for example by about 3 -fold relative to a control, such as a non-adapted CHO-derived cell line.

[0448]

[0314] Cells may overexpress genes related to cell protection against oxidative stress. This may confer to the cells a resistance against changes in oxygen concentration in a culture bioreactor. Up-regulated genes may include Selenow (about 4x upregulation).

[0449]

[0315] One or more genes participating in the pentose phosphate pathway (PPP) may be upregulated. These may include G6pd, Pgd and Rpia. PPP provides ribose-5-phosphate for nucleotide synthesis and NADPH for catabolic reactions, necessary for cell proliferation.

[0450]

[0316] Prps, which shuttles ribose -5 -phosphate into nucleotide biosynthesis, may be upregulated, for example by 30 % to 40 %.

[0451]

[0317] Many eukaryotic cells grow and proliferate through signal transduction pathways triggered by extracellular molecules binding to membrane receptors, which activate intracellular protein cascades that transmit growth and proliferation signals. One of the key pathways is the PI3K-Akt signaling pathway, which promotes nutrient uptake, particularly glucose and glutamine, to support lipid, protein, and nucleotide synthesis. Activation of PI3K-Akt signaling increases glycolysis, leading to the secretion of a high proportion of glucose -derived carbon as lactate. Other crucial pathways include the mitogen-activated protein kinase (MAPK) signaling pathway, which regulates cell proliferation and differentiation, and the TGF-beta signaling pathway, which plays a role in cell cycle regulation and extracellular matrix remodeling. Additionally, multiple other signaling pathways contribute to cell growth and proliferation, depending on the cellular context and environmental conditions.

[0452]

[0318] In one aspect of the invention, the metazoan cell may be modified to activate at least one signal transduction pathway for enhanced growth and / or proliferation.

[0453]

[0319] In another aspect of the invention, the metazoan cell with at least one signal transduction pathway activated may be further modified to improve other attributes, including immortalization, reduced telomere shortening, preservation of telomeres, maintained ability to differentiate in every or any step of cultivation, suspension growth capabilities, preservation of the epigenetic profile, temporaryor permanent loss of contact inhibition, temporary or permanent maintenance of cell divisions, enhanced nutrient metabolism (e.g. enhanced glucose metabolism, shortening of the cell cycle, switching off methylation in general or at the specific genomic loci), ability to fuse with other cells, various independence on nutritional or signaling compounds, or any other appropriate attributes.

[0454]

[0320] The metazoan cells may be derived from any animal from the group Metazoa, such as cattle (Bos taurus), chicken (Gallus domesticus), domestic pig (Sus domesticus), house cricket (Acheta domesticus), garden snail (Helix pomatia), common carp (Cyprinus carpio), horse (Equus ferus), edible crab (Cancer pagurus), marsh frog (Pelophylax ridibundus), common octopus (Octopus vulgaris), gilthead bream (Sparus aurata), roe deer (Capreolus capreolus), common sea urchin (Echinus esculentus), harbor seal (Phoca vitulina), European stag beetle (Lucanus cervus), African bush elephant (Loxodonta africana), house mouse (Mus musculus), green sea turtle (Chelonia mydas) or Southeast Asian soldier fly (Parastratiosphecomyia stratiosphecomyioides). In another aspect of the invention, the cultivated non-human metazoan cells may be derived from bovine, avian, porcine, equine, piscine, cervine or cricetine cell lines. In another aspect of the invention, metazoan cells may comprise any other metazoan cell line. The present list of animals is not limiting. The present list of cell lines is not limiting, however, human cell lines are excluded for food product applications.

[0455]

[0321] The metazoan cells may have characteristics and / or properties of: hepatocytes, myocytes, myoblasts, osteoblasts, fibroblasts, lipoblasts, odontoblasts, keratinocytes, mesenchymal stem cells, multipotent progenitor cells, embryonic stem cells, myofibroblasts, myosatellite cells and / or any combinations thereof. The present invention is not limiting by the present examples of cell types.

[0456]

[0322] As used herein, when referring to a parameter expressed as, for example a range from 0 to 1,000, it is to be understood that both the lower limit 0 and the upper limit 1,000 are explicitly included within the scope of the disclosure. The stated range encompasses all intermediate values between the specified limits, including any subranges or specific values that may be explicitly or implicitly derived from the broader range. Unless explicitly stated otherwise, all numerical values are to be interpreted as including the endpoints.

[0457]

[0323] According to the present invention, metazoan cells may be genetically modified in various ways in order to introduce specific functional adaptations or modifications that are more efficiently achieved through GM methods rather than non-GM methods. The GM output may be a stable cell line with the desired characteristics, for example: capability of continuous homogenous growth, shortened G1 phase of cell cycle in the proliferation phase, cell cycle around 24 hours in general and less than 24 hours in the proliferation phase, no structural genomic changes during the lifetime of the population, minimal impact of epigenetic changes, consistent expression profile of cells correlating with their cell type, maintaining differentiation potential with the ability to induce differentiation, reduced requirement for media composition such as reduced need for signaling factors and / or nutrition components (e.g. amino acids), maintaining endogenous signalization, or any other desired and appropriate characteristics.

[0458]

[0324] Another goal of genetic modification of non-human metazoan cells used in processes according to the invention is to improve their ability to be used to create a food product, for example cultivated meat. Examples of those improved attributes are immortalization, reduced telomere shortening and preservation of telomeres, maintaining the ability to differentiate in every or any step of cultivation, suspension growth capabilities, preservation of the epigenetic profile, temporary or permanent loss of contact inhibition, temporary or permanent maintenance of cell divisions, enhanced nutrient metabolism (e.g. enhanced glucose metabolism), shortening of the cell cycle, switching off methylation in generalor at specific genomic loci, ability to fuse with other cells, various independence on nutritional or signaling compounds, or any other appropriate attributes.

[0459]

[0325] The process of improving cell attributes may be represented by the gain of a specific function where the effect of the specific function could be an addition or reduction of functions or traits. The process of gain of function may comprise thawing of the primary cells of the desired type from a primary cell bank and performing the desired genetic modification.

[0460]

[0326] The methods used for cell modification may comprise permanent and / or transient GM. Introduction of new genomic and transcriptomic elements include for example: the introduction of new sequences as well as genome editing mediated via Clustered regularly interspaced short palindromic repeats (CRISPR) combined with CRISPR-associated protein 9 (Cas9), Zinc finger nucleases, transcription activator-like effector nucleases (TALEN), Cre-Lox recombination, FLP-FRT recombination, Recombinase-mediated cassette exchange (RMCE) and / or other genome editing tools. The generation of single or few nucleotide indels or substitutions may be sufficient to achieve the desired GM.

[0461]

[0327] To achieve permanent or transient GM, a nucleic acid (NA) sequence may be introduced into the cells and / or their genomes by various means. These means may comprise viral vectors based on adenoviruses, adeno-associated viruses, retro / lentiviruses, or vectors derived and built on the above-mentioned. Other non-viral means may comprise use of NA carriers such as cationic polymers or proteins, liposomes, non-cationic polymers, nanoparticles, etc.

[0462]

[0328] Both permanent and transient GM may be achieved by introduction of NA comprising of one or more specific functional coding or noncoding elements, such as promoter, coding DNA sequence, selection marker, or reporter marker or any other functional element. The insertion of functional elements may alter the endogenous gene expression or drive the expression of the inserted DNA per se. The recombinant NA introduced into the target cells might be of cisgenic or transgenic origin (in this document we use single-letter abbreviations defining the species of the particular DNA element, for example, “bTERT” stands for bovine telomerase reverse transcriptase). The introduced recombinant NA of cisgenic origin might encode for the amino acid sequence identical to its native counterpart or might encode for a specific allelic variant, modified native protein by addition of specific linkers, signaling peptides, or other functional elements. To further increase the expression levels of the recombinant NA, a codon-optimized NA sequence might be used.

[0463]

[0329] Stable GM may be mediated by the introduction of NA into a specific or random locus of the target genome. The targeted locus might be a specific functional element regulating the expression of the gene of interest such as its promoter or DNA sequences transcribed into the untranslated region (UTR). Another specific targeted locus might be a so-called genomic safe harbor, offering a long-term stable expression of the inserted DNA sequence, while not interfering with any endogenous coding or noncoding elements. According to the invention, the genomic safe harbors used in the process may be defined as orthologues of previously described genomic safe harbors based on sequence similarity or genomic positions, namely bROSA26, bovine Adeno-associated virus integration site 1 (bAAVS1), bovine C-C motif chemokine receptor 5 (bCCR5), bovine Hipp11 locus (bH11), bovine Glyceraldehyde-3-phosphate dehydrogenase (bGAPDH), bovine Engorgement factor alpha (bEFalpha), or bovine myosin heavy chain (bMYH9).

[0464]

[0330] In one aspect of the invention, the introns or other non-coding parts of specific genes may be used as genomic safe harbors. These genes may be ubiquitously expressed across the cell types ofdifferent tissues and may have at least one long (>10,000 bp) span of non-coding DNA with no gene or chromatin regulatory function. The insertion itself (of even large approximately 10,000 bp long DNA fragments) into these loci does not directly affect the expression of any endogenous genes.

[0465]

[0331] In one aspect of the invention, one such genomic safe harbor called PGrandom, located within the bovine gene Phosphodiesterase 4D (bPDE4D) on chromosome 20, specifically the interval from Ch20:19513000 to Ch20:19553000 may be used. This also applies to orthologous sequences of PGrandom in other species, while excluding the known coding and regulatory DNA elements. The area of the safe harbor according to the invention in the bPDE4D gene located on chromosome 20 may be in the range of 100,000 bp in both directions from the position Ch20: 19533000, or in the range of 50,000 bp, or in the range of 25,000 bp, or in the range of 20,000 bp, or in the range of 10,000 bp, or in the range of 5,000 bp in both directions from the position Ch20: 19533000. The bovine PGrandom (bPGrandom), similarly to other genomic safe harbors, may serve for knock-ins of any coding or regulatory DNA elements and may also be used for multiple tandem insertions.

[0466]

[0332] In one aspect of the invention, one such genomic safe harbor site called PGrandom, located within the bovine gene Phosphodiesterase 4D (bPDE4D) on chromosome 20, specifically the interval from Ch20: 19,373,000 to Ch20: 19,693,000 may be used. This also applies to orthologous sequences of PGrandom in other species, while excluding the known coding and regulatory DNA elements. The area of the safe harbor site according to the invention in the bPDE4D gene located on chromosome 20 may be in the range of 250,000 bp in both directions from the position Ch20: 19,533,000, or in the range of 200,000 bp, or in the range of 150,000 bp, or in the range of 50,000 bp, or in the range 25,000 bp, or in the range of 20,000 bp, or in the range of 10,000 bp, or in the range of 5,000 bp in both directions from the position Ch20: 19,533,000. The bovine PGrandom (bPGrandom) site, similarly to other genomic safe harbor sites, may serve for knock-ins of any coding or regulatory DNA elements and may also be used for multiple tandem insertions.

[0467]

[0333] At least one insertion cassette may comprise gene encoding protein from the group comprising at least one of: Telomerase reverse transcriptase (TERT), Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA or P110α), its fusion version myristoylation signal -attached PIK3CA (myr-PIK3CA), Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB or P110β), its fusion version myristoylation signal-attached PIK3CB (myr-PIK3CB), Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Delta (PIK3CD or P110δ), its fusion version myristoylation signal-attached PIK3CD (myr-PIK3CD), Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Gamma (PIK3CG), its fusion version myristoylation signal-attached PIK3CG (myr-PIK3CG), Phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1 or p85a), its fusion version myristoylation signal-attached PIK3R1 (myr-PIK3R1), Phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2 or p85β), its fusion version myristoylation signal -attached PIK3R2 (myr-PIK3R2), Phosphoinositide-3 -kinase regulatory subunit 3 (PIK3R3), its fusion version myristoylation signal-attached PIK3R3 (myr-PIK3R3), Phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4), its fusion version myristoylation signal-attached PIK3R4 (myr-PIK3R4), Phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5), its fusion version myristoylation signal-attached PIK3R5 (myr-PIK3R5), Phosphoinositide-3-kinase regulatory subunit 6 (PIK3R6), its fusion version myristoylation signal-attached PIK3R6 (myr-PIK3R6), Fibroblast growth factor 1 (FGF-1), FGF-2, FGF-5, FGF-8, FGF-9, Insulin growth factor 1 (IGF-1), IGF-2, Cyclin-dependent kinase 4 (CDK4), Transferrin (TF), Epidermal growth factor (EFG), Transforming growth factor β 1 (TGFB-1), TGFB-2, TGFB-3, Transferrin receptor (TFRC), TGFBR1, TGFBR2, Protein kinase B (PKB or Akt) or its fusion version myristoylation signal-attached Akt (myr-Akt), Myoblast determination protein (MyoD), Pair-box protein 7 (Pax7), Sterol regulatory element binding protein (SREBP), Platelet derived growth factor 1(PDGF-1), PDGF-2, PDGFR1, PDGFR2, insulin (INS), INSR, IGF1R, IGF2R, FGFR1, FGFR2, FGFR3, FGFR4, Peroxisome proliferator-activated receptor gamma (PPARy), Solute carrier family 40 member 1 (SLC40A1), Sodium leak channel (NALCN), Cluster of differentiation 2 (CD2), Focal adhesion kinase (FAK), Myogenin (MyoG), Myostatin (MSTN), Myogenic factor 5 (Myf5), or any other appropriate gene. For example the insertion cassette may comprise genes encoding TERT and myr-PIK3CA, genes encoding TERT, myr-PIK3CA and FGF-2, genes encoding myr-PIK3CA and FGF-2, genes encoding PIK3CA and FGF-2, genes encoding TERT, PIK3CB and TGF-B1, genes encoding TERT, PIK3CB and IGF1R, genes encoding TERT, myr-PIK3CB and IGF1R, genes encoding TERT, myr-Akt and FGF-5, genes encoding myr-Akt and FGF-5 and genes encoding Akt, FGF-8 and PDGFR2.

[0468]

[0334] In another aspect of the invention, the genetically modified metazoan cells may comprise at least one insertion cassette in the genome, wherein the insertion cassette may comprise at least one gene flanked by recombination sites.

[0469]

[0335] In another aspect, the invention may relate to an insertion cassette comprising at least two genes encoding proteins from the group of proteins previously described, wherein said genes may be separated by sequences encoding self-cleaving peptides from the group of T2A and / or P2A or any other appropriate self-cleaving peptide. The presence of said self-cleaving peptide may facilitate a hierarchical expression pattern in which the gene positioned first in the insertion cassette exhibits a higher expression level relative to each subsequent gene, with progressively decreasing expression levels for each gene in the order in which they are positioned within the insertion cassette.

[0470]

[0336] The controlled decrease in expression levels of downstream genes may be utilized to modulate protein production ratios, ensuring preferential expression of a primary protein while maintaining lower levels of secondary protein or subsequent proteins. The use of at least one of T2A and / or P2A sequences in this context may enable co-expression of at least two genes within a single transcriptional unit, facilitating efficient recombinant protein production and regulatory control over expression levels.

[0471]

[0337] In another aspect of the invention, genes encoding recombinant antimicrobial proteins may be part of the insertion cassette. The genes encoding recombinant antimicrobial protein may comprise at least one of allicin, nisin, surfactin, defensin (e.g. α- defensins, β-defensins, for example human defensin 5 (HD5), beta defensin 3 (HBD3), or bovine beta defensin 123 (bBD123)), a lysozyme, a cathelicidin, a histidine, bioactive peptide derived from Abalone Viscera, lactoferrin, a C-type lectin, or a host defense-related ribonuclease.

[0472]

[0338] In another aspect of the invention, the insertion cassette may encode a recombinant protein, wherein the insertion cassette comprises at least one gene encoding human collagenase (MMP-1, MMP-8, MMP-13, MMP-18) and / or at least one gene encoding a protein from the spidroin family (MaSpl, MaSp2, MiSp, Flag, TuSp, AcSp, PySp).

[0473]

[0339] In another aspect of the invention, the genes present in the insertion cassette, as previously described, may also be utilized for recombinant protein production.

[0474]

[0340] In another aspect, the genes encoding proteins within the insertion cassette may be present in genome of the cell or on one or more mobile genetic elements (MGEs) within the cell, wherein the mobile genetic element may be at least one of plasmid vectors, transposons, retrotransposons or any other mobile element. The MGE may be incorporated into the cells via transformation, transduction or any other suitable genetic transfer method.

[0341] Precise regulation of expression levels of target genes of a particular GM is an inevitable step of a successful GM and a crucial part of the present invention. In order to fine-tune the expression levels and to decrease the risk of silencing the expression of the target gene in the modified cells, species-specific promoters of housekeeping genes may be used. For cells of bovine origin, such examples may include the promoter of bovine glyceraldehyde-3-phosphate dehydrogenase (bGAPDH), the promoter of bovine eukaryotic translation Elongation factor 1 a (bEF1a; SEQ ID NO: 1), or the promoter of bovine phosphoglycerate kinase 1 (bPGK1; SEQ ID NO: 2). The metazoan cell may also comprise a promoter of human cytomegalovirus (CMV), SV40, PGK1, Ubc, tetracycline response element (TRE), UAS, GDS, ADH1 or any other appropriate promoter.

[0475]

[0342] An inducible promoter system may be used in the genetic modification processes according to the invention. To control the expression of desired target genes used in gain of function genetic modification, inducible promoter systems may be used. Expression of accompanied target genes in an inducible promoter-target gene complex may be controlled in terms of switching on and switching off the target gene expression. Ongoing expression might be dependent on a continuous signal delivery or, alternatively, it could be stopped by signal delivery. Small interacting molecules such as protein, saccharide, nucleic acid and / or other various compounds in the culture medium may serve as signals. Examples of signaling compounds might be, for example, rapamycin, abscisic acid, auxin or auxin derivatives or auxin-like analogues, various antibiotics such as tetracycline or corticoid hormones or glucocorticoids or combination of the above mentioned compounds, or any other appropriate signaling compounds. Physical conditions optimized for a specific promoter may also be used as an induction trigger, starting or stopping expression of a target gene. Examples include promoters whose ability to regulate expression of accompanied target genes is dependent on a specific temperature condition or exposure to a physical condition such as light of a specific wavelength, exposure to a magnetic or electromagnetic field, ultrasonic application or other external stimulation.

[0476]

[0343] The bovine Growth hormone polyadenylation signal (bGH-PolyA) is a specialized termination sequence for protein expression in eukaryotic cells. The bGH-polyA may be used in all expression constructs intended for knock-in mediated by, for instance, CRISPR / Cas9, Zinc finger nucleases, TALEN, Cre-Lox recombination, FLP-FRT recombination, RMCE and / or other genome editing tool. The signal may regulate termination of transcription, stabilize the transcripts and / or increase the expression.

[0477]

[0344] The genetically modified non-human metazoan cells used for a food product may be immortalized. The genetically modified non-human metazoan cells may have overcome the Hayflick limit via genetic modification.

[0478]

[0345] In one aspect of the invention, stable long-term expression of bTERT might be used to prevent cells from the gradual shortening of telomeres concomitant with aging. The expression levels of bTERT may or may not match the levels of gene expression in native bTERT -positive cells. This is an important modification usable for all cell types. The TERT gene may be truncated such that its stability and expression levels are improved.

[0479]

[0346] In one aspect of the invention, metazoan cells may be immortalized by the introduction of at least one native and / or modified gene from the group of TERT, Bcl-2, p53, p21, SV40LT or their combinations thereof or any other appropriate genes generating immortalized metazoan cells.

[0480]

[0347] In one aspect of the invention, the at least one gene used for cell immortalization may be from the group of the bovine telomerase reverse transcriptase gene (bTERT), truncated rbTERT variant withdeletion of the bases 1228 - 1287 characterized by coding sequence SEQ ID NO: 3 encoding protein characterized by SEQ ID NO: 4 with deletion of amino acids 410 - 429, a coding sequence having at least 80 %, or at least 85 %, or at least 90 %, or at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 3.

[0481]

[0348] In this aspect of the invention, the product of rbTERT comprises a truncated protein variant with the deletion of twenty amino acids (410-429) characterized by SEQ ID NO: 4, or a protein having at least 80 %, or at least 85 %, or at least 90 %, or at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 4.

[0482]

[0349] In other aspects of the invention, the gene used for cell immortalization may be the bTERT gene with at least 80 %, or at least 85 %, or at least 90 %, or at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 5. In other aspects of the invention, the gene used for cell immortalization may be a bTERT gene with the sequence of SEQ ID NO: 5.

[0483]

[0350] In this aspect of the invention, the product of the bTERT gene may be a protein with at least 80 %, or at least 85 %, or at least 90 %, or at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 6. In this aspect of the invention, the product of the bTERT gene may be a protein with the sequence of SEQ ID NO: 6.

[0484]

[0351] The abovementioned TERT constructs may be introduced into the cells via both viral and non-viral means. The expression of the coding sequence may be driven by an endogenous promoter or by recombinant introduced promoter such as the bGAPDH promoter, bEF1a promoter (SEQ ID NO: 1), or bPGK1 promoter (SEQ ID NO: 2). The genome editing may be done via CRISPR / Cas9, Zinc finger nucleases, TALEN, Cre-Lox recombination, FLP-FRT recombination, RMCE and / or another genome editing tool.

[0485]

[0352] Introduction of the full-length bovine telomerase gene sequence and / or introduction of full-length telomerase gene sequence from other mammals may be one of the approaches used to immortalize cells according to the invention. The introduction of a codon-optimized sequence of the telomerase gene or a coding sequence or reduced coding sequence may be another method used to immortalize cells. These sequences of NA introduced into the genome result in the translation of bTERT or its allelic or species-specific allelic variation. The sequences may be inserted at random sites of the genome or safe harbors or may be in specific safe harbor PGrandom. Introduction of an alternative promoter, additional regulatory DNA element or modification of the native bTERT promoter may be performed to induce native TERT expression.

[0486]

[0353] Alternatively, various modified sequences of bTERT may be used, including recombinant sequences fulfilling native bTERT protein function. Alternatively, different promoters may be used. Alternatively, different safe harbors may be used.

[0487]

[0354] In one aspect of the invention GM of the native bTERT promoter or respective orthologs in other species may be used. This GM may comprise indels or substitutions of the native TERT promoter.

[0488]

[0355] In one aspect of the invention, the cell cultivation process may comprise the introduction of TERT gene sequence or modified TERT gene sequence into the safe harbor PGrandom located in gene bPDE4D on chromosome 20 in order to immortalize cells. The process may comprise introduction of full-length telomerase gene sequence, for example bovine full length telomerase gene sequence, or full-length telomerase gene sequence from other mammals. Introduction of allelic or species-specific allelic variation of TERT gene sequence, codon optimized telomerase gene sequence or coding sequence, orreduced coding sequence, may be used and introduced into the gene bPDE4D in order to immortalize cells. GM (indels, substitutions) of native TERT promoter may be used to induce native TERT expression. Examples of those genetic modifications may be introduction of transcription factors or cis-regulatory elements. Any other appropriate variant or modified TERT gene may be introduced to immortalize cells. The safe harbor according to the invention may be PGrandom. Other target genes for immortalization may be, for example, Bcl-2, p53, p21, SV40LT, or any other appropriate target genes.

[0489]

[0356] In one aspect of the invention, introduction of a modification comprising an insertion cassette encoding one of the existing splicing variants of a target gene and, therefore, changing the balance between the transcribed splicing variants may be performed. An example of this target gene may be Bcl-2.

[0490]

[0357] In one aspect of the invention, the immortalized metazoan cells may be kept in a production cell bank and the immortalization cassette may be removed before inoculation to a cultivation device, for example formed by a production bioreactor. This would serve the purpose of eliminating risks associated with the new genetic structures in the genome, as the cells would be genetically identical to their wild-type counterparts. Those cells may survive for many passages after TERT expression ceases, as the cultivation with the overexpressed TERT will have elongated their telomeres substantially.

[0491]

[0358] In one aspect of the invention, the non-human metazoan cells may be spontaneously immortalized. The immortalization process may comprise culturing and sub-culturing a population of non-human metazoan cells under conditions that promote spontaneous immortalization, wherein the cells may be exposed, during at least a portion of the sub-culturing period, to an effective amount of one or more chemical agents capable of increasing replicative lifespan and / or promoting acquisition of an immortalized phenotype. The non-human metazoan cells immortalized by this process may be referred to as pseudo-immortalized non-human metazoan cells.

[0492]

[0359] The non-human metazoan cells may be cultured and / or sub-cultured in the presence of at least one chemical agent capable of increasing replicative lifespan and / or promoting acquisition of an immortalized phenotype, wherein the at least one chemical agent may be from the group of: N-(3-chloro-4-fluorophenyl)-4-fluoro-3,5-dimethylbenzenesulfonamide (TAC) and / or a derivative thereof, 2-propylpentanoic acid (valproic acid; VP A) and / or a derivative thereof, sodium butanoate (sodium butyrate; NaB) and / or a derivative thereof, 6-((2-((4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile (CHIR99021) and / or a derivative thereof, mirdametinib (PD0325901) and / or a derivative thereof, 5-(l-piperazinylsulfonyl)isoquinoline (HA-100) and / or a derivative thereof, 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A-83-01) and / or a derivative thereof, 2,5 -furandione (maleic anhydride; MA), (3β,6α,16β,24R)-20,24-Epoxy-9,19-cyclolanostane-3,6,16,25-tetrol (cycloastragenol) and / or its derivatives, or a combination thereof.

[0493]

[0360] In another aspect of the invention, the chemical agent may further comprise one or more plant extracts and / or phytochemicals, for example, but not limited to, Centella asiatica extract, Vaccinium myrtillus (bilberry / blackberry) extract, Camellia sinensis (green tea) extract, Vitis vinifera (grape seed or grape skin) extract, Punica granatum (pomegranate) extract, Rosmarinus officinalis (rosemary) extract, Curcuma longa (turmeric) extract, Panax ginseng extract, Aloe barbadensis (aloe vera) extract, Glycyrrhiza glabra (licorice) extract, Scutellaria baicalensis extract, Polygonum cuspidatum (Japanese knotweed) extract, Olea europaea (olive leaf / fruit) extract, Ginkgo biloba extract, Chamomilla recutita (chamomile) extract, Calendula officinalis extract, Hibiscus sabdariffa extract, Morus alba (mulberry)extract, and / or Oryza sativa (rice) extract, or fractions, isolates, purified compounds, and / or derivatives thereof, or combinations thereof.

[0494]

[0361] In one aspect of the invention, the effective concentration of the one or more chemical agents for the culturing and / or subculturing process to spontaneously immortalize the non-human metazoan cells in the culture medium may be in the range of about 0.01 μM to about 50 μM, about 0.05 μM to about 20 μM, or about 0.1 μM to about 10 μM.

[0495]

[0362] In one aspect of the invention, the effective concentration of the TAC for the culturing and / or subculturing process to spontaneously immortalize the non-human metazoan cells may be in a range of 0.05 μM to 2.5 μM, or in a range of 0.1 μM to 1.0 μM, or in a range of 0.2 μM to 0.5 μM.

[0496]

[0363] In one aspect of the invention, the effective concentration of the CAG for the culturing and / or subculturing process to spontaneously immortalize the non-human metazoan cells may be in a range of 0.1 μM to about 10 μM, about 0.5 μM to about 8 μM, or about 1 μM to about 5 μM.

[0497]

[0364] In one aspect of the invention, the effective concentration of the MA for the culturing and / or subculturing process to spontaneously immortalize the non-human metazoan cells may be in a range of 0.1 μM to about 10 μM, about 0.5 μM to about 8 μM, or about 1 μM to about 5 μM.

[0498]

[0365] In another aspect of the invention, the non-human metazoan cell subpopulations that exhibit sustained proliferation beyond the subculture number at which corresponding untreated cells undergo senescence or growth arrest may be selected and isolated. In further aspects of the invention, the chemical agent may be withdrawn after a selection period and the resulting pseudo-immortalized non-human metazoan cells maintain long-term proliferative capacity in the absence of the chemical agent.

[0499]

[0366] In one aspect of the invention, the sequences of proteins artificially designed through computational methods, including machine learning approaches, that can perform substantially the same function as the protein described herein, with at least partially similar sequence identity.

[0500]

[0367] As used herein, the term “sequence identity” may refer to the degree of similarity between two polynucleotide or polypeptide sequences, expressed as a percentage. The percentage may be calculated by using local or global alignment algorithms, optimally aligning the two polynucleotide or two polypeptide sequences.

[0501]

[0368] The local sequence alignment may be calculated as follows:

[0502] Sequence identity (%) = (Number of identical residues / Defined length in given alignment) × 100 In this context, residues refer to amino acids or nucleotides, depending on the type of compared sequences. Identical residues refer to positions where the residues (amino acids or nucleotides) are the same in both sequences at that alignment position. The defined length in the given alignment includes gaps and mismatches but may exclude overhangs. The local alignment may be calculated by using BLAST, FASTA, or any other appropriate tool for the local alignment.

[0503]

[0369] As used herein, the term “sequence identity” may refer to the degree of similarity between two polynucleotide or polypeptide sequences, expressed as a percentage. The percentage may be calculated by using local or global alignment algorithms, optimally aligning the two polynucleotide or two polypeptide sequences.

[0504]

[0370] The local alignment may be calculated alternatively as follows:Sequence identity (%) = (Number of identical residues aligned by local alignment / Total number of residues in the longer sequence) × 100

[0505] In this context, residues refer to amino acids or nucleotides, depending on the type of compared sequences. Identical residues are those where the same amino acid or nucleotide is present at corresponding positions within the aligned region identified through local alignment.

[0506] This calculation method differs from traditional local alignment methods that calculate identity based on the length of the given alignment itself. Instead, it normalizes the sequence identity by considering the total number of residues in the longer sequence involved in the comparison, including any gaps introduced during the alignment. This approach retains the characteristics of local alignment while allowing the resulting identity value to reflect the proportion of similarity in the context of the entire longer sequence. Suitable algorithms for this method may include customized local alignment tools or modified versions of established software like BLAST or FASTA.

[0507]

[0371] The global sequence alignment may be calculated as follows:

[0508] Sequence identity (%) = (Number of identical residues / Total number of residues in the sequence) × 100

[0509] In this context, residues refer to amino acids or nucleotides, depending on the nature of the compared sequences. Identical residues refer to positions where the residues (amino acids or nucleotides) are the same in both sequences at that alignment position. The total number of residues in the sequence represents the final length of the alignment after both sequences have been aligned. If the sequences differ in length, gaps are introduced to make both sequences equal in length. Therefore, the total number of residues corresponds to the length of the aligned sequences, which is always the same for both sequences in the global alignment. The global alignment may be calculated by using EMBOSS Needle, EMBOSS Stretcher, MUSCLE, BLAST using Needleman-Wunsch algorithm or any other appropriate tool for the global alignment.

[0510]

[0372] The local sequence alignment may be calculated as follows:

[0511] Sequence identity (%) = (Number of identical residues / Defined length in given alignment) × 100 In this context, residues refer to amino acids or nucleotides, depending on the type of compared sequences. Identical residues refer to positions where the residues (amino acids or nucleotides) are the same in both sequences at that alignment position. The defined length in the given alignment includes gaps and mismatches but may exclude overhangs. The local alignment may be calculated by using BLAST, FASTA, EMBOSS Water using Smith-Waterman algorithm, or any other appropriate tool for the local alignment.

[0512]

[0373] The local alignment may be calculated alternatively as follows:

[0513] Sequence identity (%) = (Number of identical residues aligned by local alignment / Total number of residues in the longer sequence) × 100

[0514] In this context, residues refer to amino acids or nucleotides, depending on the type of compared sequences. Identical residues are those where the same amino acid or nucleotide is present at corresponding positions within the aligned region identified through local alignment.

[0374] This calculation method differs from traditional local alignment methods that calculate identity based on the length of the given alignment itself. Instead, it normalizes the sequence identity by considering the total number of residues in the longer sequence involved in the comparison, including any gaps introduced during the alignment. This approach retains the characteristics of local alignment while allowing the resulting identity value to reflect the proportion of similarity in the context of the entire longer sequence. Suitable algorithms for this method may include customized local alignment tools or modified versions of established software like BLAST, FASTA, or EMBOSS Water.

[0515]

[0375] The global sequence alignment may be calculated as follows:

[0516] Sequence identity (%) = (Number of identical residues / Total number of residues in the sequence) × 100

[0517] In this context, residues refer to amino acids or nucleotides, depending on the nature of the compared sequences. Identical residues refer to positions where the residues (amino acids or nucleotides) are the same in both sequences at that alignment position. The total number of residues in the sequence represents the final length of the alignment after both sequences have been aligned. If the sequences differ in length, gaps are introduced to make both sequences equal in length. Therefore, the total number of residues corresponds to the length of the aligned sequences, which is always the same for both sequences in the global alignment. The global alignment may be calculated by using EMBOSS Needle, EMBOSS Stretcher, MUSCLE, BLAST using Needleman-Wunsch algorithm or any other appropriate tool for the global alignment.

[0518]

[0376] The global sequence alignment may be calculated alternatively as follows:

[0519] Sequence identity (%) = (Number of identical residues / Total number of residues in the shorter sequence) × 100

[0520] This calculation method differs from traditional global alignment methods that calculate identity based on the final length of the alignment after both sequences have been aligned. Instead, it normalizes the sequence identity by considering the total number of residues in the shorter sequence involved in the comparison. This approach retains the characteristics of local alignment while allowing the resulting identity value to reflect the proportion of similarity in the context of the entire longer sequence. Suitable algorithms for this method may include customized local alignment tools or modified versions of established software like EMBOSS Needle, EMBOSS Stretcher, MUSCLE, BLAST using Needleman-Wunsch algorithm or any other appropriate tool for the global alignment.

[0521]

[0377] In another aspect of the invention, the sequence identity may be calculated alternatively, as follows:

[0522] Sequence identity (%) = Number of identical residues / Total number of residues in the claimed sequence) × 100

[0523] The claimed sequence may be the full sequence of amino acids or nucleotides claimed or a part of this sequence such as a domain or a motif relevant to the function of the claimed sequence. Global or local alignment algorithms may be used to calculate the alignment itself.

[0524]

[0378] In one aspect of the invention, the alignment algorithms used for the calculation of sequence identity may apply alignment parameters comprising scoring matrices and gap penalties (gap-open, gap-extension).

[0379] In addition to the choice of alignment algorithm, it may utilize scoring matrices to evaluate the similarity between residues. The scoring matrix scheme assigns numerical values to matches, and mismatches. At its most basic level, both nucleotide and protein alignments are scored by assigning a positive value for a match (e.g., +1) and a negative value for a mismatch (e.g., -1). Gaps are not scored as neutral events but are penalized separately by the combination of gap open and gap extension parameters. The final alignment score may be calculated as follows:

[0525] Alignment score = Sum of substitution scores - sum of gap penalties of all gaps, wherein the gap penalty follows an affine model and may be calculated as:

[0526] Gap penalty = (gap open) + (gap extension) x (k-1),

[0527] where k represents the length of the gap.

[0528] For nucleotide sequence alignments (DNA or RNA), substitution matrices such as EDNAFULL, DNAfull, DNAmat, or NUC.4.4 may be employed, with the choice of nomenclature depending on the software package (e.g., EMBOSS, BLAST, or FASTA). These matrices provide a more biologically realistic evaluation of nucleotide similarity, particularly in analyses where mutational bias or evolutionary distance is relevant.

[0529] For protein sequence alignments, substitution matrices such as PAM (Point Accepted Mutation) and BLOSUM (Blocks Substitution Matrix) may be employed. BLOSUM62 or variance of the matrix (such as EBLOSUM62) is a commonly accepted standard that assigns empirically derived log-odds scores to amino acid substitutions observed in conserved protein blocks. Other matrices such as BLOSUM50, BLOSUM80, or PAM250 may be selected depending on the expected evolutionary distance between the compared proteins.

[0530]

[0380] In sequence alignment, insertions and deletions are modeled as gaps that are penalized to prevent overfitting of alignments. Two parameters are typically distinguished: a “gap open” penalty, which is applied when a new gap is introduced into the alignment, and a “gap extension” penalty, which is applied for each additional residue that extends an already existing gap. The selection of gap open and gap extension values may depend on the alignment algorithm used and the nature of the sequences compared. For example, typical gap open penalties may range from about 5 to 15 for protein sequences and from about 10 to 20 for nucleotide sequences, while gap extension penalties may typically range from about 0.1 to 2 for proteins and from about 0.5 to 5 for nucleotides. In most alignment algorithms, the total gap penalty follows an affine model, in which the penalty for a gap of length k residues is calculated as previously mentioned:

[0531] Gap penalty = (gap open) + (gap extension) x (k-1).

[0532] Higher gap open penalties tend to discourage introduction of new gaps, favoring alignments with fewer insertions or deletions, whereas lower penalties permit more flexible alignment accommodating potential evolutionary changes.

[0533]

[0381] By way of example, the sequence identity of protein sequences may be calculated by using scoring matrix EBLOSSUM62, with gap open 10 and gap extend 0.5 by implementation of local sequence alignment or global sequence alignment:Sequences of beta-defensin 6 of Gallus gallus:

[0534] Sequence A: ILYLLLSVLFVVLQGVA (17 aa, SEQ ID NO: 26)

[0535] Sequence B (truncated and modified at the end): ILYLLLSVLGVADCED (16 aa, SEQ ID NO: 27) The sequence identity is calculated as the number of identical residues divided by the defined alignment length. In this case, there are 12 identical residues out of 17 positions in the local alignment, corresponding to 70.6 % identity, and 12 identical residues out of 21 positions in the global alignment, corresponding to 57.1 % identity. The alignment of the sequences for local and global alignment are visualized in Fig. 17.

[0536]

[0382] In one aspect of the invention, a genetic modification that reduces growth factor requirements in the culture medium, optionally combined with the immortalization of metazoan cells, may be achieved through activation of the PI3K-Akt signaling pathway, or TGF-beta signaling pathway or any other signaling pathway or a combination thereof.

[0537]

[0383] In one aspect of the invention, a genetic modification that reduces growth factor requirement in the culture medium, optionally combined with the immortalization of metazoan cells, may be achieved through activation of the PI3K-Akt signaling pathway, or TGF-beta signaling pathway or any other appropriate signaling pathway or a combination thereof by at least one genetic modification. Said genetic modification may comprise the addition of nucleotide sequences encoding amino acid sequences, at the N-terminal and / or C-terminal region of a protein, wherein said sequences induce post-translational lipid modifications, from the group of myristoylation, prenylation, and / or palmitoylation, and wherein these modifications facilitate covalent lipid attachment to proteins, thereby enhancing their membrane localization, stability, and signaling activity.

[0538]

[0384] The genetic modification may comprise nucleotide sequences encoding amino acid sequences at the N-terminal and / or C-terminal region of a protein, wherein said sequences induce post-translational lipid modifications, from the group of myristoylation, prenylation, and / or palmitoylation, and wherein the amino acid sequences may be selected from at least one of SEQ ID NO: 7-10.

[0539]

[0385] In one aspect of the invention, a genetic modification that reduces growth factor requirements in the culture medium, optionally combined with the immortalization of metazoan cells, may be achieved through activation of the PI3K-Akt signaling pathway, or TGF-beta signaling pathway or any other appropriate signaling pathway or a combination thereof by at least one genetic modification. Said genetic modification may comprise at least one single point mutation (SNP) in the nucleotide sequence encoding protein participating in PI3K-Akt signaling pathway, or TGF-beta signaling pathway or any other appropriate signaling pathway or combinations thereof.

[0540]

[0386] In another aspect of the invention, the PI3K-Akt signaling pathway may be activated directly or indirectly. Direct activation of the PI3K-Akt signaling pathway may involve the stimulation of membrane-bound receptors, such as receptor tyrosine kinases (RTKs) and / or G protein-coupled receptors (GPCRs), and / or intracellular signaling proteins that directly phosphorylate and activate PI3K and / or its downstream effectors. Indirect activation of the PI3K-Akt signaling pathway may be achieved through the modulation of upstream signalingcascades, including, but not limited to, pathways that engage cross-talk with the PI3K-Akt pathway, leading to its activation via secondary messengers or intermediary proteins.

[0541]

[0387] In one aspect of the invention, a metazoan cell, optionally immortalized, may be genetically modified to activate the PI3K-Akt signaling pathway through modification of at least one gene encoding subunits of PI3K resulting in a modified form of PI3K, wherein the genes encoding subunits of PI3K may be at least one of genes encoding PIK3CA, PIK3CB, PIK3CG, PI3KCD, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5, PIK3R6.

[0542]

[0388] As used herein, a “modified form of PI3K” may refer to a phosphoinositide 3-kinase (PI3K) in which at least one PI3K subunit is genetically altered relative to a corresponding wild-type PI3K subunit by at least one genetic modification and / or at least one genetic modification resulting in activation of PI3K-Akt signaling pathway. In one aspect of the invention, the at least one genetic modification may comprise an insertion, deletion, and / or substitution in a gene encoding the at least one PI3K subunit, thereby producing a modified PI3K subunit and a modified form of PI3K.

[0543]

[0389] In one aspect of the invention, the terms PI3K signalling pathway and PI3K-Akt signalling pathways may be interchangeable.

[0544]

[0390] In another aspect of the invention, the activation of the PI3K-Akt signaling pathway may be achieved by modifying a gene encoding PIK3CA, PIK3CB, PIK3CG and / or PIK3CD, which are subunits of PI3K. The modification may involve the introduction of at least one SNP mutation and / or the addition of a nucleotide sequence encoding an amino acid sequence to facilitate myristoylation, prenylation or palmitoylation of PIK3CA, PIK3CB, PIK3CG and / or PIK3CD, and / or a combination thereof, to force localization to the intracellular side of the cell membrane or any other appropriate modification.

[0545]

[0391] The SNP mutation of PIK3CA, resulting in an amino acid substitution in the PIK3CA protein (SEQ ID NO: 11), may be at least one SNP mutation resulting in at least one substitution from the group of H1047R (SEQ ID NO: 12), E545K (SEQ ID NO: 13), E542K (SEQ ID NO: 14) and / or R88Q (SEQ ID NO: 15), a combination thereof or any other appropriate SNP mutation.

[0546]

[0392] In one aspect, the nucleotide sequence encoding a modified form of PIK3CA has at least 90% sequence identity to the reference sequence of PIK3CA (SEQ ID NO: 11), wherein said nucleotide sequence may comprise at least one SNP mutation resulting in at least one substitution from the group of H1047R (SEQ ID NO: 12), E545K (SEQ ID NO: 13), E542K (SEQ ID NO: 14), and / or R88Q (SEQ ID NO: 15), or a combination thereof.

[0547]

[0393] In one aspect, the nucleotide sequence encoding a modified form of PIK3CA has at least 90% sequence identity to the reference sequence of PIK3CA (SEQ ID NO: 11), wherein said nucleotide sequence may comprise at least one SNP mutation resulting in at least one amino acid substitution from the group of H1047R (SEQ ID NO: 12), E545K (SEQ ID NO: 13), E542K (SEQ ID NO: 14), and / or R88Q (SEQ ID NO: 15), or a combination thereof.

[0548]

[0394] In one aspect, the nucleotide sequence encoding a modified form of PIK3CA has at least 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % or 99 % sequence identity to the reference sequence of PIK3CA (SEQ ID NO: 11), wherein said nucleotide sequence may comprise at least one SNP mutation resulting in at least one amino acid substitution from the group ofH1047R (SEQ ID NO: 12), E545K (SEQ ID NO: 13), E542K (SEQ ID NO: 14), and / or R88Q (SEQ ID NO: 15), or a combination thereof.

[0549]

[0395] The amino acid sequence to facilitate the myristoylation, prenylation, and / or palmitoylation of PIK3CA, PIK3CB, PIK3CG or Akt to force localization to the intracellular side of the cell membrane may be at least one of SEQ ID NO: 7-10.

[0550]

[0396] In one aspect of the invention, the genetic modification of genes encoding subunits of PI3K or gene encoding Akt results in activation of the PI3K-Akt signaling pathway, leading to enhanced growth, stability, and / or proliferation of metazoan cells.

[0551]

[0397] In one aspect of the invention, the genetic modification of genes encoding subunits of PI3K or gene encoding Akt results in activation of the PI3K-Akt signaling pathway, leading to enhanced growth, stability, and / or proliferation of non-human metazoan cells in the absence of at least one growth factor within the culture medium.

[0552]

[0398] In another aspect of the invention, a genetically modified metazoan cell, which may be optionally immortalized and / or may express a modified form of PI3K or modified form of Akt, may be further genetically modified to express at least one additional gene encoding a protein that provides an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell in the absence of growth factors in the culture medium. The additional gene may be at least one of FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGF-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFRA, PDGFRB, mucin or a combination thereof, or any other appropriate growth factor, wherein the expressed protein may contribute to the activation or enhancement of PI3K-Akt signaling pathway.

[0553]

[0399] In another aspect of the invention, a genetically modified metazoan cell, which may be optionally immortalized and / or may express a modified form of PI3K or modified form of Akt, may be further genetically modified to express at least one additional gene encoding a protein that provides an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell under low concentrations of growth factors in the culture medium. The additional gene may be at least one of FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGF-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFRA, PDGFRB, mucin or a combination thereof, wherein the expressed protein may contribute to the activation or enhancement of PI3K-Akt signaling pathway.

[0554]

[0400] The growth factors at low concentrations within the culture medium may be at least one of INS, TGF-beta, FGF and TF. The low concentration of INS may be in the concentration range of 0 to 20 mg / L, or 0 to 1, or 0 to 0.1 mg / L within the culture medium. The low concentration of TGF-beta may be in a concentration range of 0 to 0.001 mg / L, or 0 to 0.0001 mg / L, or 0 to 0.00001 mg / L within the culture medium. The low concentration of FGF may be in a concentration range of 0 to 0.01 mg / L, or 0 to 0.001 mg / L, or 0 to 0.0001 mg / L within the culture medium. The low concentration of TF may be in a concentration range of 0 to 2.5 mg / L, or 0 to 0.25 mg / L, or 0 to 0.01 mg / L within the culture medium.

[0555]

[0401] In other aspects of the invention, the gene used for overexpression of insulin may be the INS gene characterized by SEQ ID NO: 16 or nucleotide sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 16.

[0402] In other aspect of the invention, the protein product of a gene used for overexpression of insulin may be the protein characterized by SEQ ID NO: 17 or an amino acid sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 17.

[0556]

[0403] In other aspect of the invention, the gene used for overexpression of an FGF ligand may be an FGF2 gene characterized by SEQ ID NO: 18 or a nucleic acid sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 18.

[0557]

[0404] In other aspect of the invention, the protein product of the gene used for overexpression of FGF2 may be characterized by SEQ ID NO: 19 or an amino acid sequences having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 19.

[0558]

[0405] In another aspect, the present invention provides a genetically modified metazoan cell that expresses a modified form of PI3K or a modified form of Akt and at least one additional gene encoding a protein providing an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell in the absence of growth factors in the cultivation medium, wherein the combined expression of these modifications may result in a synergistic effect on cell viability and proliferation.

[0559]

[0406] In another aspect of the invention, a metazoan cell (alternatively non-human metazoan cell), optionally immortalized, may be subjected to an adaptation process to activate the PI3K- Akt signaling pathway.

[0560]

[0407] In one aspect of the invention, a non-human metazoan cell, optionally immortalized, may be genetically modified to indirectly activate the PI3K-Akt signaling pathway through modification of at least one gene encoding protein from MAPK signaling pathway, wherein the gene encoding protein from MAPK signaling pathway may be at least one of gene encoding Growth factor receptor-bound protein 2 (GRB2), Son of sevenless homolog 1 (SOS1), SOS2, SH2 domain-containing protein tyrosine phosphatase-2 (SHP2), RAS (HRAS, KRAS, NRAS) or a combination thereof. The activation of the PI3K-Akt signaling pathway may occur through signal transduction from RAS protein of the MAPK signaling pathway. The indirect activation of the PI3K signaling pathway may result in enhanced growth, stability and / or proliferation of metazoan cells in the absence of at least one growth factor within the culture medium.

[0561]

[0408] In one aspect of the invention, a metazoan cell, optionally immortalized, may be genetically modified to activate the PI3K-Akt signaling pathway through modification of at least one gene encoding an upstream activator of the PI3K-Akt signaling pathway, wherein the gene encoding an upstream activator of the PI3K-Akt signaling pathway may be at least one of gene encoding Epidermal growth factor (EGF), EGFR, FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFRA, PDGFRB, IGF-1, IGF-2, IGF1R, IGF2R or a combination thereof. The activation of the PI3K signaling pathway through the genetic modification of at least one upstream activator may result in enhanced growth, stability and / or proliferation of metazoan cells in the absence of at least one growth factor within the culture medium.

[0562]

[0409] In one aspect of the invention, a metazoan cell, optionally immortalized, may be genetically modified to activate the TGF-beta signaling pathway through modification of atleast one gene encoding TGF-beta receptor, wherein the gene may be from the group of genes encoding TGFBR1, TGFBR2.

[0563]

[0410] In another aspect of the invention, the activation of the TGF-beta signaling pathway may be achieved by modifying the gene encoding TGFBR1. This modification may involve the introduction of at least one SNP mutation resulting in continuous activation of this receptor without binding TGFBR2 to transfer the signal.

[0564]

[0411] The SNP mutation of TGFBR1, resulting in an amino acid substitution in the TGFBR1 protein (SEQ ID NO: 20), may encode a TGFBR1 protein with an T200D substitution (SEQ ID NO: 21).

[0565]

[0412] In another aspect of the invention, the activation of the TGF-beta signaling pathway may be achieved by modifying the gene encoding TGFBR2. This modification may involve the introduction of at least one single nucleotide polymorphism (SNP) mutation resulting in continuous activation of this receptor without binding TGFBR1 to transfer the signal.

[0566]

[0413] The SNP mutation of TGFBR2, resulting in an amino acid substitution in the TGFBR2 protein (SEQ ID NO: 23), may encode a TGFBR2 protein with a R537P substitution (SEQ ID NO: 24).

[0567]

[0414] In one aspect of the invention, the genetic modification of genes encoding TGF-beta receptors results in activation of the TGF-beta signaling pathway, leading to enhanced growth, stability, and / or proliferation of metazoan cells in the absence of TGF-beta or low concentration of TGF-beta within the culture medium.

[0568]

[0415] The concentration of TGF-beta in the culture medium for cultivating metazoan cells with an activated TGF-beta signaling pathway may be within a range of 0 to 0.001 mg / L, 0 to 0.0001 mg / L, or 0 to 0.00001 mg / L.

[0569]

[0416] In another aspect of the invention, a genetically modified metazoan cell, which may be optionally immortalized and / or may express a modified form of TGFBR, may be further genetically modified to express at least one additional gene encoding a protein that provides an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell in the absence of growth factors or low concentration of growth factors in the culture medium. The additional gene may be at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2, or mucin or a combination thereof.

[0570]

[0417] In other aspects of the invention, the gene used for overexpression of TGFBR 1 with a SNP mutation may be characterized by SEQ ID NO: 22 or a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 13.

[0571]

[0418] In other aspect of the invention, the protein product of the gene used for overexpression of TGFBR1 is characterized by SEQ ID NO: 21 or a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 21.

[0572]

[0419] In other aspects of the invention, the gene used for overexpression of TGFBR2 with a SNP mutation may be characterized by SEQ ID NO: 25 or a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 25.

[0420] In other aspect of the invention, the protein product of the gene used for overexpression of TGFBR1 is characterized by SEQ ID NO: 24 or a sequence having at least 80 %, at least 85 %, at least 90 %, at least 95 %, or at least 99 % sequence identity to SEQ ID NO: 24.

[0573]

[0421] The invention also encompasses genetically modified cells with activated PI3K-Akt signaling pathway or TGF-beta signaling pathway, or a combination thereof, that may be cultivated in a culture medium that does not comprise at least one of insulin, FGF, TGF-beta, TF, IGF, EGF, PDGF or combination thereof, or any other appropriate growth factor.

[0574]

[0422] In the present invention as depicted in Fig. 8, metazoan cells (001) are genetically modified to activate the PI3K-Akt signaling pathway and / or TGF-beta signaling pathway (002), enabling enhanced stability, growth, and / or proliferation in serum-free and / or protein-free culture medium. These genetically modified metazoan cells are capable of growing in serum- free and protein-free culture medium making them suitable as cheap expression systems for the production of many recombinant proteins (003). These genetically modified metazoan cells may produce recombinant proteins that are then isolated and purified (004).

[0575]

[0423] The genetically modified non-human metazoan cells are processed (005) and used as a consumable product for human and animal use (006).

[0576]

[0424] In another aspect of the invention, an optionally immortalized genetically modified metazoan cell with activated PI3K-Akt signaling pathway and / or activated TGF-beta signaling pathway, or any other appropriate signaling pathway, optionally comprising at least one additional gene providing an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell with absence of growth factors or low concentration of growth factors within the culture medium, wherein the cell is used as a producer of exosome vesicles. The exosome vesicles produced may be utilized in the pharmaceutical, food, medical, or any other industry.

[0577]

[0425] The exosomes may be endogenously loaded with recombinant proteins through genetic modifications that facilitate the expression of recombinant proteins and their incorporation into the vesicular cargo.

[0578]

[0426] In further aspect, the invention may comprise genetically modified non-human cells, optionally immortalized, that may produce exosomes either naturally or through genetic modification. The cell may be further genetically modified to comprise at least one insertion cassette and / or mobile genetic element (MGE) including at least one gene encoding recombinant protein participating in exosome production, wherein the gene encoding a recombinant protein participating in exosome production may be at least one of CD63, CD81, HSP70, HSP90 or a combination thereof, or any other appropriate gene.

[0579]

[0427] The term metazoan cells may be used interchangeably with the term genetically modified metazoan cells with an activated PI3K-Akt signaling pathway and / or TGF-beta signaling pathway or any appropriate signaling pathway.

[0580]

[0428] In another aspect of the invention, a genetically modified metazoan cell, which may be optionally immortalized, comprises an activated PI3K-Akt signaling pathway and / or an activated TGF-beta signaling pathway, wherein said activation may be achieved by at least one genetic modification.

[0429] The genetically modified metazoan cell may further comprise at least one additional gene providing an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of or in low concentration of growth factors in the culture medium.

[0581]

[0430] The genetically modified metazoan cell may be utilized as an expression system for the production of at least one recombinant protein encoded by at least one gene, wherein said recombinant protein is suitable for applications in at least one industry selected from the group of pharmaceutical, food, medical, or any other relevant industry.

[0582]

[0431] The recombinant protein may be selected from those previously mentioned in the insertion cassette or in the MGE.

[0583]

[0432] In a further aspect, the invention may provide the expression system made from the genetically modified cells that may be cultivated in serum-free and / or protein-free medium.

[0584]

[0433] The invention also encompasses the expression system made from the genetically modified cells that may be cultivated in the culture medium that does not comprise at least one of insulin, FGF, TGF-beta, TF, IGF, EGF, PDGF or a combination thereof, or any other appropriate growth factor.

[0585]

[0434] In another aspect of the invention, the expression system may not comprise immortalized metazoan cells. The insertion cassette comprising at least one native and / or modified gene generating immortalized non-human metazoan cell may be removed from its genome by CRISPR / Cas9, Zinc finger nucleases, TALEN, Cre-Lox recombination, FLP-FRT recombination, RMCE, or any other appropriate genome editing tool.

[0586]

[0435] In one aspect, the invention may provide a method for isolating and purifying a recombinant protein from genetically modified non-human metazoan cells. The process may comprise the steps of cell lysis, protein extraction, and purification using chromatography - based or filtration-based separation techniques.

[0587]

[0436] The genetically modified non-human metazoan cells may be lysed using mechanical disruption (e.g., high-pressure homogenization, sonication, or bead milling), chemical lysis (e.g., detergents or chaotropic agents), or enzymatic digestion (e.g., lysozyme or protease treatment). Following lysis, the cellular debris and pellet may be separated from the supernatant comprising recombinant proteins by centrifugation or filtration to remove cellular debris and pellet from the supernatant.

[0588]

[0437] The recombinant protein from the supernatant may be purified using affinity chromatography, wherein an affinity tag (e.g., His-tag, GST-tag, FLAG-tag, or Strep-tag) facilitates selective binding to a chromatography resin (e.g., Ni-NTA, glutathione, or streptavidin beads). As an alternative, ion-exchange chromatography may be used to separate proteins based on charge differences, while size -exclusion chromatography may be used for fractionation based on molecular weight.

[0589]

[0438] Further purification may involve hydrophobic interaction chromatography or reverse - phase chromatography for enhanced resolution. If required, additional polishing steps such as ultrafiltration / diafiltration, dialysis, or precipitation methods (e.g., ammonium sulfate precipitation, PEG precipitation) may be employed to concentrate and further purify the recombinant protein.

[0439] The purified recombinant protein may then be subjected to the additional analyses for biochemical characterization and quality assessment, including SDS-PAGE, Western blotting, Mass spectrometry, Isoelectric focusing, and ELISA or any other appropriate analysis to confirm purity, structural integrity, and / or functional activity of the purified recombinant protein. The final product may be optionally lyophilized or stored in a formulation buffer for downstream applications in pharmaceutical, food, medical industry, or any other industry.

[0590]

[0440] In another aspect, the invention may provide, optionally immortalized genetically modified non-human metazoan cell with an activated PI3K-Akt signaling pathway and / or activated TGF-beta signaling pathway, optionally comprising at least one additional gene, for use in the production of a food product for human and / or animal consumption.

[0591]

[0441] In another aspect, the invention may provide, optionally immortalized genetically modified non-human metazoan cells with an activated FGFR that may be capable of growing in a protein-free and / or serum-free culture medium.

[0592]

[0442] In another aspect of the invention, the genetically modified non-human metazoan cell may comprise an active TERT and an activated FGFR, wherein the combination may provide improved long-term proliferative capacity and / or reduced senescence of the genetically modified non-human metazoan cell when cultured in a protein-free and / or serum-free culture medium.

[0593]

[0443] In another aspect of the invention, the genetically modified non-human metazoan cell may comprise an active TERT and an activated FGFR, wherein the combination may provide improved long-term proliferative capacity and / or reduced senescence of the genetically modified non-human metazoan cell when cultured in a culture medium in absence with at least one growth factor.

[0594]

[0444] In another aspect, “activated FGFR” may refer to an FGFR exhibiting increased receptor signaling relative to a corresponding wild-type FGFR, optionally in a ligandindependent manner, as determined by increased receptor phosphorylation and / or increased downstream signaling through MAPK / ERK and / or PI3K-Akt signaling pathways.

[0595]

[0445] In one aspect of the invention, a metazoan cell, optionally immortalized, may be genetically modified to activate the PI3K-Akt signaling pathway through modification of at least one gene encoding FGFR resulting in activated FGFR.

[0596]

[0446] In one aspect of the invention, a metazoan cell, optionally immortalized, may be genetically modified to activate the PI3K-Akt signaling pathway through modification of at least one gene encoding FGFR resulting in activated FGFR, wherein the FGFR may be from the group of FGFR1, FGFR2, FGFR3, FGFR4, FGFR6.

[0597]

[0447] In another aspect of the invention, optionally immortalized genetically modified non- human metazoan cells with an activated FGFR may result in enhanced growth, stability and / or proliferation of metazoan cells in the absence of at least one growth factor within the culture medium.

[0598]

[0448] The modification of the FGFR may involve the introduction of at least one SNP mutation of the FGFR, wherein the FGFR may be from the group of FGFR family, specificallyFGFR1, FGFR2, FGFR3, FGFR4, Fibroblast growth factor receptor -like 1 (FGFRL1), FGFR6, or combinations thereof.

[0599]

[0449] The SNP mutation of FGFR1, resulting in an amino acid substitution in the FGFR1 protein, may be at least one SNP mutation resulting in at least one substitution from the group of K656E, N546K, a combination thereof or any other appropriate SNP mutation.

[0600]

[0450] The SNP mutation of FGFR1, resulting in an amino acid substitution in the FGFR1 protein (SEQ ID NO: 28), may be at least one SNP mutation resulting in at least one substitution from the group of K656E (SEQ ID NO: 29), N546K (SEQ ID NO: 30), a combination thereof or any other appropriate SNP mutation.

[0601]

[0451] The food product according to the invention intended for human consumption and / or as a pet food may comprise a different amount of cultured non-human metazoan cells. The amount of cultured non-human metazoan cells in the food product may be in the range of 0.1 % to 100 % by weight, or in the range of 5 % to 90 %, or in the range of 10 % to 80 %, or in a range of 20 % to 60 %.

[0602]

[0452] The final food product may comprise genetically modified non-human metazoan cells with other non-cellular compounds. Non-cellular compounds may be edible and may bring additional sensory and structural properties as well as additional nutritional values. The resulting food product may be used as a substitute for conventional food in food applications, including fresh, frozen, or pre-cooked meals.

[0603]

[0453] The final food product may comprise non-human metazoan cells with other non- cellular compounds. Non-cellular compounds may be edible and may bring additional sensory and structural properties as well as additional nutritional values. The resulting food product may be used as a substitute for conventional food in food applications, including fresh, frozen, or pre-cooked meals.

[0604]

[0454] The final food product may comprise genetically modified non-human metazoan cells may be mixed with at least one component of saccharide source, protein source, fat source, vitamin source, mineral source, palatant source, colorant source, preservative source, antioxidant source or any other compound or any other appropriate component.

[0605]

[0455] The final food product may comprise non-human metazoan cells may be mixed with at least one component of saccharide source, protein source, fat source, vitamin source, mineral source, palatant source, colorant source, preservative source, antioxidant source or any other compound or any other appropriate component.

[0606]

[0456] The final food product may comprise at least one saccharide source, wherein the saccharide source may comprise at least one of rice, corn, potatoes, sweet potatoes, barley, oats, peas, tapioca, lentils, chickpeas, sorghum, quinoa, millet, wheat, cassava, yams, pumpkin, carrots, beet pulp, apples, bananas, blueberries, cranberries, apricots, butternut squash, chia seeds, flaxseed, sunflower seeds, pumpkin seeds, carrageenan and / or any combination thereof.

[0607]

[0457] The final food product may comprise at least one protein source, wherein the protein source may comprise at least one of soy protein, pea protein, potato protein, rice protein, lentil protein, chickpea protein, fava beans protein, hemp protein, pumpkin seed protein, sunflowerseed protein, alfalfa protein, oat protein, corn gluten protein, quinoa protein, wheat protein, yeast protein, microbial protein and / or any combination thereof.

[0608]

[0458] The final food product may comprise at least one fat source, wherein the fat source may comprise at least one of olive oil, coconut oil, avocado oil, canola oil, sunflower oil, flaxseed oil, sesame oil, almonds, walnuts, pea oil, lupin oil, walnut oil, spirulina oil, peanut oil, cashews, pecans, macadamia nuts, hazelnuts, flaxseeds, sunflower seeds, pumpkin seeds, hemp seeds, sesame seeds, avocado, olives, almond butter, cashew butter, seaweed, tahini, hummus and / or any combination thereof.

[0609]

[0459] The final food product may comprise at least one vitamin source, wherein the vitamin source may comprise at least one of ascorbic acid, ascorbic acid phosphate, biotin, choline chloride, D-calcium pantothenate, folic acid, i-inositol, niacinamide, para-aminobenzoic acid, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and / or any combination thereof.

[0610]

[0460] The final food product may comprise at least one mineral source, wherein the mineral source may comprise at least one of any compound capable of providing minerals in ionic form, wherein the mineral component comprises at least one element of Ca, Cl, Cr, Cu, F, Fe, I, K, Mn, Co, Na, Ni, Se, Sn, Zn, or any combination thereof.

[0611]

[0461] The final food product may comprise at least one palatant source, wherein the palatant source may comprise at least one of animal-derived or plant-derived palatants, wherein the palatants may comprise at least one of artificial and natural flavors, hydrolyzed proteins, fat sprays, Maillard’s reaction products or any combination thereof.

[0612]

[0462] The final food product may comprise at least one colorant source, wherein the colorant source may comprise at least one of beta-carotene, beet juice powder, turmeric, caramel color, spinach powder, spirulina extract, paprika extract, annatto extract, annatto seeds, chlorophyll, saffron, gardenia extract, red beet powder, carrot juice concentrate, purple sweet potato, hibiscus extract, cochineal extract, curcumin, cabbage extract, paprika, grape skin, caramelized onion, anthocyanins or any combination thereof.

[0613]

[0463] The final food product may comprise at least one preservative source, wherein the preservative source may comprise at least one of vitamin E, rosemary extract, citric acid, mixed tocopherols, ascorbic acid, green tea extract, cranberry extract, clove oil, oregano oil, butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin, propyl gallate, sorbic acid, calcium propionate, potassium sorbate, sodium benzoate, tert-butylhydroquinone or any combination thereof.

[0614]

[0464] The final food product may comprise at least one antioxidant source, wherein the antioxidant source may comprise at least one of butylated hydroxyanisole, ethoxyquin, tert- butylhydroquinone, vitamin C, vitamin E, lycopene or any combination thereof.

[0615]

[0465] The final food product may comprise at least one other compound, wherein such compounds may comprise at least one of beneficial microorganisms, emulsifiers, sweeteners, acidity regulators and digestibility enhancers or any combination thereof.

[0616]

[0466] In another aspect of the invention, the food product may not comprise immortalized non-human metazoan cells. The insertion cassette comprising at least one native and / ormodified gene generating immortalized non-human metazoan cell may be removed from its genome by CRISPR / Cas9, Zinc finger nucleases, TALEN, Cre-Lox recombination, FLP-FRT recombination, RMCE, or any other appropriate genome editing tool.

[0617]

[0467] In the context of food product, the term “non-human metazoan cells” may be used interchangeably with the term “genetically modified non-human metazoan cells with an activated PIK3-Akt signaling pathway and / or TGF-beta signaling pathway or any other appropriate signaling pathway”.

[0618]

[0468] In the context of food product, the term “non-human metazoan cells” may be used interchangeably with the term “genetically modified non-human metazoan cells with an activated PI3K-Akt signaling pathway and / or TGF-beta signaling pathway or any other appropriate signaling pathway”.

[0619]

[0469] In one aspect of the invention, these genetically modified non-human metazoan cells may be used for the preparation of cultured meat products for human consumption, including but not limited to meat chunks, whole-cut fillets, structured marbled meat, minced meat, and processed meat products. The cultured meat may be formulated into steaks, fillets, poultry cuts, or seafood analogs, or restructured into processed forms such as meat patties, sausages, nuggets, meatballs, and deli slices.

[0620]

[0470] In one aspect of the invention, non-human metazoan cells may be used for the preparation of cultured meat products for human consumption, including but not limited to meat chunks, whole-cut fillets, structured marbled meat, minced meat, and processed meat products. The cultured meat may be formulated into steaks, fillets, poultry cuts, or seafood analogs, or restructured into processed forms such as meat patties, sausages, nuggets, meatballs, and deli slices.

[0621]

[0471] In another aspect of the invention, these genetically modified non-human metazoan cell may be used for preparation of pet food products, including but not limited to dry kibbles, dry snacks, meaty chunks, meaty chunks with gravy, pate, wet snacks and / or any other products.

[0622]

[0472] In another aspect of the invention, non-human metazoan cells may be used for preparation of pet food products, including but not limited to dry kibbles, dry snacks, meaty chunks, meaty chunks with gravy, pate, wet snacks and / or any other products.

[0623]

[0473] In one aspect of the invention, the genome of metazoan cells may be modified using Cre-Lox recombination system and / or the FLP-FRT recombination system, optionally referred to herein as an exchange system for site-specific genetic modifications.

[0624]

[0474] For the Cre-Eox recombination system, the system may comprise MGE encoding Cre recombinase enabling the site-specific exchange of at least one inserted cassette for another at a genomic locus flanked by non-identical lox sites oriented in the same direction on the same DNA strand within the genome. The lox sites may be from the group of loxP, loxM, loxN, loxl71, lox2272, lox511, loxM2, loxM3, loxM7, lox71, lox66 or combinations thereof or any other appropriate lox site.

[0625]

[0475] For the FLP-FRT recombination system, the system may comprise MGE encoding FLP recombinase enabling the site-specific exchange of at least one inserted cassette for another at a genomic locus flanked by non-identical FRT sites oriented in the same direction on the sameDNA strand within the genome. The FRT sites may be from the group of wild type FRT, FRT3, FRT5, FRT7, FRT14, F3, F5 or combinations thereof or any other appropriate FRT site.

[0626]

[0476] The term “oriented in the same direction” may imply that both lox sites or FRT sites may be positioned in the 5' to 3' direction on the same DNA strand. If the lox sites or FRT sites are in the opposite direction, one would be in the 5' to 3' direction, and the other in the 3' to 5' direction on the same DNA strand.

[0627]

[0477] In one aspect of the invention, the Cre-Lox recombination or FLP-FRT recombination system may enable the site-specific removal of at least one insertion cassette from a genetically modified metazoan cell if the lox sites or FRT sites are identical and are oriented in the same direction on the same DNA strand within the genome.

[0628]

[0478] In another aspect of the invention, the exchange system may be utilized for the preparation of non-human metazoan cells for the production of cultivated meat and / or for the modification of metazoan cells used as expression systems.

[0629]

[0479] In another aspect of the invention, the exchange system may comprise, but is not limited to, Cre-Lox recombination or FLP-FRT recombination system.

[0630]

[0480] The exchange system may be utilized for the generation of non-genetically modified cells from previously genetically modified cells. The application of this system is not limited to metazoan cells and may include, but is not limited to:

[0631] the removal of at least one insertion cassette involved in immortalization in previously immortalized genetically modified metazoan cells,

[0632] the removal of at least one insertion cassette involved in the production of a recombinant protein responsible for cellular differentiation, wherein the cassette may be eliminated postdifferentiation, or

[0633] the exchange of at least one insertion cassette comprising at least one gene encoding recombinant protein, allowing for the replacement of this insertion cassette for another insertion cassette comprising at least one gene encoding a different recombinant protein.

[0634]

[0481] In one aspect of the invention, the exchange system may be utilized for the generation of de-modified metazoan cells (alternatively non-human metazoan cells) from previously genetically modified cells. The application of this system is not limited to metazoan cells and may include, but is not limited to:

[0635] the removal of at least one insertion cassette involved in immortalization in previously immortalized genetically modified metazoan cells,

[0636] the removal of at least one insertion cassette involved in the production of a recombinant protein responsible for cellular differentiation, wherein the cassette may be eliminated postdifferentiation, or

[0637] the exchange of at least one insertion cassette comprising at least one gene encoding a recombinant protein, allowing for the replacement of this insertion cassette for another insertion cassette comprising at least one gene encoding a different recombinant protein.

[0482] In one aspect, the invention may provide a strategy for generating a de-modified metazoan cell derived from a genetically modified metazoan cell by removal of one or more previously introduced insertion cassettes. In another aspect of the invention, a genetically modified metazoan cell may comprise at least one insertion cassette integrated into the genome, wherein the insertion cassette may be flanked by site-specific recombination sites.

[0638]

[0483] In another aspect of the invention, the exchange system may be utilized for the exchange of at least one insertion cassette located in the genome of a genetically modified metazoan cell (alternatively non-human metazoan cell).

[0639]

[0484] In one aspect of the invention, the insertion cassette involved in immortalization of the metazoan cell (alternatively non-human metazoan cell) may comprise a gene encoding TERT, wherein this insertion cassette in the genome of genetically modified metazoan cell (alternatively non-human metazoan cell) may be removed by the exchange system, wherein the system may comprise, but is not limited to, Cre-Lox recombination or FLP-FRT recombination system.

[0640]

[0485] In another aspect of the invention, the metazoan cells with activated PI3K-Akt signaling pathway and / or TGF-beta signaling pathway and / or cells subjected to Cre-Lox recombination or FLP-FRT recombination may be cultivated in a cultivation system.

[0641]

[0486] In another aspect of the invention, the metazoan cells may be subjected to Cre-Lox recombination or FLP-FRT recombination either prior to cultivation in the cultivation system and / or during cultivation within the cultivation system. For example, the metazoan cells subjected to Cre-Lox recombination or FLP-FRT recombination may include, but are not limited to, metazoan cells with an activated PI3K-Akt signaling pathway and / or TGF-beta signaling pathway, or any other genetically modified cells.

[0642]

[0487] In one aspect, the invention may provide a genetically modified metazoan cell, comprising a PI3K-Akt signaling pathway activated by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K.

[0643]

[0488] In one aspect of the invention, a metazoan cell, comprising a PI3K-Akt signaling pathway activated by at least one adaptation process.

[0644]

[0489] In, one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0645] (a) a PI3K-Akt signaling pathway activated by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K, and

[0646] (b) at least one additional gene encoding a protein, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0647]

[0490] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0648] a PI3K-Akt signaling pathway activated by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K,wherein the genetically modified metazoan cell may be capable of growth in a serum-free medium, a protein-free medium or a combination thereof.

[0649]

[0491] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0650] (a) a PI3K-Akt signaling pathway activated by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K, and

[0651] (b) at least one additional gene encoding a protein, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin, and

[0652] wherein the genetically modified metazoan cell may be capable of growth in a serum-free medium, a protein-free medium or a combination thereof.

[0653]

[0492] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0654] (a) a PI3K-Akt signaling pathway activated by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K, and

[0655] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0656]

[0493] In one aspect, the invention may provide a metazoan cell comprising:

[0657] (a) a genetic modification reducing growth factor requirements in the culture medium achieved through activation of the PI3K-Akt signaling pathway, wherein the activation of PI3K-Akt signaling pathway may be achieved by at least one genetic modification of subunits of PI3K, and

[0658] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium.

[0659]

[0494] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0660] at least one insertion cassette (or for example MGE) comprising:

[0661] (a) a gene encoding a modified form of PI3K; and(b) at least one additional gene encoding a protein that may enhance growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium;

[0662] wherein at least one of said genes (for example PIK3CA, TGFB-1, FGF-2, FGF-5, PDGFR-1) in the insertion cassette (or for example MGE) may be separated by a nucleotide sequence encoding a self-cleaving peptide, wherein said self-cleaving peptide sequence may facilitate a hierarchical expression pattern in which the gene positioned first in the insertion cassette may exhibit a higher expression level relative to each subsequent gene, with progressively decreasing expression levels for each gene in the order in which they are positioned within the insertion cassette.

[0663]

[0495] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0664] (a) a gene encoding a modified form of PI3K; and

[0665] (b) at least one additional gene encoding a protein that may enhance growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, and

[0666] wherein at least one of said genes in the insertion cassette (or for example MGE) may be separated by a nucleotide sequence encoding a self-cleaving peptide, wherein said self -cleaving peptide sequence facilitates a hierarchical expression pattern in which the gene positioned first in the insertion cassette may exhibit a higher expression level relative to each subsequent gene, with progressively decreasing expression levels for each gene in the order in which they are positioned within the insertion cassette.

[0667]

[0496] In one aspect, the invention may provide a method for genetic modification of a metazoan cell (alternatively non-human metazoan cell), the method comprising:

[0668] (a) activating the PI3K-Akt signaling pathway by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K, and

[0669] (b) introducing at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0670]

[0497] In one aspect, the invention may provide a method for genetic modification of a metazoan cell (alternatively non-human metazoan cell), the method comprising:

[0671] (a) activating the PI3K-Akt signaling pathway by at least one genetic modification in a gene encoding a subunit of PI3K resulting in a modified form of PI3K, and(b) introducing at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium.

[0672]

[0498] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell) comprising:

[0673] (a) a TGF-beta signaling pathway activated by at least one genetic modification in a gene encoding a TGF-beta receptor, and

[0674] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0675]

[0499] In one aspect, the invention may provide a genetically modified metazoan cell, comprising:

[0676] (a) a TGF-beta signaling pathway by at least one genetic modification in a gene encoding TGF-beta receptor, and

[0677] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium.

[0678]

[0500] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human metazoan cell), comprising:

[0679] (a) a PI3K-Akt signaling pathway and / or TGF-beta signaling pathway activated by at least one genetic modification, and

[0680] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0681]

[0501] In one aspect, the invention may provide a genetically modified metazoan cell, comprising:(a) a PI3K-Akt signaling pathway and / or TGF-beta signaling pathway activated by at least one genetic modification, and

[0682] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium.

[0683]

[0502] In one aspect, the invention may provide a method for genetic modification of a metazoan cell (alternatively non-human metazoan cell), the method comprising:

[0684] (a) activating the TGF-beta signaling pathway by at least one genetic modification in a gene encoding a TGF-beta receptor, and

[0685] (b) introducing at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0686]

[0503] In one aspect, the invention may provide a method for genetic modification of a metazoan cell (alternatively non-human metazoan cell), the method comprising:

[0687] (a) activating the TGF-beta signaling pathway by at least one genetic modification in a gene encoding a TGF-beta receptor, and

[0688] (b) introducing at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium.

[0689]

[0504] In one aspect, the invention may provide an immortalized genetically modified metazoan cell (alternatively non-human metazoan cell) used as an expression system, comprising a PI3K-Akt signaling pathway or TGF-beta signaling pathway activated by at least one genetic modification, or a combination thereof, wherein the immortalized genetically modified metazoan cell further comprise:

[0690] (a) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2, or mucin, and / or(b) at least one gene encoding a recombinant protein, wherein said gene may be located in an insertion cassette and / or MGE, wherein the metazoan cell (alternatively non-human metazoan cell) may function as an expression system for recombinant protein production.

[0691]

[0505] In one aspect, the invention may provide a food product comprising a non-human metazoan cell suitable for human and / or animal consumption, wherein the non-human metazoan cell further comprises:

[0692] (a) a PI3K-Akt signaling pathway or TGF-beta signaling pathway activated by at least one genetic modification, or a combination thereof, and

[0693] (b) at least one additional gene encoding a protein that may provide an additive effect on enhanced growth, stability, and / or proliferation of the non-human metazoan cell in the absence of growth factors or in a concentration of INS in a range of 0 to 20 mg / L, TGF-beta in a range of 0 to 0.001 mg / L, FGF in a range of 0 to 0.01 mg / L, or TF in a range of 0 to 2.5 mg / L or a combination thereof within the culture medium, wherein said additional gene may comprise at least one of: FGF-1, FGF-2, FGF-5, FGF-8, FGF-9, FGFR1, FGFR2, TGFB-1, TGFB-2, TGFB-3, TGFBR1, TGFBR2, PDGF-1, PDGF-2, PDGFR1, PDGFR2 or mucin.

[0694]

[0506] In one aspect, the invention may provide a method for modifying the genome of a metazoan cell (alternatively non-human metazoan cell), which may comprise at least one insertion cassette in a genomic locus flanked by lox sites and wherein the insertion cassette may be exchanged for another insertion cassette at the same genomic locus through Cre-Lox recombination, wherein the Cre recombinase may enable site-specific excision and replacement of the cassette.

[0695]

[0507] In one aspect, the invention may provide a method for modifying the genome of a genetically modified metazoan cell (alternatively non-human metazoan cell), comprising subjecting the genetically modified metazoan cell to Cre-Lox recombination to remove at least one insertion cassette from its genome, wherein the Cre-Lox recombination may be utilized for at least one of the following modifications:

[0696] (a) removal of at least one insertion cassette involved in immortalization, or

[0697] (b) removal of at least one insertion cassette involved in the recombinant production of a protein responsible for cellular differentiation, wherein the insertion cassette may be eliminated postdifferentiation, or

[0698] (c) exchange of at least one insertion cassette comprising at least one gene encoding a recombinant protein, allowing for its replacement with another insertion cassette comprising at least one gene encoding a different recombinant protein,

[0699] wherein the genetically modified metazoan cell may be subjected to Cre-Lox recombination either prior to cultivation in a cultivation system and / or during cultivation within the cultivation system.

[0700]

[0508] In one aspect, the invention may provide a method for modifying the genome of a metazoan cell (alternatively non-human metazoan cell), which may comprise at least one insertion cassette in a genomic locus flanked by FRT sites and wherein the insertion cassette may be exchanged for another insertion cassette at the same genomic locus through FLP-FRTrecombination, wherein the FLP recombinase may enable site-specific excision and replacement of the cassette.

[0701]

[0509] In one aspect, the invention may provide a method for modifying the genome of a genetically modified metazoan cell (alternatively non-human metazoan cell), comprising subjecting the genetically modified metazoan cell to FLP-FRT recombination to remove at least one insertion cassette from its genome, wherein the FLP-FRT recombination may be utilized for at least one of the following modifications:

[0702] (a) removal of at least one insertion cassette involved in immortalization, or

[0703] (b) removal of at least one insertion cassette involved in the recombinant production of a protein responsible for cellular differentiation, wherein the insertion cassette may be eliminated postdifferentiation, or

[0704] (c) exchange of at least one insertion cassette comprising at least one gene encoding a recombinant protein, allowing for its replacement with another insertion cassette comprising at least one gene encoding a different recombinant protein,

[0705] wherein the genetically modified metazoan cell may be subjected to FLP-FRT recombination either prior to cultivation in a cultivation system and / or during cultivation within the cultivation system.

[0706]

[0510] In one aspect, the invention may provide a genetically modified metazoan cell (alternatively non-human), wherein the genome of the cell may be modified by Cre-Lox recombination or FLP-FRT recombination to remove and / or exchange at least one insertion cassette from the genome, wherein said Cre-Lox recombination or FLP-FRT may achieve at least one modification from the group of:

[0707] (a) removal of at least one insertion cassette involved in immortalization;

[0708] (b) removal of at least one insertion cassette involved in recombinant production of a protein responsible for cellular differentiation, wherein the insertion cassette is eliminated post-differentiation; and

[0709] (c) exchange of at least one insertion cassette comprising at least one gene encoding a recombinant protein with another insertion cassette comprising at least one gene encoding a different recombinant protein.

[0710]

[0511] In some aspects, the present invention provides bovine cell lines with improved properties that may be adapted to various conditions and may be able to grow on a large scale usable for industrial applications.

[0711]

[0512] In one aspect, the invention may provide bovine cells adapted to specific culture conditions as described below. The adaptation may result from at least one mutation in the genome of the cell, wherein the mutation may enhance adaptation, may alter cellular function, or may affect at least one biological pathway or result in any other beneficial trait for the cell. The mutations are described further in the description.

[0712]

[0513] The mutations described herein may result in the enhanced adaptation of the bovine cell to the adaptation processes described below and / or result in any other beneficial trait for the cell.

[0514] In one aspect of the invention, the cell cultivation processes may comprise steps of: obtaining the bovine cells;

[0713] preparing primary cell bank;

[0714] preparing working cell bank;

[0715] modification of cells to provide a gain of function and / or loss of function to the cells;

[0716] preparing production cell bank;

[0717] inoculation of cells to the seeding tank or to the cultivation device:

[0718] cultivation of cells in the cultivation device;

[0719] harvesting the cultured cells; and / or

[0720] preparing the food product.

[0721]

[0515] The step of obtaining bovine cells may optionally comprise one or more processes such as cell isolation, tissue disintegration, separation, purification, preparation of a primary cell bank, preparation of a production cell bank, and / or any other suitable or functionally equivalent procedures.

[0722]

[0516] In one aspect of the invention, the processes may comprise steps of mixing different cell lines before or after the harvesting.

[0723]

[0517] In one aspect of the invention, the processes may comprise the step of differentiation of cells.

[0724]

[0518] The processes according to the invention may optionally comprise the step of preparing a food product for human and / or animal consumption. The food product may be, for example, in the form of pet food or cultured meat product for human consumption, with the desired shape and sensory properties.

[0725]

[0519] In one aspect of the invention, the term "primary cell bank" may refer to a cell bank comprising cells that may be isolated from a biological material and that have not been genetically modified and / or adapted to specific culture conditions. The term "working cell bank" may refer to a cell bank comprising cells that have been genetically modified and / or adapted to specific culture conditions in order to confer a gain of function and / or loss of function phenotype. The term "production cell bank" may refer to a cell bank comprising cells that have been genetically modified and / or adapted to specific culture conditions and may be suitable for use in the production of recombinant proteins, such as antibodies, and / or for the production of food products.

[0726]

[0520] A tissue sample may be taken for the purpose of isolation of cells. The sample may be taken post-mortem, by biopsy from a live animal or from the tissue that was previously frozen. The tissue may be frozen in pieces of various sizes ranging from 0.1 mm2to 5 cm2, or ranging from 1 cm3to 5 cm3, or ranging from 1 mm3to 5 mm3and kept under constant conditions, for example, at temperature in the range of -20 °C to -196 °C, or in the range of -80 °C to -110 °C,or in the range of -85 °C to -100 °C, alternatively the cells of the tissue may be stored in liquid nitrogen, for example at the temperature, in the range of -100 °C to -196°C, or in the range of -130 °C to -190 °C, or in the range -150 °C to -175 °C.

[0727]

[0521] In the case of a post-mortem sample collection, the tissue may be taken from Bos taurus, various breeds may be used, for example, Czech Fleckvieh Cattle (Red Pied, Spotted), Charolais, Angus Aberdeen, Holstein, Belgian blue, Hereford, Simmental, Longhoen, Gelbvieh, Limousin, Highlands, Wagyu or any other appropriate breed of Bos taurus. The anatomical location of tissue sample may be, for example, muscle (semimembranosus, sternomandibularis), connective tissue (connective tissue under the skin above the main muscle at the hind leg, connective fascia cover of muscle segments of the hind leg), or fat tissue (above sternum under the skin, or any other appropriate location). The tissue samples may be taken, for example, in the range of 1 min to 60 mins, in the range of 3 min to 45 min, or in the range of 5 min to 20 min after the animal is slaughtered, The sample size may be in the range of 0.5 g to 30 g, in the range of 2 g to 15 g, in the range of 3 g to 10 g, or in the range of 2 g to 15 g. Immediately after extraction, the samples may be sprayed with ethanol and transferred to Phosphate -Buffered Saline (PBS) with antibiotics and / or antimycotics (e.g. Penicillin, Streptomycin, Amphotericin, and / or any other suitable antibiotics and / or antimycotics) to prevent contamination. Samples may be placed, for example, into glass containers with a volume of 200 mL to 1 L, with 100 mL to 500 mL of PBS, and then transported for further processing, while maintaining a constant temperature. The temperature may be, for example, in the range of 2 °C to 6 °C.

[0728]

[0522] In case of biopsy from live Bos taurus, various breeds may be used, for example, Czech Fleckvieh Cattle (Red Pied, Spotted), Charolais, Angus Aberdeen, Holstein, Belgian blue, Hereford, Simmental, Longhoen, Gelbvieh, Limousin, Highlands, Wagyu or any other appropriate breed of Box taurus. The amount of tissue sample may be in the range of 0.1 g to 5 g, in the range of 0.2 g to 2 g, or in the range of 0.3 g to 1 g. The sample may be taken, for example, from the hind leg with a biopsy needle, which is valid for muscle tissue, connective tissue, and fat tissue as well.

[0729]

[0523] The samples may be then transferred to colder environments, for example, at 2 °C to 6 °C, for further processing, and then optionally isolation.

[0730]

[0524] The tissue sample may be mechanically homogenized, and subsequently, the homogenized tissue may be subjected to enzymatic dissociation in order to obtain dissociated single cells. The enzyme used for dissociation of cells from the tissue may be, for example, collagenase, trypsin, or any other appropriate enzyme. The homogenized tissue may be placed on a shaker at, for example, 0.1 G to 3 G; maintained at a temperature in a range of, for example, 34 °C to 38 °C for the time required for enzyme digestion such as 10 min to 60 min. The cells may then be filtered from tissue residues. The cells may be selected on adherent surfaces (subculture T) and expanded. The cells may then be collected (tissue based) and sorted. The sorted cell types may be expanded (subculture 2). The cell stocks may be frozen, for example, at -75 °C to -196 °C, in order to obtain a primary cell bank. The frozen, uniform cells may be stored in cryovials, wherein each cryovial may contain an amount of cells in a range of 200,000 to 4 million, or in a range of 0.5 million to 3 million, or in a range of 0.7 million to 2 million. The volume of cryovials may be, for example, in a range of 1 mL to 5 mL, or any other appropriate volume.

[0525] Cells may be stored, for example, in cryovials or in other appropriate containers, in liquid nitrogen or in a freezer, while maintaining a constant temperature, for example, in a range of -75 °C to -196 °C.

[0731]

[0526] The cell types used in cultivation processes according to the invention may include various types of bovine cells. These may comprise stem cells, including embryonic stem cells (ESCs) and other cell types derived from blastocysts or early-stage embryos. Muscle stem cells, such as myosatellite cells and mesenchymal stem cells, as well as cells derived from bone marrow, adipose tissue, subcutaneous tissue, or other tissues, may also be utilized. Additionally, the invention encompasses cells in which sternness is induced or established post-isolation, such as induced pluripotent stem cells (iPSCs). Other applicable cell types include myoblasts, myocytes, fibroblasts, fibro-adipogenic progenitors, preadipocytes, and adipocytes. Furthermore, epithelial cells, cartilage cells, tendon-derived cells (such as chondroblasts and chondrocytes), macrophages, keratinocytes, hepatocytes, testicular cells, Sertoli cells, osteoblasts, lipoblasts, odontoblasts, multipotent progenitor cells, myofibroblasts, and endothelial cells may also be employed. The invention further allows for the use of any other suitable cell types or combinations thereof.

[0732]

[0527] In one aspect of the invention, the isolated bovine cells may be spontaneously immortalized. In another aspect of the invention, the cells may be immortalized through adaptation and / or genetic modification.

[0733]

[0528] In one aspect of the invention, the adaptation process may be defined as the gradual exposure of a bovine cell line to changing environmental conditions. This controlled progression may facilitate the selection of cells naturally capable of withstanding environmental stress, thereby enabling their adaptation to newly established conditions. The mechanism underlying such adaptation may involve modulation at both the phenotypic and genotypic levels. As a result, the adapted bovine cell line may exhibit enhanced properties in comparison to the original bovine cell line, wherein the adapted bovine cell line may maintain the key parameters of a cell line, such as phenotypic and genotypic homogeneity.

[0734]

[0529] The bovine cells according to the invention may be adapted to at least one of the following conditions: low concentration and / or absence of signaling proteins, e.g. growth factors in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to the mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0735]

[0530] In one aspect of the invention the bovine cell line may be adapted to at least one specific condition, wherein the specific condition may be from the group of low or zero concentration of signaling proteins (protein-free conditions), e.g. growth factors in culture medium, low concentration of glutamine, proline, or thymidine in culture medium, resilience to a mechanical stress caused by stirring or sparging of the cell culture, suspension cultivation conditions, high concentration of metabolites, for example lactate or ammonium ions in culture medium, defined pH, defined osmolality, e.g. high osmotic pressure, culture medium comprising protein hydrolysate as a source of amino acids, altered oxygen concentration, e.g. lowered oxygen concentration or increased oxygen concentration, serum-free conditions, or any other appropriate conditions, or a combination thereof.1531] The bovine cells according to the invention may be adapted to one or more of the above mentioned conditions and may obtain one or more new modified properties. The cells according to the invention may be adapted independently to any one of the above mentioned conditions, or may be gradually adapted to more than one of the above mentioned conditions and may obtain a combination of more than one new modified properties.

[0736]

[0532] The bovine cells according to the invention may be adapted to growth in alternative cultivation environments such as, for example, culture media comprising protein hydrolysate as the main source of amino acids.

[0737]

[0533] The bovine cells according to the invention may be adapted to at least two of the following conditions: low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0738]

[0534] The bovine cells according to the invention may be adapted to at least three of the following conditions: low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0739]

[0535] The bovine cells according to the invention may be adapted to at least four of the following conditions: low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0740]

[0536] The bovine cells according to the invention may be adapted to at least five of the following conditions: low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0741]

[0537] The bovine cells according to the invention may be adapted to at least six of the following conditions: low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0742]

[0538] The bovine cells according to the invention may be adapted to at least seven of the following conditions: low concentration and / or of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, altered, e.g. increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0743]

[0539] The bovine cell lines according to the invention may comprise:

[0744] (i) at least one adaptation from the group of: suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, or low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium; and

[0745] (ii) at least one adaptation from the group of: low concentration of L-glutamine, increased mechanical stress resistance caused by stirring or sparging of the cell culture, increased oxygen concentration, any other appropriate conditions, or a combination thereof.

[0746]

[0540] The bovine cell lines according to the invention may comprise:

[0747] (i) at least two adaptations from the group of: suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids or low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium: and

[0748] (ii) at least two adaptations from the group of: low concentration of L-glutamine, increased mechanical stress resistance caused by stirring or sparging of the cell culture, increased oxygen concentration, any other appropriate conditions, or a combination thereof.

[0749]

[0541] The bovine cell lines according to the invention may comprise:

[0750] (i) at least three adaptations from the group of: suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids or low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium; and

[0751] (ii) at least three adaptations from the group of:: low concentration of L-glutamine, increased mechanical stress resistance caused by stirring or sparging of the cell culture, increased oxygen concentration, any other appropriate conditions, or a combination thereof.

[0752]

[0542] The bovine cell lines according to the invention may comprise:

[0753] (i) adaptation to suspension cultivation conditions,

[0754] (ii) adaptation to culture medium comprising protein hydrolysate as a source of amino acids, (iii) adaptation to low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium; and

[0755] (iv) at least one adaptation from the group of: low concentration of L-glutamine, increased mechanical stress resistance caused by stirring or sparging of the cell culture, increased oxygen concentration, any other appropriate conditions, or a combination thereof.

[0756]

[0543] The bovine cell lines according to the invention may comprise:

[0757] (i) adaptation to suspension cultivation conditions,

[0758] (ii) adaptation to culture medium comprising protein hydrolysate as a source of amino acids,(iii) adaptation to low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium; and

[0759] (iv) at least two adaptations from the group of: low concentration of L -glutamine, increased mechanical stress resistance caused by stirring or sparging of the cell culture, increased oxygen concentration, any other appropriate conditions, or a combination thereof.

[0760]

[0544] In one aspect of the invention, for the purpose of adaptation, the bovine cells may be cultivated in a culture medium from the group of Skeletal muscle cell growth media (SKGM), Dulbecco's Modified Eagle Medium (DMEM), DMEM7F12, Minimum Essential Medium (MEM), HAM’s F10, HAM’s Fl 2, Iscove’s Modified Dulbecco’s Medium (IMDM), McCoy’s Media and RPMI (Roswell Park Memorial Institute), to be adapted to at least one condition from the group of: low or zero concentration of signaling protein (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, altered (e.g. increased) oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof.

[0761]

[0545] For the adaptation processes, the bovine cell lines may be cultivated at a temperature in a range from 30 °C to 40 °C, or 32 °C to 38 °C, or 33 °C to 36 °C.

[0762]

[0546] The bovine cells according to the invention may be suitable for industrial scale cultivation and may be used for the production of cultivated bovine cell biomass for preparing food products for animal and / or human consumption, for example, for preparing cultivated meat.

[0763]

[0547] The phenotypic characteristics of the bovine cells according to the invention, along with results from genomic and transcriptomic analyses, as well as the processes for deriving the final cell lines from precursor cell lines, are also provided.

[0764]

[0548] In one aspect of the invention, bovine cells may undergo subculturing during the adaptation process to specific culture conditions and may subsequently be cryopreserved in M030 cryopreservation medium (“cryo-medium”) for later use, such as continued adaptation or further application such as, for example, in the preparation of a food product.

[0765]

[0549] The M030 cryo-medium for the storage of cells, including but not limited to bovine cells, may comprise

[0766] (a) methyl cellulose (MC), including its derivatives, substituted forms, chemically modified forms, or functional equivalents thereof;

[0767] (b) trehalose, including derivatives, substituted forms, chemically modified forms, or functional equivalents thereof;

[0768] (c) dimethylsulfoxide (DMSO), including derivatives, substituted forms, chemically modified forms, or functional equivalents thereof; and

[0769] (d) a basal medium.

[0770] The cryo-medium may comprise MC in a range of 0.02 % to 0.5 %, or in a range of 0.05 % to 0.4 %, or in a range of 0.09 % to 0.3 %. The cryo-medium may comprise trehalose in a range of 0.5 % to 3 %, or in a range of 0.7 % to 2.3 %, or in a range of 1 % to 1.9 %. The cryo-medium may comprise DMSO in a range of 5 % to 20 %, or in a range of 8 % to 17 %. or in a range of 11 % to 14 %.

[0550] The M030 cryo-medium for the storage of cells, including but not limited to bovine cells, may comprise methyl cellulose (MC), trehalose, dimethylsulfoxide (DMSO) and culture medium. The cryo-medium may comprise MC in a range of 0.02 % to 0.5 %, or in a range of 0.05 % to 0.4 %, or in a range of 0.09 % to 0.3 %. The cryo-medium may comprise trehalose in a range of 0.5 % to 3 %, or in a range of 0.7 % to 2.3 %, or in a range of 1 % to 1.9 %. The cryo-medium may comprise DMSO in a range of 5 % to 20 %, or in a range of 8 % to 17 %, or in a range of 11 % to 14 %.

[0771]

[0551] In one aspect of the invention, commercially available serum-free cryomedia for eukaryotic cells include, by way of example and without limitation, cryopreservation media intended for mammalian and other eukaryotic cells. Such commercially available cryomedia may include, for example, CryoStor® formulations, Synth-a-Freeze®, FreezIS®, CellBanker® serum-free formulations, or other chemically defined or serum-free cryopreservation media commonly used for the storage of eukaryotic cells. These cryomedia are typically based on basal culture media supplemented with cryoprotective agents, such as dimethyl sulfoxide, sugars, polymers, or other stabilizing components.

[0772]

[0552] For the purposes of the present invention, the term “basal medium” may refer to a conventionally used, commercially available cell culture medium that may provide essential nutrients required for the maintenance and growth of cells. Basal media typically comprise inorganic salts, carbohydrates, amino acids, vitamins, and buffering agents, and may optionally include additional supplements. Exemplary basal media may include, without limitation, Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F-12, DMEM / F-12, RPMI 1640, Iscove’s Modified Dulbecco’s Medium (IMDM), Minimum Essential Medium (MEM), and other standard commercially available basal culture media commonly used for the cultivation of eukaryotic cells.

[0773]

[0553] In one aspect of the invention, the M030 cryo-medium for the storage of cells, including but not limited to bovine cells, may be free of exogenously added FBS and / or growth factors.

[0774]

[0554] The bovine cells after the adaptation process may be centrifuged at Relative Centrifugal Force (RCF) in the range of 10 G to 1000 G, in the range of 80 G to 600 G, or in the range of 100 G to 300 G, for a time period in the range of 1 min to 20 min, or in the range of 2 min to 10 min, and resuspended in cryo-medium. The amount of bovine cells may be, for example, in a range of 0.5 million cells to 100 million per 1 mL, or in a range of 1 million cells to 50 million cells per 1 mL, or 1.5 million cells to 25 million cells per 1 mL of the freezing stock. The stock may be then transferred into a suitable freezing container.

[0775]

[0555] In another aspect of the invention, cells, including but not limited to bovine cells, may be stored in the M030 cryo-medium in a suitable freezing container at a temperature ranging from -80 °C to -196 °C, or ranging from -100 °C to -166 °C, or ranging from -135 °C to -180 °C, or -135 °C to -160 °C.

[0776]

[0556] In another aspect of the invention, cells, including but not limited to bovine cells, may be subjected to cryopreservation in M030 cryo-medium, which may involve slow cooling rate freezing and storage of the frozen cells in M030 cryo-medium. The cooling rate of cells may be in a range of -1 °C to -10 °C per min, or in a range of -2 °C to -8 °C per min, or in a range of -4 °C to - 6 °C per min, alternatively the cooling rate may be in a range of -1 °C to -10 °Cper min, or in a range of -1 °C to -8 °C per min, or in a range of -1 °C to -6,:’C, or in a range of -1 °C to -4 °C.

[0777]

[0557] The cells, including but not limited to bovine cells, may be stored in the M030 cryo¬ medium in a suitable freezing container for long-term storage.

[0778]

[0558] The cell lines, including but not limited to bovine cells, may be stored in the M030 cryo-medium in a suitable freezing container for long-term storage, wherein the cells may be stored in the M030 cryo-medium in the suitable freezing container, for example, for 1 to 1825 days, or 1 to 1095 days, or 1 to 730 days, or 1 to 365 days.

[0779]

[0559] The cell recovery (%) of cells after thawing may be calculated as the ratio of the number of cells after thawing (post-freeze) and before freezing (pre-freeze) multiplied by 100, as depicted in Fig. 12.

[0780]

[0560] In one aspect of the invention, the cell recovery (%) of cells after thawing may be in a range of 55 % to 67 %, or 50 % to 71 %, or 84 % to 92 %, or 80 % to 95 %, and cell viability after thawing may be in a range of 60 % to 95 %, or 75 % to 85 %, wherein the cell recovery of cells after thawing may depend on the type of cell line, duration and / or temperature of storage.

[0781]

[0561] In one aspect of tire invention, cell recovery (%) of cells after thawing when using the M030 cryo-medium is from about 50 % to about 95 %. Preferably, cell recovery may be from about 55 % to about 71 %, more preferably from about 80 % to about 95 %, and most preferably from about 84 % to about 92 %, depending on the cell type and freezing / thawing protocol.

[0782]

[0562] In some embodiments, cell recovery is within the lower preferred window (about 55 % to about 71 %) for cell types that are particularly sensitive to cryostress or when using standard freezing protocols. In other embodiments, for more robust cell types or when optimized freezing and thawing protocols are employed, cell recovery is within the higher preferred window (about 80 % to about 95 %), optionally most preferably about 84 % to about 92 %.

[0783]

[0563] Cell viability after thawing may be from about 60 % to about 95 %, preferably from about 75 % to about 85 %, wherein both cell recovery and cell viability after thawing may depend on the cell type, the duration and temperature of storage, cell density, cryovial volume, cooling rate, thawing procedure, and other process parameters.

[0784]

[0564] The M030 cryo-medium may demonstrate improved and / or comparable performance relative to commercially available serum-free cryomedia. In another aspect of the invention, the cell recovery (%) of cells after thawing using M030 cryo-medium may be increased in comparison to the commercially available serum-free cryomedia, whose cell recovery rate is in the range of 0 % to 30 %.

[0785]

[0565] In one aspect of the invention, the M030 cryo-medium may be utilized for the cryopreservation of cells, including but not limited to bovine cells, wherein said cryo-medium may enhance cell recovery and cell viability upon thawing compared to commercially available serum-free cryomedia. The cell recovery achieved using M030 cryo-medium may be higher than that obtained with currently used commercial serum-free cryomedia, by approximately 0 % to 95 %, or 5 % to 90 %, or 20 % to 80 %, or 30 % to 70 %, or 40 % to 60 %.

[0566] The cells, including but not limited to bovine cells, may be subjected to freezing in M030 cryo-medium and may be stored in commercially used cryogenic storage vials, for example in Nalgene™ and Nunc™ Cryogenic Vials, Eppendorf CryoStorage Vials, Corning® Cryogenic Vials or any other commercially used container.

[0786]

[0567] In one aspect of the invention, the bovine cells adapted to at least one of the following conditions: low concentration and / or absence of signaling proteins (e.g. growth factors) in culture medium, suspension cultivation conditions, culture medium comprising protein hydrolysate as a source of amino acids, increased oxygen concentration, serum-free conditions, resilience to mechanical stress, low concentration of L-glutamine, any other appropriate conditions, or a combination thereof and / or genetically modified bovine cells may be subjected to cryopreservation in the cryo-medium.

[0787]

[0568] In one aspect of the invention, bovine cells may be adapted for growth in serum-free conditions through different strategies:

[0788] (a) sequential adaptation e.g, by stepwise reduction in the volume of culture medium supplemented with FBS while simultaneously increasing the volume of a culture medium supplemented with at least one growth factor, or

[0789] (b) direct adaptation e.g, cultivation of cells in the culture medium not supplemented with FBS.

[0790]

[0569] Initially, the adaptation to serum-free conditions through sequential adaptation may begin with a ratio of 9:1, wherein 90 % of the total medium volume may comprise a culture medium supplemented with FBS, and 10 % of the total medium volume may comprise a culture medium supplemented with at least one growth factor. The growth factor may be from the group of insulin (INS), transferrin (TF), fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), or transforming growth factor beta (TGF-beta), insulin-like growth factor 1 (1GF- 1), insulin-like growth factor 2 (IGF-2), any other appropriate growth factor or a combination thereof. As the adaptation progresses, the ratio of total medium may be adjusted by decreasing volume of culture medium supplemented with FBS and increasing volume of culture medium supplemented with at least one growth factor, until the bovine cells are fully adapted to serum- free conditions and capable to grow in the culture medium without FBS.

[0791]

[0570] In another aspect of the invention, the initial concentration of FBS in the culture medium may be in a range from 5 % to 30 % by volume, or in a range of 7 % to 20 % by volume, or in a range of 8 % to 15 % by volume.

[0792]

[0571] Furthermore, the initial concentration of at least one growth factor in the culture medium may be within the following ranges:

[0793]

[0572] The initial concentration of INS in the culture medium may be in a range of 15 mg / L to 27 mg / L, or in a range of 19 mg / L to 25 mg / L, or in a range of 20 mg / L to 23 mg / L.

[0794]

[0573] The initial concentration of TF in the culture medium may be in a range of 8 mg / L to 16 mg / L, or in a range of 9 mg / L to 13 mg / L, or in a range of 10 mg / L. to 11 mg / L,

[0795]

[0574] The initial concentration of FGF-1 in the culture medium may be in a range of 0.01 mg / L to 3 mg / L, or in a range of 0.09 mg / L to 2.5 mg / L, or in a range of 1.3 mg / L to 1.8 mg / L.

[0575] The initial concentration of FGF-2 in the culture medium may be in a range of 0.01 mg / L to 3 mg / L, or in a range of 0.09 mg / L to 2.5 mg / L, or in a range of 1.3 mg / L to 1.8 mg / L.

[0796]

[0576] The initial concentration of TGF-beta in the culture medium may be in a range of 0.001 mg / L to 1 mg / L, or in a range of 0.05 mg / L to 0.8 mg / L, or in a range of O.lmg / L to 0.5 mg / L.

[0797]

[0577] The initial concentration of IGF-1 in the culture medium may be in the range of 0 ng / mL to 1000 ng / mL, or in the range of 0.01 ng / mL to 700 ng / mL, or in the range of 0.1 ng / mL to 250 ng / mL.

[0798]

[0578] The initial concentration of IGF-2 in the culture medium may be in the range of 0 ng / mL to 1000 ng / mL, or in the range of 0.01 ng / mL to 700 ng / mL, or in the range of 0.1 ng / mL to 250 ng / mL.

[0799]

[0579] Furthermore, the initial concentration of at least one growth factor in the culture medium may be within the following ranges:

[0800] The initial concentration of INS in the culture medium may be in a range of 15 mg / L to 27 mg / L, or in a range of 19 mg / L to 25 mg / L, or in a range of 20 mg / L to 23 mg / L.

[0801] The initial concentration of TF in the culture medium may be in a range of 8 mg / L to 16 mg / L, or in a range of 9 mg / L to 13 mg / L, or in a range of 10 mg / L to 11 mg / L.

[0802] The initial concentration of FGF-1 in the culture medium may be in a range of 0.01 mg / L. to 3 mg / L, or in a range of 0.09 mg / L to 2.5 mg / L, or in a range of 1.3 mg / L to 1.8 mg / L.

[0803] The initial concentration of FGF-2 in the culture medium may be in a range of 0.01 mg / L to 3 mg / L, or in a range of 0.09 mg / L to 2.5 mg / L, or in a range of 1.3 mg / L to 1.8 mg / L.

[0804] The initial concentration of TGF-beta in the culture medium may be in a range of 0.001 mg / L to 1 mg / L, or in a range of 0.05 mg / L to 0.8 mg / L, or in a range of O.lmg / L to 0.5 mg / L.

[0805] The initi al concentration of IGF- 1 in the culture medium may be in the range of 0 ng / mL to 1000 ng / mL, or in the range of 0.01 ng / mL to 700 ng / mL, or in the range of 0.1 ng / mL to 250 ng / mL.

[0806] The initial concentration of IGF-2 in the culture medium may be in the range of 0 ng / mL to 1000 ng / mL, or in the range of 0.01 ng / mL. to 700 ng / mL, or in the range of 0.1 ng / mL to 250 ng / mL,

[0807]

[0580] During the adaptation process, bovine cells may be monitored for growth and survival performance, wherein adaptation to a serum-free condition is assessed based on population doubling time (PDT), population doubling level (PDL), and / or cell viability.

[0808]

[0581] In one aspect of the invention, the direct adaptation may comprise cultivating bovine cells in a culture medium that does not comprise FBS, until the growth rate of the cells is comparable to that observed in a culture medium comprising FBS.

[0809]

[0582] In one aspect of the invention, the direct adaptation process may comprise cultivating bovine cells in a culture medium comprising FBS, followed by replacing the culture medium with a serum-free culture medium supplemented with at least one growth factor, and subsequently subculturing the cells until their growth rate becomes comparable or higher to that observed in the culture medium comprising FBS. The growth factor may be from the group ofINS, TF, FGF-1, FGF-2, or TGF-beta, IGF-1, IGF-2, any other appropriate growth factor or a combination thereof.

[0810]

[0583] Bovine cells may be cultivated in adherent culture for a number of sub-cultures ranging from 1 to 100, or ranging from 3 to 80, or ranging from 5 to 65, or ranging from 7 to 52, or ranging from 9 to 35, or ranging from 11 to 24 in order to verify that cells are able to grow in serum-free conditions.

[0811]

[0584] In one aspect of the invention, bovine cells may be adapted to growth in protein-free conditions, or conditions with low concentration of protein.

[0812]

[0585] The protein may comprise for example at least one signaling protein, such as growth factor. The signaling protein may comprise at least one of: FGF family growth factor (e.g. FGF- 1, FGF-2), INS, IGF-1, IGF-2, TGF beta family ligands, TF, or any other appropriate signaling protein.

[0813]

[0586] In another aspect of the invention, the bovine cells may be cultivated in the culture medium supplemented with at least one of sodium selenite, ethanolamine, ferric citrate, ferric ammonium citrate, or with any other appropriate supplement. This step may serve as confirmation that bovine cells are able to grow in the absence of exogenous signaling proteins.

[0814]

[0587] Furthermore, the concentration of sodium selenite in the culture medium may be in a range of 0.01 mg / L to 0.11 mg / L, or in a range of 0.02 mg / L to 0.09 mg / L, or in a range of 0.04 mg / L to 0.06 mg / L.

[0815]

[0588] Furthermore, the concentration of ethanolamine in the culture medium may be in a range of 0.001 mg / L. to 15 mg / L., or in a range of 0.1 mg / L. to 13.5 mg / I., or in a range of 2 mg / L to 7 mg / L, or in a range of 4 mg / L to 6 mg / L.

[0816]

[0589] Furthermore, the concentration of ferric citrate in the culture medium may be in a range of 0.5 mg / L to 500 mg / L, or in a range of 20 mg / L. to 450 mg / L, or in a range of 100 mg / L to 400 mg / L, or in a range of 145 mg / L to 325 mg / L, or in a range of 230 to 300 mg / L.

[0817]

[0590] Furthermore, the concentration of ferric ammonium citrate in the culture medium may be in a range of 0.5 mg / L to 500 mg / L, or in a range of 20 mg / L to 450 mg / L, or in a range of 100 mg / L. to 400 mg / L., or in a range of 145 mg / L to 325 mg / L, or in a range of 230 to 300 mg / L.

[0818]

[0591] The initial concentration of protein (e.g. signaling protein) in the culture medium for cell adaptation to low or protein-free conditions may be in the range of 0.001 mg / L to 2,000 mg / L, or in the range of 0.01 mg / L to 250 mg / L, or in the range of 0.1 mg / L to 50 mg / L.

[0819]

[0592] The initial concentration of protein (e.g. signaling protein) in the culture medium for cell cultivation may be in the range of 0 mg / L to 2000 mg / L, or in the range of 0.01 mg / L to 250 mg / L, or in the range of 0.1 mg / L. to 50 mg / L.

[0820]

[0593] The initial concentration of IGF-1 in the culture medium may be in the range of 0 ng / mL to 1,000 ng / mL, or in the range of 0.01 ng / mL to 700 ng / mL, or in the range of 0.1 ng / mL to 250 ng / mL.

[0594] The initial concentration of IGF-2 in the culture medium may be in the range of 0 ng / mL to 1,000 ng / mL, or in the range of 0.01 ng / mL to 700 ng / mL, or in the range of 0.1 ng / mL to 250 ng / mL.

[0821]

[0595] The initial concentration of TGF beta in the culture medium may be in the range of 0 mg / L to 0.002 mg / L.

[0822]

[0596] The initial concentration of TF in the culture medium may be in the range of 0 mg / L to 10 mg / L, or in the range of 0.1 mg / L to 8 mg / L, or in the range of 0.5 mg / L to 5 mg / L. In one aspect of the invention, the reduced amount of TF may be in the range of 0 mg / L to 0.01 mg / L.

[0823]

[0597] The initial concentration of INS in the culture medium may be in the range of 0 g / L to 2 g / L, or in the range of 0.1 mg / L to 1 g / L, or 0.5 mg / L to 500 mg / L. In one aspect of the invention, the bovine cells may be adapted to grow in the culture medium comprising a reduced amount of INS that may be in the range of 0 mg / L to 0.1 mg / L.

[0824]

[0598] The initial concentration of FGF-1 in the culture medium may be in the range of 0 mg / L.

[0825] to 1 mg / L, or in the range of 0.05 mg / L to 0.8 mg / L, or 0.1 mg / L to 0.5 mg / L. In one aspect of the invention, the bovine cells may be adapted to grow in the culture medium comprising a reduced amount of FGF-1 that may be in the range of 0 mg / L to 0.01 mg / L.

[0826]

[0599] The initial concentration of FGF-2 in the culture medium may be in the range of 0 mg / L to 1 mg / L, or in the range of 0.05 mg / L to 0.8 mg / L, or 0.1 mg / L to 0.5 mg / L. In one aspect of the invention, the bovine cells may be adapted to grow in the culture medium comprising a reduced amount of FGF-2 that may be in the range of 0 mg / L to 0.01 mg / L.

[0827]

[0600] The initial concentration of TGF beta in the culture medium may be in the range of 0 mg / L to 0.2 mg / L, or in the range of 0.01 mg / L to 0.15 mg / L, or 0.05 mg / L to 0.1 mg / L. In one aspect of the invention, the bovine cells may be adapted to grow in the culture medium comprising a reduced amount of TGF beta that may be in the range of 0 mg / L to 0.001 mg / L.

[0828]

[0601] In one aspect of the invention, the adapted bovine cells may be able to grow in the culture medium without at least one signaling protein, for example growth factors, or in the culture medium with a low-concentration of at least one signaling protein, for example growth factors.

[0829]

[0602] During the adaptation process, bovine cells may be monitored for growth and survival performance, wherein adaptation to a reduced concentration of at least one growth factor is assessed based on PDT, PDL, and / or cell viability.

[0830]

[0603] Metazoan cells can commonly be cultivated in adherent culture as a cell layer upon a suitable cultivation surface, such as plasma-treated polystyrene, gelatin or matrigel. Without a suitable cultivation surface, most metazoan cell types fail to proliferate or die from lack of attachment (anoikis). In order to produce sufficient biomass of cells for cultivated meat applications, the cultivation surface would need to be impractically large. Therefore, it is necessary to adapt the cells to suspension cultivation conditions, enabling them to proliferate without attachment to a solid surface, thereby facilitating scalable, high-density cell cultivation.

[0831]

[0604] In one aspect of the invention, bovine cells may be adapted to growth in suspension cultivation conditions. Under these conditions, bovine cells may be able to grow with minimaladhesion to the culture vessel as a suspension of single cells and / or small cell aggregates, and may be subcultured with a simple transfer of cell suspension to a new culture vessel.

[0832]

[0605] Adaptation to suspension culture may enable the cultivation of these cells in large-scale processes (in a bioreactor), unlike adherent culture methods. The adaptation process to suspension cultivation conditions may start with adherent cells or with mixed culture of adherent and suspension cells. Agitation may be used in order to encourage suspension growth, for example, in spinner flasks or bioreactors.

[0833]

[0606] In one aspect of the invention, the adaptation process of bovine cells to suspension cultivation conditions in Erlenmeyer flasks and / or 6-well plates (clear bottom, flat) may involve exposure to agitation conditions, wherein the agitation rate may be initiated at a speed, expressed in revolutions per minute (rpm), ranging from 74 rpm to 350 rpm, or 75 rpm to 125 rpm, or 120 rpm to 330 rpm, or 150 rpm to 300 rpm, or 200 rpm to 250 rpm. During this adaptation process, bovine cells may be subcultured, and the adaptation may continue. In another aspect of the invention, during the adaptation process, bovine cells may either directly transition into a single-cell suspension or initially form size-defined cell aggregates, or a combination thereof.

[0834]

[0607] In another aspect of the invention, the size-defined cell aggregates may comprise spheroids, organoids, myospheres or any other appropriate size-defined cell aggregate, wherein the preferable size-defined cell aggregate may have a size ranging from 20 μm to 400 μm, from 25 μm to 320 μm, or from 50 μm to 170 μm.

[0835]

[0608] In another aspect of the invention, during the adaptation of the bovine cells to suspension conditions, the cells may be cultivated in a material from the group of plastic (for example polypropylene, polystyrene, polycarbonate, polyethylene terephthalate glycol (PETG)), glass, and / or bio-glass, wherein the material may be coated to prevent bovine cells from attaching to the solid surface. The material may be coated with polyethylene glycol (PEG), polyvinyl alcohol (PVA), polysulfone (PSU), any other appropriate coating material, or combinations thereof.

[0836]

[0609] For the cultivation of bovine cells in suspension conditions, the culture medium may be supplemented with a shear protectant such as Pluronic F-68 or with any other appropriate shear protectant.

[0837]

[0610] A shear protectant may refer to a substance or compound that protects cells, biomolecules, or biological materials from mechanical stress during culture conditions. Shear stress may result from agitation, flow forces, pipetting, or mixing in culture vessels, leading to cell membrane damage, protein denaturation, or reduced cell viability. Shear protectants may function by absorbing, reducing, or redistributing mechanical forces, thereby preserving the structural and functional integrity of the biological material. In another aspect of the invention, shear stress may be considered a subset of mechanical stress.

[0838]

[0611] The shear protectant may comprise at least one of: polyethylene glycol, Pluronic F68, Pluronic F127, methyl cellulose, (hydroxypropyl)methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), or dextran sulfate, or a combination thereof.

[0612] The total input of the shear protectant to culture medium may be in the range of 0.005 g / L to 10 g / L, or in the range of 0.02 g / L to 8 g / L, or in the range of 0.03 g / L to 5 g / L, or in the range 0.04 g / L to 3 g / L.

[0839]

[0613] Cell clumping may be prevented by optimizing agitation rates and culture medium composition, and / or adding anti-clumping agents into the culture medium, thereby maintaining a uniform suspension culture.

[0840]

[0614] An anti-clumping agent may refer to a substance or compound that prevents the aggregation or clustering of cells in suspension cultures. Cell clumping can hinder uniform growth, reduce mass transfer efficiency, and complicate downstream processing.

[0841]

[0615] The total input of anti-clumping agent to culture medium may be in the range of 0.0005 g / L to 12 g / L, or in the range of 0.001 g / L to 10 g / L, or in the range of 0.1 g / L to 8 g / L, or in the range of 0.3 g / L to 5 g / L.

[0842]

[0616] The anti-clumping agent may comprise PEG, Pluronic F68, Pluronic F127, MC, HPMC, HPC, CMC, dextran sulfate, xantham gum, gellan gum, Tween20, Tween80, Tween- X-100, lecithin or a combination thereof.

[0843]

[0617] In another aspect of the invention, optimizing the composition of the culture medium, for example by chelation of divalent ions by addition of trisodium citrate dihydrate, ethylenediaminetetraacetic acid, nitrilotriacetic acid or gluconic acid or any other chelating agent into the culture medium, may influence the degree of cell aggregation within the medium.

[0844]

[0618] In another aspect of the invention, bovine cells may be adapted to mechanical stress induced by stirring and / or shear stress during cultivation under suspension conditions. The adaptation may be achieved by cultivating the cells in the presence of at least one shear protectant and / or at least one anti-clumping agent.

[0845]

[0619] In bioreactor-based cultivation of metazoan cells, including bovine cells, oxygen is typically supplied at low concentrations to approximate physiological conditions. However, due to aeration and mechanical agitation used to ensure adequate mixing and nutrient distribution, the effective oxygen concentration within the culture medium may exceed physiological levels. This is particularly evident during early cultivation phases when viable cell density is low and oxygen transfer efficiency is high.

[0846]

[0620] Higher agitation rates further contribute to increased oxygen transfer, leading to elevated dissolved oxygen (DO) levels. The increased oxygen levels, combined with variations in oxygen distribution throughout the bioreactor, create heterogeneous microenvironments where some metazoan cells, for example bovine cells, may experience oxygen oversaturation while others may be exposed to lower oxygen levels. These fluctuations may induce oxidative stress in non-adapted cells, which may result in reduced cell viability, altered metabolism, and impaired overall process performance. Therefore, adapting bovine cells to elevated oxygen concentrations is critical for large scale cultivation processes, ensuring their survival, maintaining metabolic stability, and improving overall process robustness. This adaptation may minimize adverse effects caused by oxygen fluctuations within the bioreactor and may enable cells to function optimally under process-relevant conditions.

[0621] The adaptation of bovine cells to elevated oxygen concentrations in a cultivating incubator environment (gas composition in terms of volumetric fraction of oxygen) may be achieved by one or more of the following strategies:

[0847] (i) direct adaptation, wherein bovine cells are immediately exposed to an oxygen concentration in the range of 3.5 % to 25 % oxygen by volume, or in the range of 5 % to 20 % oxygen by volume, or in the range of 10 % to 15 % oxygen by volume, and subcultured until stable adaptation is reached, (ii) gradual adaptation, wherein bovine cells may be initially cultivated at oxygen volumetric fraction ranging from 3.5 % to 7 %, followed by incremental increase in oxygen concentration until reaching 18 % to 25 % of oxygen,

[0848]

[0622] In addition to incubator-based systems, adaptation may be implemented also in a cultivation vessel. The elevated agitation rates may further enhance oxygen transfer, and consequently raise DO levels, particularly during early growth phases. To promote cellular adaptation to elevated oxygen concentrations, bovine cells may be cultivated under controlled DO set-points, defined as relative values of dissolved oxygen expressed as a percentage of the saturation concentration.

[0849]

[0623] The DO set-points may be maintained within defined operational ranges, such as from 5 % to 80 %, or from 15 % to 40 %, or from 17.5 % to 25 %, depending on the phase of cultivation, cell type and / or the specific process design, The DO level is actively regulated by a Proportional-Integral-Derivative (PID) control loop, which dynamically adjusts stirring speed and aeration rate in response to the oxygen mass transfer demands imposed by increasing cell density.

[0850]

[0624] This control strategy may ensure that sufficient oxygen concentrations are delivered to meet cellular metabolic needs while avoiding excessive DO levels that could negatively impact cell physiology. Through this approach, bovine cells may be progressively adapted to elevated oxygen concentrations, enhancing their resilience and improving overall process robustness under industrially relevant conditions.

[0851]

[0625] In one aspect of the invention, the selection of the adaptation strategy may depend on the specific bovine cell line, target production characteristics, and process scalability requirements in bioreactor-based cultivation systems.

[0852]

[0626] The cell specific oxygen consumption in continuous culture may be in the range of 1 pmol / cell / day to 15 pmol / cell / day, or in the range of 5 pmol / cell / day to 10 pmol / cell / day, or in the range of 6 pmol / cell / day to 9 pmol / cell / day, or in the range of 7 pmol / cell / day to 8 pmol / cell / day.

[0853]

[0627] Adapted bovine cells may exhibit equal or improved culture performance, measured in terms of growth rate and viability, at dissolved oxygen concentrations in the range of 3 % to 200 %, or in the range of 5 % to 100 %, or in the range of 10 % to 50 %, or in the range of 15 % to 25 %, where dissolved oxygen concentration is defined as a ratio of dissolved concentration to the saturation concentration obtained at the atmospheric pressure (values higher than 100 % indicate that the cultivating system is pressurized). Moreover, cell-specific oxygen consumption, glucose consumption, and lactate production may remain comparable to baseline at the adapted oxygen partial pressures, indicating maintenance of metabolic function under elevated DO conditions.

[0628] In one aspect of the invention, the bovine cells may be adapted to elevated oxygen concentration, the method of adaptation comprising:

[0854] (i) cultivating bovine cells in the culture medium with oxygen concentration exceeding physiological levels; and

[0855] (ii) maintaining the cells under either a direct adaptation or a gradual adaptation approach until the cells exhibit stable growth and viability at the elevated oxygen concentration, wherein the oxygen concentration is defined as follows:

[0856] (a) for incubator-based cultivation using a direct adaptation approach, the oxygen concentration may be in a range of 3.5 % to 25 %, or in a range of 5 % to 20 %, or in the range of 10 % to 15 % of oxygen volumetric fraction;

[0857] (b) for incubator-based cultivation using gradual adaptation approach, the oxygen concentration may initially be in a range of 3.5 % to 7 % of oxygen volumetric fraction, followed by incremental increase in oxygen concentration until reaching 18 % to 25 %;

[0858] (c) for cultivation in a culture vessel, the dissolved oxygen concentration may be maintained at defined set-points such as, expressed as a percentage of relative oxygen saturation concentration (%), in a range of 10 % to 80 %, or in a range of 15 % to 40 %, or in a range of 17.5 % to 25 %.

[0859]

[0629] All or the vast majority of the amino acids may be supplied to the culture medium in the form of a protein hydrolysate. Additionally, protein hydrolysates may contain amino acids and / or peptides which may also be consumed by the cells or have other beneficial effects, for example, short peptides may serve as signaling compounds and promote cell growth.

[0860]

[0630] One of the approaches to characterize protein hydrolysates may be size-exclusion chromatography (SEC) combined with mass spectrometry (MS) or photo diode array (PDA) which allows for separation of the peptides from hydrolysate based on their molecular weights. The results may vary according to mobile phase composition, column type and its specification, flow rate, temperature, sample injection parameters, detector settings and / or any other parameter used.

[0861]

[0631] In one aspect of the invention, the protein hydrolysate may comprise peptides with molecular weights higher than 17 kDa, with a relative fraction size in a range 0 % to 10 % of the protein hydrolysate, in a range of 0 % to 8 % of the protein hydrolysate, or in a range of 0 % to 6 % of the protein hydrolysate.

[0862]

[0632] In one aspect of the invention, proteolytic enzymes within a source of protein may yield peptides in purified protein hydrolysate with a molecular weights ranging from 6.7 to 17 kDa, with a relative fraction size in a range 1 % to 35 % of the purified protein hydrolysate, in a range of 4 % to 30 % of the purified protein hydrolysate, or in a range of 5 % to 25 % of the purified protein hydrolysate.

[0863]

[0633] In one aspect of the invention, proteolytic enzymes within a source of protein may yield peptides in purified protein hydrolysate with a molecular weights ranging from 1.7 kDa to 6.7 kDa, with a relative fraction size in a range 1 % to 40 % of the purified protein hydrolysate, ina range of 10 % to 35 % of the purified protein hydrolysate, or in a range of 15 % to 30 % of the purified protein hydrolysate.

[0864]

[0634] In one aspect of the invention, proteolytic enzymes within a source of protein may yield amino peptides in purified protein hydrolysate with a molecular weights ranging from 1 kDa to 1.7 kDa, with a relative fraction size in a range 1 % to 40 % of the purified protein hydrolysate, in a range of 10 % to 35 % of the purified protein hydrolysate, or in a range of 20 % to 30 % of the purified protein hydrolysate.

[0865]

[0635] In one aspect of the invention, proteolytic enzymes within a source of protein may yield free amino acids and / or peptides in purified protein hydrolysate with a molecular weights less than 1 kDa, with a relative fraction size in a range 20 % to 100 % of the purified protein hydrolysate, in a range of 22 % to 70 % of the purified protein hydrolysate, or in a range of 25 % to 60 % of the purified protein hydrolysate.

[0866]

[0636] In one aspect of the invention, the protein hydrolysate may comprise peptides with a relative fraction size in a range of 30 % to 50 % of the protein hydrolysate, in a range of 35 % to 45 % of the protein hydrolysate, or in a range of 38 % to 42 % of the protein hydrolysate.

[0867]

[0637] In one aspect of the invention, an additional method may be used to further separate the small peptides with molecular weights ranging from 1,000 Da to 6,500 Da, 600 Da to 1,000 Da, 300 Da to 600 Da, or molecular weights lower than 300 Da.

[0868]

[0638] In one aspect of the invention, proteolytic enzymes within a source of protein may yield amino acids and / or peptides in purified protein hydrolysate with a molecular weights ranging from 1,000 Da to 6,500 Da, with a relative fraction size in a range of 1 % to 70 %, in a range of 5 % to 40 %, or in a range of 10 % to 35 % of the measured fractions.

[0869]

[0639] In one aspect of the invention, proteolytic enzymes within a source of protein may yield amino acids and / or peptides in purified protein hydrolysate with a molecular weights ranging from 600 Da to 1,000 Da, with a relative fraction size in a range of 1 % to 60 %, in a range of 2 % to 40 %, or in a range of 5 % to 30 % of the measured fractions.

[0870]

[0640] In one aspect of the invention, proteolytic enzymes within a source of protein may yield amino acids and / or peptides in purified protein hydrolysate with a molecular weights ranging from 300 Da to 600 Da, with a relative fraction size in a range of 1 % to 60 %, in a range of 2 % to 40 %, or in a range of 5 % to 30 % of the measured fractions.

[0871]

[0641] In one aspect of the invention, proteolytic enzymes within a source of protein may yield amino acids and / or peptides in purified protein hydrolysate with a molecular weights less than 300 Da, with a relative fraction size in a range of 20 % to 100 %, in a range of 30 % to 80 %, or in a range of 40 % to 70 % of the measured fractions.

[0872]

[0642] The bovine cells according to the invention may be adapted to culture media comprising protein hydrolysate as a source of amino acids. The culture medium according to the invention may comprise soy protein hydrolysate, or any other appropriate protein hydrolysate. Examples of suitable industrially scalable protein sources for hydrolysis may include soy, pea, rice, wheat, corn, fava beans, alfalfa, hemp, chickpea, potato, pumpkin, rapeseed, red lentil, Spirulina, Chlorella, sunflower, water lentil, mung bean, flax or baker'syeast, or any other appropriate protein source. The present invention is not limited to the listed exemplary sources of protein,

[0873]

[0643] The cells may be adapted to hydrolysate gradually in several steps in order to avoid drastic change in cell cultivation conditions. The number of steps of hydrolysate concentration change or change of concentration of free amino acids added to the medium separately may be in a range of 1 to 10, or in the range of 2 to 8, or in the range of 3 to 6.

[0874]

[0644] For example, the concentration of soy protein hydrolysate in the culture medium may be increased in several steps, wherein the concentration of soy protein hydrolysate in the culture medium in the first step may be in a range of 0.2 g / L to 1.5 g / L, or in the range of 0.4 g / L to 1.2 g / L, or in the range of 0.6 g / L to 1 g / L. In the second step, the concentration of soy protein hydrolysate may be in a range of 1 g / L to 3 g / L, or in the range of 1.5 g / L to 2.5 g / L, or in the range of 1.7 g / L to 2.2 g / L. In the third step, the concentration of soy protein hydrolysate may be in a range of 2 g / L to ...

Claims

1. A cell line adapted for cultivation under at least one culture condition from the group of:(a) culture in a culture medium comprising one or more signaling proteins in a concentration in a range of 0 mg / L to 2 mg / L; or(b) culture in suspension cultivation conditions; or(c) culture in a culture medium comprising a protein hydrolysate as a source of amino acids; or any combination thereof.

2. The cell line according to claim 1, wherein the cell line is adapted to be resistant to negative effects of lactate during cultivation by culturing in a culture medium comprising lactate in a concentration in a range of 10 mg / L to 500 mg / L.

3. The cell line according to claim 1, wherein the cell line is a Chinese hamster ovary (CHO) or bovine cell line.

4. The cell line according to claim 3, wherein the cell line is a CHO-derived cell line adapted to synthesize L-proline, wherein the CHO-derived cell line is proline-prototrophic and is capable of proliferation in a culture medium that is free of L-proline or comprises a low concentration of L- proline.

5. The cell line according to claim 3, wherein the cell line is a CHO-derived cell or a cell line comprising a plurality of CHO-derived cells, wherein the CHO-derived cell comprises at least 80% of the small mutations listed in Table 1 or 80% of the small mutations listed in Table 2, wherein said small mutations are defined relative to a CHO-K1 reference genome and comprise nucleotide- level sequence variations between a reference genome allele and an adapted cell allele.

6. The cell line according to claim 3, wherein the cell line is an adapted bovine cell line exhibiting equal or improved culture performance at dissolved oxygen concentrations in a range of 3 % to 200 %, as measured by growth rate and / or cell viability.

7. The cell line according to claim 3, wherein the cell line is an adapted bovine cell line exhibiting a stable genomic profile and comprising at least 70 % of the intergenic mutations listed in Table 15, wherein said intergenic mutations are defined relative to the Bos taurus reference genome.

8. A method of cultivating a cell line, comprising culturing the cell line in a culture medium under at least one culture condition from the group of:(a) culture in a culture medium comprising one or more signaling proteins in a concentration in a range of 0 mg / L to 2 mg / L; or(b) culture in suspension cultivation conditions; or(c) culture in a culture medium comprising a protein hydrolysate as a source of amino acids, or any combination thereof.

9. The method according to claim 8, comprising culturing the cell line in a culture medium comprising lactate in a concentration in a range of 10 mg / L to 500 mg / L, thereby adapting the cell line to resist negative effects of lactate.

10. The method according to claim 8, wherein the cell line is a proline-prototrophic CHO-derived cell line, and wherein the method comprises culturing the cell line in a culture medium that is free of L-proline or comprises L-proline in a range of 0 mg / L to 10 mg / L.

11. The method according to claim 8, wherein the cell line is an adapted bovine cell line and wherein the method comprises culturing the cell line a dissolved oxygen concentration in a range of 3 % to 200 %, and wherein the adapted bovine cell line exhibits equal or improved growth rate and / or cell viability under said dissolved oxygen conditions.

12. A genetically modified metazoan cell line comprising at least one genetic modification, wherein the genetic modification results in activation of PI3K–Akt signaling, TGF-beta signaling pathway, or both relative to an otherwise identical cell line lacking said genetic modification.

13. The genetically modified metazoan cell line according to claim 12, wherein activation of the PI3K–Akt signaling pathway is provided by at least one genetic modification in a gene encoding a phosphoinositide 3-kinase (PI3K) subunit, thereby producing a modified form of PI3K, or by at least one genetic modification in a gene encoding fibroblast growth factor receptor (FGFR).

14. The genetically modified metazoan cell line according to claim 12, wherein activation of the TGF-beta signaling pathway is provided by at least one genetic modification in a gene encoding a TGF-beta receptor.

15. A method for generating a genetically modified metazoan cell line, the method comprising: introducing at least one genetic modification into a metazoan cell,wherein the genetic modification results in activation of a PI3K-Akt signaling pathway, a TGF-beta signaling pathway, or both, relative to an otherwise identical cell lacking said genetic modification.

16. The method according to claim 15, wherein the genetic modification comprises at least one genetic modification in a gene encoding a phosphoinositide 3-kinase (PI3K) subunit or at least one genetic modification in a gene encoding fibroblast growth factor receptor (FGFR), thereby activating the PI3K-Akt signaling pathway.

17. The method according to claim 15, wherein the genetic modification comprises a modification in a gene encoding a TGF-beta receptor, thereby activating the TGF-beta signaling pathway.

18. A food product comprising a non-human metazoan cell line, wherein the cell line:(a) is adapted to at least one culture condition from the group of:(i) culture in a culture medium comprising one or more signaling proteins in a concentration in a range of 0 mg / L to 2 mg / L; or(ii) culture in a suspension cultivation conditions; or(iii) culture in a culture medium comprising a protein hydrolysate as a source of amino acids;or any combination thereof;(b) comprises at least one genetic modification, wherein the genetic modification results in activation of a PI3K-Akt signaling pathway, a TGF-beta signaling pathway, or both relative to an otherwise identical cell line lacking said genetic modification; or (c) a combination of (a) and (b).

19. A food product according to claim 18, wherein the food product does not comprise immortalized non-human metazoan cells.

20. A food product according to claim 18, wherein the food product comprises non-human metazoan cells in a range of 0.1 % to 100 %.

21. A genetically modified metazoan cell or de-modified metazoan cell obtained by a method comprising subjecting a genetically modified metazoan cell to an exchange system to remove or exchange at least one insertion cassette from the genome.

22. The genetically modified metazoan cell or de-modified metazoan cell according to claim 21, wherein the exchange system comprises a Cre–Lox recombination system, an FLP–FRT recombination system, or both.

23. The genetically modified metazoan cell or de-modified metazoan cell according to claim 21, wherein the de-modified metazoan cell is obtained by a method comprising subjecting an immortalized genetically modified metazoan cell comprising at least one insertion cassette that is involved in immortalization to an exchange system to remove the at least one insertion cassette involved in immortalization from the genome.

24. A method for modifying the genome of a genetically modified metazoan cell, the method comprising subjecting the genetically modified metazoan cell to an exchange system to remove or exchange at least one insertion cassette from the genome of the genetically modified metazoan cell.

25. The method according to claim 24, wherein the exchange system comprises a Cre–Lox recombination system, an FLP–FRT recombination system, or both.

26. The method according to claim 24, comprising subjecting an immortalized genetically modified metazoan cell comprising at least one insertion cassette that is involved in immortalization to an exchange system to remove the at least one insertion cassette involved in immortalization from the genome.

27. A cryo-medium for storage of metazoan cells, comprising:(a) methyl cellulose (MC), or a derivative, substituted form, chemically modified form, or functional equivalent thereof; and(b) trehalose, or a derivative, substituted form, chemically modified form, or functional equivalent thereof; and(c) dimethyl sulfoxide (DMSO), or a derivative, substituted form, chemically modified form, or functional equivalent thereof; and(d) a basal medium,wherein the cryo-medium is free of exogenously added fetal bovine serum (FBS), exogenously added growth factors, or both.

28. The cryo-medium according to claim 27, wherein cell recovery of cells stored in the cryo- medium after thawing is in a range of about 50 % to about 95 %.

29. A method for cryopreserving metazoan cells, the method comprising:(a) contacting the cells with a cryo-medium to form a cell suspension; and(b) freezing the cell suspension for storage,wherein the cryo-medium is free of exogenously added fetal bovine serum (FBS), exogenously added growth factors, or both.

30. The method according to claim 29, further comprising thawing the cells after storage, wherein cell recovery of cells after thawing is in a range of about 50 % to about 95 %.

Images